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(12) United States Patent (10) Patent No.: US 8,758,584 B2 Kahn et al. (45) Date of Patent: Jun. 24, 2014

(54) ELECTROCHEMICAL SENSORS 422/68.1, 83.02, 82.03; 428/149, 1.23, 428/405; 324/438: 257/4 (75) Inventors: Carolyn R. Kahn, San Francisco, CA See application file for complete search history. (US); Elicia Wong, Foster City, CA (US); James A. Wilkins, San Francisco, (56) References Cited CA (US); Vern Norviel, San Francisco, U.S. PATENT DOCUMENTS CA (US) 3.682,160 A 8, 1972 Murata (73) Assignee: Sensor Innovations, Inc., Burlingame, 3,926,764 A 12/1975 Ruzicka et al. CA (US) 3,982,960 A 9/1976 Hoekje et al. (Continued) (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 FOREIGN PATENT DOCUMENTS U.S.C. 154(b) by 0 days. DE 10062044 A1 6, 2002 (21) Appl. No.: 13/329,135 DE 10.108539 A1 9, 2002 (Continued) (22) Filed: Dec. 16, 2011 OTHER PUBLICATIONS (65) Prior Publication Data Ward et al. (Biosensors & Bioelectronics 17, 2002, 181-189).* US 2012/O187OOO A1 Jul. 26, 2012 (Continued) Primary Examiner — Jennifer Dieterle Related U.S. Application Data (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich & (60) Provisional application No. 61/424,040, filed on Dec. Rosati 16, 2010, provisional application No. 61/550,355, (57) ABSTRACT filed on Oct. 21, 2011. Systems and methods are provided for detecting the presence (51) Int. Cl. ofan analyte in a sample. A solid state electrochemical sensor GOIN 27/333 (2006.01) can include a redox active moiety having an oxidation and/or GOIN 27/26 (2006.01) reduction potential that is sensitive to the presence of an GOIN 27/30 (2006.01) analyte immobilized over a surface of a working electrode. A GOIN 27/216 (2006.01) redox active moiety having an oxidation and/or reduction (52) U.S. Cl. potential that is insensitive to the presence of the analyte can CPC ...... G0IN 27/302 (2013.01); G0IN 27/4167 be used for reference. Voltammetric measurements made (2013.01); G0IN 27/3335 (2013.01) using Such systems can accurately determine the presence USPC ...... 204/416; 204/433; 204/400; 324/438: and/or concentration of the analyte in the sample. The Solid 422/82.03; 422/68.1: 548/406; 428/149 state electrochemical sensor can be robust and not require (58) Field of Classification Search calibration or re-calibration. USPC ...... 204/400 435: 205/775 792: 548/406; 23 Claims, 36 Drawing Sheets

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(56) References Cited Lafitte, et al. Anthraquinone ferrocene film electrodes: Utility in pH and oxygen sensing. Electrochemistry Communications. 2008; OTHER PUBLICATIONS 10(12): 1831-1834. Lawrence, et al. Triple Component Carbon Epoxy pH Probe. Shinwari, et al. Microfabricated reference electrodes and their Electroanalysis. 2007; 19(4):424-428. biosensing applications. Sensors. 2010; 10:1679-1715. Leventis, et al. Derivatised carbon powder electrodes: reagentless pH sensors. Talanta. 2004; 63(4): 1039-1051. Dicks, et al. The application of ferrocene-modified n-type silicon in MaoZ. et al. On the formation and structure of self-assembling glucose biosensors. Electroanalysis. Jan. 1993; 5(1): 1-9. monolayers. I: Comparative ATR-wettability study of Langmuir U.S. Appl. No. 13/787,300, filed Mar. 6, 2013, Kahn et al. Blodgett and adsorbed films on flat Substrates and glass microbeads. U.S. Appl. No. 13/787.534, filed Mar. 6, 2013, Kahn et al. Journal of colloid and interface science. 1984; 100(2):465-496. U.S. Appl. No. 13/923,214, filed Jun. 20, 2013, Kahn et al. Medina, et al. Ferrocenyldimethyl-2.2-cryptand: Solid State Struc Lim, et al. Synthesis, charaterization and catalytic performance of ture of the External Hydrate and Alkali and Alkaline-earth-depen porous nafion resin/silica nanocomposites for esterification of lauric dent Electrochemical Behaviour. J. Chem. Soc. Chem. Commun. acid and methanol. Journal of Physical Science. 2009; 20(2):23–36. 1991:290-292. Liu, et al. Nafion/PTFE composite membranes for fuel cell applica Pandurangappa, etal. Homogeneous Chemical Derivatisation of Car tions. Journal of Membrane Science. 2003; 212:213-223. bon Particles: A Novel Method for Functionalizing Carbon Surfaces. U.S. Appl. No. 13/241,171, filed Sep. 22, 2011, Kahn et al. The Analyst. 2002; 127:1568-1571. Bateman, et al. Alkylation of Porous Silicon by Direct Reaction with Pandurangappa, et al. 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Low-Temperature Preparation of Oxygen- and Carbon Seymour, et al. Reaction with N,N-Diethyl-p-phenylenediamine: A Free Silicon and Silicon-Germanium Surfaces for Silicon and Sili Procedure for the Sensitive Square-Wave Voltammetric Detection of con-Germanium Epitaxial Growth by Rapid Thermal Chemical Chlorine. Electroanalysis. 2003; 15(8):689-694. Vapor Deposition. J. ElectrochemSoc. 2000; 147(12):4652-4659. Shu, et al. Synthesis and Charge-Transport Properties of Polymers Casimiri, et al. Co-immobilized L-lactate oxidase and L-lactate Derived from the Oxidation of 1-Hydro-1-46-(pyrrol-1-yphexyl)- dehydrogenase on a film mounted on oxygen electrode for highly 4,4'-bipyridinium Bis(hexafluorophosphate) and Demonstration of sensitive L-lactate determination. Biosensors and Bioelectronics. a pH-Sensitive Microelectrochemical Transistor Derived from the 1996; 11(8):783-789. Redox Properties of a Conventional Redox Center. J. Phys. Chem. Chou, et al. 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An electrochemical immunosensor using ferrocenyl tethered dendrimer. Analyst. Mar. 2006; 131(3):402-6. * cited by examiner U.S. Patent Jun. 24, 2014 Sheet 1 of 36 US 8,758,584 B2

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strae |?? !”, strae () No.surae (sun) Hid ddaedaeaeduaedº0 US 8,758,584 B2 1. 2 ELECTROCHEMICAL SENSORS In some embodiments the analyte is hydrogen, and the redox-active moiety that is sensitive to the presence of the CROSS-REFERENCE hydrogen ion comprises a Substituent selected from the group consisting of anthracenes, quinones, anthroquinones, This application claims priority to U.S. Provisional Patent 5 phenanthroquinones, phenylene diamines, catechols, phe Application Ser. No. 61/424,040, filed Dec. 16, 2010, and nothiazinium dyes, monoquaternized N-alkyl-4,4'-bipyri U.S. Provisional Patent Application Ser. No. 61/550,355, dinium, RuOx, and Ni(OH)2. In some embodiments the filed Oct. 21, 2011, which are entirely incorporated herein by redox-active moiety that is sensitive to the presence of hydro reference. gen ion comprises a Substituent comprising anthracene. In 10 Some embodiments the redox-active moiety that is sensitive BACKGROUND OF THE INVENTION to the presence of hydrogen ion comprises a Substituent com prising an anthraquinone or a phenanthraquinone. The measurement of analyte concentration, in particular, In some embodiments the redox-active moiety that is sen hydrogenion concentration or pH is important in a number of sitive to the presence of an analyte is sensitive to the concen research, industrial, and manufacturing processes. For 15 tration of the analyte. In some embodiments the oxidation instance, the measurement of pH is routinely practiced in potential and/or reduction potential of the redox-active moi food and beverage, biofuel, biophamaceuticals, as well as in ety is sensitive to the concentration of the analyte in a range the treatment of water and waste. from 10 M to 10' M. In some embodiments oxidation Many conventional pH sensors use a potentiometric potential and/or reduction potential of the redox-active moi approach which involves the use of glass electrode to measure ety is sensitive to the concentration of the analyte in a range pH. The potentiometric approach suffers from several draw from 10 M to 10' M. In some embodiments the sensor backs. One limitation of potentiometric sensors is the need for detects hydrogen ion concentration from pH 3 to pH 10. In constant calibration. Potentiometric pH electrodes, like bat Some embodiments the sensor detects hydrogen ion concen teries, tend to run down with time and use. As the potentio tration from pH 1 to pH 14. metric electrode ages, its glass membrane tends to change in 25 In some embodiments the sensor measures hydrogen ion resistance, which in turn will alter the electrode potential. For concentration within an accuracy of plus or minus 0.1 pH this reason, the glass electrodes require calibration on a regu unit. In some embodiments the sensor measures hydrogenion lar basis. The need for constant recalibration to provide an concentration within an accuracy of plus or minus 0.01 pH accurate pH output significantly impedes industrial applica units. tions especially where constant in-line pH measurements are 30 In some embodiments the redox-active moiety is required. Recalibration is particularly cumbersome in a bio covalently bound to the surface of the electrode. In some tech environment where pH measurement is conducted in embodiments the redox-active moiety is bound to a polymer medium containing biological materials. Another significant that is immobilized onto the surface of the electrode. drawback of conventional pH sensors is that the glass elec In Some embodiments the semiconductor electrode is trodes have internal Solutions, which in some cases can leak 35 doped. In some embodiments the semiconductor electrode is out into the Solution being measured. The glass electrodes can P-doped. In some embodiments the semiconductor electrode also become fouled by species in the measuring Solution, e.g., comprises a silicon electrode doped with boron. In some proteins, causing the glass electrode to foul. ISFET devices embodiments the semiconductor electrode is N-doped. In have been developed which use a field effect transistor struc Some embodiments the semiconductor electrode comprises a ture on a silicon Surface to measure pH (Bergveld E met al., 40 silicon electrode is doped with phosphorous. IEEE Sensor Conference, Toronto, October 2003, 1-26). In some embodiments the semiconductor electrode com These devices also have limitations. Thus, there remains a prises a monolithic piece of silicon. considerable need for reliable and consistent analyte sensors, In some embodiments the semiconductor electrode com and in particular, pH sensors. prises a composite electrode, the composite electrode com 45 prising semiconductor particles in a matrix. In some embodi SUMMARY OF THE INVENTION ments the semiconductor electrode comprises a composite electrode bound to a conductive substrate. In some embodi An aspect of the invention provides a sensor for detecting ments the semiconductor particles are present in an amount the presence of an analyte comprising: a semiconductor elec Such that a conductive path is created across the composite trode having a surface having immobilized thereon a redox 50 electrode. active moiety, wherein the redox-active moiety exhibits an In some embodiments the electrode comprises silicon. In oxidation potential and/or a reduction potential that is sensi Some embodiments the electrode comprises unpolished sili tive to the presence of the analyte. In some embodiments the con. In some embodiments the electrode comprises polished analyte is hydrogen ion and the redox-active moiety is sensi silicon. tive to hydrogen ion concentration. In some embodiments the 55 In some embodiments the semiconductor electrode has a sensor comprises a plurality of redox-active moieties, resistivity within the range of 0.01 to 1000 S2-cm. In some wherein at least one of the redox-active moieties is sensitive embodiments the semiconductor electrode has a resistivity to the presence of an analyte, and at least one other redox within the range of 1 to 100 G.2-cm. active moiety is substantially insensitive to the presence of the In some embodiments the sensor is capable of measuring analyte. 60 analyte concentration without calibration with an external In some embodiments the analyte is hydrogen ion, and the standard. In some embodiments the sensor remains sensitive moiety that is Substantially insensitive to the presence of to the analyte without calibration after a first use by an end hydrogen ion has a Substituent selected from the group con user. In some embodiments the analyte is hydrogen ion and sisting of ferrocene, polyvinylferrocene, viologen, polyviolo the sensor remains sensitive to hydrogen ion after exposure to gen, and polythiophene. In some embodiments the moiety 65 a cell culture medium for at least 6 days. In some embodi that is substantially insensitive to the presence of hydrogen ments the sensor is capable of measuring pH with an accuracy ion is ferrocene or a derivative of ferrocene. of plus or minus 0.2 pH units after exposure to the cell culture US 8,758,584 B2 3 4 medium for at least 6 days. In some embodiments the analyte In some embodiments immobilized thereon is also a sec is hydrogen ion and the sensor is capable of measuring pH ond redox-active moiety having an oxidation potential and/or with an accuracy of plus or minus 0.2 pH units after autoclave a reduction potential that is substantially insensitive to the treatment at 121°C. for 40 minutes. In some embodiments the presence of the analyte. sensor is capable of measuring pH with an accuracy of plus or 5 In some embodiments the analyte is an ion. In some minus 0.2 units after autoclave treatment at 121° C. for 400 embodiments the analyte is hydrogen ion. In other embodi minutes. ments, the analyte is a polarizable molecule. In other embodi In some embodiments the semiconductor Substrate has a ments, the analyte is an ionizable molecule. Such as upon the plurality of Zones wherein at least a first Zone is sensitive to an application of an external source of energy. 10 In some embodiments the semiconductor comprises an analyte, and a second Zone that is insensitive to an analyte. inorganic semiconductor. In some embodiments the semicon Another aspect of the invention provides an analyte-sens ductor comprises an organic semiconductor. In some embodi ing system comprising: a working electrode having a semi ments the inorganic semiconductor comprises silicon or gal conductor Surface that has immobilized thereon a redox-ac lium arsenide. In some embodiments the organic tive moiety, wherein the redox-active moiety has an oxidation 15 semiconductor comprises polyacetylene, polythiophene, or potential and/or reduction potential that is sensitive to the polypyrrole. presence of the analyte; a counter electrode and optionally a In some embodiments the semiconductor comprises sili reference electrode; a source for Supplying a plurality of con. In some embodiments the semiconductor comprises potentials to the working electrode; and a device for measur unpolished silicon. In some embodiments the semiconductor ing current through the working electrode at the plurality of comprises polished silicon. potentials. In some embodiments the redox-active moiety is directly In some embodiments the invention further comprises a bound to the surface. In some embodiments the redox-active second working electrode comprising a second semiconduc moiety is covalently bound to the surface. In some embodi tor Substrate comprising a second redox-active moiety having ments the redox-active moiety is covalently bound to the an oxidation potential and/or reduction potential that is insen 25 Surface through a linker. In some embodiments the redox sitive to the presence of the analyte. In some embodiments the active moiety is covalently bound to a polymer that is immo Source for Supplying a plurality of potentials is a potentiostat bilized onto the surface of the semiconductor substrate. In capable of applying square waves for square wave Voltamme Some embodiments the redox-active moiety is covalently try. bound to a polymer that is covalently bound to the surface of In some embodiments the invention further comprises a 30 the semiconductor Substrate computation system that communicates with the device for In some embodiments the semiconductor is doped. In some measuring current, and that calculates a reduction or oxida embodiments the semiconductor is N-doped. In some tion potential from the measured current at a plurality of embodiments the semiconductor is P-doped. potentials. In some embodiments substrate comprises crystalline sili 35 con wherein the Surface displaying predominantly one crys In some embodiments the system is used as an in-line talline plane. sensor in a process. In some embodiments the Substrate has a plurality of Zones In some embodiments the currents measured at a plurality wherein each Zone comprises a redox-active moiety immobi of potentials are used to determine analyte concentration, and lized thereon, the redox-active moiety exhibiting an oxidation the determined analyte concentration is used to control a 40 potential and/or a reduction potential that is sensitive to the process parameter. presence of an analyte. In some embodiments a first Zone In some embodiments the sensor measures hydrogen ion comprises a redox moiety sensitive to a first analyte, and a concentration within an accuracy of plus or minus 0.1 pH second Zone comprises a redox moiety sensitive to a second unit. In some embodiments the sensor measures hydrogenion analyte. In some embodiments the invention further com concentration within an accuracy of plus or minus 0.01 pH 45 prises a third redox-active moiety exhibiting an oxidation units. potential and/or a reduction potential that is sensitive to the In some embodiments the sensor is capable of measuring presence of a second analyte. In some embodiments the sec analyte concentration without calibration with an external ond analyte is ammonia, oxygen or carbon dioxide. standard. In some embodiments the sensor remains sensitive Another aspect of the invention provides a method for to the analyte without calibration after a first use by an end 50 forming an analyte-sensitive semiconductor electrode, the user. In some embodiments the analyte is hydrogen ion and electrode having a surface, the method comprising immobi the sensor remains sensitive to hydrogen ion after exposure to lizing a redox-active moiety that is sensitive to the presence of a cell culture medium for at least 6 days. In some embodi an analyte onto the Surface. In some embodiments immobi ments the sensoris capable of measuring pH with an accuracy lizing the redox-active moiety covalently binds the redox of plus or minus 0.2 units after exposure to the cell culture 55 active moiety to the Surface. In some embodiments the redox medium for at least 6 days. In some embodiments the analyte active moiety is covalently bound to the Surface through a is hydrogen ion and the sensor is capable of measuring pH linker. In some embodiments immobilizing the redox-active with an accuracy of plus or minus 0.2 units after autoclave moiety comprises hydrosilation, a free radical reaction, car treatment at 121°C. for 40 minutes. In some embodiments the bodiimide coupling, a Diels-Alder reaction, a Michael addi sensor is capable of measuring pH with an accuracy of plus or 60 tion, or click chemistry. minus 0.2 units after autoclave treatment at 121° C. for 400 In some embodiments the redox-active moiety is minutes. covalently bound to a polymer that is immobilized onto the Another aspect of the invention provides a semiconductor Surface. Substrate having a Surface, wherein the Surface comprises a In some embodiments the step of immobilizing comprises redox-active moiety immobilized thereon, the redox-active 65 polymerization including a monomer or oligomer comprising moiety exhibiting an oxidation potential and/or a reduction a redox-active moiety. In some embodiments the polymeriza potential that is sensitive to the presence of an analyte. tion of a monomer or oligomer comprising a redox-active US 8,758,584 B2 5 6 moiety includes a reaction with a functional group covalently or adjacent to the layer of the redox-active moiety. In some bound to the surface, whereby the polymer formed by poly cases, the layer of the composite material covers the layer of merization is covalently bound to the Surface. In some the redox-active moiety. embodiments the monomer or oligomer is electropolymer In some embodiments, the composite material comprises ized onto the surface. Nafion. In some cases, the composite material comprises a In some embodiments the step of immobilizing comprises porous material. Such as a porous polymeric material (e.g., coating or casting the polymer onto the Surface. plastic), impregnated with Nafion. In some embodiments the semiconductor Surface com In some situations, a working electrode comprises a layer prises a composite electrode that comprises semiconductor of a polymeric material for shielding light-sensitive moieties particles within a matrix. 10 on or over the working electrode from light. In some cases, the Another aspect of the invention provides a method for layer of the polymeric material comprises polyetherSulphone forming a semiconductor Surface derivatized with one or (PES). more redox-active moieties comprising: contacting an H-ter In another aspect of the invention, an electrochemical sen minated semiconductor Surface with the one or more redox Sor comprises a Solid state (e.g., a semiconductor, Such as active moieties wherein at least one redox active moiety is 15 silicon, or carbon) electrode that is equipped with (or opera sensitive to the presence of an analyte, and wherein each tively coupled to) a light emitting device (also “light source” redox-active moiety comprises a functional group that will herein). react with the H-terminated semiconductor surface to form a In some embodiments, a sensor comprises a light-emitting covalently bond, thereby forming a derivatized semiconduc device. Such as a light-emitting diode, and a layer of a semi tor Surface. conducting or a non-semiconducting material over the light In some embodiments the semiconductor Surface com emitting device. The layer of the semiconducting or non prises silicon. semiconducting material may have a redox sensitive moiety In some embodiments at least two redox active moieties are thereon. used, and one of the redox active moieties is insensitive to the In some embodiments, the light-emitting diode is an presence of the analyte. 25 organic light-emitting diode. In some embodiments, the In some embodiments the H-terminated semiconductor semiconducting material is silicon. In some embodiments, surface is formed by treatment with hydrofluoric acid. the non-semiconducting material is carbon, such as activated In some embodiments the functional group is a vinyl carbon. group. In some embodiments the functional group is an alde In another embodiment, a sensor for detecting the presence hyde group. In some embodiments the functional group is a 30 or absence of an analyte comprises an electrode having a diazonium group. Surface having immobilized thereon a redox-active moiety, Another aspect of the invention provides a method of deter wherein the redox-active moiety exhibits an oxidation poten mining the concentration of an analyte, comprising placing an tial and/or a reduction potential that is sensitive or insensitive electrode in contact with the analyte, the electrode compris to the presence of the analyte. A light-emitting device is ing a semiconductor Substrate with a semiconductor Surface 35 adjacent to the electrode. The light-emitting device is config having immobilized thereon an analyte-sensitive redox-ac ured to generate light. tive moiety, the analyte-sensitive redox-active moiety exhib In some embodiments, a Solid state sensor for detecting the iting an oxidation potential and/or reduction potential that is presence or absence of an analyte, comprises a solid State sensitive to the concentration of the analyte; applying a plu electrode configured to detect the presence or absence of the rality of potentials to the electrode; and measuring the current 40 analyte, and a light emitting device adjacent to the Solid state through the electrode at the plurality of potentials to deter electrode. mine a reduction and/or oxidation potential of the analyte In some embodiments, a sensor for detecting the presence sensitive redox-active moiety, thereby determining the con or absence of an analyte, comprising a working electrode centration of the analyte. having a redox active moiety formed adjacent a light emitting In some embodiments the measuring of the current at the 45 device, is provided. plurality of potentials provides a peak current, and whereby In another aspect of the invention, a sensor for detecting the the peak current is used to determine reduction and/or oxida presence or absence of an analyte comprises a semiconductor tion potential of the analyte-sensitive redox-active moiety. electrode having a Surface having immobilized thereon a In some embodiments the analyte is hydrogen ion. layer of a redox-active moiety, wherein the redox-active moi In some embodiments the invention further comprises an 50 ety exhibits an oxidation potential and/or a reduction poten analyte-insensitive redox-active moiety bound to a an elec tial that is sensitive or insensitive to the presence of the trode comprising a semiconductor Substrate, such redox-ac analyte. A light blocking layer is adjacent to the layer of the tive moiety having a reduction and/or oxidation potential that redox-active moiety. is Substantially insensitive to the analyte, further comprising In some embodiments, a Solid state sensor for detecting the determining the oxidation and/or reduction potential of the 55 presence or absence of an analyte, comprises a solid State analyte-insensitive redox-active moiety, and determining the electrode and a light blocking layer adjacent to the solid state concentration of the analyte from the difference in the oxida electrode. The light blocking layer may be formed of a poly tion and/or reduction potentials of the analyte-sensitive and meric material. In some situations, the light blocking layer analyte-insensitive moieties. transmits less than 10%, 5%, or 1% of light incident on the In Some embodiments the analyte is provided in a solution. 60 light blocking layer. Another aspect of the invention provides a sensor for Another aspect of the invention is a method comprising: detecting the presence of an analyte. The sensor comprises a measuring a pH value of a step in a water or waste treatment semiconductor electrode having a surface having immobi process with a voltammetric pH sensor, wherein the pH sen lized thereon a layer of a redox-active moiety, wherein the Sor comprises a redox-active moiety that is sensitive to hydro redox-active moiety exhibits an oxidation potential and/or a 65 gen ion concentration, and a redox-active moiety that is Sub reduction potential that is sensitive or insensitive to the pres stantially insensitive to hydrogen ion; and using the pH value ence of the analyte, and a layer of composite material on, over to monitor or control the treatment process. US 8,758,584 B2 7 8 Another aspect of the invention provides a method com sensor. In an embodiment, the redox-active moiety-contain prising measuring a pH value of a reaction mixture in a ing analyte sensor comprises one or more redox-active moi biopharmaceutical process with a Voltammetric pH sensor, eties. In another embodiment, the redox-active moiety-con wherein the pH sensor comprises a redox-active moiety that is taining analyte sensor comprises aredox-active moiety that is sensitive to hydrogen ion concentration, and a redox-active sensitive to the presence of an analyte and another redox moiety that is substantially insensitive to hydrogen ion con active moiety that is insensitive to the presence of the analyte. centration to obtain a pH value. The pH value is used to In another embodiment, the redox-active moiety-containing monitor the biopharmaceutical process. analyte sensor is disposed in a probe body having a form In some embodiments the pH value is measured on a factor configured for insertion into a container for use with a sample obtained from the reaction mixture. 10 glass probe analyte sensor. Another aspect of the invention provides a reactor for car rying out a biopharmaceutical process wherein the reactor Another aspect of the invention provides a method for comprises a pH sensor having a redox-active moiety that is detecting the presence or absence of an analyte, comprising sensitive to hydrogen ion concentration, and a redox-active using a sensor System, as described above, to detect the pres moiety that is substantially insensitive to hydrogen ion con 15 ence or absence of the analyte. centration. Another aspect of the invention provides a method for In some embodiments the pH sensor is a voltammetric pH forming an analyte sensor, comprising inserting a sensor sys SSO. tem as described herein into a container configured for use In an embodiment, the pH sensor is a disposable pH sensor. with a glass probe analyte sensor. In an embodiment, the In another embodiment, the pH sensor is a single-use pH method further comprises removing a glass probe analyte sensor. In another embodiment, the pH sensor is a disposable sensor from the container prior to inserting the sensor System and single-use pH sensor. into the container. In some embodiments the reactor is a disposable bioreac Another aspect of the invention provides a method for tor. In some embodiments the reactor is a bioprocess flexible forming an analyte sensor, comprising inserting a sensor sys container. 25 tem as described herein into a container configured for use Another aspect of the invention provides a method for with a reactor, in-line flow system, or sample preparation, or carrying out an industrial process comprising: measuring a analysis. pH value of a step of an industrial process with a voltammetric Another aspect of the invention provides a sensor for pH sensor having a redox-active moiety that is sensitive to detecting the presence or absence of an analyte, comprising a hydrogenion concentration, and a redox-active moiety that is 30 semiconductor electrode having a surface having immobi Substantially insensitive to hydrogen ion concentration; and lized thereon a redox-active moiety, wherein the redox-active using the pH value to carry out the industrial process. moiety exhibits an oxidation potential and/or a reduction Another aspect of the invention provides a sensor for mea potential that is sensitive to the presence of the analyte. The suring ion concentration in a bodily fluid within a body. The sensor includes a form factor for insertion into a container of sensor comprises an electrode configured to be in contact 35 a glass probe analyte sensor. with a bodily fluid, the electrode comprising a semiconductor In an embodiment, the analyte is hydrogen ion and the surface that has immobilized thereon a redox-active moiety. redox-active moiety is sensitive to hydrogen ion concentra The redox-active moiety has an oxidation potential and/or tion. In another embodiment, the sensor comprises a plurality reduction potential that is sensitive to concentration of the of redox-active moieties, wherein at least one of the redox ion. 40 active moieties is sensitive to the presence of an analyte, and Another aspect of the invention provides a method for at least one other redox-active moiety is substantially insen measuring concentration in a bodily fluid within a body, com sitive to the presence of the analyte. In another embodiment, prising placing Such a sensor in contact with the bodily fluid the analyte is hydrogen ion, and the moiety that is Substan and operating the sensor to yield a value of the concentration tially insensitive to the presence of hydrogen ion has a Sub of the ion present in the bodily fluid. 45 stituent selected from the group consisting offerrocene, poly Another aspect of the invention provides a bioreactor com vinylferrocene, viologen, polyviologen, and polythiophene. prising a reservoir for containing a reaction mixture and a pH In another embodiment, the moiety that is Substantially insen probe wherein the pH probe comprises an electrode having a sitive to the presence of hydrogen ion is ferrocene or a deriva semiconductor Surface, the semiconductor Surface having tive of ferrocene. In another embodiment, the analyte is immobilized thereon a redox active moiety having a reduc 50 hydrogen, and the redox-active moiety that is sensitive to the tion and/or oxidation potential that is sensitive to the presence presence of the hydrogen ion comprises a Substituent selected of hydrogen ion. from the group consisting of anthracenes, quinones, anthro In some embodiments the invention further comprises a quinones, phenanthroquinones, phenylene diamines, cat semiconductor Surface having immobilized thereon a redox echols, phenothiazinium dyes, monoquaternized N-alkyl-4, active moiety having a reduction and/or oxidation potential 55 4'-bipyridinium, RuOx, and Ni(OH). In another that is insensitive to the presence of hydrogen ion. embodiment, the redox-active moiety that is sensitive to the In some embodiments the semiconductor Surface on which presence of hydrogen ion comprises a Substituent comprising the redox active moiety having a reduction and/or oxidation anthracene. In another embodiment, the redox-active moiety potential that is sensitive to the presence of hydrogen ion is that is sensitive to the presence of hydrogen ion comprises a immobilized is the same semiconductor surface on which the 60 Substituent comprising an anthraquinone or a phenan redox active moiety having a reduction and/or oxidation thraquinone. In another embodiment, the redox-active moiety potential that is insensitive to the presence of hydrogen ion is that is sensitive to the presence of an analyte is sensitive to the immobilized on. In some embodiments the probe further concentration of the analyte. In another embodiment, the comprises a counter electrode. oxidation potential and/or reduction potential of the redox Another aspect of the invention provides a sensor System, 65 active moiety is sensitive to the concentration of the analyte in comprising a redox-active moiety-containing analyte sensor a range from 10 M to 10' M. In another embodiment, the for insertion into a container for use with a glass probe analyte oxidation potential and/or reduction potential of the redox US 8,758,584 B2 10 active moiety is sensitive to the concentration of the analyte in are sensitive to an analyte. Such as hydrogen. In another a range from 10 M to 10' M. embodiment, the redox-active moieties are insensitive to the In an embodiment, the sensor detects hydrogen ion con analyte. centration from pH 1 to pH 14. In another embodiment, the Another aspect of the invention provides a sensor for sensor detects hydrogen ion concentration from pH 3 to pH detecting the presence or absence of an analyte, comprising a 10. In another embodiment, the sensor measures hydrogen semiconductor electrode having a surface having immobi ion concentration within an accuracy of plus or minus 0.1 pH lized thereon a layer of a redox-active moiety, wherein the units. In another embodiment, the sensor measures hydrogen redox-active moiety exhibits an oxidation potential and/or a ion concentration within an accuracy of plus or minus 0.01 reduction potential that is sensitive or insensitive to the pres pH units. 10 ence of the analyte. The sensor further comprises a light In an embodiment, the redox-active moiety is covalently blocking layer adjacent to the layer of the redox-active moi bound to the Surface of the electrode, Such as through an ety. In an embodiment, the light blocking layer comprises a oxygen-to-surface, carbon-to-Surface or Sulfur-to-Surface polymeric material. In another embodiment, the polymeric bond. In another embodiment, the redox-active moiety is 15 material comprises a fluoropolymer-copolymer. In another bound to a polymer that is immobilized onto the surface of the embodiment, the polymeric material comprises Nation. In electrode. another embodiment, the sensor further comprises a protec In an embodiment, the semiconductor electrode is doped. tive layer adjacent to the light blocking layer. In another In another embodiment, the semiconductor electrode is embodiment, the protective layer comprises a polymeric p-doped. In another embodiment, the semiconductor elec material. In another embodiment, the polymeric material trode is doped with boron. In another embodiment, the semi comprises polyetherSulphone. conductor electrode is n-doped. In another embodiment, the In an embodiment, the light blocking layer comprises a semiconductor electrode is doped with phosphorous. In composite material. In another embodiment, the composite another embodiment, the semiconductor electrode comprises material comprises Nation. In another embodiment, the com a monolithic piece of silicon. In another embodiment, the 25 posite material comprises a porous plastic. In another semiconductor electrode comprises a composite electrode, embodiment, the composite material is a porous membrane. the composite electrode comprising semiconductor particles Another aspect of the invention provides a redox-active in a matrix. In another embodiment, the semiconductor elec moiety-containing analyte sensor for use with a glass probe trode comprises a composite electrode bound to a conductive analyte detection system. The redox-active moiety-contain Substrate. In another embodiment, the semiconductor par 30 ing analyte sensor can have any of the features and charac ticles are present in an amount Such that a conductive path is teristics of sensors described above. created across the composite electrode. In another embodi Another aspect of the invention provides a redox-active ment, the electrode comprises silicon. In another embodi moiety-containing analyte sensor for use in a time period of ment, the electrode comprises unpolished silicon. In another about 1 day, or 5 days, or 10 days, or 20 days, or 25 days, or embodiment, the electrode comprises polished silicon. In 35 30 days. The redox-active moiety-containing analyte sensor another embodiment, the semiconductor electrode has a can have any of the features and characteristics of sensors resistivity within the range of about 0.01 to 1000 G.2-cm. In described above. another embodiment, the semiconductor electrode has a Another aspect of the invention provides a redox-active resistivity within the range of 1 to 100 G.2-cm. moiety-containing analyte sensor having a sensitivity In an embodiment, the sensor is capable of measuring 40 between about 20 mV perpH unit and 60 mV perpH unit. The analyte concentration without calibration with an external redox-active moiety-containing analyte sensor can have any standard. In another embodiment, the sensor remains sensi of the features and characteristics of sensors described above. tive to the analyte without calibration after a first use by an Another aspect of the invention provides a redox-active end user. In another embodiment, the analyte is hydrogen ion moiety-containing analyte sensor having a shelf life between and the sensor remains sensitive to hydrogen ion after expo 45 about 3 months and 3 years. The redox-active moiety-con sure to a cell culture medium for at least 6 days. In another taining analyte sensor can have any of the features and char embodiment, the sensor is capable of measuring pH with an acteristics of sensors described above. accuracy of plus or minus 0.2 pH units after exposure to the Another aspect of the invention provides a sensor for cell culture medium for at least 6 days. In another embodi detecting an analyte having an accuracy to within 0.001 pH ment, the analyte is hydrogen ion and the sensor is capable of 50 units while in use or storage for at least 2 years. The redox measuring pH with an accuracy of plus or minus 0.2 pH units active moiety-containing analyte sensor can have any of the after autoclave treatment at 121°C. for 40 minutes. In another features and characteristics of sensors described above. embodiment, the sensor is capable of measuring pH with an Another aspect of the invention provides a sensor for accuracy of plus or minus 0.2 pH units after autoclave treat detecting an analyte having an accuracy to within 0.001 pH ment at 121°C. for 400 minutes. In another embodiment, the 55 units while in use or storage for at least 4 years. The redox semiconductor Substrate has a plurality of Zones, wherein at active moiety-containing analyte sensor can have any of the least a first Zone is sensitive to an analyte and a second Zone features and characteristics of sensors described above. is insensitive to an analyte. Another aspect of the invention provides a sensor for In an embodiment, the container is cylindrical in shape. In detecting an analyte having an accuracy to within 0.001 pH another embodiment, the container has a circular cross-sec 60 units while in use or storage for at least 8 years. The redox tion. In another embodiment, the container is formed of one or active moiety-containing analyte sensor can have any of the more metals. In another embodiment, the one or more metals features and characteristics of sensors described above. include stainless steel. Another aspect of the invention provides a sensor for Another aspect of the invention provides a sensor compris detecting an analyte having an accuracy to within 0.001 pH ing a solid state electrode having a Surface immobilized 65 units while in use or storage for at least 10 years. The redox thereon a mixed layer of hydrocarbon molecules and redox active moiety-containing analyte sensor can have any of the active moieties. In an embodiment, the redox-active moieties features and characteristics of sensors described above. US 8,758,584 B2 11 12 Another aspect of the invention provides a sensor for Another aspect of the invention provides a method for detecting the presence or absence of an analyte, comprising detecting the presence or absence of an analyte, comprising an electrode having a Surface having immobilized thereon a using a sensor, as described above, to detect the presence or redox-active moiety, wherein the redox-active moiety exhib absence of the analyte. In some embodiments, the analyte is its an oxidation potential and/or a reduction potential that is hydrogen ion. sensitive or insensitive to the presence of the analyte, and a Another aspect of the invention provides a sensor having a light-emitting device adjacent to the electrode, the light-emit Solid state working electrode having disposed thereon a ting device configured to generate light. In an embodiment, redox-active moiety exhibiting an oxidation potential and/or the light-emitting device is configured to generate light that is a reduction potential that is sensitive to the presence of an i) incident on the Surface, ii) incident on another Surface of the 10 analyte. The working electrode has a size and shape for use in electrode, the another Surface opposite from the Surface, and/ glass probe sensor, a reactor, a flow system, or a sample or iii) directed through the electrode. In another embodiment, separation system. In an embodiment, the reactor is a biore the electrode is formed of a solid state material. In another actor. In another embodiment, the sensor further comprises an embodiment, the electrode is formed of a semiconductor additional working electrode having disposed thereon a material. In another embodiment, the semiconductor material 15 redox-active moiety exhibiting an oxidation potential and/or includes silicon. In another embodiment, the electrode is reduction potential that is insensitive to the presence of the formed of a non-semiconductor material. In another embodi analyte. In another embodiment, the working electrode is ment, the non-semiconductor material includes carbon. In doped p-type and the additional working electrode is doped another embodiment, the light-emitting device is a light-emit n-type or p-type. In another embodiment, the working elec ting diode having an active region configured to generate light trode has a resistivity is greater than or equal to about 1 S2-cm upon the recombination of electrons and holes. In another and the additional working electrode has a resistivity greater embodiment, the light-emitting diode is an organic light than or equal to about 5 LS2-cm. emitting diode. In another embodiment, during use, light Another aspect of the invention provides an analyte sensor, from the light-emitting device is incident on the Surface. comprising a first solid state working electrode and a second Another aspect of the invention provides a sensor for 25 solid state working electrode. The first solid state working detecting the presence or absence of an analyte, comprising a electrode has disposed thereon a redox-active moiety exhib working electrode having a redox active moiety formed adja iting an oxidation potential and/or a reduction potential that is cent a light emitting device. The sensor can have any of the sensitive to the presence of an analyte, the first solid state features and characteristics of sensors described above. working electrode doped p-type. The second Solid State work Another aspect of the invention provides a Solid state sen 30 ing electrode has disposed thereon a redox-active moiety sor for detecting the presence or absence of an analyte, com exhibiting an oxidation potential and/or a reduction potential prising a solid state electrode and a light blocking layer adja that is insensitive to the presence of the analyte, the second cent to the solid state electrode. In an embodiment, the light Solid state working electrode doped n-type or p-type. In an blocking layer is formed of a polymeric material. In another embodiment, the first solid state working electrode is dis embodiment, the light blocking layer transmits less than 10% 35 posed adjacent to the second solid State working electrode. In of light incident on the light blocking layer. In another another embodiment, the first solid state working electrode is embodiment, the light blocking layer transmits less than 5% electrically isolated from the second solid state working elec of light incident on the light blocking layer. In another trode. In another embodiment, the first solid state working embodiment, the light blocking layer transmits less than 1% electrode has a resistivity greater than or equal to 1 S2-cm of light incident on the light blocking layer. 40 (also “G2cm herein). In another embodiment, the second Another aspect of the invention provides a Solid state sen Solid state working electrode has a resistivity greater than or sor for detecting the presence or absence of an analyte, com equal to about 5 LS2-cm. In another embodiment, the first and prising a Solid state electrode configured to detect the pres second solid State working electrodes are formed of a semi ence or absence of the analyte, and a light emitting device conductor. In another embodiment, the semiconductor is sili adjacent to the Solid state electrode. The sensor can have any 45 con. In another embodiment, the second Solid State working of the features and characteristics of sensors described above. electrode is doped n-type and has a resistivity greater than or Another aspect of the invention provides a sensor for equal to about 1 S2-cm. In another embodiment, the resistivity detecting the presence or absence of an analyte, comprising a of the second solid state working electrode is between about working electrode having a redox active moiety formed adja 1 S.2-cm and 90 S2-cm. In another embodiment, the solid state cent a light emitting device. The sensor can have any of the 50 working electrodes are disposed on a Substantially flat Surface features and characteristics of sensors described above. of the analyte sensor. Another aspect of the invention provides a method for Another aspect of the invention provides a method for detecting the presence or absence of an analyte, comprising forming an analyte sensor, comprising inserting a sensor as bringing an analyte sensor in contact with a sample, the described herein into a container that is for use with a glass analyte sensor having an electrode having immobilized 55 probe analyte sensor. In an embodiment, the method further thereon a redox-active moiety. The redox-active moiety comprises removing a glass probe analyte sensor from the exhibits an oxidation potential and/or a reduction potential container prior to inserting the sensor into the container. that is sensitive to the presence of the analyte. Next, with the Another aspect of the invention provides a method for aid of the analyte sensor, the analyte is detected at an accuracy forming an analyte sensor, comprising inserting a sensor as within at least about 5% without re-calibration for a period of 60 described herein into a container that is for use with a reactor, at least about 1 day. In an embodiment, the accuracy is within in-line flow system, or sample preparation, or analysis. at least about 1%. In another embodiment, the accuracy is Additional aspects and advantages of the present disclo within at least about 0.1%. In another embodiment, the period sure will become readily apparent to those skilled in this art is at least about 7 days. In another embodiment, the period is from the following detailed description, wherein only illus at least about 1 month. In another embodiment, the period is 65 trative embodiments of the present disclosure are shown and at least about 1 year. In another embodiment, the period is at described. As will be realized, the present disclosure is least about 2 years. capable of other and different embodiments, and its several US 8,758,584 B2 13 14 details are capable of modifications in various obvious solutions of 1.23, 4.65, 5.52 and 9.32 (VA circle, peaks going respects, all without departing from the disclosure. Accord from right to left); and (b) a plot of peak potential difference ingly, the drawings and description are to be regarded as against pH using the VA+VFc derivatized silicon sample, in illustrative in nature, and not as restrictive. accordance with an embodiment of the invention; FIG. 12 depicts square wave Voltammograms showing the INCORPORATION BY REFERENCE effect of 10 autoclave cycles on FcA+VA derivatized silicon sample, in accordance with an embodiment of the invention. All publications and patent applications mentioned in this The electrochemical measurements were conducted at pH specification are herein incorporated by reference to the same 6.52 buffer prior to autoclave and after autoclave; extent as if each individual publication or patent application 10 FIG. 13 depicts square wave Voltammograms showing was specifically and individually indicated to be incorporated minimal fouling on FcA+AnA derivatized silicon samples, in by reference. accordance with an embodiment of the invention. The elec trochemical measurements were conducted at pH 6.52 buffer BRIEF DESCRIPTION OF THE DRAWINGS before and after six days exposure in the cell culture at four 15 different FcA+AnA derivatized silicon samples (a), (b), (c) The features and advantages of the invention may be fur and (d); ther explained by reference to the following detailed descrip FIG. 14 depicts a square wave Voltammogram obtained at tion and accompanying drawings (or figures, also "Fig.” and FcA+AnA derivatized silicon sample in cell culture medium “FIG. herein) that sets forth illustrative embodiments. after Sterilization and 6 days exposure, in accordance with an FIG. 1(a) shows a blow up drawing illustrating an embodi embodiment of the invention; ment of the invention comprising a semiconductor electrode FIG. 15(a) depicts square wave voltammetric responses sensor in a housing assembly, in accordance with an embodi FcA on Si (100, N-type, 1-5 mS2 cm) in pH 7.33 buffer ment of the invention. FIG. 1(b) shows an exemplary housing medium, showing every 50" scan of the 2,500 consecutive assembly comprising the semiconductor electrode sensor, in runs, in accordance with an embodiment of the invention. accordance with an embodiment of the invention; 25 FIG. 15(b) depicts voltammetric responses of VFc on Si(111, FIG. 2 depicts an embodiment of the invention comprising N-type, 0.02-0.05 G2cm in pH 7.33 buffer medium, showing a unit that electrically connects to the semiconductor elec every 50" scan of the 2,500 consecutive runs, in accordance trode sensor and comprising a source for Supplying a plurality with an embodiment of the invention; of potentials and a current measuring device, in accordance FIG. 16 depicts the square wave voltammetric response of with an embodiment of the invention 30 Ac derivatized silicon surface at various temperatures (8, 17. FIG.3 depicts an embodiment of the invention comprising 2844, 56°C.) in pH 7.33 buffer medium, in accordance with a probe for measuring analytes within a reactor comprising an embodiment of the invention; two working silicon electrodes, in accordance with an FIG. 17 is a drawing of an embodiment of a bioreactor of embodiment of the invention; the invention comprising a silicon-based Voltammetric sen FIG. 4 illustrates a method of preparing of H-terminated 35 Sor, in accordance with an embodiment of the invention; silicon Surface (Si-H), in accordance with an embodiment FIG. 18(a) depicts Voltammograms taken on an anthracene of the invention; derivatized silicon sensor over the 7 day period in cell culture FIG. 5 illustrates a silicon surface derivatization with fer medium (every 250" scan of the 10,000 consecutive runs), in rocene moieties, vinyl-ferrocene (VFc) and ferrocene car accordance with an embodiment of the invention. FIG. 18(b) boxaldehyde (FCA) by covalent attachment, in accordance 40 depicts a plot of the anthracene peak potential over the 7 day with an embodiment of the invention; time period, in accordance with an embodiment of the inven FIG. 6 illustrates a silicon surface derivatized with tion; anthracene moieties, vinyl anthracene (VA) and anthralde FIG. 19 has charts showing the peak current of silicon hyde (AnA) by covalent attachment, in accordance with an substrates derivatized with (a) vinyl ferrocene and (b) fer embodiment of the invention; 45 rocene carboxaldehyde in pH 1.63 solution for four types of FIG. 7 illustrates a silicon surface derivatized with both the doped silicon, in accordance with an embodiment of the anthracene (VA) and ferrocene (VFc) moieties by covalent invention; attachment, in accordance with an embodiment of the inven FIG. 20 depicts square wave voltammograms obtained tion; with a four electrode system having highly-doped N-type FIG.8 depicts a schematic diagram and picture of an exem 50 silicon derivatized with vinyl ferrocene and at a lightly-doped plary electrochemical cell, in accordance with an embodi p-type silicon derivatized with anthracene carboxaldehyde, in ment of the invention; accordance with an embodiment of the invention; FIG. 9 depicts square wave Voltammograms showing the FIG.21 depicts an exemplary embodimentofa probe of the effect of pH on VFc derivatized silicon sample at pH solution present invention having a four electrode configuration with a of 1.23, 4.61, 7.33 and 9.33, in accordance with an embodi 55 ring reference electrode (RE) and a ring counter electrode ment of the invention; (CE), in accordance with an embodiment of the invention. FIG. 10(a) depicts square wave Voltammograms showing FIG. 21(a) is a shaded drawing, and FIG. 21(b) is a line the effect of pH on VA derivatized silicon sample at pH drawing: solutions of 1.23, 4.61, 7.33 (not shown; peak maximum FIG.22A schematically illustrates an electrochemical sen between -0.4 and -0.6 V, to the left of the pH 4.61 peak and 60 sor having three modules: FIG. 22B is an enlarged view of a to the right of the pH 13.63 peak) and 13.63, in accordance portion of the electrochemical sensor of FIG. 22A, in accor with an embodiment of the invention. FIG. 10(b) depicts a dance with an embodiment of the invention; plot of peak potential against pH using the VA derivatized FIG. 23 schematically illustrates a printed circuit board, in silicon sample, in accordance with an embodiment of the accordance with an embodiment of the invention; invention; 65 FIG. 24 schematically illustrates a sensor formed on a FIG. 11 depicts: (a) Square wave Voltammograms showing printed circuit board and mounted on a head assembly, in the effect of pH on VA+VFc derivatized silicon sample at pH accordance with an embodiment of the invention; US 8,758,584 B2 15 16 FIG. 25A schematically illustrates an electrochemical sen relates to the measurement of the concentration of hydrogen Sor mounted on a wall of a disposable container, in accor ion, or pH using the Subject devices or systems. The Surface dance with an embodiment of the invention; FIG. 25B illus modified solid state sensors of the present invention can be trates an electrochemical sensor comprising electrodes used to measure the pH of a variety of solutions. The surface formed on a printed circuit board, in accordance with an 5 modified sensors of the present invention are robust, reliable, embodiment of the invention; accurate, and/or can be made such that they do not require FIG. 26 schematically illustrates an electrochemical sensor calibration. mounted on a chamber of a flow-through tube, in accordance In some embodiments, solid state electrodes are formed of with an embodiment of the invention; semiconductors. Semiconductors have advantages as Sub FIG. 27A schematically illustrates an electronics unit for 10 permitting integration of an electrochemical sensor into cur strates and sensors for the methods of the present invention. rent probe Systems, in accordance with an embodiment of the Semiconductors have band gaps that may be modified with invention; the aid of chemical dopants, which can aid in preparing sen FIG. 27B illustrates a probe attached to the electronics unit sitive and accurate sensors. In addition, semiconductors can of FIG. 27A, in accordance with an embodiment of the inven- 15 be less prone to fouling and degradation than other Substrates. tion; FIG. 27C shows a probe attached to an electronic unit, Semiconductor Surfaces, for example, inorganic semicon which is in turn attached to a reader, in accordance with an ductors such as silicon and organic semiconductors, can be embodiment of the invention; amenable to Surface modification, e.g., covalent modifica FIG. 28 schematically illustrates an electrochemical probe tion. Semiconductors generally have electronic band struc mounted on a bioreactor, in accordance with an embodiment 20 tures, the characteristics of which can be modified, for of the invention; example, by doping. In some cases, the semiconductor that is FIG. 29 shows a working electrode having a light-emitting used is silicon. An advantage of using silicon as a Substrate device and an electrode adjacent to the light-emitting device, and as an electrode is that silicon is amenable to mass pro in accordance with an embodiment of the invention; duction. In particular, semiconductor processing techniques FIG. 30 shows sensor output (y-axis, mV) with time 25 are readily available for producing silicon electrodes in large (X-axis) during pH measurements for a shielded (top) and quantities at low cost. In addition, existing semiconductor unshielded (bottom) sensors; processing techniques make it feasible to integrate electronic FIGS. 31A-31F show sensor output (y-axis; current, arbi functionality into the material comprising the silicon elec trary units) at various pHs and under light and dark condi trode. Another advantage of silicon is that it can form strong tions as a function of Voltage (mV); 30 covalent bonds, for example with carbon, nitrogen, oxygen, FIGS. 32A-32E show exemplary sensors having form fac thus allowing for the facile and robust modification of the tors suited for various applications; and surface in a manner required for its intended uses. For FIG.33 shows the pH of a fermentation reactor as a func instance, a silicon Surface can be modified to attachment of tion of time as measured by a glass electrode sensor and a any Suitable redox-active agent. Silicon is also an advanta redox-active moiety-based pH sensor. 35 geous Surface for carrying out Voltammetry in that it is stable to a wide range of electrical potential without undergoing DETAILED DESCRIPTION OF THE INVENTION degradation. An aspect of the invention is a Surface modified Solid state While preferable embodiments of the invention have been (e.g., semiconductor) redox sensor that can measure analyte shown and described herein, it will be obvious to those skilled 40 concentration reliably and consistently with minimal inter in the art that such embodiments are provided by way of vention, such as re-calibration. Another aspect of the inven example only. Numerous variations, changes, and Substitu tion is a semiconductor redox sensor that does not require tions will now occur to those skilled in the art without depart calibration or re-calibration. The ability to sense analytes, ing from the invention. It should be understood that various Such as hydrogen ion without calibration has a number of alternatives to the embodiments of the invention described 45 advantages for analyte measurements, for example, for in herein may be employed in practicing the invention. line monitoring. For example, it allows for ease of operator The invention relates to compositions, devices, systems, handling for single point measurements. In some embodi and methods for producing and using Solid state electrodes ments, the sensors of the present invention are included in modified with redox-active agents as sensors. The Subject in-line operator-independent control measurements. Such in devices and systems are particularly useful for Voltammetri- 50 line measurements can be made independent of the operator cally measuring concentrations of an analyte of interest. The and can be used for process control, for example for pH sensors of the present invention utilize a solid state electrode measurements for process control. The Subject sensors can be comprising redox-active species on its surface. At least one set to provide real-time measurements of an analyte, includ redox-active species on the Solid sate (e.g., semiconductor) ing, but not limited to real time-measurements of hydrogen Surface has a redox potential (reduction potential or oxidation 55 ion concentration. potential) that is sensitive to the presence and or amount of an Semiconductor Substrates analyte of interest. Voltammetry can be performed on the An aspect of the invention provides an electrochemical Solid state electrode and used to measure the redox potential sensor having a working electrode formed of a semiconductor of the analyte-sensitive redox groups on the Surface of the Substrate. The semiconductor Substrate can comprise any electrode. The measured value of the redox potential can then 60 Suitable semiconductor material including those known in the be used to determine the concentration of an analyte, for art and those described herein. The semiconductor substrate example an analyte in solution. In some embodiments, the can be an inorganic semiconductor or an organic semicon solid state electrode of the present invention has more than ductor. The semiconductor substrate can be doped or one redox-active species. In an embodiment, at least one undoped. In some embodiments the semiconductor Substrate redox-active species is sensitive to the presence of an analyte 65 comprises silicon. (e.g., protons) and another redox-active species is insensitive A semiconductor Substrate of the invention is generally a to the presence of the analyte. Another aspect of the invention solid material that has electrical conductivity in between that US 8,758,584 B2 17 18 of a conductor and that of an insulator. The conductivity can mers). Examples of semiconducting Small molecules (e.g., vary over that wide range either permanently or dynamically. unsaturated and aromatic hydrocarbons) are pentacene, Inorganic semiconductor Substrates of the invention can anthracene, rubrene, tetracene, chrysene, pyrene, perylene, comprise, for example, Group IV elemental semiconductors, coronene, metal complexes of porphine and phthalocyanine, Such as diamond (C), silicon (Si), germanium (Ge): Group IV compounds such as zinc 1,10,15,20-tetraphenyl-21H,23H compound semiconductors, such as silicon carbide (SiC), porphine, copper phthalocyanine, lutetium bisphthalocya silicon nitride (SiN), silicon germanide (SiGe), Group III-V nine, and aluminum phthalocyanine chloride can be used. semiconductors, such as aluminum antimonide (AISb), alu Suitable derivatives of these small molecules can also be minum arsenide (AlAS), aluminum nitride (AIN), aluminum used. In some embodiments, the organic semiconductor Sub phosphide (AlP), boron nitride (BN), boron phosphide (BP), 10 strates of the invention can comprise thin films. boron arsenide (BAS), gallium antimonide (GaSb), gallium Examples of semiconducting polymers or oligomers arsenide (GaAs), gallium nitride (GaN), gallium phosphide include Suitable conjugated hydrocarbon or heterocyclic (GaP), indium antimonide (InSb), indium arsenide (InAs), polymers or oligomers. Suitable polymers or oligomers indium nitride (InN), indium phosphide (InP); Group III-V include: polyaniline, polypyrrole, and polythiophene, poly(3- ternary semiconductor alloys, such as aluminum gallium ars 15 hexylthiophene), poly(p-phenylene vinylene), F8BT poly enide (AlGaAs, AlxGa1-xAs), indium gallium arsenide (In acetylene, polydiacetylene, polyacene, polyphenylene, poly GaAs, InxGa1-xAs), indium gallium phosphide (InGalP), alu (phenylene vinylene), polyfuran, polypyridine, poly minum indium arsenide (AlInAs), aluminum indium (thienylene vinylene), poly(ferrocenyl vinylene phenylene antimonide (AlInSb), gallium arsenide nitride (GaAsN), gal vinylene), poly(fluorine), and poly(carbazole), and combina lium arsenide phosphide (GaAsP), aluminum gallium nitride tions thereof. Derivatives of these polymers, for instance (AlGaN), aluminum gallium phosphide (A1GaP), indium gal derivatives having functional side chains amenable to the lium nitride (InGaN), indium arsenide antimonide (InAsSb). attachment of redox active species, can be used. indium gallium antimonide (InGaSb), Group III-V quater Other examples of semiconducting polymers are poly(a- nary semiconductor alloys, such as aluminum gallium indium nilinesulfonic acid), poly(ferrocenyl vinylene phenylene phosphide (AlGalnP, also InAlGaP. InGaAlP, AlInGaP), alu 25 vinylene), poly(fluorine), and poly(carbazole). The organic minum gallium arsenide phosphide (AlGaAsP), indium gal semiconductor Substrates of the invention can comprise, for lium arsenide phosphide (InGaAsP), Aluminum indium ars example, organic charge-transfer complexes, and "linear enide phosphide (AIInASP), aluminum gallium arsenide backbone' polymers derived from polyacetylene, such as nitride (AlGaAsN), indium gallium arsenide nitride (In polyacetylene itself, polypyrrole, and polyaniline. Charge GaAsN), indium aluminum arsenide nitride (InAlAsN), gal 30 transfer complexes can exhibit similar conduction mecha lium arsenide antimonide nitride (GaAsSbN), Group III-V nisms to inorganic semiconductors. This includes the pres quinary semiconductor alloys, such as gallium indium nitride ence of a hole and electron conduction layer and a band gap. arsenide antimonide (GalinNAsSb), gallium indium arsenide The materials can exhibit tunneling, localized states, mobility antimonide phosphide (GainAsSbP), II-VI semiconductors, gaps, and phonon-assisted hopping. Organic semiconductors cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium 35 can be doped. In some embodiments, the invention can utilize telluride (CdTe), zinc oxide (ZnO), Zinc selenide (ZnSe), Zinc highly doped organic semiconductors, for example Polya sulfide (ZnS), Zinc telluride (ZnTe), Group II-VI ternary alloy niline (Ormecon) and PEDOT:PSS. In some cases organic semiconductors, such as cadmium Zinc telluride (CdZnTe, semiconductors can be produced such that they are transpar CZT), mercury cadmium telluride (HgCdTe), mercury Zinc ent and/or flexible, which can be useful in some embodi telluride (HgZnTe), mercury Zinc selenide (HgZnSe), Group 40 mentS. I-VII semiconductors, such as cuprous chloride (CuCl). The semiconductors of the invention can typically be char Group IV-VI semiconductors, such as lead selenide (PbSe), acterized as having a band gap, the band gap representing the lead sulfide (PbS), lead telluride (PbTe), tin sulfide (SnS), tin amount of energy separating the Valence and conduction telluride (SnTe), Group IV-VI ternary semiconductors, such bands of the semiconductor. The addition of dopants makes as lead tin telluride (PbSnTe), Thallium tin telluride 45 the band gap Smaller, tending to allow more facile promotion (Tl2SnTe5), thallium germanium telluride (T12GeTe5), of electrons from the valence band to the conduction band. A Group V-VI semiconductors, such as bismuth telluride Smaller band gap can result in higher conductivity for the (Bi2Te3), and Group II-V semiconductors, such as cadmium semiconductor Substrate. The band gap and conductivity phosphide (CdSP2), cadmium arsenide (Cd5As2), cadmium characteristics of the semiconductor Substrates can be con antimonide (Cd5Sb2), Zinc phosphide (Zn3P2), Zinc arsenide 50 trolled in Some cases by the introduction of dopants. In some (Zn3As2), and zinc antimonide (Zn3Sb2). cases upon the addition of a Sufficiently large proportion of The inorganic semiconductor Substrates of the invention dopants, the semiconductor Substrates of the invention can can also comprise layered semiconductors, such as lead(II) conduct electricity nearly as well as metals. Depending on the iodide (Pb2), molybdenum disulfide (MoS2), gallium kind of dopant or impurity, a doped region of semiconductor selenide (GaSe), tin sulfide (SnS), bismuth sulfide (Bi2S3). 55 can have more electrons or holes, and is named N-type or other semiconductors, such as copper indium gallium P-type (herein also “n-type' and “p-type’) semiconductor selenide (CIGS), platinum silicide (PtSi), bismuth(III) iodide material, respectively. Junctions between regions of N- and (Bil3), mercury(II) iodide (Hg12), thallium(I) bromide P-type semiconductors create electric fields, which cause (TlBr), and miscellaneous oxides, such as titanium dioxide: electrons and holes to be available to move away from them, anatase (TiO2), copper(I) oxide (Cu2O), copper(II) oxide 60 and this effect is critical to semiconductor device operation. (CuO), uranium dioxide (UO2), and uranium trioxide (UO3). Also, a density difference in the amount of impurities pro In some embodiments of the invention, the semiconductor duces a small electric field in the region which is used to Substrate can comprise an organic semiconductor. The accelerate non-equilibrium electrons or holes. organic semiconductor is any Suitable organic material that In some embodiments the presence of the band gap can be has semiconductor properties. The organic semiconductor 65 advantageous. For example, when performing electrochem Substrates of the invention can comprise, for example, Small istry, when the Fermi energy of a doped semiconductor lies at molecules, short chain (oligomers) and long chain (poly the same energy as the Solution/molecular redox potential at US 8,758,584 B2 19 20 a certain potential, generally no net transfer of charge/current antimony, and bismuth. In some embodiments the dopant is will flow from the redox species (immobilized on the surface phosphorous. In some embodiments the dopant is antimony. or in the solution) to the substrate or from the substrate to the Where the semiconductor is a Group IV semiconductor such redox species. This potential is sometimes referred to as the as silicon or germanium, Suitable electron acceptors include flatband potential. The location of the flatband potential can 5 boron, aluminum, gallium, and the like. In some embodi be influenced by dopant densities. The Mott-Schottky equa ments, the dopant is boron. Where the semiconductor com tion (see P. Schmuki, H. Bohni and J. A. Bardwell, J. Elec prises an element in another group than Group IV, as is known trochem. Soc., 1995, 142, 1705) can be used to estimate the in the art, elements that are outside of that group can act as flat band potential. eithern type of p type dopants. A conductor electrode generally does not have a flatband 10 In addition to modification through doping, the resistance potential thus has a broad potential window where current can of semiconductors can in Some cases, for example, be modi flow from the redox species to the substrate. For example, fied dynamically by applying electric fields. The ability to when the conductor electrode is exposed to an aqueous solu control resistance/conductivity in semiconductor Substrate or tion containing a mixture of several redox active species, within regions of semiconductor Substrate dynamically currents corresponding to these redox species (i.e., non-spe 15 through the application of electric fields can be useful in some cific adsorption) may be recorded unless additional efforts are embodiments. made to Screen off these non-specific interactions, e.g., put The semiconductor Substrate can be in any form that is ting a diluent layer onto the conductor electrode. The non amenable to the production of a semiconductor electrode. The specific interactions can be reduced or eliminated by using a semiconductor Substrate can comprise a monolithic piece of semiconductor electrode wherein the semiconductor has a the the semiconductor, a coating of the semiconductor deposited band gap that has a limited potential window, Such that the onto another material, or a powder of semiconductor par semiconductor can only conduct current for the electrochemi ticles. The semiconductor substrate can be a monolithic form cal reaction occurring within that limited window. For Such as a chip, wafer, rod, needle, block, ingot, or the like. The example, the limited window can be between -1.0 to 0 V. semiconductor Substrate can alternately be in particulate Thus, in some embodiments, non-specific interactions in the 25 form, for example in the form of powder comprised of par Solution can be reduced or eliminated by using a semicon ticles. The particles can be of arbitrary shape or can be in the ductor electrode of the invention with the appropriate dopant form of fibers, sheets, beads, discs, or balls. Where the sub density (which can be estimated using the Mott-Schottky strate is in the form of a powder made up of particles, it will equation). generally be formed into a composite electrode as described One useful aspect of the semiconductors of the invention is 30 in more detail below. that their conductivity can be modified by introducing impu The semiconductor Substrate of the present invention can rities (dopants) into their crystalline lattice or amorphous be a thin layer of semiconductor that is formed upon another regions. The process of adding controlled impurities to a material, for example a thin layer of semiconductor formed semiconductor can be referred to as doping. The amount of on glass would constitute a semiconductor Substrate. impurity, or dopant, added to an intrinsic (pure) semiconduc 35 In an embodiment, the semiconductor Substrate can toralters its level of conductivity. Doped semiconductors may include a layer of semiconductor material having a thickness be referred to as extrinsic. The semiconductors of the present of about 0.1 nanometers (nm) and 5000 nm, or between invention can be either intrinsic or extrinsic semiconductors. about 1 nm and 1000 nm, or between about 10 nm and 500 Suitable dopants can be chosen, as is known in the art, on nm. In another embodiment, the semiconductor Substrate can the atomic properties of the dopant and the material to be 40 include a layer of silicon having a thickness of about 0.1 doped. In general, dopants that produce the desired controlled nanometers (nm) and 5000 nm, or between about 1 nm and changes are classified as either electron acceptors or donors. 1000 nm, or between about 10 nm and 500 nm. A donor atom that activates (e.g., becomes incorporated into In some embodiments the semiconductor Substrate used to the crystal lattice) donates weakly-bound valence electrons to make the electrode is a composite material comprising semi the material, creating excess negative charge carriers. These 45 conductor particles dispersed in a matrix or binder. The semi weakly-bound electrons can move about in the semiconduc conductor Substrate can be made of a composite material tor relatively freely and thus can facilitate electrical conduc comprising a powder of semiconductor dispersed in a binder tion in the presence of an electric field. In some cases, the to make a composite semiconductor Substrate. The semicon donor atoms introduce Some states below, but very close to, ductor powder can be in the form of spheres, crystallites, rods, the conduction band edge. Electrons at these states can be 50 fibers, or any other arbitrary shape. In an embodiment the thermally excited to conduction band, becoming free elec composite electrode is made of semiconductor crystallites trons, in some cases, at room temperature. In some embodi dispersed in a polymeric matrix. The matrix or binder can be ments an activated acceptor dopant is utilized. The activated an organic, inorganic, or organometallic polymer. Non-lim acceptor can produce a hole. Semiconductors doped with iting examples of useful inorganic polymeric materials donor impurities are typically called N-type, while those 55 include polyphosphaZenes, polysilanes, polysiloxane, doped with acceptor impurities are typically known as P-type. polygermanes, polymeric Sulfur, polymeric selenium, sili In some embodiments of the invention, the semiconductor of cones, and mixtures of any of the foregoing. the invention can have both donor and acceptor dopants. In In some embodiments the polymer can be an organic poly Some embodiments the semiconductor can have both in type mer. Non-limiting examples of Suitable organic polymeric and p type charge carriers. The n or p type designation gen 60 materials include, but are not limited to, thermoset materials erally indicates which charge carrier acts as the materials and thermoplastic materials. Non-limiting examples of poly majority carrier. The opposite carrier is generally called the mers useful in the invention include polyesters such as poly minority carrier, which, in some cases, exists due to thermal ethylene terephthalate, polybutylene terephthalate, and poly excitation at a lower concentration than the majority carrier. ethylene naphthalate, polycarbonates, polyolefins such as As described herein, where the semiconductor is a Group 65 polyethylene, polypropylene, and polyisobutene, acrylic IV semiconductor Such as silicon or germanium, Suitable polymers such as copolymers of styrene and an acrylic acid electron donors include, for example, phosphorous, arsenic, monomer, and polymers containing methacrylate, polya US 8,758,584 B2 21 22 mides, thermoplastic polyurethanes, vinyl polymers, polyim form a conductive junction for connecting conductors for ides, polyamides, polytetrafluoroethelene and other fluo transfer of current to and from the composite electrode. ropolymers, and mixtures of any of the foregoing. The semiconductor Substrates of the invention generally The binder can be insulating, semiconductive, or conduc have a high enough electrically conductivity to act as elec tive. In an embodiment, the binder is a material. Such as a 5 trodes, to transmit current for the oxidation and/or reduction polymer, which is an insulating material. Where an insulating of the bound redox-active moieties. To make the semiconduc binder is used, the current will tend to only flow through the tor Substrate more conductive, the semiconductor Substrate dispersed semiconductor powder. In some embodiments, the can include impurities or dopants to increase electrical con binder includes conductive components. In some embodi ductivity and reduce the resistivity. ments, the binder comprises a conductive polymer Such as 10 The electrical resistivity (reciprocal of conductivity) can be polyaniline, polyacetylene, poly(alkylthiophene), poly(alky for example 0.1 (ohm-centimeters), 1 (ohm-centimeters), 10 lpyrrole), and the like. In some embodiments, the conductive (ohm-centimeters), 100 (ohm-centimeters), to in excess of component can comprise conductive particles such as metal 1000 or even 10,000 (ohm-centimeters) or even higher which particles, such as nickel particles other conductive particles is comparable to graphite and conventional metallic conduc including carbon particles. In some embodiments, the con- 15 tOrS. ductive component is chosen such that the conductive com In some embodiments the resistivity of the semiconductor ponent Such as the conductive polymer exhibits reduction substrate is the range of 0.0001 to 100,000 G.2-cm (or ohm and/or oxidation potentials that are outside of the reduction centimeters). In some embodiments the resistivity of the and/or oxidation potentials of the redox active moieties. semiconductor substrate is the range of 0.001 to 10,000 The composite semiconductor substrate can be formed by 20 S2-cm. In some embodiments the resistivity of the semicon mixing the semiconductor powder with a monomer, oligo ductor substrate is the range of 0.01 to 1000 G.2-cm. In some mer, or prepolymer and curing the monomer, oligomer or embodiments the resistivity of the semiconductor substrate is prepolymer to form a polymeric matrix. The polymerization the range of 0.1 to 100 G.2-cm. In some embodiments the can be initiated in any manner known in the art or disclosed resistivity of the semiconductor substrate is within the range herein. The polymerization can be initiated, for example, 25 of 1 to 100 G.2-cm. In some embodiments the resistivity of the thermally or photochemically in the presence of an initiator. semiconductor substrate is within the range of 10 to 90.2-cm. The polymerization can be carried out with one or more In some embodiments the semiconductor Substrate is single crosslinkers. The cross-linkers can be chosen to adjust the crystal semiconductor is Si(100) that is P-type with a resis physical properties of the polymeric matrix and thus adjust tivity of 10 to 90 S2-cm. In some cases several semiconductor the properties of the composite semiconductor substrate. The 30 substrates with different resistivities may be used. For composite semiconductor Substrate can be formed by mixing example, a system of the invention may comprise one lightly the semiconductor powder with a molten thermoplastic poly doped semiconductor substrate having one redox active spe mer, forming the Substrate, and allowing the mixture to cool. cies bound to it, and also a more highly doped semiconductor The composite semiconductor substrate can be formed by Surface having another redox active species bound to it. For mixing the semiconductor powder with a polymer or prepoly- 35 example, a system of the invention may comprise one lightly merina Solvent, and allowing the solvent to evaporate to form doped semiconductor Surface having a pH sensitive redox the composite. Combinations of any of the above methods can active moiety Such as anthraquinone bound thereto, and a be used. second semiconductor Surface that is more highly doped hav The electrical properties of the composite semiconductor ing a hydrogen ion insensitive redox active moiety Such as substrate can be affected by the amount of semiconductor, the 40 ferrocene bound to it. particle size, and the particle shape. In general, the amount of In some cases different regions of the semiconductor Sub semiconductor in the composite is high enough to create strate can be doped at different levels. For example, it is well conductive pathways throughout the material. This amount of known in the semiconductor processing art that a mask can be material necessary to provide conductive paths across the used to cover some regions of the semiconductor, while leav material is sometimes called the percolation threshold. The 45 ing other regions exposed. The exposed regions can be treated amount of semiconductor particles for conductivity can also selectively with dopants resulting in a semiconductor Surface depend on the processing conditions such as the Viscosity of wherein some regions are doped differently than other the binder and the amount of mixing. The amount of semi regions. By using multiple steps with various masks, the conductor is generally set at a level at which the physical conductivity properties of different regions of the semicon properties of the material. Such as mechanical strength and 50 ductor Surface can be controlled. Thus, Some regions can have flexibility will not suffer to the point that the material is not high conductivity, some low, Some regions can have a large useful. The amount of semiconductor will generally be from band gap, and other regions can have Small band gaps, some 0.1 volume percent to 70 volume percent of the composite regions can have N-doping, Some P-doping, and some no material. In some embodiments the amount of semiconductor doping. In addition, the various regions can be connected with may be from 1 volume percent to 50 volume percent. In some 55 conducting regions, for example deposited metal, e.g., gold in embodiments the amount of semiconductor may be from 10 to be able to electrically address the various regions. volume percent to 40 volume percent. The amount of semi Silicon Substrates conductor can be from 1% to 5%, 5% to 10%, 10% to 15%, Another aspect of the invention provides an electrochemi 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% or 50% cal sensor having a semiconductor Substrate that includes to 60%. 60 silicon. The silicon Substrate can have a surface onto which The composite semiconductor substrate can be formed by are attached redox-active moieties. The silicon Substrate can methods used for shaping polymeric materials such as coat comprise amorphous silicon or silicon comprising a variety of ing, molding, and casting into shapes that are useful as elec crystalline forms. The silicon Substrate can also be polycrys trodes. The composite semiconductor substrate electrode will talline. In some embodiments the silicon Substrate can have generally be connected to an electrically conductive wire to 65 both amorphous and crystalline regions. In some embodi apply current and potential. The material can be cast, coated, ments, the silicon can be nanocrystalline or microcrystalline and/or molded onto a conductive substrate Such as a metal to silicon. Nanocrystalline silicon and microcrystalline silicon US 8,758,584 B2 23 24 can be used to describe an allotropic form of silicon with the form of powder comprised of particles. The particles can paracrystalline structure having Small grains of crystalline be of arbitrary shape or can be in the form of fibers, sheets, silicon within the amorphous phase. Where the silicon sub beads, discs, polyhedra, or balls. Where the substrate is in the strate is crystalline, the Surface of the silicon Substrate can form of a powder made up of particles, it will generally be have various crystalline faces on the surface. Crystalline sili formed into a composite electrode as described in more detail con is generally in a face centered cubic (fcc) form. In some below. embodiments. Such as where a polycrystalline silicon is used, In some embodiments the semiconductor electrode is made the Surface may have multiple crystalline planes exposed. from single crystal silicon. The single crystal silicon can be Where single crystal silicon is used, in some cases, the silicon made by Zone melting, also called Zone refining, a process in Substrate can be made to have one or more crystal planes 10 which rods of metallurgical grade silicon are first heated to represented or predominantly represented on the Surface. In melt at one end. Then, the heater is typically slowly moved some embodiments, the surface of the silicon substrate will down the length of the rod, keeping a small length of the rod comprise one or more crystal planes having a crystalline molten as the silicon cools and re-solidifies behind it. Since lattice of (XXX) wherein X is an integer corresponding to the most impurities tend to remain in the molten region rather lattice defining the crystal plane. In some embodiments the 15 than re-solidify, when the process is complete, most of the crystal planes (100), (010), (001), (110), (101), or (112) may impurities in the rod will typically have been moved into the be predominantly represented at the Surface. In some embodi end that was the last to be melted. This end is then cut off and ments a silicon substrate has the (100) plane predominantly discarded, and the process repeated if a still higher purity is represented at the Surface. desired. The single crystal silicon of the invention can also be The silicon electrode of the present invention can comprise produced via the Czochralski process, (CZ Si) which tends a polished or an unpolished silicon Substrate. Silicon is gen to be inexpensive and is capable of producing large size erally polished prior to silicon processing, for example, build crystals. ing features such as transistors. In some embodiments, such In some embodiments the silicon electrode is polycrystal as those embodiments where electronic functionality is incor line. As used herein, the term “polysilicon' is used inter porated into a silicon sensor, a polished silicon Surface may be 25 changeably with the term “polycrystalline silicon'. In some desirable. In other embodiments, an unpolished silicon sub embodiments, the polysilicon is deposited. The polycrystal strate can be used. An unpolished silicon Substrate can be less line silicon can be deposited by low pressure chemical vapor expensive than a silicon Substrate that has gone through a deposition (LPCVD), plasma-enhanced chemical vapor polishing step. In addition, an unpolished silicon Substrate deposition (PECVD), or solid-phase crystallization (SPC) of can have a higher Surface area for a given area of silicon than 30 amorphous silicon in certain processing regimes These pro a polished silicon Substrate. cesses can require relatively high temperatures, for example, The silicon electrode of the present invention can comprise above 300° C. The polycrystalline silicon electrodes can also porous silicon. An advantage of porous silicon is an increase be made, for example on polymeric Substrates, using laser of the effective surface area. An increased Surface area can be crystallization to crystallize a precursor amorphous silicon advantageous for providing a higher signal from the oxidation 35 (a-Si) material on a plastic Substrate without melting or dam and reduction of the surface bound redox moieties due to a aging the plastic. In some cases, the for example, short, high higher number of such moieties in contact with the sample. As intensity ultraviolet laser pulses are used to heat the deposited is known in the art, if the Surface is too porous, it can become a-Si material to above the melting point of silicon, without less robust. Therefore the level of porosity can be controlled melting the entire Substrate. By controlling the temperature to maximize important properties for the particular applica 40 gradients, the crystal size on the electrodes can be controlled. tions. The porous silicon can be prepared by, for example, Grain sizes can be, for instance from 10 nanometer to 1 galvanostatic, chemical, or photochemical etches from sili micrometer. Another method to produce polysilicon at low con wafers. In some embodiments, chemical etching with temperatures for the electrodes of the present invention is a hydrofluoric acid (HF) can be used to produce a porous sili metal-induced crystallization in which an amorphous silicon con Substrate. In some embodiments, the average pore size of 45 thin film is crystallized, for example at temperatures at or the silicon substrate ranges from 1 nm to 500 nm. Pore size above 150° C., while in contact of a metal film such as can be measured by, for example, nitrogen gas adsorption or aluminum, gold, or silver. The polycrystalline silicon elec Hg porosimetry. In some embodiments, the amount of poros trodes can also be formed onto a metal structure Such as a ity ranges between 1% and 98%. In some embodiments, the wire. For example, the end of a cylindrical wire can be coated amount of porosity ranges between 5% and 75%. In some 50 with polysilicon, which can be derivatized with redox active embodiments, the amount of porosity ranges between 10% species as described herein. The structure can be used as an and 50%. In some embodiments, the amount of porosity electrode or portion of an electrode with silicon portion ranges between 20% and 40%. In some embodiments the accessible to the medium containing the analyte, and the wire porosity is between 1% to 5%, 5% to 10%, 10% to 30%, 20% acting to connect the silicon electrode to the parts of the to 40%, 30% to 50%, or 40% to 60%. The porosity measure 55 system providing Voltage and allowing for the flow of current. ment can be made on an area percent basis or a volume An advantage of polysilicon over amorphous silicon (a-Si) percent basis. is that the mobility of the charge carriers can be orders of In addition, porous silicon could be readily integrated with magnitude larger than in single crystal silicon and the mate existing silicon-based integrated circuit (IC) manufacturing rial also can show greater stability under electric field and processes. 60 light-induced stress. The silicon substrate can be in any form that is amenable to The silicon substrate of the present invention can be a thin the production of a silicon electrode. The silicon substrate can layer of silicon that is formed upon another material, for comprise a monolithic piece of silicon, a coating of silicon example a thin layer of silicon formed on glass would con deposited onto another material, or a powder of silicon par stitute a silicon Substrate. ticles. The silicon substrate can be a monolithic form such as 65 In some embodiments the silicon Substrate used to make a chip, wafer, rod, needle, block, ingot, or the like. The silicon the electrode is a composite material comprising silicon par Substrate can alternately be in particulate form, for example in ticles dispersed in a matrix orbinder. The silicon substrate can US 8,758,584 B2 25 26 be made of a composite material comprising a powder of amount of mixing. The amount of silicon is generally set at silicon dispersed in a binder to make a composite silicon level at which the physical properties of the material, such as substrate. The silicon powder can be in the form of spheres, mechanical strength and flexibility will not suffer to the point crystallites, rods, fibers, or any other arbitrary shape. In an that the material is not useful. The amount of silicon will embodiment the composite electrode is made of silicon crys generally be from 0.1 volume percent to 70 volume percent of tallites dispersed in a polymeric matrix. The matrix or binder the composite material. In some embodiments the amount of can be an organic, inorganic, or organometallic polymer. silicon may be from 1 volume percent to 50 volume percent. Non-limiting examples of useful inorganic polymeric mate In some embodiments the amount of silicon may be from 10 rials include polyphosphaZenes, polysilanes, polysiloxane, volume percent to 40 volume percent. The amount of silicon polygermanes, polymeric Sulfur, polymeric selenium, sili 10 cones, and mixtures of any of the foregoing. can be from 1% to 5%, 5% to 10%, 10% to 15%, 10% to 20%, In some embodiments the polymer can be an organic poly 20% to 30%, 30% to 40%, 40% to 50% or 50% to 60%. mer. Non-limiting examples of Suitable organic polymeric The composite silicon substrate can be formed by methods materials include, but are not limited to, thermoset materials used for shaping polymeric materials such as coating, mold and thermoplastic materials. Non-limiting examples of poly 15 ing, and casting into shapes that are useful as electrodes. The mers useful in the invention include polyesters such as poly composite silicon Substrate electrode will generally be con ethylene terephthalate, polybutylene terephthalate, and poly nected to an electrically conductive wire to apply current and ethylene naphthalate, polycarbonates, polyolefins such as potential. The material can be cast, coated, and/or molded polyethylene, polypropylene, and polyisobutene, acrylic onto a conductive Substrate Such as a metal to form a conduc polymers such as copolymers of styrene and an acrylic acid tive junction for connecting conductors for transfer of current monomer, and polymers containing methacrylate, polya to and from the composite electrode. mides, thermoplastic polyurethanes, vinyl polymers, polyim The silicon substrates of the invention generally have a ides, polyamides, polytetrafluoroethelene and other fluo high enough electrically conductivity to act as electrodes, and ropolymers, and mixtures of any of the foregoing. to transmit current for the oxidation and/or reduction of the The binder can be insulating, semiconductive, or conduc 25 bound redox-active moieties. To make the silicon substrate tive. In an embodiment, the binder is a material. Such as a more conductive, the silicon Substrate will generally include polymer, that is an insulating material. Where an insulating impurities or dopants to increase electrical conductivity. binder is used, the current will tend to only flow through the Where polycrystalline silicon is used, the polycrystalline sili dispersed silicon powder. In some embodiments, the binder con electrode can either be deposited as doped polycrystalline includes conductive components. In some embodiments, the 30 silicon (in situ doped) or can be deposited undoped and Sub binder comprises a conductive polymer Such as polyaniline, sequently doped with an impurity dopant such as phosphorus polyacetylene, poly(alkylthiophene), poly(alkylpyrrole), and or boron by ion implantation or a thermal diffusion process. the like. In some embodiments, the conductive component Dopant impurities, such as phosphorus and boron, tend to can comprise conductive particles such as metal particles, diffuse much more rapidly along the grain boundaries than Such as nickel particles other conductive particles including 35 they do through the silicon itself. carbon particles. In some embodiments, the conductive com The dopant for a type IV semiconductor Such as silicon, ponent is chosen Such that the conductive component Such as can be, for example, either an electron donor or an electron the conductive polymer exhibits reduction and/or oxidation acceptor. Suitable electron donors are phosphorous, arsenic, potentials that are outside of the reduction and/or oxidation antimony, and bismuth. In some embodiments the dopant is potentials of the redox active moieties. 40 phosphorous. In some embodiments the dopant is antimony. The composite silicon Substrate can be formed by mixing Suitable electron acceptors are boron, aluminum, gallium, the silicon powder with a monomer, oligomer, or prepolymer and the like. In some embodiments, the dopant is boron. In and curing the monomer, oligomer or prepolymer to form a Some embodiments, electronacceptors can impart a chemical polymeric matrix. The polymerization can be initiated in any resistance to the silicon electrode. manner known in the art or disclosed herein. The polymer 45 Where the silicon substrate is a monolithic material such as ization can be initiated, for example, thermally or photo a wafer, the dopant can be either distributed throughout the chemically in the presence of an initiator. The polymerization bulk of the silicon, or can be limited to the surface region of can be carried out with one or more crosslinkers. The cross the silicon wafer. In some embodiments, for example where linkers can be chosen to adjust the physical properties of the the silicon substrate comprises multiple Zones with different polymeric matrix and thus adjust the properties of the com 50 redox active moieties, the dopant can be distributed such that posite silicon Substrate. The composite silicon Substrate can isolated regions of the surface of the silicon substrate are be formed by mixing the silicon powder with a molten ther conductive. moplastic polymer, forming the Substrate, and allowing the The dopant is generally present in an amount greater than mixture to cool. The composite silicon Substrate can be 0.01 weight percent of the silicon, and generally in an excess formed by mixing the silicon powder with a polymer or 55 of 0.1 percent of the silicon. Generally, the dopant is less than prepolymerina solvent, and allowing the solvent to evaporate 3 weight percent of the silicon, and almost always less than 6 to form the composite. Combinations of any of the above weight percent of the silicon. The presence of small amounts methods can be used. of the dopant can increase the electrical conductivity. The The electrical properties of the composite silicon substrate dopant may not be homogenously distributed throughout the can be affected by the amount of silicon, the particle size, and 60 silicon, and the local concentration may vary between differ the particle shape. In general, the amount of silicon in the ent regions of the silicon material. composite is high enough to create conductive pathways The electrical resistivity (reciprocal of conductivity) can be throughout the material. This amount of material necessary to for example 0.1 (ohm-centimeters), 1 (ohm-centimeters), 10 provide conductive paths across the material is sometimes (ohm-centimeters), 100 (ohm-centimeters), to in excess of called the percolation threshold. The amount of silicon par 65 1000 or even 10,000 (ohm-centimeters) or even higher which ticles required for conduction can also depend on the process is comparable to graphite and conventional metallic conduc ing conditions such as the Viscosity of the binder and the tOrS. US 8,758,584 B2 27 28 In some embodiments the resistivity of the silicon substrate presence and or the level of a substance in the Solution. In is the range of 0.0001 to 100,000 G.2-cm. In some embodi Some embodiments, the semiconductor Surface can have at ments the resistivity of the silicon substrate is the range of least one redox-active functional group sensitive to an ana 0.001 to 10,000 G.2-cm. In some embodiments the resistivity lyte, and at least one redox-active functional group that is of the silicon substrate is the range of 0.01 to 1000 S2-cm. In substantially insensitive to the analyte to be tested. When some embodiments the resistivity of the silicon substrate is used in this manner, the Substantially insensitive group can the range of 0.1 to 100 G.2-cm. In some embodiments the act as a reference, allowing for greater accuracy and repro resistivity of the silicon substrate is within the range of 1 to ducibility of the measurements. 100 G.2-cm. In some embodiments the resistivity of the silicon In Some situations, an electrochemical sensor can include a substrate is within the range of 10 to 90 S2-cm. In some 10 layer of a nitride. Such as silicon nitride, having immobilized embodiments the silicon Substrate is single crystal silicon is thereon a redox-active moiety that is sensitive to the presence Si(100) that is P-type with a resistivity of 10 to 90 S2-cm. In of an analyte. Such as H+, and/or a redox-active moiety that is some cases several silicon substrates with different resistivi insensitive to the presence of the analyte. ties may be used. For example, a system of the invention may In some situations, redox active moieties that are sensitive comprise a one lightly doped silicon Substrate having one 15 and/or insensitive to the presence or absence of an analyte are redox active species bound to it, and also a more highly doped bound to a surface of a solid State working electrode, such as silicon Surface having another redox active species bound to a semiconductor (e.g., silicon) Surface, through a Surface-to it. For example, a system of the invention may comprise one carbon interaction, Such as, e.g., a silicon-to-carbon bond in lightly doped silicon Surface having a pH sensitive redox cases in which the working electrode is formed of silicon. The active moiety Such as anthraquinone bound thereto, and a silicon-to-carbon interaction can be a covalent interaction. second silicon Surface that is more highly doped having a The carbon atom in Such a case is a carbon atom of a redox hydrogen ion insensitive redox active moiety such as fer active moiety. In other situations, redox active moieties can be rocene bound to it. bound to a Surface of a working electrode through Surface-to In some cases different regions of the silicon Substrate can oxygen, Surface-to-sulfur, and/or surface-to-carbon interac be doped at different levels. For example, it is well known in 25 tions, which can be covalent interactions. the semiconductor processing art that a mask can be used to The redox groups can be chemically or physically bound to cover some regions of the silicon, while leaving other regions the Surface. The redox groups can be attached to the semicon exposed. The exposed regions can be treated selectively with ductor covalently (e.g., via Si C, Si O, or Si S bonds), dopants resulting in a silicon Surface wherein some regions can be adsorbed to the semiconductor, or can be attached to are doped differently than other regions. By using multiple 30 polymers that are either covalently or non-covalently bound steps with various masks, the conductivity properties of dif to the surface. Covalent binding of either the redox group or ferent regions of the silicon surface can be controlled. Thus, the polymer to which the redox group is a part can be benefi Some regions can have high conductivity, some low, some cial in improving the lifetime and stability of the electrode. regions can have a large band gap, and other regions can have Semiconductor materials such as silicon and germanium can Small band gaps, some regions can have N-doping, some 35 form covalent bonds with carbon, and thus is a desirable P-doping, and some no doping. In addition, the various substrate for functionalizing with carbon based molecules. regions can be connected with conducting regions, for The covalent binding to the surface can be through a bond example deposited metal, e.g., gold to be able to electrically between the semiconductor, e.g., silicon, and carbon, oxygen, address the various regions. nitrogen, Sulfur, or other atom. In some embodiments the In some embodiments, Nisil is used. Nisil is an alloy of 40 bondis between silicon and carbon. In some embodiments the nickel and silicon. In some embodiments Nisil with 4%-5% bond is between silicon and oxygen. The physical bonding silicon is used. In some embodiments Nisil with 4.4% silicon can occur through adsorption, and can include, for example, is used. In some embodiments Nicrosil is used. Nicrosil is a spontaneous self assembly onto the semiconductor Surface of nickel alloy. In some cases Nicrosil comprising 14.4% chro molecules such as those derived from fatty acids which com mium, 1.4% silicon, and 0.1% magnesium is used. 45 prise redox active moieties. In some cases, for example, where the silicon Substrate is Where a linker group is used, the linker can be small, for cast silicon, the silicon Substrate will include, for example, a example one to 3 atoms, or can be longer, e.g., 20 to 100 silicide of a transition metal to provide castability. The sili atoms, or can be any size between large and a small linker. cide of the transition metal can provide favorable mechanical Where a short linker is used, the redox-active moiety is held properties to the cast alloy. Typical metals useful in providing 50 close to the Surface. Where a longer group is used, the redox the transition metal silicide present in the silicon electrode of active moiety may be able to move away from the surface, for the secondary cell of this invention include titanium, Zirco example into the solution. Linker groups can comprise hydro nium, hafnium, Vanadium, columbium, chromium, molybde philic, hydrophobic groups, or mixtures thereof. Linker num, tungsten, manganese, iron, cobalt, nickel, copper, ruthe groups can comprise, for example, hydrocarbons, esters, nium, rhodium, palladium, osmium, iridium, platinum, gold, 55 ethers, amides, amines, carbonyls, thiols, olefins, silicones, or and silver. Most commonly, the transition metal present as the other organic, inorganic or organometallic groups. The linker silicide in the silicon alloy may be a silicide of manganese, groups can be formed by polymerization or oligomerization chromium, iron, cobalt, nickel, or molybdenum. The amount reactions such as free radical, cationic, or anionic polymer of the silicide may be sufficient to provide satisfactory casta ization. The linker group can comprise, for example, ethylene bility but not great enough to deleteriously effect the proper 60 oxide, propylene oxide, vinyl ether, or acrylamide repeat ties of the silicon, i.e., from 2 percent or more up to as high as units. Linkers can have ring structures including aromatic 30 percent transition metal, elemental basis. rings. The variation in the linker structure can be used to vary Redox-Active Moieties the mobility of the redox-active moiety in the solution. In some embodiments, an electrochemical sensor com As used herein, the term moiety generally refers to a por prises a Solid state (e.g., semiconductor) Surface that is modi 65 tion of a molecule or Substituent. A redox-active moiety may fied with redox-active functional groups. At least one redox be highly substituted, and can still act as a redox-active moi active functional group on the Surface is sensitive to the ety. As used herein, the terms “redox active moiety”, “redox US 8,758,584 B2 29 30 active group”, “redox active functional group', and “redox 5 shows examples of the hydrogen ion insensitive redox group' are used interchangeably. Thus, for example, the active moiety ferrocene bound to a silicon Surface. redox-active moiety ferrocene includes substituted fer Non-limiting redox-active moieties that are sensitive to rocenes, ferrocene polymers, and ferrocene covalently hydrogen ion include: quinones, anthroquinones, phenanth attached to the surface with or without linker molecules. roquinones, phenylene diamines, catechols, phenothiazinium In some embodiments, the redox-active moiety can be dyes, and monoquaternized N-alkyl-4,4'-bipyridinium. In incorporated into a polymer, and the polymer comprising the Some embodiments the redox-active moiety that is sensitive redox active moiety can be immobilized onto the semicon to the presence of hydrogen ion can include inorganic mate ductor surface. The immobilization of the polymer can be rials and metal oxides. Hydrogen ion sensitive inorganic 10 redox-active inorganic moieties include Prussian Blue, either chemical or physical. The immobilization of the poly Ni(OH), and RuO. FIG. 6 shows examples of the hydrogen mer can be through covalent bonds, or through adsorption of ion sensitive redox-active moiety anthracene covalently the polymer to the semiconductor Surface. bound to a silicon surface. FIG. 7 shows an example of a In Some embodiments, the redox-active moiety is bound to silicon surface having covalently bound thereto both the a particle that is bound to semiconductor. The particle is 15 hydrogen ion sensitive redox-active moiety ferrocene and the generally an electrically conductive particle. The particle hydrogen ion insensitive redox-active agent anthracene. attached to the semiconductor Surface in a manner that allow In some embodiments the analyte is carbon monoxide for current to flow between the semiconductor surface and the (CO). An example of a CO sensitive redox-active agent is particle. The particles can be attached chemically or physi ferrocenyl ferrazetine disulfide or ferrazetine-ferrazetine dis cally to the Surface. For example, the redox-active moiety can ulfide. A CO insensitive redox-active agent can be, for be attached to a carbon particle, and the carbon particle example, ferrocene. attached to the semiconductor Surface. In some embodiments, In some embodiments the analyte is an alkali metal. Alkali the carbon particle can be a carbon nanotube. In some metal sensitive redox-active agents include, for example: embodiments of the invention, carbon nanotubes can be 1,1'-(1,4,10,13-tetraoxa-7, 16-diazacyclooctadecane-7, 16 attached to the surface of the semiconductor, where there are 25 diyl dimethyl), ferrocenylthiol, and other ferrocene deriva redox-active groups attached to the carbon nanotubes. For tives containing covalently attached cryptands. These mate instance, attachment of well-aligned single-walled carbon rials are described, for example, Hammond, et al., J. Chem. nanotubes architecture to a single-crystal silicon Surface can Soc. Perkin. Trans. 1707 (1983); Medina, et al., J. Chem. Soc. be used. In some embodiments, for example, ferrocenemetha Chem. Commun. 290 (1991); Shu and Wrighton, J. Phys. nol molecules are attached to single walled carbon nanotube 30 Chem. 92, 5221 (1988). Included are examples such as the (SWCNT) arrays that are directly anchored to the silicon above ferrocenyl ferrazetine and ferrocenyl cryptand, in surface, for example, a (100) surface. For example, single which an ordinarily chemically insensitive redox center (fer wall carbon nanotubes can be coupled to the Surface using this rocene) is covalently linked to a chemical recognition site in method as described in Yu et al., Electrochimica Acta 52 Such a way as to make the redox center chemically sensitive. (2007) 6206-6211. 35 Also suitable are molecules or polymers in which the sensor The redox-active moieties generally have reversible redox and reference functionalities are covalently linked such as activity with well-defined cyclic voltammetry oxidation and/ 1-hydro-1'-(6-(pyrrol-1-yl)hexyl)-4,4'-bipyridinium bis or reduction peaks. A Suitable reference redox reagent can (hexafluorophosphate), as described by Shu and Wrighton, J. vary from application to application or medium to medium Phys. Chem. 92,5221 (1988). depending on the intended use. 40 In some cases, more than one analyte-sensitive redox-ac The position of the reduction and/or oxidation potentials of tive moiety can be used. For example, there can be one redox the redox active moiety can be chosen to improve the accu active moiety that is sensitive to hydrogen ion, and another racy and quality of the measurement of redox potential. In that is sensitive to a second analyte Such as CO, oxygen, Some cases, the reduction and or oxidation potential can be ammonia, or an alkali metal. This approach allows for the chosen to be away from other redox active species. The sili 45 simultaneous measurement of several analytes. In some cases con Surface, for example, generally has a wide window in redox-active agents sensitive to 3 or more analytes can be which to perform measurement of reduction or oxidation used. Where there are redox agents sensitive to multiple ana potential without interfering with the measurement of the lytes, in many cases, it is also desired to have one or more reduction and or oxidation of the redox active moieties bound analyte-insensitive redox-active moieties bound to the semi to the Surface. The silicon Surface can generally be used to 50 conductor Surface as well to provide a reference, to improve measure oxidation and/or reduction potentials from between accuracy, and to minimize or avoid calibration. negative 2V to positive 2V. In some cases, for example where Where more than one redox-active moiety is used it can be the medium is an aqueous medium, the reduction and/or important to ensure there is not significant interference oxidation potential of the redox-active moiety can be chosen between the peaks. This can be desirable especially when the so as not to fall within the reduction or oxidation potential of 55 multiple redox-active moieties are on the same electrically the medium to minimize interference. This can be useful addressable Zone on the semiconductor substrate. This can be where cyclic Voltammetry is used, and is less important when achieved, for example, by ensuring that there is sufficient square wave Voltammetry is used. separation of the oxidation and reduction potentials, or by Redox-active moieties that are insensitive to the presence physically separating the redox moieties. of analytes should show little or no change in their oxidation 60 In some embodiments, the semiconductor Substrate has a and/or reduction potentials in the presence or absence of such plurality of isolated separately electrically addressable Zones. analytes. Redox-active moieties that are generally insensitive In some embodiments, the different Zones will comprise dif to the presence of analytes, and in particular are insensitive to ferent redox-active moieties. The use of separate Zones can be the presence of hydrogen ion include: ferrocene, polyvinyl beneficial in that the Voltammetric measurements can be car ferrocene, Os(bpy)Cl. Ru(bpy)Cl. Viologen, polyviolo 65 ried out separately, allowing for the use of multiple redox gen, and polythiophene. Redox reagents having a high degree active agents with that have similar reduction and/or oxida of electrochemical reversibility are generally preferred. FIG. tion potentials. US 8,758,584 B2 31 32 The separately electrically addressable Zones can be made simultaneously or sequentially address the Zones, for by conventional semiconductor processing methods, for example, by mutiplexing (MUX). example masking to create structures in specific areas on the Solid State Electrode Sensors Surface, for example specific areas on the Surface having Another aspect of the invention provides a Solid State sen certain levels of doping. Conventional semiconductor pro- 5 Sor having a solid State working electrode formed of a solid cessing can also be used to incorporate conductive intercon state (e.g., semiconductor) Substrate. The Solid state electrode nects allowing the Zones to be separately addressable. Mask sensor comprising the Solid state Substrate can be used for the ing can also be used during the attachment of the redox-active measurement of the presence or absence of one or more moieties to the semiconductor Surface to attach specific redox analytes, or can be used to accurately measure the concentra active moieties to different regions of the surface. 10 tion of analyte in a sample. The semiconductor substrate with separately addressable In some embodiments, a solid state electrode sensor is Zones can effectively create an electrochemical sensor array. formed of a semiconductor, Such as silicon. The semiconduc Another aspect of the invention provides a semiconductor tor electrode sensor can be used to detect the presence or electrochemical sensor array wherein a plurality of Zones, absence and/or the measure the concentration of analytes, each Zone comprising a redox active moiety. The array can 15 including hydrogen ion, alkali metals, CO, or O. In some have multiple Zones with analyte-sensitive redox-active moi embodiments, the semiconductor electrode sensor is used to eties, and one or more Zones with analyte-insensitive redox measure the concentration of hydrogen ion, or pH. active moieties. A Zone can have a single redox-active moiety, The semiconductor electrode sensor comprises a semicon or multiple redox active moieties. The array can be con ductor Substrate as described above comprising a redox-ac structed, for example, to measure both pH and O, wherein 20 tive moiety that is sensitive to the presence of an analyte. The one Zone comprises a redox-active moiety sensitive to hydro semiconductor electrode sensor may also comprise a redox gen ion, another Zone has a redox active moiety sensitive to active moiety that is insensitive to the presence of an analyte. O, and a third Zone with a redox-active moiety that is insen The semiconductor electrode sensor can comprise more than sitive to both hydrogen ion and O. one semiconductor Substrate. For example, the sensor may In some embodiments, an array of separately addressable 25 comprise one semiconductor Substrate that has an analyte Zones or an array of electrodes is used where there are a sensitive redox active moiety and another semiconductor Sub plurality of Zones or electrodes that are each constructed to strate having an analyte insensitive redox active moiety. measure the same analyte and used in a redundant matter, The semiconductor electrode sensor is configured to be wherein another Zone capable of measuring the same analyte incorporated into a system that will Supply Voltage, and can is used either simultaneously or in place of the other Zone or 30 drive current to the sensor to perform voltammetry. Thus, the electrode. In some cases, more than one Zone or electrode, for semiconductor Substrate or Substrates within the sensor may example in an array, is used simultaneously to improve the be electrically connected in a manner which will allow for quality of the measurement. In some cases, more than one connection to a device for Supplying and measuring current Zone or electrode, for example in an array, is used sequen and Voltage. tially, wherein if one Zone or electrode shows degraded per- 35 The semiconductor electrode sensor is generally the work formance, the measurement of that analyte is performed on ing electrode in an electrochemical system that will also another Zone or electrode constructed to have similar charac comprise a counter electrode, and in Some embodiments, a teristics. The sequential use of similar Zones or electrodes can reference electrode. provide reliability of measurement over time. While the elec The sensor may be put into contact with a sample having trodes of the present invention can be prepared to be robust 40 the analyte to be detected. The sample is generally a liquid and to resist fouling, in some circumstances, degradation of sample. In some cases the sample can be a gel, Suspension, the measurement overtime may occur. In some embodiments, molten, or semi-solid medium. The sample can be, for the maximum current (I) can be monitored over time. A example, a foodstuff. The sample can be any type of liquid system can be configured, for example, such that when the including hydrocarbons, oils, fluorocarbons, silicones, and maximum current drops below a certain level, a Switch is 45 aqueous Solutions. Where the analyte is hydrogen ion, an made to a redundant Zone or electrode for further measure aqueous medium is generally used, but in Some case a polar ment of that analyte. There can be multiple redundant ele protic medium or polar aprotic medium can be used. The ments, e.g., 1, 2, 3, 4, 5, 10, 20 or more redundant elements. sensor is useful for measuring pH in aqueous solutions. The semiconductor substrate of the invention can also In some embodiments, the sensor of the invention can comprise circuitry. The circuitry can be used, for example for 50 accurately measure analyte concentrations where the analyte controlling the current and potential provide to the redox is presentina concentration range from 10' M to 10''M. In active moiety. The circuitry can also be used for analyzing Some embodiments, the sensor of the invention can accurately signals or for processing data related to the Voltammetric measure analyte concentrations where the analyte is present measurement. The circuitry can also have other functionality, in a concentration range from 10 M to 10' M. In some Such as the ability to measure other parameters such as tem- 55 embodiments, the sensor of the invention can measure the perature, the ability to store date, or the ability to send data concentration to an accuracy of plus or minus 100%, 50%, and receive instructions from a remote location. The circuitry 30%, 20%, 10%, 5%, 2% or 1%. In some embodiments, the can contain, for example, an amplifier Such as an operational sensor of the invention can measure the concentration within amplifier. The circuitry can contain, for example, an analog to a range of 10M to 10' M to an accuracy of plus or minus digital converter (ADC). In some cases having the amplifying 60 100%, 50%, 30%, 20%, 10%, 5%, 2% or 1%. and ADC functions incorporated into the semiconductor sub In some embodiments, the analyte is hydrogenion, and the strate can provider higher quality and reliability of the trans sensor of the invention can accurately measure the pH in a mission of the signal from the sensor. The use of circuitry on range from pH 1 to pH 14. In some embodiments, the analyte the semiconductor substrate can be particularly useful when is hydrogenion, and the sensor of the invention can accurately an array of Zones is utilized. The circuitry can assist in man- 65 measure the pH in a range from pH 3 to pH 10. In some aging the passage of current in and out of the semiconductor embodiments, the sensor of the invention can accurately mea Substrate. In some cases, the circuits can allow for Schemes to sure pH to an accuracy of plus or minus 0.5,0.3, 0.2,0.1, 0.07, US 8,758,584 B2 33 34 0.05, 0.03, 0.02, or 0.01 pH units. In some embodiments, the is hydrogen ion and the sensor is capable of measuring pH sensor of the invention can accurately measure pH in a range with an accuracy of 0.2 units after autoclave treatment at 121° from pH 3 to pH 10 to an accuracy of plus or minus 0.5,0.3, C. for 400 minutes. 0.2,0.1, 0.07, 0.05, 0.03, 0.02, or 0.01 pH units. A subject sensor that does not require calibration over long The semiconductor electrode sensors of the invention can 5 periods of time in a medium that can change characteristics is accurately measure analyte concentration in a wide variety of useful, for example, as an implantable sensor. The implant sample types. The sensors can be made to be robust, and able sensor can be placed under the skin or within the body in resistant to fouling, and therefore reliable for long-term mea contact with a bodily fluid such as blood, saliva, breast milk, SurementS. amniotic fluid, lymph, Sweat, tears, or urine. The sensor can Another aspect of the invention is a sensor which does not 10 measure the concentration of analytes such as hydrogen ion, require routine calibration (or re-calibration), or in some Sodium, potassium, calcium, or oxygen. cases any calibration at all. Conventional potentiometric sen The implantable sensor has an electrode configured to be in sors rely on a glass membrane to sense, for example, hydro contact with a bodily fluid, said electrode comprising a semi genion. These types of sensors generally need to be calibrated conductor surface that has immobilized thereon a redox ona regular basis, usually by placing the sensorinto standards 15 active moiety, wherein the redox active moiety has an oxida of known analyte concentration. These types of sensors gen tion potential and/or reduction potential that is sensitive to erally need calibration when going from one solution to concentration of said ion. The implantable sensor can be another solution, and will also need calibration with time, included in an implantable medical device Such as described even if kept within the same solution and upon standing in U.S. Pat. No. 6,738,670. For example, the implantable outside of a solution. The situation is made worse if there is a medical device in which the sensor resides could include change in the composition of the medium over the time that pacemakers, defibrillators, drug delivery pumps, diagnostic the sensor is monitoring the medium, for example, when recorders, cochlear implants, and the like. The implantable monitoring a chemical reaction, biochemical reaction, or fer medical device is typically programmed with a therapy and mentation. In these cases, potentiometric sensors may drift then implanted in the body typically in a Subcutaneous pocket and need calibration due to the accumulation of some species 25 at a site selected after considering clinician and patient pref in the reaction, or due to fouling of the sensor by species erences. In some embodiments the implanted device is in a present. form which can be swallowed, allowing the measurement of In some embodiments, the sensors of the present invention the properties of the regions encountered as it passes through do not need to be calibrated under any of these situations. In the body Such as the digestive tract including the stomach, the Some embodiments, the sensors of the invention do not need 30 upper and lower intestines, and the colon. The information to be calibrated over time in solution. In some embodiments, obtained by the sensor in the implanted device can either be the sensors of the invention do not need to be calibrated after accessed in real time, for example, by wireless communica an hour, 10 hours, 1 day, 2 days, 5 days, a week, two weeks, tion, or can be retrieved from the device after passage through a month, 6 months, 1 year, 2 years or longer while in a solution the body. A wide variety of programmers, also known as or in storage. In some embodiments the sensors or the present 35 downlink transmitters, can be used to transmit data to and invention are accurate at measuring analyte concentration to receive data from the implantable medical device. Examples 50%, 40%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2% or 0.1% after of downlink transmitters include devices such as physician the times above. In some embodiments where the sensors programmers, patient programmers, programming Wands, measure pH, the sensors are accurate to 1, 0.8, 0.5, 0.3, 0.2, telemetry access units, and the like. The clinician, for 0.1, 0.08, 0.05, 0.03, 0.02, or 0.01 pH units after the times 40 example, can periodically use a physician programmer to above. In some embodiments where the sensors measure pH, communicate with the implantable medical device to manage the sensors are accurate to within 0.1 pH units after one week the patient’s therapy and collect implantable medical device in Solution or in storage. data. The semiconductor electrode sensor can be incorporated In some embodiments, the sensor is capable of measuring into or attached to the implantable medical device and can analyte concentration without any calibration with an exter 45 provide data on analyte concentration within the region of the nal standard. In some embodiments, the sensor remains sen body into which it is implanted. The patient can use the sitive to the analyte without calibration after a first use by an patient programmer to communicate with the implanted end user. device to make therapy adjustments that have been pro In some embodiments the analyte is hydrogen ion and the grammed by the clinician. Both the physician programmer sensor remains sensitive to hydrogen ion after exposure to a 50 and patient programmer can have an antenna locator that cell culture medium for at least 1, 3, 6, 9, 12, 18, or 24 hours indicates when a telemetry head is aligned closely enough or 2, 3, 4, 6, 8, 12, 24, 48, 60, 90, or more days. In some with the implanted device for adequate telemetry. embodiments the analyte is hydrogen ion and the sensor Another aspect of the invention is a method for measuring remains sensitive to hydrogen ion after exposure to a cell concentration in a bodily fluid within a body, the method culture medium for at least 3 days. In some embodiments the 55 comprising placing a semiconductor electrode sensor com analyte is hydrogen ion and the sensor remains sensitive to prising a redox active moiety in contact with the bodily fluid, hydrogen ion after exposure to a cell culture medium for at and operating the sensor to yield a value of the concentration least 6 days. In some embodiments, the sensor is capable of of the analyte present in said bodily fluid. measuring pH with an accuracy of 0.2 units after exposure to Systems for Measuring the Concentration and/or Presence or the cell culture medium. 60 Absence of an Analyte In some embodiments, the analyte is hydrogen ion and the Another aspect of the invention provides a system for mea sensor is capable of measuring pH with an accuracy of 0.2 Suring analyte concentration. In some embodiments, the sys units after autoclave treatment at 121° C. for 10, 20, 40, 80, tem comprises a working electrode having a solid state (e.g., 100, 200, 400, or 800 minutes. In some embodiments, the semiconductor) Surface that has immobilized thereon a redox analyte is hydrogen ion and the sensor is capable of measur 65 active moiety, wherein the redox active moiety has an oxida ing pH with an accuracy of 0.2 units after autoclave treatment tion potential and/or reduction potential that is sensitive to the at 121°C. for 40 minutes. In some embodiments, the analyte presence of an analyte; a counter electrode and optionally a US 8,758,584 B2 35 36 reference electrode. The system further comprises a source and 90 S2-cm, or 10 S.2-cm and 4.0 G.2-cm. In some embodi for Supplying a plurality of potentials to the working elec ments, an N-type semiconductor (e.g., silicon wafer) is used trode, and a device for measuring current through the working for a working electrode having an H+ insensitive moiety (e.g., electrode at the plurality of potentials. The working electrode ferrocene). In some situations, an undoped or lightly doped referred to herein can comprise the solid state electrochemi p-type semiconductor (e.g., silicon wafer) is used for a work cal sensor described above. It is desirable in some embodi ing electrode having an H-- sensitive moiety (e.g., ments that the solid state surface also has immobilized anthracene). In other embodiments, a P-type semiconductor thereon a second redox active moiety having an oxidation (e.g., silicon wafer) may be used for both the pH sensitive and potential and/or reduction potential that is insensitive to the the pH insensitive moieties. presence of said analyte. The redox active moiety that is 10 In some embodiments, the system will have 3 or more insensitive to the presence of the analyte can be on the same working electrodes. For example, in some embodiments, the Solid state surface, or can be on another Solid state surface in system will have one working electrode comprising a semi electrical communication with the system and in contact with conductor surface that has immobilized thereon a redox the sample. In some embodiments, the redox active moiety active moiety that is sensitive to the presence of a first analyte, that is sensitive to the presence of the analyte is on first solid 15 a second working electrode comprising a semiconductor Sur state (e.g., semiconductor) working electrode, and the redox face that has immobilized thereon a redox active moiety that active moiety that is insensitive to the presence of the analyte is sensitive to the presence of a second analyte, and a third is on a second solid state working electrode that is electrically working electrode comprising a semiconductor Surface that isolated (or electrically insulated) from the first working elec has immobilized thereon a redox active moiety that is insen trode. The system is configured such that the working elec sitive to the presence of either the first analyte nor the second trode(s), the counter electrode, and optionally the reference analyte. The system can also have more than 3 working elec electrode are in contact with the sample. In many embodi trodes, for example having 4, 5, 6, 7, 8, 9, 10, 12, 20, 50 or ments, the sample is a liquid sample, and the electrodes are more working electrodes, each having redox active moieties each in contact with the liquid. In some cases, the sample will sensitive to and analyte. These systems can also have one or not be a liquid, but may be a Solid, generally comprising a 25 more than one semiconductor working electrode having a Solid electrolyte, a semi-solid (e.g., Solid-liquid mixture), or a redox species that is insensitive to the analytes, for example to gas, or a sample having a viscosity characteristic of a gas or provide a reference. In some cases more than one redox liquid. In some embodiments, the first Solid state working species that is insensitive to the analyte can be used. electrode is separately and independently addressable from In some embodiments the system of the present invention the second solid state working electrode, enabling a reading 30 comprises a probe that comprises the 2 or more electrodes. from the first working electrode independently from the sec The probe can physically hold the electrodes such that the ond working electrode. electrodes can be brought into contact with the sample. The In some embodiments, the system will have two or more probe allows the working electrodes to be held close to the working electrodes. For example, in some embodiments, the reference and/or counter electrode. FIG. 21 shows an exem system will have one working electrode comprising a semi 35 plary embodiment of a probe of a system of the present conductor surface that has immobilized thereon a redox invention having 4 electrodes: a first working electrode active moiety whose oxidation potential and/or reduction (WE1), a second working electrode (WE2), a reference elec potential is sensitive to the presence of said analyte, and a trode (RE), and a counter electrode (CE). WE1 and WE2 can second working electrode comprising redox active moiety each be formed of a solid state material. Such as a semicon whose oxidation potential and/or reduction potential is insen 40 ductor. FIG. 21(a) shows a shaded drawing of the probe. FIG. sitive to the presence of said analyte. An example of a system 21(b) shows a line drawing of the probe. As shown in FIG.21, with two working electrodes is a system having two semicon in some cases it can be beneficial in the present invention for ductor wafers or chips, one of which has a redox active moiety the reference electrode and the counter electrode to have a which is sensitive to pH. Such as anthracene, and another ring configuration. In other embodiments, only one of the redox active moiety which is insensitive to pH. Such as a 45 reference electrode or counter electrode will have a ring con ferrocene. In some cases the semiconductor wafer or chip on figuration. A ring electrode can in Some cases provide signal which each redox active species is immobilized may be a stability. While this embodiment shows one configuration, different type of semiconductor wafer or chip. For instance, there are other ring electrode configurations that can be used the semiconductor wafer or chip to which the pH sensitive with the present invention. moiety is bound may have one doping level, and the semi 50 In some embodiments it is useful to use undoped or lightly conductor wafer or chip on which the pH insensitive moiety is doped semiconductor (e.g., silicon) Substrates for a moiety bound may have a different doping level. This type of con such as anthracene. While not being bound by theory, the struction can be beneficial because, in some cases, one type of bandgap of the semiconductor, e.g., silicon can be influenced redox active species will perform better in terms of amplitude, by the level of doping of the semiconductor, and it is believed sensitivity or stability with one type of doping, while another 55 that in some cases, the use of a semiconductor with the appro redox active species will perform better on a semiconductor priate level of doping can be useful to tailor the appropriate wafer orchip with a different type of doping. In some embodi redox active moiety with the appropriate band gap. Thus in ments, the pH sensitive moiety, e.g., anthracene, is bound to a Some cases it is desirable to use lightly doped semiconductor, semiconductor wafer that has a low level of doping, and the e.g., silicon with a moiety Such as anthracene. Thus in some pH insensitive moiety, e.g., ferrocene is bound to a silicon 60 embodiments it is useful to use two semiconductor working wafer that has a higher level of doping. In some embodiments electrodes: one optimized for ferrocene moieties and the the semiconductor wafer onto which the pH sensitive moiety, other optimized for the anthracene moieties. When two work e.g., anthracene is bound has a resistivity between 1 S.2-cm an ing electrodes are used, in some embodiments, two sequential 1000 S2-cm, or between 109.2-cm and 90 S2-cm, or between 10 electrochemical measurements (e.g., with square wave Volta S.2-cm and 40 S.2-cm, while semiconductor wafer onto which 65 mmetry) may be carried out using the same counter and the pH insensitive moiety, e.g., ferrocene, is bound has a reference electrode. For instance, the first measurement can resistivity between 1 milliohm-cm and 1000S.2-cm, or 1 S.2-cm be conducted using reference, counter and working electrode US 8,758,584 B2 37 38 1 (e.g., anthracene derivatized) between -1.2 to -0.5 V, fol Where an analyte insensitive redox active moiety is used, lowed by the second measurement which may be conducted detection is generally accomplished by measuring the poten using reference, counter and working electrode 2 (e.g., fer tial difference, delta E, associated with current peaks for rocene derivatized) between 0 to 0.5 V. The peaks potential oxidation (or reduction) of the immobilized redox active moi detected in the first and second measurements can then be eties, where the magnitude of delta E can be related to the stored and processed to get a pH reading. This type of system concentration of analyte, e.g., hydrogen ion (H+) in solution. and method can be accomplished through the use of a bipo The analyte insensitive redox active moiety has an electro tentiostat or a two-channel multiplexer. A similar approach chemical response that is insensitive to variations in the can be applied to multiple working electrodes with a multipo medium and serves as the reference. Current peaks for oxi tentiostat or a multi-channel multiplexer. 10 dation or reduction of the reference and indicator are deter The system is configured to carry out Voltammetric mea mined from a Voltammogram using a counter electrode. Surements on the sample. Some embodiments provide a In some embodiments, the system further comprises a method which includes the measurement of pH with a volta computation system that communicates with the device for mmetric pH sensing system comprising the semiconductor measuring current. The computation system can have algo electrode sensor described above, a potentiostat for providing 15 rithms for calculating reduction or oxidation potential from Voltage to the electrodes, and a meter for detecting the current the measured current at a plurality of potentials from the as a function of Voltage. Voltammetry measurements. The computing systems can be The counter electrode typically is needed to complete the part of the sensing system, in some cases allowing the sensing electrochemical circuit to make the measurements described system to be self-contained. The computing system can com herein. The counter electrode is generally made of a material prise memory for storing raw and/or processed data from the which is electrochemically inert to the medium so that current sensors. The computing system can be connected to a trans overloading does not occur during the course of measure mission device that will wirelessly, by wire, fiber or other ment. Suitable materials in many applications include plati means, transmit processed data to an external device. The num, silver, gold, stainless steel, and carbon. computing system can provide signals and measurements A reference electrode is optional and is used as a third 25 which can be transmitted in Some cases in real time, allowing electrode in some embodiments of the invention. In the case the system to alert end users of conditions which may require of a three-electrode system, the counter electrode generally attention. The transmitted signals and measurements can, for completes the circuit, allowing current to flow through the example, provide the information required for adjusting a cell, while the reference electrode maintains a constant inter manufacturing process such as a chemical or biochemical facial potential difference regardless of the current. In the 30 process. case where the system comprises an analyte sensitive redox In some embodiments the system is made up of a housing active moiety and an analyte insensitive redox active moiety, that holds the semiconductor electrode sensor that is electri the analyte insensitive redox active moiety can act as a refer cally connected to a unit comprising the source for Supplying ence, allowing the potential difference to be used to determine a plurality of potentials and the current measuring device. In analyte concentration. Even where the system comprises an 35 Some embodiments the unit also comprises the computing analyte insensitive moiety, in some embodiments, a reference system described above for at least partially analyzing the electrode will still be used. In some embodiments, pseudo data. The unit can be self powered, e.g., with a battery, or can reference electrodes can also be utilized. Reference elec have a connection to an outside power Source. The unit can trodes that can be employed include: Standard hydrogen elec have a display and input buttons to allow the user to control trode (SHE), also known as “normal hydrogen electrode' 40 the measurement and to read the output from the sensor. The (NHE), saturated calomel electrode (SCE), copper-copper(II) unit can have transmission capability for sending out data, sulfate electrode, and silver/silver chloride (Ag/AgCl) elec and for receiving instructions or to be tested by an external trode. In some embodiments a silver electrode or a polyure device. thane coated silver electrode can act as a silver/silver chloride FIG. 1 shows drawings of an embodiment of the connec electrode where sufficient chloride is present at or near the 45 tion of a semiconductor sensor electrode into a sensor hous silver electrode. In some cases, e.g., in non-aqueous medium, ing for use in measuring analytes in a fluid Such as the fluid in a metal electrode Such as a platinum or a silver electrode, or a a biochemical reactor. FIG. 1(a) shows a blow up drawing of polyurethane coated silver electrode can be used as the ref an assembly that holds the semiconductor electrode sensor erence electrode. and provides electrical connections to the semiconductor To carry out Voltammetry, the system generally has a 50 electrode sensor for voltammetry measurements. In FIG. 1(a) Source for Supplying a plurality of potentials. The Voltamme the semiconductor electrode sensor (I), is held in place by the try can be, for example cyclic Voltammetry, pulse Voltamme end cap (II), in contact with the metallized ceramic disk (III). try, normal pulse Voltammetry, square wave Voltammetry, The ceramic disk can be metallized in specific areas on both differential pulse Voltammetry, linear Voltammetry, or square the front and the back of the disk, with vias connecting the wave Voltammetry. The Source for Supplying a plurality of 55 specific metallized areas. On one side of the ceramic disk, the potentials can be a potentiostat, for example, a potentiostat semiconductor working electrodes as well as the counter and capable of applying square waves for square wave Voltamme optionally reference electrodes can be present. For example, try. electrodes such as the semiconductor working electrodes can Generally, the analyte concentration is determined by each being mounted to a specific metallized area. The disk is using Voltammetry to identify the position of current peaks, 60 then mounted into the housing such that the side of the disk which current peaks indicate the reduction or oxidation having the electrodes is exposed to the medium into which the potential of a redox active moiety. In some embodiments, the probe is immersed, and the other side of the disk is away from position of the reduction and/or oxidation potential of the the medium, allowing for electrical connection to the metal analyte sensitive redox active moiety is used to determine the lized areas on to the disk Such that Voltage can be applied and concentration of the analyte. This method can be used, for 65 current can flow to and from the electrodes. The sealing example, where no analyte insensitive redox active moiety is gasket (IV) provides sealing from the fluid in which the employed. sensor is immersed while allowing electrical contact with the US 8,758,584 B2 39 40 pin contacts (V) on the shaft of the housing (VI). FIG. 1(b) ments the sensor is in a vessel, in other embodiments the shows the housing assembled for insertion into the fluid to be sensor is in a conduit or pipe through which a process fluid measured. Pipe fittings are used to seal the wires that provide flows. In some embodiments, the currents measured at a electrical connection to the semiconductor electrode for Vol plurality of potentials by voltammetry are used to determine tammetric measurements. analyte concentration, and the determined analyte concentra FIG. 2 shows a drawing of an embodiment of the unit tion is used to control a process parameter. The systems of the comprising the source for Supplying a plurality of potentials present invention are valuable in in-line sensing in that they and the current measuring device. FIG. 2(a) shows a top view can be made to be robust, to resist fouling, and are able to and FIG. 2(b) shows a back side view. The unit has an elec measure analyte concentration for long periods of time in trical input/output connector for connecting to the electrodes 10 media that changes its properties, as in a process such as a for carrying out Voltammetry. The unit has a connection for chemical reaction. AC power. The unit has a universal serial bus interface (USB Methods for Forming Solid State Electrochemical Sensors I/F) and an RS-232 Serial port for transmitting data, for Another aspect of the invention provides a method for receiving instructions, and for testing and debugging by an forming a solid state electrochemical sensor. The method external device. The unit also comprises a liquid crystal dis 15 generally comprises having a solid state (e.g., semiconductor) play (LCD) and has user interface buttons to allow the user to Substrate with a Surface and immobilizing a redox active control the measurements and to read the output from the moiety with a reduction and/or oxidation potential that is SSO. sensitive to an analyte onto the Solid state surface. FIG.3 shows another exemplary embodiment of a system, Some embodiments provide a method for forming an ana in accordance with an embodiment of the invention. FIG. 3 lyte-sensitive semiconductor electrode, the electrode having shows a probe that contains two semiconductor (silicon) a semiconductor Surface. The method comprises immobiliz working electrodes, a reference electrode, and a counter elec ing a redox-active moiety that is sensitive to the presence of trode. The electrodes can be electrically connected through an analyte onto the semiconductor Surface. the probe to the source for Supplying a plurality of potentials Any Suitable method such as those known in the art or to the working electrode, the counter electrode, and option 25 disclosed herein can be used to construct a semiconductor ally the reference electrode, and a device for measuring cur surface as described above that is useful as part of a subject rent through the working electrode at the plurality of poten sensor. The redox groups can be immobilized onto the Surface tials. In this embodiment, the two working electrodes, the chemically or physically. The redox groups can be reacted reference electrodes and the counter electrode are contained with the semiconductor surface to attach them to the semi on the end of the probe on a disk which allows the electrodes 30 conductor covalently. Alternatively, the redox active groups to be in contact with the medium comprising the analyte(s) to can be adsorbed to the semiconductor. The redox active be measured. The areas of the various electrodes can be varied groups can also be immobilized by attaching the groups to a to improve the performance of the system. While this embodi polymer that is either covalently or non-covalently bound to ment shows two working electrodes, in some embodiments, the surface. Covalent binding of either the redox group or the there may be one working electrode, and in other embodi 35 polymer to which the redox group is a part to the Surface can ments, there are 3, 4, 5, 10, 20, or more working electrodes. In be beneficial in improving the lifetime and stability of the Some embodiments one working electrode can comprise a electrode. semiconductor Surface with a redox active moiety that is Functional groups can be covalently attached to semicon sensitive to pH. Such as anthracene, and the other working ductors, such as silicon or germanium. Silicon can, for electrode can comprise a redox active moiety that is insensi 40 example, form covalent bonds with carbon, and thus is a tive to pH. Such as ferrocene. In some embodiments, the desirable substrate for functionalizing with carbon based semiconductor Surface on which the pH sensitive moiety, molecules. The covalent binding to the Surface can be through Such as anthracene, is bound comprises a silicon wafer that is a bond between the semiconductor, e.g., silicon, and carbon, lightly doped, and the silicon Surface on which the pH insen oxygen, nitrogen, Sulfur, or other atom. In some embodiments sitive moiety, Such as ferrocene, is bound comprises a silicon 45 the bond to the Surface is between the semiconductor, e.g., wafer that is more heavily doped. silicon, and carbon. In some embodiments the bond to the The semiconductor working electrodes can be bonded to Surface is between semiconductor, e.g., silicon, and oxygen. conductive regions on the disk. Conductive vias through the In some embodiments, the immobilization of the redox disk allow electrical connection to electrode pins which are active moiety by covalent binding to the semiconductor Sur contained within the housing. The electrode pins are in turn, 50 face is accomplished by reaction with a semiconductor electrically connected, for example to wires, which can run hydride, for example, a silicon hydride (Si-H) surface. A through the probe housing, and then out of the housing to semiconductor-hydride, e.g., silicon-hydride Surface can be electrically connect the electrodes to the source for applying obtained, for example by treatment of the semiconductor, potentials to the electrode, and to the device for measuring the e.g., silicon Surface, for example a surface that is in the native current that passes through the electrodes. The threads, 55 oxide state, with hydrofluoric acid (HF). For example, dilute o-ring, and hex body allow for the probe to be mounted into a (1-3%) aqueous HF treatment, or, a 40% aqueous NHF treat reactor such as a bioreactor or fermentor. The threads allow ment can be used to create a Si-H terminated Surface. for the probe to mate with a corresponding threaded hole Porous silicon, when etched through standard procedures through the wall of the reactor, the hex body allows for tight involving HF, can also be used as a Si-H surface. FIG. 4 ening the probe into the reactor, and the gasket assists in 60 shows a schematic illustrating the conversion of a native establishing a seal. In some embodiments the unit is mounted oxide surface to a Si-H Surface on a silicon wafer through into the reactor without the use of threads, mounting without the treatment of the wafer with a 2.5% aqueous solution of threads can provide resistance to some failure modes that are HF. A Si-H surface can also be formed by other processes, present when threads are used. for example by decomposition of silanes as described in U.S. In some embodiments, the system is configured to be used 65 Pat. No. 6,444,326. A Si-H surface can also be formed as an in-line sensor in a process. An in-line sensor can be a through reacting Surface silanol moieties with reagents such sensor that is used in an on-going process. In some embodi as trihydroxyhydridosilane via Sol-gel type methods (see e.g., US 8,758,584 B2 41 42 U.S. Pat. Nos. 5,017,540, and 5,326,738) or through dry-etch chemically. In some embodiments, ultrahigh vacuum tech processes with plasmas of sulfur hexafluoride or Freon 23. A niques can be used to prepare the functionalized Surfaces of Germanium hydride (Ge—H) Surface can undergo the same the invention, for example by 2+2 Reactions of alkynes and types of the reactions of Si-H to create covalently bonded alkenes or Diels-Alder (4+2) reactions of dienes with recon redox active reagents. Suitable reactions to covalently bond to structed Si surfaces. the Si or Ge surface are described, for example, in J. M. Capping of semiconductor oxide Surfaces such as silica Buriak, Chem. Review 2002, 102 (5), 1271. The hydrides of and glass Surfaces with alkyl-, alkoxy- and chloro-silanes other semiconductor Substrates can also be prepared and used may also be used to functionalize the semiconductor Surface. to covalently bond redox active moieties. Suitable semicon The semiconductor Surface can contain oxide functionality ductor hydrides include for example, hydrides of siliconger 10 including hydroxy functionality (native oxide). In some manium (M. S. Carroll, et al., J. Electrochem Soc. 2000, 147 embodiments, the semiconductor electrodes of the invention (12), 4652), gallium arsenide (P. E. Gee, et al., J. Vacuum Sci. are modified by covalent attachment to this oxide functional Tech. A. Vacuum Surf. Film 1992, 10 (4), 892), gallium ity. For example, the hydroxy groups of silicon or other semi nitride, diamond film (S. Yamashita, et al., U.S. Pat. No. conductor elements can be coupled to Surface bound groups 5,786,604 (1998)), and indium phosphide (Y. Sun, et al., J. 15 using the many reactions known in organic chemistry for Appl. Phys. 2005, 97, 124902). carbon bound hydroxy groups, including for example, the The semiconductor-hydride surface, e.g., Si-H surface, formation of esters and ethers. One derivatization method can be reacted with a variety of functional groups to create involves the use of a carbodiimide for coupling to the surface. covalent bonds and thereby attacha redox active moiety to the Exemplary carbodiimides include, for example, dicyclohexy semiconductor surface. The Si-H surface, Ge Hsurface or lcarbodiimide (DCC), or (1-ethyl-3-3-dimethylaminopro other Semiconductor-H Surface can participate in hydrosily pylcarbodiimide hydrochloride) (EDC). lation reactions, involving the addition of for example, the The redox active moieties can also be attached covalently Si-H across an unsaturated site to form a Si C, Si-O, or to the surface by reactions with the native oxide, —O or Si N bond to the surface. Functional groups which can be —OH on the semiconductor surface. Many methods are used in these reactions including hydrosilylation include alk 25 known for carrying such reactions to form covalent bonds enes, alkynes, imines, carbonyls and oximes. These reactions, through various functional groups (see MaoZetal. J. Colloid. including hydrosilylation can be carried out thermally, pho Interface Sci., 1984, 100, 465-496). In some embodiments an tochemically, with a metal catalyst or with a radical initiator indirect approach can be employed in which an alkoxysilane (see Buriak, Chem. Commun., 1999, 1051-1060). The Si-H comprising other reactive functionality is reacted with the Surface or other semiconductor-H surface can also be reacted 30 —O or —OH groups on the semiconductor Surface to with alkyl oraryl carbanions through, for example, Grignard, covalently attach the alkoxysilane to the semiconductor Sur or lithium reagents. In some embodiments, a Si-H surface or face. The other reactive functionality on the alkoxysilane can other semiconductor-H Surface can react with azido, diazo, then be used to covalently attach a redox active moiety to the and diazonium groups. Suitable diazonium reactions are Surface. In these embodiments, the alkoxysilane can become described, for example, in Stewart et al., J. Am. Chem. Soc. 35 a linker or portion of a linker. The other reactive functionality 126, 2004, 370-378. can be any reactive functionality that can be used to attach a FIG. 5 illustrates the reaction of a surface Si-H with an redox active moiety. The functionality can be, for example, an aldehyde functionality attached to a ferrocene redox active olefin, an acetylene, an amine, a mercaptan, or an epoxy moiety to create a covalently bound ferrocene through a group. The reaction used to couple the alkoxysilane to the Si-O bond. FIG. 5 also illustrates the reaction of a surface 40 redox active moiety can be, for example a, Diels-Alder reac Si-H with a vinyl functional group attached to a ferrocene tion, Michael addition, click chemistry, or epoxy chemistry. redox active moiety to create a covalently bound ferrocene Semiconducting polymers comprising redox active moieties through a Si-C bond. FIG. 6 illustrates the reaction of a can be polymerized onto a surface, graft polymerized onto a surface Si-H with an aldehyde functionality attached to an Surface, photo-polymerized, or pre-formed and cast onto a anthracene redox active moiety to create a covalently bound 45 Surface. anthracene through a Si-O bond. FIG. 6 also illustrates the In some embodiments, the reactions described above for reaction of a surface Si-H with a vinyl functional group covalently attaching functional groups to the semiconductor attached to an anthracene redox active moiety to create a Surface are used to attacha linker group or portion of a linker covalently bound anthracene through a Si C bond. FIG. 7 group having a chemical functionality that can be used to illustrates the reaction of both a ferrocene redox active moiety 50 covalently bind the redox active moiety to the surface in a and an anthracene redox active moiety through vinyl func Subsequent step. tionality to produce a silicon Surface with a covalently Reactions that can be used to covalently attach a redox attached redox group that is sensitive to hydrogen ion (an active moiety to the semiconductor Surface include hydrosi thracene) and a covalently attached redox group that is insen lylation, free radical reactions, carbodiimide coupling, Diels sitive to hydrogen ion (ferrocene). In some embodiments, a 55 Alder reactions, Michael addition, epoxy reactions, or click carbonyl group Such as an aldehyde group is Substituted for chemistry (see, e.g., Evans et al. Australian Journal of Chem the vinyl functionality for providing attachment to the sur istry 60 (6): 384-395 (2007). face. Organic semiconductor Substrates of the invention can in The redox active moieties can alternatively be attached Some embodiments be modified to attach redox active species covalently to the Surface by direct reactions with a semicon 60 after the formation of the organic semiconductor. For ductor, e.g., silicon Surface from which all functionality has example, an organic semiconductor can be cast into a film. In been removed, typically by high temperature and vacuum. Some embodiments the organic semiconductive polymers, for The pure silicon Surface, for example, can react directly, for example, polyaniline, polypyrrole, and polythiophene are example with alkenes and alkynes to form Si-C covalent prepared by chemical or electrochemical oxidation. The attachment. (see Bateman, et. al., Angew. Chem. Int. Ed. 65 semiconductive polymer can be Subsequently modified using, 1998, 37(19), 2683-2685). Diazonium species can also be for example, the chemistry described for the attachment of used to functionalize the surface either thermally or electro redox active agents to the inorganic semiconducting Sub US 8,758,584 B2 43 44 strates. In some embodiments, the semiconductive polymers In some embodiments, the redox-active moiety can be can comprise functional groups that can be used to attach incorporated into a polymer, and the polymer comprising the redox active species to the semiconductive polymer. For redox active moiety can be immobilized onto the semicon example, the Sulfonic acid groups in poly(anilinesulfonic ductor surface. The immobilization of the polymer can be acid) can be used to immobilize aminoferrocene, 1,1'-diami either chemical or physical. The immobilization of the poly noferrocene, and aminoanthracene through sulfamide forma mer can be through covalent bonds, or through adsorption of tion. In some embodiments, a pyrrole, thiophene or aniline the polymer to the semiconductor Surface. monomer can have a functional group Such as an N-hydroxy The redox active moieties can be incorporated into any Succinimide ester, a carboxylic acid, or a reactive vinyl or type of polymer that can be immobilized onto the surface of allyl group. The monomers can then be polymerized, for 10 the semiconductor Surface. Types of polymers that the redox example electropolymerized, and the redox active moiety can active moieties can be incorporated into include biopolymers be attached to the functional group on the polymerized mono such as RNA, DNA or proteins, conductive polymers, fluo C. ropolymers, polyterpenes, inorganic polymers, phenolic res In some embodiments, the redox active moiety can be 15 ins polyanhydrides, polyesters, polyolefins, polysiloxanes, incorporated into the semiconductive polymer itself, for polyamides, polyimides, polyethers, polyketones, polysul example as part of the backbone of the polymer. For example, fones, and vinyl polymers. redox active groups within poly(ferrocenyl vinylene phe The polymer comprising the redox active moieties can, in nylene vinylene), poly(fluorine), and poly(carbazole) that Some cases, be produced at the semiconductor Surface. For comprise redox active moieties in their main chains, which example monomers or oligomers comprising the redox active can be used for analyte, e.g., pH sensing. moieties can be polymerized in the region of the Surface to In Some cases the Surface of the semiconductor is modified product the polymer near the Surface. In some cases, the by coating the Surface with conductive compounds Such as polymerization can be initiated at the semiconductor Surface, metals or metal oxides. In some embodiments, the semicon resulting in polymer covalently bound to the surface. The ductoris coated with gold, silver, palladium, copper, platinum 25 polymerization can be initiated at the Surface can be initiated, or other metals. The metals can be coated from solution, for for example by a free radical reaction initiated by a diazo example by electrodeposition, or can be coated onto the Sur group attached to the semiconductor Surface. In other cases, face with vacuum techniques such as plasma deposition, or the polymerization can be initiated in Solution, for example metal vaporization. The semiconductor Surface can be coated near the Surface. Such that the nascent polymer is immobi with conductive or semiconductive metal oxide compounds 30 lized onto the surface as it is formed. Methods for determin such as indium-tin oxide. When these materials are coated ing the appropriate solvent conditions are known. For onto the semiconductor electrode surface, the redox-active example by establishing that the monomer and/or oligomer agents are attached to the semiconductor electrode by attach are soluble, while the polymer is insoluble, allowing for sur ment to the layer on the semiconductor electrode. face deposition to occur. The semiconductor Surface can com Where a linker group is used, the linker can be small, for 35 prise polymerizable functional groups that are capable of example one to 3 atoms, or can be longer, e.g., 20 to 100 copolymerizing with the monomers or oligomers comprising atoms. The linker can also be any size between the small or the redox active moieties resulting in covalently binding the longer linker. In some embodiments, the linker is relatively redox active polymer onto the semiconductor Surface. short allowing for the redox active moiety to be close to the In some embodiments, a polymer comprising the redox surface, which can be beneficial for electron transfer. In some 40 active moieties can be electropolymerized at the semiconduc embodiments the linker is provided such that the redox active tor Surface. For example, monomers comprising the redox agent is held 1,2,3,4, 5, 6, or 7 atoms from the surface of the active moieties are added to a solution, and current is pro semiconductor. Where a short linker is used, the redox-active vided through the semiconductor Surface causing electropo moiety is held close to the Surface. Where a longer group is lymerization of the monomers. In some embodiments, the used, the redox active moiety may be able to move away from 45 electropolymerization can result in the covalent attachment of the surface, for example further out into the solution. Linker the electropolymerized polymer to the semiconductor Sur groups can comprise hydrophilic, hydrophobic groups, or face. In other embodiments, the electropolymerization can mixtures thereof. Linker groups can comprise, for example, result in polymerization, for example, at the semiconductor hydrocarbons, alkenes, alkynes, esters, ethers, amides, Solution interface, and the polymer that is formed can deposit amines, carbonyls, thiols, olefins, silicones, or other organic, 50 onto the semiconductor Surface, resulting immobilization of inorganic or organometallic groups. The linker groups can be the polymer by physisorption to the surface. The polymers formed by polymerization or oligomerization reactions such can be polymerized onto a surface, graft polymerized onto a as free radical or anionic polymerization. The linker group Surface, photo-polymerized, or pre-formed and cast onto a can comprise, for example, ethylene oxide, propylene oxide, Surface. or acrylamide repeat units. Linkers can have ring structures 55 The polymers can be chemically or electrochemically including aromatic rings. The variation in the linker structure deposited on individual microelectrodes, polymerized, as can be used to vary the mobility of the redox-active moiety in respond to a signal in a reversible manner, in a way that can be the solution. If the linker is too long and densely packed the electrochemically detected. Other such materials are redox active moiety can be far enough away from the Surface described by R. W. Murray in Electroanalytical Chemistry, such that the electron transfer to the electrode surface may be 60 vol. 13, edited by A. J. Bard (Marcel Dekker, NY 1984), the compromised. In these cases, having a linker that has electri teachings of which are specifically incorporated herein. cal conductivity can be useful. In some embodiments, the polymer is formed away from A redox-active moiety may be highly substituted, and can the semiconductor Surface, and Subsequently immobilized still act as a redox-active moiety. Thus, for example, the thereto. The polymer can be immobilized onto the surface by redox-active moiety ferrocene includes substituted fer 65 a variety of methods including, adsorption from solution, rocenes, ferrocene polymers, and ferrocene covalently coating including spin coating and dip coating, spraying, attached to the surface via linker molecules. printing, electropainting, or electrodeposition. US 8,758,584 B2 45 46 In some embodiments the semiconductor electrode is current can shift, for example to more negative potentials formed by contacting an H-terminated semiconductor Surface upon increasing the operating frequency. In some cases with the one or more redox-active moieties wherein at least changing the potential at higher frequencies can result in one redox active moiety is sensitive to the presence of an more noise in the square wave Voltammograms. In some cases analyte, wherein each redox-active moiety comprises a func it is advantageous to shield the electrode from the light tional group that will react with the H-terminated semicon because in Some cases, light can contribute to the background ductor surface to form a covalently bond, thereby forming a noise. derivatized semiconductor Surface. In some embodiments, In some embodiments, the electrode further comprises an the Surface comprises at least two redox active moieties and analyte-insensitive redox-active moiety having a reduction one of the redox active moieties is insensitive to the presence 10 and/or oxidation potential that is Substantially insensitive to of the analyte. the analyte, and the method further comprising determining Methods for forming semiconductor electrochemical sen the oxidation and/or reduction potential of the analyte-insen sors can be applied to forming other Solid State electrochemi sitive redox-active moiety, and determining the concentration cal sensors, such as carbon sensors. It will be understood that of the analyte from the difference in the oxidation and/or in the case of other Solid State materials, the doping configu 15 reduction potentials of the analyte-sensitive and analyte-in ration can be changed to effect desirable sensor properties, sensitive moieties. The redox-active moiety having a reduc such as sensitivities and reliability. tion and/or oxidation potential that is Substantially insensitive Uses of the Compositions and Devices to the analyte can be on the same electrode as the redox-active Another aspect of the invention provides a method for moiety exhibiting an oxidation potential and/or reduction determining the concentration of an analyte. In an embodi potential that is sensitive to the analyte, or a different elec ment, the method comprises bringing an electrode of a sensor trode (e.g., separate and electrically isolated electrodes). in contact with the analyte, the electrode comprising a solid Generally, the analyte concentration is determined by state (e.g., semiconductor) Substrate with Surface having using Voltammetry to identify the position of current peaks, immobilized thereon an analyte-sensitive redox-active moi which current peaks indicate the reduction or oxidation ety. The analyte-sensitive redox-active moiety exhibits an 25 potential of a redox active moiety. In some embodiments, the oxidation potential and/or reduction potential that is sensitive position of the reduction and/or oxidation potential of the to the analyte. Next, a plurality of potentials are applied to the analyte sensitive redox active moiety is used to determine the electrode. The current through the electrode is measured at concentration of the analyte. For example, the position of the the plurality of potential to determine a reduction and/or current peak with respect to the potential at a reference elec oxidation potential of the analyte-sensitive redox-active moi 30 trode can be used. This method can be used, for example, ety, thereby determining the concentration of the analyte. where no analyte insensitive redox active moiety is In some embodiments, the method comprises determining employed. the concentration of an analyte by (a) placing an electrode in Where an analyte insensitive redox active moiety is used, contact with said analyte, said electrode comprising a solid detection is generally accomplished by measuring the poten state (e.g., semiconductor) Substrate with Surface having 35 tial difference, delta E, associated with current peaks for immobilized thereon an analyte-sensitive redox-active moi oxidation (or reduction) of the immobilized redox active moi ety, said analyte-sensitive redox-active moiety exhibiting an eties, where the magnitude of delta E can be related to the oxidation potential and/or reduction potential that is sensitive concentration of analyte, e.g., hydrogen ion (H+) in solution. to the concentration of the analyte; (b) applying a plurality of That is, in many embodiments, delta E represents the poten potentials to the electrode; and (c) measuring the current 40 tial difference between the reduction and/or oxidation poten through the electrode at the plurality of potentials, and deter tial between a redox active analyte sensitive moiety and a mining a reduction and/or oxidation potential of the analyte redox active analyte insensitive redox active moiety. The sensitive redox-active moiety, thereby determining the con analyte insensitive redox active moiety which has an electro centration of the analyte. chemical response that is insensitive to variations in the The method of determining the concentration of the ana 45 medium serves as the reference. Current peaks for oxidation lyte can be used to measure pH by utilizing redox active or reduction of the reference and indicator can be determined moieties that are sensitive to hydrogen ion as described from a Voltammograms using a counter electrode, and with above. out the need for a reference electrode. The measurement of current at a plurality of potentials In some embodiments the measured current through the allows for carrying out Voltammetry for determining the oxi 50 electrode at the plurality of potentials is used to determine the dation and or reduction potential of the redox active moiety or concentration of the analyte. The determination of the con moieties immobilized on the surface. The voltammetry used centration using the measured current (e.g., current peaks) in the method can be, for example cyclic Voltammetry, pulse can be accomplished by using a computation system that Voltammetry, normal pulse Voltammetry, square wave Volta communicates with the device for measuring current. The mmetry, differential pulse voltammetry linear voltammetry, 55 computation system can apply algorithms for calculating or square wave Voltammetry. The source for Supplying a reduction or oxidation potential from the measured current at plurality of potentials can be a potentiostat, for example, a a plurality of potentials from the Voltammetry measurements. potentiostat capable of applying square waves for square The computing systems can be part of the sensing system, in wave Voltammetry. Some cases allowing the sensing system to be self contained. The frequency of the measurement can affect the quality of 60 The computing system can utilize its memory for storing raw the data. In some embodiments, square wave Voltammograms or processed data from the sensors. The method can further are currently at a step height of 2 mV. amplitude of 25 mV and comprise communication between the computing system and a frequency of 10 Hz. In some cases, it is advantageous to the sensor via transmission device that will wirelessly or by increase the frequency. For example, increasing the fre wire transmit processed data to an external device. quency to 500 Hz can result in a faster scan rate. We have 65 Carrying out the method typically requires the use of at observed that in Some cases, a higher frequency results in a least one other electrode (the counter electrode). The counter higher level of observed current. In some cases, the peak electrode is used to complete the electrochemical circuit to US 8,758,584 B2 47 48 make the measurements described herein. The counter elec an embodiment, the pH value from the voltammetric pH trode is generally made of a material that is chemically inert measurement with a semiconductor electrode is used to to the medium So that its potential does not change signifi decide whether or not to add one or more components to the cantly during the course of measurement. Suitable materials process, and/or to decide how much of the component to add. in many applications include platinum, gold, stainless steel, In some embodiments, the pH value is used to control the pH and carbon. In some cases, the counter electrode can be incor in a part of the process, for example, as input into the decision porated into the chip that also comprises the semiconductor on the addition of either acidic or basic components. In some sensor electrode. embodiments, the pH value is used to determine whether a A reference electrode is optional and is used as an addi process has reached a certain stage, for instance, whether a tional electrode in some embodiments of the method of mea 10 reaction is at completion. In some embodiments, the pH value Suring analyte concentration. In the case of a three-electrode is used to determine the addition of nutrients or other com system, the counter electrodegenerally completes the circuit, ponents to a reaction containing an organism to maintain the allowing current to flow through the cell, while the reference health and productivity of the organism. electrode maintains a constant interfacial potential difference The process control step can be automated Such that a given regardless of the current. In the case where the system com 15 pH measurement value from the sensor results in the change prises an analyte sensitive redox active moiety and an analyte of a process parameter without the intervention of a person. In insensitive redox active moiety, the analyte insensitive redox other embodiments, the pH measurement is viewed by a active moiety can act as a reference, allowing the potential person who uses the information to make the decision the difference to be used to determine analyte concentration. change of a process parameter. Even when an analyte insensitive moiety is also used, in some The process control step can be controlled by a voltammet embodiments, a reference electrode will still be used. In some ric pH system with a semiconductor electrode that has a embodiments, pseudo-reference electrodes can also be uti sensor, a Voltage source, a current measuring detector, and a lized. Reference electrodes which can be employed are computer for determining the pH from the current measure described above. ments. The Voltammetric pH system can be in communica In some embodiments, the sample is a liquid sample, and 25 tion either with a process control system, or with an operator, the electrodes are each in contact with the liquid. In some by analog or digital means, either with a wire, wireless con cases, the sample will not be a liquid, but may be a solid, nections, fibers or combinations thereof. generally comprising a solid electrolyte, or a gas. Another aspect of the invention is a method of voltammet In some embodiments, the method involves the in-line ric pH sensing with a semiconductor electrode wherein the sensing of a process. An in-line sensor can be a sensor that is 30 pH sensor requires little calibration. In some embodiments, used in an on-going process. In some embodiments the the pH sensor is substantially free of the need for calibration method comprise the use of a sensor is in a vessel, in other or re-calibration. Substantially accurate measurements may embodiments the sensor is in a conduit or pipe through which be made without re-calibration. a process fluid flows. In some embodiments, the method The use of the Voltammetric pH sensing with a semicon comprises using currents measured at a plurality of potentials 35 ductor electrode has a number of advantages. For example, by Voltammetry to determine analyte concentration, and the the sensors of the present invention generally comprise Solid determined analyte concentration is used to control a process state sensors. The sensors of the present invention have a parameter. The systems of the present invention are valuable built-in internal standard Such that calibration is not required. in in-line sensing in that they can be made to be robust, to The sensors of the present invention can be constructed to be resist fouling, and are able to measure analyte concentration 40 physically robust, such that they are not prone to breakage. for long periods of time in media that changes its properties, The sensors of the present invention can be made to be rela as in a process Such as a chemical reaction, biochemical tively insensitive to fouling. The sensors of the invention can reaction, or fermentation. be constructed to be resistant to chemical sterilization Such as In some cases, under remote monitoring the sensor can be exposure to ethylene oxide, UV stabilization, gamma irradia programmed to automatically take readings. The automatic 45 tion, electron beam irradiation, and temperature treatment. readings can be programmed to occur on a periodic basis, to The sensors of the invention can be constructed to be resistant occur upon the happening of some event, or to occur when the to high humidity and high temperature treatment under pres sensor is prompted. The periodic events can be separated on Sure Such as experienced in an autoclave. the order of seconds to on the order of months. The happening The Voltammetric pH sensing methods with a semiconduc of some event could be, for instance at the point when the 50 tor electrodes comprise reactions carried out in stainless Steel measured solution reaches a certain Volume level, or at given reactors, glass reactors (e.g., for product development), and points in the steps of a manufacturing process (e.g., at the disposable reactors (e.g., plastic reagent bags), for example beginning of or end of a step, or upon the addition of a reagent reactors described by manufacturers such as Wave Biotech, to a vessel). Hyclone, Xcellerex, and Stedim. Remote monitoring generally includes communication 55 Another aspect of the invention provides methods for vol from the remote sensing unit, and/or communication to the tammetric pH sensing with a semiconductor electrode for remote sensing unit. The communication to and from the processing including chromatography and tangential flow remote sensing unit can be done with transmission lines, ultrafiltration. and/or wirelessly. Any type of signal including, for example, Another aspect of the invention is as a sensor in a remote digital, analog, wideband, narrowband, or combinations 60 monitoring system such as a drug (pharmaceutical agent) thereof, can be used. delivery system. Such a system is described, for example, in Another aspect of the invention provides voltammetric U.S. Patent Application 2003/0153900, which is entirely monitoring of pH with a semiconductor electrode as part of incorporated herein by reference. The analyte monitoring process control in processes. In an embodiment, a Voltam system or monitoring and drug (pharmaceutical agent) deliv metric pH measurement is made in an industrial process 65 ery system can be partitioned into a disposable module, a stream, and the pH value from that measurement is used to as reusable module and a personal digital assistant (PDA) mod input to a decision on the adjustment a process parameter. In ule. APDA is typically a portable, e.g., handheld device that US 8,758,584 B2 49 50 has computing and networking capability, and a user inter user more optimally adjust either drug levels or behaviors to face, with output, e.g., a display, and input, e.g., stylus, key more optimal levels. The PDA configuration provides a user board, and/or touchscreen capability. This configuration opti interface and preferably allows users the ability to program mally distributes functionality among these three and or control testing. The user can view individual analyte configurations to achieve certain advantages. However the measurements and graphically display analyte level trends by invention is not limited to this configuration. For example, a the day, week or custom time period. The PDA can be used to one-unit disposable device including all electronics, micron display any and all of the measurements recorded by the eedles, chemistry, sensors, mechanics and user interface may system. Using the proper software, the user can be provided be alternatively employed. Or, more relevantly, the design of with recommendations for drug regiment modification. In the invention allows for any distribution of components 10 Some cases, the user can program the times when their analyte between one or more system modules. For example, compo tests are to be taken. Preferably, the user can also set the upper nents may be partitioned among one or more system modules and lower limits for alerts. based on the overall system cost, user safety and/or perfor The system can be programmed Such that whenevera user aCC COCCS. makes changes and with verification from the user, the infor The disposable module contains those components that 15 mation can be wirelessly downloaded to the system. During once used must be discarded to maintain safety and accuracy. the day the user may not need to use the PDA unless alerted by This module preferably includes any structural or mechanical the system to check for an analyte reading. The user can elements to maintain integrity, Sterility and/or an electrome initiate a test from the PDA if wanting to make an immediate chanical interface to any reusable components. Therefore this measurement. Once the user selects this command, Verifies it, system module can include, for example: microneedles, a and transmits it to the reusable module, a confirmation is microfluidic assembly, membrane, reagent chemistry and made back to the PDA. associate housing materials. The portion of a sensor which is Another aspect of the invention comprises drug dispenser in contact with a biological fluid, for example, may be part of capsule comprising a Voltammetric sensor. In some embodi the disposable module. This module can also include retain ments, the drug dispenser capsule comprises a semiconductor ing mechanisms for establishing and maintaining intimate 25 based voltammetric sensor as described herein. The drug contact with the body thereby providing mechanical retention dispenser capsule of the present invention internally senses a of the analyte monitoring/drug (pharmaceutical agent) deliv biologic condition by the detection of the presence or amount ery system. of an analyte, and internally dispenses drugs within the diges The reusable module generally contains those components tive tract of a body (e.g., a human body or animal body) based that control, automate motion, measure the analyte concen 30 upon the sensed level of analyte. The capsule is inert and is tration, alarm the user, transmit data to the PDA module. This therefore swallowable and passable through the digestive module can also include retaining mechanisms. Generally, tract without being consumed. By sensing the level of one or this module includes: a microprocessor with associated cir more analytes, the Swallowable drug dispenser capsule senses cuitry (e.g., memory, Supporting electronics and the like), information the digestive tract or senses conditions within the sensing circuitry, including, for example, a Voltage Supply 35 digestive tract that are indicative of conditions in other organs and current measuring device, drive mechanisms such as (e.g., skin). In addition to the Voltammetric analyte sensor, the motors or the like, a power Supply (e.g., battery) and an capsule contains one or more other sensors (e.g., chemical, interface operable to communicate with a portable computing electrical, etc.) so that more types of biologic data can be device or PDA. The interface can be RF, magnetic or induc tracked through the digestive system. In response to that tive, optical or the like. The reusable module can also have an 40 sensed information, the capsule dispenses a bioactive Sub audible or vibration alarm to notify the user that user action stance within the digestive tract without the need to transmit intervention is required. or receive signals from a remote transmitter/receiver, and The PDA module generally includes a separate user inter without active human or computer management. Drug dis face via a portable computing device Such as a personal penser capsules are described, for example, in U.S. Pat. No. digital assistant (PDA), handheld computer or the like for 45 6,929,636, which is entirely incorporated herein by reference. controlling and/or interacting with the device. A typical por The Swallowable drug dispensing capsule comprising a table computing device includes a processor, memory, asso Voltammetric sensor can include, for example, sensors, a ciated circuitry, a display (e.g., monochrome or color LCD) controller, memory, optional programmable logic, a power and an input device Such as a key pad, touch screen (e.g., Supply, a microactuator, a drug storage module, and commu integrated with the display) or the like and an operating sys 50 nication interface having at least one of the following types of tem. The display can show the value of the analyte to be communication modules: radiofrequency; ultrasonic; and/or measured, could provide the user with instructions on how to infrared. In one preferred embodiment, at least memory, and respond to the measured level of analyte, or may tell the user preferably also controller and/or programmable logic are what automatic actions have been taken in response to a embodied on a semiconductor-based, e.g., silicon-based, measured level of analyte. 55 module in one or more semiconductor chips. Today, portable computing devices with improved operat In some embodiments, the Swallowable drug dispensing ing system Software and user interfaces are readily available. capsule has multiple sensors that are arranged an outer Sur These devices provide the potential for rich and extended face of capsule in a desired predetermined orientation that is functionality. For example a typical PDA includes a relatively expected to expose each sensor to a targeted bodily condition large viewing screen and can also include wireless commu 60 or landmark within the human body. Each sensor can com nications mechanisms, a Sophisticated operating system and a prise a single type of sensor Such as an image detector or a variety of business and personal Software (calendars, Sched different type of sensor (e.g., chemical, electrical, tempera uling, etc.). The invention preferably includes the use of a ture, etc.). Chemical detectors detect the presence of many PDA to provide the proprietary software (programs) for analytes, such as pH, or other analytes. autonomous operation with an improved user interface. 65 The Swallowable drug dispensing capsule of the invention For example, the PDA module can provide the user with can have a controller that regulates communication between software that facilitates informed decisions to help a patient sensors and memory, communication between memory and US 8,758,584 B2 51 52 any remote controllers outside of the human body, and com the present invention do not require a direct electrical con munication with programmable logic component(s). Finally, nection to the reinforcement steel within the structure, using controller can operably control both communication interface the structural steel as one of the referencing materials. Since and a microactuator. The controller typically is a logic con the disclosed instruments do not require proximity to the Steel troller and includes a microprocessor. The controller may also within the structure, the instruments can be dispersed at criti comprise one or more logical devices (e.g., a logic gate) cal locations within the structure, regardless of steel place capable of performing a sequence of logical operations. ment. In some embodiments, the systems and devices are The Swallowable drug dispensing capsule generally has a self-contained, incorporating all required sensing electrodes memory or storage device that is typically an ultra-high and electronics. The devices can be deployed as described in capacity storage device, and which is often based on a semi 10 U.S. Pat. No. 6,690,182, which is entirely incorporated herein conductor chip, e.g., a silicon chip. by reference. The Swallowable drug dispensing capsule generally has a The semiconductor electrode voltammetric pH sensors of drug storage module and a microactuator. The drug storage the present invention can be used in winemaking. Measure module represents a container for holding a drug or bioactive ments of various properties including pH are taken through Substance that may be released, for example, into the diges 15 out the process, including during (1) pressing, (2) primary tive tract. Accordingly, the drug storage module also includes fermentation, which often takes between one and two weeks, one or more selectively activated dispensing ports that openin where yeast converts most of the Sugars in the grape juice into an outer Surface of capsule. The microactuator can have a ethanol (alcohol) (3) secondary fermentation. chemically activated or electromechanically activated The semiconductor electrode voltammetric pH sensors of mechanism for causing the drug storage module to release its the present invention can be used in brewing. The measure contents. The Swallowable drug dispensing capsule has a ment of pH can be important at the various stages of brewing, Suitable power Supply, such as a lithium-ion battery, which is for example, at mashing, lautering, lauter tun, mash filter, relatively non-toxic. Alternatively, other power supplies that boiling, whirlpool, wort cooling, fermenting, conditioning, are suitable for in vivo environments can be used. filtering, and secondary fermentation. The semiconductor The Swallowable drug dispensing capsule generally has a 25 electrode voltammetric pH sensors of the present invention communication interface that includes any suitable wireless can be particularly important during fermentation, where the transmission technology (e.g., ultrasonic, radiofrequency, Voltammetric pH sensors of the present invention are advan etc.) that readily permits communication to and from the tageous as they require little to no calibration, and can be capsule while the capsule is in digestive tract and the remote made to resist fouling during the fermentation process. transmitter/receiver which is located remotely outside of the 30 The semiconductor electrode voltammetric pH sensors of body. However, a wireless port is preferably used for com the present invention can be used in the production of biofu municating with capsule after capsule is captured from the els, including the production of biodiesel, ethanol, butanol, body. Likewise, a wireless port may be used for programming and Substitutes for gasoline, diesel, jet fuel, and additives to the controller, memory, and/or logic component prior to be used in any of the forgoing. The production of ethanol insertion of capsule within the body to determine the manner 35 includes both the process of converting the cellulose to Sug that the sensors will operate and communicate with the ars, and the process of converting the Sugars to ethanol. memory, as well as the manner that microactuator will oper Although there are several key technological differences in ate and communicate with memory via controller. how ethanol is produced from corn or cellulosic feedstock, In use, the sensors, including the Voltammetric sensor of both paths to ethanol production typically require a fermen the capsule sense analyte concentrations and biologic data 40 tation step that involves the conversion of glucose and other within the digestive tract and the sensed data is passed Sugars to ethanol. Currently, baker's yeast, Saccharomyces through the controller for storage in memory and/or compari cerevisiae, provides the primary microbiological system used son with a stored data profile in memory and/or logic. After by the corn-based ethanol industry. The methods of the the predetermined criteria are met, controller activates micro present invention relate to ethanol production for fuel from actuator to dispense the drug from drug storage module into 45 Saccharomyces cerevisiae and other organisms. The control digestive tract. The sensed data optionally is stored in of pH can be useful in catalytic biofuel production processes, memory and retrieved via the communication interface after such as for the production of biofuel. capture of capsule upon exiting the digestive tract. Finally, a The semiconductor electrode voltammetric pH sensors of wireless communication system optionally can be used in the present invention can be used in oil recovery and refining. addition to, or as an alternative to, controller and memory to 50 The sensors can be incorporated into down-hole devices for facilitate evaluating and storing sensed data and to dispense measuring the analytes present in the down-hole environ drugs upon selective activation at the appropriate time. ment. The sensors can be used in other aspects of processing The semiconductor electrode voltammetric pH sensors of the oil such as in oil refining. the present invention can be used in manufacturing operations The semiconductor electrode voltammetric pH sensors of Such as the manufacture of coatings, cleaners and sealers that 55 the present invention can be used in the production of biop enhance paint and finish bonding, metal passivation to protect harmaceuticals, for example, medical drugs produced using Substrates during shipment and storage, paint spray booth biotechnology. They include, for example, proteins (includ treatments that enhance quality and efficiency, and air scrub ing antibodies), nucleic acids (DNA, RNA or antisense oli bers that limit pollutant emissions. In these applications, the gonucleotides) used for therapeutic or in vivo diagnostic pur reliable measurement of pH can be an integral part of the 60 poses. Biopharmaceuticals are produced by means other than process. direct extraction from a native (non-engineered) biological In some embodiments, sensors can be used as embeddable Source. An example is recombinant human insulin (rII, trade corrosion measuring instruments that are capable of provid name Humulin), which was developed by Genentech and ing information related to corrosion rate, corrosion potential, marketed by Eli Lilly. conductivity and chloride concentration, and/or pH levels of 65 Another aspect of the invention is a semiconductor elec steel rebar reinforced structures. The devices can be used to trode voltammetric pH sensor for the production of biophar monitor the integrity of the steel. The devices and systems of maceuticals including: blood factors (e.g., Factor VIII and US 8,758,584 B2 53 54 Factor IX), thrombolytic agents (e.g., tissue plasminogen bioreactor comprising a semiconductor based sensor wherein activator), hormones (e.g., insulin, growth hormone, gona the semiconductor sensor comprises a semiconductor Surface dotrophins), haematopoietic growth factors (e.g., erythropoi having immobilized thereon a redox active moiety that is etin, colony stimulating factors), interferons (e.g., interfer sensitive to the presence of an analyte. Such as hydrogen ion, ons-C, -3, -8), interleukin-based products (e.g., interleukin 5 FIG. 17 shows an example of a bioreactor of the invention 2), vaccines (Hepatitis B surface antigen), monoclonal comprising a probe for measuring pH, and thereby control antibodies (e.g., infliximab, basiliximab, abciximab, dacli ling the pH in the reactor during the reaction. The probe Zumab, gemtuzumab, alemtuzumab, rituximab, palivizumab, comprises an electrode having a semiconductor Surface hav trastuzumab (herceptin), and etanercept) and other products ing immobilized thereon a redox active moiety that is sensi Such as tumor necrosis factor, and therapeutic enzymes. 10 Another aspect of the invention is a method for forming a tive to hydrogen ion. In some embodiments the probe com protein comprising carrying out a fermentation reaction that prises two electrodes, each comprising a semiconductor produces such protein, wherein the pH of the fermentation surface, one of the electrodes having immobilized thereto a reaction is controlled by measuring the pH with a pH sensor redox active moiety that is sensitive to hydrogen ion, and one comprising a solid state (e.g., semiconductor) electrode with 15 of the electrodes having attached thereto a redox active moi a surface having immobilized thereon a redox active moiety ety that is insensitive to hydrogen ion. In some embodiments that is sensitive to the presence of hydrogenion, and using the the probe further comprises a counter electrode, and in some measured pH to control the pH of the fermentation reaction. embodiments it further comprises a reference electrode. The solid state electrode in some cases is formed of a semi The semiconductor electrode voltammetric pH sensors of conductor, Such as silicon. In some embodiments, the control the present invention can be used to assist in the Successful of the pH can be manual, for instance, where an operator reads manipulation of cultured cells. As cells generally continue to the pH from the pH sensor and uses the measured pH to divide in culture, they generally grow to fill the available area determine whether or how much to adjust the pH, and in other or volume. This can generate several issues that the reliable embodiments, the control can be automatic, where the pH measurement of pH can assist with, Such as: Nutrient deple measurement is read by instruments that can adjust the pH 25 tion in the growth media; accumulation of apoptotic/necrotic based on the value of the measurement received. (dead) cells; cell-to-cell contact stimulating cell cycle arrest, The semiconductor electrode voltammetric pH sensors of causing cells to stop dividing known as contact inhibition; the present invention can be used for biopharmaceuticals cell-to-cell contact stimulating promiscuous and unwanted produced from microbial cells (e.g., recombinant E. coli), cellular differentiation Sometimes these issues can be iden mammalian cell lines and plant cell cultures in bioreactors of 30 tified by monitoring pH, alone or in combination with other various configurations. measurements, and can then be controlled or remediated by Cell culture requires cells to be grown, often under a strict adjusting tissue culture conditions that often rely on sterile set of conditions to maintain the health of the cells and maxi techniques. These methods aim to avoid contamination with mize the production of the culture. Cells are grown and main bacteria or yeast that will compete with mammalian cells for tained at an appropriate temperature and gas mixture (for 35 nutrients and/or cause cell infection and cell death. The pH example, 37°C., 5% CO2) in a cell incubator. Culture con measurements of the present invention are amenable to being ditions vary widely for each cell type, and variation of con carried out in a biosafety hood or laminar flow cabinet to ditions for a particular cell type can result in different pheno exclude contaminating micro-organisms. types being expressed. The semiconductor electrode voltammetric pH sensors of Aside from temperature and gas mixture, the most com 40 the present invention can be used for pH sensing in plant monly varied factor in culture systems is the growth medium. tissue culture, bacterial and yeast cell culture, and viral cell Recipes for growth media can vary in pH, glucose concentra culture. tion, growth factors, and the presence of other nutrient com Another aspect of the invention is a semiconductor elec ponents. The effect of changes in pH can be dramatic in some trode Voltammetric pH sensor for sensing pH in plant tissue cases, and it can be important to maintain the pH. The devices, 45 culture. The pH measurements of the present invention can be systems, and methods of the invention allow for control of pH used at any step of plant cell culture. Plant tissue culture is within a range of 1,0.5,0.02, 0.1, 0.05, 0.02, 0.01 pH units or typically performed under aseptic conditions under filtered less to maintain the growth and health of the cells. The semi air. Living plant materials from the environment are naturally conductor electrodes of the present invention allow for the contaminated on their Surfaces (and sometimes interiors) accurate measurement of pH with limited fouling, and in 50 with microorganisms, so Surface sterilization of starting Some embodiments, no need for calibration. materials (explants) in chemical solutions (usually alcohol The semiconductor electrode voltammetric pH sensors of and mercuric chloride) is an important first step. Explants are the present invention can be used for cells that are grown then usually placed on the Surface of a solid culture medium, completely in Solution, and for cells that are grown on a but are sometimes placed directly into a liquid medium, par substrate. Some cells naturally live without attaching to a 55 ticularly when cell suspension cultures are desired. Solid and surface, such as cells that exist in the bloodstream. Others liquid media are generally composed of inorganic salts plus a require a surface. Such as most cells derived from Solid tis few organic nutrients, vitamins and plant hormones. Solid Sues. Cells grown unattached to a surface are referred to as media are prepared from liquid media with the addition of a suspension cultures. Other adherent cultures cells can be gelling agent, usually purified agar. The pH measurements of grown on tissue culture plastic, which may be coated with 60 the present invention can be made in the liquid, in the moist extracellular matrix components (e.g., collagen or fibronec soil, or in the agar. The composition of the medium, particu tin) to increase its adhesion properties and provide other larly the plant hormones and the nitrogen source (nitrate signals needed for growth. Versus ammonium salts oramino acids), and the pH, can have Another aspect of the invention is a bioreactor or fermentor profound effects on the morphology of the tissues that grow in which the reaction or fermentation occurring therein is 65 from the initial explant. For example, an excess of auxin will controlled by a semiconductor based voltammetric sensor of often result in a proliferation of roots, while an excess of the invention. In an embodiment, the invention comprises a cytokinin may yield shoots. US 8,758,584 B2 55 56 The semiconductor electrode voltammetric pH sensors of cheaper, but it also adds to the resulting water hardness. the present invention can be used with any cell line including: Neutralizing with soda ash, however, increases the Sodium National Cancer Institute’s cancer cell lines, Zebrafish ZF4 content of the water. Making the water slightly alkaline helps and AB9 cells, Madin-Darby Canine Kidney MDCK epithe ensure that coagulation and flocculation processes work lial cell line, Chinese Hamster Ovary CHO cells, Insect cell 5 effectively and also helps to minimize the risk of corrosion in line Sf21, MCF-7 (breast cancer), MDA-MB-438 (breast can pipes and pipe fittings. The pH value can be used to determine cer), U87(glioblastoma), A172 (glioma), HeLa (cervical can whether water is likely to be hard or soft. In general, water cer), HL60 (promyelocytic leukemia), A549 (lung cancer), with a low pH (<6.5) is acidic, and tends to be soft, and HEK 293 cells (kidney—original HEK line is contaminated corrosive. Therefore, the water could contain metalions, such with HeLa), SHSY5Y Human neuroblastoma cells, cloned 10 as iron, manganese, copper, lead, and zinc. In some cases this from a myeloma, Jurkat cell line, derived from a patient with results in elevated levels of toxic metals. This can cause T cell leukemia, BCP-1 cells (PEL), Primate cell lines, Vero premature damage to metal piping, and have associated aes (African green monkey Chlorocebus kidney epithelial cell thetic problems such as a metallic or sour taste, staining of line initiated 1962), COS-7 (African Green Monkey Kidney laundry, and the characteristic "blue-green” staining of sinks Cells), Rattumor cell lines, GH3 (pituitary tumor), 9L (glio 15 and drains. More importantly, there are health risks associ blastoma), Mouse cell lines, 3T3 cells, MC3T3 (embryonic ated with these ions or contaminants. The primary way to treat calvarial), C3H-10T1/2 (embryonic mesenchymal). NIH the problem of low pH water is with the use of a neutralizer. 3T3 (embryonic fibroblast), Invertebrate cell lines, C6/36 The neutralizer feeds a basic solution into the water to prevent Aedes albOpictus (Asian tiger mosquito) larva, Plant cell the water from reacting with the household plumbing or con lines, Tobacco BY-2 cells (kept as cell suspension culture, tributing to electrolytic corrosion. Water with a pH>8.5 could they are model system of plant cell). indicate that the water is hard. Hard water does not pose a Another aspect of the invention is a semiconductor elec health risk, but can cause aesthetic problems. These problems trode voltammetric pH sensor for use in water purification. include an alkali taste to the water, formation of a deposit on Water purification is the process of removing contaminants dishes, utensils, and laundry basins, difficulty in getting soaps from a raw water source, the goal is generally to produce 25 and detergents to lather, and formation of insoluble precipi water for a specific purpose with a treatment profile designed tates on clothing. to limit the inclusion of specific materials. Water purification Another aspect of the invention is sensing of analyte levels is not only water purified for human consumption or drinking such at the pH of bodies of water for example for resource water. The semiconductor electrode voltammetric pH sensors control. The body of water can be, for example, a lake, ocean, of the present invention can also be used water purified to 30 stream, or river. The ability of the invention to be used meet the requirements of medical, pharmacology, chemical remotely and to be used without the need for frequent cali and industrial applications. The semiconductor electrode vol bration or any calibration at all allows the systems, devices, tammetric pH sensors of the present invention can be used in and electrodes to be deployed remotely in bodies of water to water purification processes including, but not limited to provide information on analytes such as hydrogen ion, etc. in ultraviolet light; filtration; water softening; reverse osmosis, 35 such remote bodies. ultrafiltration; molecular stripping; deionization; and carbon Another aspect of the invention is a semiconductor elec treatment. Water purification may remove particulate sand, trode voltammetric pH sensor for the measurement of pH in Suspended particles of organic material; parasites, such as processes related to sewage treatment. Sewage treatment can giardia; cryptosporidium; bacteria; algae; virus; fungi, etc.; have the same steps as described above, but may refer to water minerals such as calcium, silica, magnesium; and toxic met 40 that has a higher level of contamination. Raw influent (sew als such as lead, copper, and chrome. Some purification may age) can be the liquid and semi-solid waste from toilets, baths, be elective in its inclusion in the purification process; showers, kitchens, sinks etc. Household waste that is dis examples, Smell (hydrogen Sulfide remediation), taste (min posed of via sewers can compose the sewage. In some areas eral extraction), and appearance (iron encapsulation). sewage also includes some liquid waste from industry and Water from any source is applicable to the present inven 45 COCC. tion. Groundwater (well water) is an economical choice for Stability Control drinking water, as it is inherently pre-filtered, by the aquifer In some cases, traditional electrochemical sensors can Suf from which it is extracted. Water from an aquifer will have a fer from IV fluctuations during measurement, which can lead limited output and can take thousands of years to recharge. to inaccurate measurements and low measurement times as Surface water, (rivers, lakes, streams) is far more abundant 50 more measurements would have to be taken to obtain a time and is the typical raw water source used to make drinking averaged measurement. Such fluctuations can lead to insta water, as a water source it is carefully monitored for the bility in square wave voltammetry (SWV). presence of a variety of contaminants. The methods of the In some cases, exposing a ferrocene functionalized silicon present invention encompass the Voltammetric measurement surface to an alkaline environment can shift the reaction Fe'“ of pH of these types of water where the pH value of the 55

Stability During Active Measurement in Cell Culture 15 Medium Example 13 A fouling testing was carried out using anthracene deriva Derivatization of Silicon Wafers with Anthracene tized silicon (100) wafer in the incubator (under controlled Using a Semiconductor Oxide Surface temperature of 37° C. and CO, content of 5%) with continu Si(100) wafer pieces were cleaned using “Piranha' solu ous electrochemical measurements in cell culture (LPVA) tion (3:1 v/v concentrated H2SO4/30%H2O2) for 30 minutes medium for 7 days using a three-electrode setup connected to at 100° C. and rinsed thoroughly with deionized water. Sub anuautolab type III potentiostat. The three-electrode setup (in sequently, the wafers were oxidized in 2:1:8 HC/H2O2/H2O an electrochemical cell) was autoclaved in a Consolidated for 15 minutes at 80° C. followed by 2:1:8 NH4OH/H2O2/ Stills & Sterilizers autoclave for 40 min prior to adding 5 mL 25 H2O for 15 minutes at 80° C. and then rinsed with copious of the cell culture medium to the autoclaved setup. The elec amounts of water. The oxidized silicon wafers were then trochemical cell containing the medium was then transferred immersed in 2% 3-APTES solution in acetone for 2 minutes to the incubator, where continuous electrochemical measure followed by rinsing with acetone to form an amino-termi ments were performed. nated monolayer. This was then allowed to react with 10 Voltammograms were taken repeatedly over 7 days result 30 mg/mL dicyclohexyl-carbodiimide (DCC) and 50 mg/mL ing in 10,000 consecutive voltammograms. FIG. 18(a) shows anthracene carboxylic acid in DMSO for 12 hours. Following voltammograms taken over the 7 day period (every 250" scan the reaction, the anthracene derivatized silicon wafer was of the 10,000 consecutive runs). The anthracene peak was rinsed with dichloromethane, acetone and methanol and then observed at ~-0.71V vs. Ag and remained consistent through dried under a stream of nitrogen. out the 7 days of in-situ electrochemical measurements in the 35 cell culture medium. A plot of the anthracene peak potential Example 14 over the 7 day time period is shown in FIG. 18(b). These findings demonstrate that the anthracene derivatized silicon is Ferrocene Covalently Attached to Various Doped still in good working order and that the peak potential remains Si(100) Substrates consistent while the sensor is actively sensing the solution for 40 7 days. Ferrocene was covalently attached to the following four silicon Surfaces via an H-terminated silicon Substrate and Example 11 vinyl ferrocene: i) Si (100) N-type (0.02-0.05 ohm-cm)— highly-doped N-type, ii) Si (100) P-type (0.005-0.020 ohm Derivatization of H-Terminated Silicon Surfaces 45 cm) highly-doped P-type, iii) Si (100) N-type (10-40 ohm with 2-allyl-1-Hydroxy-Anthraquinone cm) lightly-doped N-type, and iv) Si (100) P-type (10-90 ohm-cm) highly-doped P-type. A well-defined ferrocene Into a round bottom flask containing 50 mL of mesitylene peak was observed for all four substrates. The highly-doped was placed 5 mg of 2-allyl-1-hydroxy-anthraquinone. Nitro Substrates in general produced a larger electrochemical cur gen was bubbled for at least 30 minutes. Subsequently, a 50 rent than the corresponding lightly-doped substrates. While H-terminated silicon wafer was placed into the flaskand was not being bound by theory, this difference may be explained allowed to react with 2-allyl-1-hydroxy-anthraquinone for 12 by the fact that the highly-doped substrates contain more hours under reflux at 150° C. in an oil bath. During the charge carriers, i.e., are more conductive. Similar observa reaction, the solution was purged with nitrogen gas to prevent tions were observed when the electrochemistry was per the substrate from being re-oxidized. Following the reaction, 55 formed in a solution of ferrocene carboxaldehyde. FIG. 19 the 2-allyl-1-hydroxy-anthraquinone derivatized silicon has charts showing the peak current of silicon Substrates wafer was rinsed with dichloromethane, hexane, then metha derivatized with (a) vinyl ferrocene and (b) ferrocene carbox nol and dried under a stream of nitrogen. aldehyde in pH 1.63 solution on the four types of doped silicon. Example 12 60 Example 15 Derivatization of Silicon Wafers with Ferrocene Using a Semiconductor Oxide Surface Four Electrode System

Si(100) wafer pieces were cleaned using “Piranha' solu 65 A system was constructed having four electrodes compris tion (3:1 v/v concentrated H2SO4/30%H2O2) for 30 minutes ing (i) a counter electrode, (ii) a reference electrode, (iii) a at 100° C. and rinsed thoroughly with deionized water. Sub highly-doped N-type silicon wafer derivatized with vinyl fer US 8,758,584 B2 85 86 rocene, and (iv) a lightly-doped P-type silicon wafer deriva working electrodes (bottom). The figure also shows the effect tized with anthracene carboxaldehyde. FIG. 20 illustrates the of either infrared (IR) or room light for both shielded and Voltammograms obtained with the four electrode system con unshielded probes. The shielded sensor is unresponsive to IR sisting of a highly-doped N-type silicon wafer derivatized or room light. with vinyl ferrocene and a lightly-doped P-type silicon wafer 5 derivatized with anthracene carboxaldehyde. The voltammo Example 19 grams were obtained in pH 7.03 solution and the peak poten tials were stable over 200 consecutive scans (every 50th scan The Effect of Light on Sensor Output is shown). 10 A sensor has a first working electrode (WE1) and second Example 16 working electrode (WE2) that are formed of silicon. WE1 includes a layer of ferrocene and WE2 includes a layer of Working Electrodes Coated with Nafion Membranes anthracene. The sensor is immersed in various Solutions, each Solution having a predetermined pH. Sensor output is A Nafion membrane was applied to a working electrode 15 recorded both with and without exposure of the sensor to having ferrocene. A 5 mil thick section of pre-formed Nafion light. FIGS. 31A-31F show sensor output (y-axis; current, membrane (N115) was pre-treated either by soaking in water arbitrary units) at various pHs and under light and dark or in an acid solution (about 5% HCl or 5M HNO) at about conditions as a function of Voltage (mV). Channel 2 corre 100° C. for about 1 hr. The treated membrane was then cut to sponds to the sensor output for WE2. Also provided in the a size appropriate to cover the Surface of the working elec figures are Channel 2 peak positions (mV). FIG. 31A shows trode. The Nafion membrane was then coated with a liquid the sensor output at pH 5 without exposure to light (i.e., the dispersion of Nafion 117 or Nafion 2020, and applied directly sensor is in the dark). FIG.31B shows the sensor output at pH to the electrode surface. The membrane-coated electrode was 5 while the sensor is exposed to light. FIG. 31C shows the then cured in a regular or vacuum oven at about 120° C. for sensor output at pH 7 without exposure to light. FIG. 31D about 1 hr. The electrode was then rehydrated and tested. 25 shows the sensor output at pH 7 while the sensor is exposed to Testing showed stability as to the peak position in SWV. light. FIG. 31E shows the sensor output at pH 10 without exposure to light. FIG. 31F shows the sensor output at pH 10 Example 17 while the sensor is exposed to light. The Channel 2 signal (current) when the sensor is exposed to light is more intense Nafion-Plastic Composites 30 than situations in which pH measurements are made without exposure to light. This indicates that, when the sensor is A porous plastic membrane (pore size 75-100 micron, 0.5 exposed to light, better signal to noise may be achieved. mm thick) is cut into a 1 cm disk. The disk is saturated with a Nafion dispersion and allowed to dry for 30 min at 50° C. Example 20 After a second cycle of Saturation and drying, the disk is 35 placed in a 120° C. oven for about 1 hour. A single electrode The Effect of Light on Sensor Output is then wetted with the Nafion dispersion and placed on the smooth side of the disk. This assembly is then cured at 120° C. A sensor has a first working electrode (WE1) and second for about 1 hour. The backside of the electrode is then coated working electrode (WE2) that are formed of silicon. WE1 with a conductive epoxy and a wire is affixed. The assembly 40 includes a layer of ferrocene and WE2 includes a layer of is potted into a waterproof fixture for electrochemical mea anthracene. The sensor is immersed in a solution with a pH of SurementS. 5. Sensor output is recorded both with and without exposure of the sensor (and WE1 and WE2) to light offixed intensities Example 18 and wavelengths. Light is exposed on sensor Surfaces having 45 the ferrocene and anthracene moieties. It is observed that Light Sensitivity peak shapes are sharper in the presence of light up to a certain intensity and wavelength. At higher intensities the signal is A sensor has a first working electrode (WE1) and second destroyed. Optimal responses are obtained in the IR or near working electrode (WE2) that are formed of silicon. WE1 IR wavelength range. Such as light having a wavelength includes a layer of ferrocene and WE2 includes a layer of 50 greater than or equal to about 750 nm, or greater than or equal anthracene. A first probe has WE1 and WE2 that are shielded to about 850 nm. from light with PES layers, and a second probe has WE1 and WE2 that are not shielded (or unshielded) from light. The first Example 21 probe and second probe are inserted into a solution having a pH of about 4. Continuous pH measurements are made and 55 Doping Configurations plotted as the difference between the peak potentials of WE1 and WE2. Sensor outputs (y-axis, mV) as a function of time Multiple sensors are formed from silicon wafers having (X-axis) during pH measurements for both the shielded varying doping configurations. Each sensor has a first work (dashed line, top) and unshielded (solid line, bottom) probes ing electrode (WE1) and second working electrode (WE2) are shown in FIG. 30. The output of the shielded sensor upon 60 that are formed of silicon. WE.1 includes a layer of ferrocene exposure to light is different than the output of the unshielded and WE2 includes a layer of anthracene. During pH measure sensor upon exposure to light. During pH measurements, the ments, electrochemical responses from anthracene-covered output of the unshielded sensor shows a sensor response that electrodes (WE2) are observed when anthracene is deriva is coincident with exposure to light; the output of the shielded tized onto the following silicon surfaces: Si (100) wafer, sensor does not exhibit such a behavior. Sensor output when 65 p-type, resistivity between about 1 and 20 G.2-cm; Si(100) the first and second working electrodes are shielded (top) wafer, p-type, resistivity between about 1 and 90 S.2-cm; does not display the light-sensitive behavior of the unshielded Si(111) wafer, p-type, resistivity between about 1 and 20 US 8,758,584 B2 87 88 S2-cm; and Si(111) wafer, p-type, resistivity between about 1 a person skilled in the art. It is therefore contemplated that the and 10 S2-cm. An anthracene signal with an appreciable sig invention shall also cover any Such modifications, variations nal-to-noise ratio is not observed when derivatized on an and equivalents. n-type silicon Substrate. An anthracene signal is not observed when derivatized on a p-type silicon substrate with resistivity 5 What is claimed is: less than about 1 S2-cm. Electrochemical responses are 1. A sensor for detecting the presence or absence of an observed for ferrocene on both p-type and n-type silicon (both analyte, comprising: Si(100) and Si(111). Signals for Ferrocene on all resistivities a first solid state electrode having a Surface having immo of p-type silicon and on n-type silicon with resistivities<5 bilized thereon a layer of a first redox-active moiety, LS2-cm are unstable over time (in Some situations, it is 10 wherein the first redox-active moiety has an oxidation observed that the signal degrades with continuous square potential and/or a reduction potential that is sensitive to wave Voltammetric scanning). Ferrocene on n-type surfaces the presence of the analyte; and with resistivities between 1 and 90 S.2-cm are stable. a second Solid state electrode having a surface having immobilized thereon a layer of a second redox-active Example 22 15 moiety and a layer of a polymeric material adjacent to the layer of the second redox-active moiety, wherein the Sensor Stability second redox-active moiety has an oxidation potential and/or reduction potential that is sensitive to the pres A test was conducted to monitor fermentation in a fermen ence of the analyte. tation reactor. The pH of the reactant (i.e., cell culture) and 2. The sensor of claim 1, wherein the layer of the polymeric product content of the fermentation reactor was monitored material is a light blocking layer. over a 10-day period using both a redox-active moiety-based 3. The sensor of claim 2, wherein the polymeric material pH sensor (see above) and a conventional glass electrode comprises polyetherSulphone. sensor (Applikon Biotechnology). Both sensors were auto 4. The sensor of claim 1, further comprising a protective claved within the bioreactor prior to the initiation of the cell 25 layer adjacent to the layer of the polymeric material. culture. The redox-active moiety-based pH sensor was not 5. The sensor of claim 4, wherein the protective layer recalibrated during the 10-day period. FIG.33 shows the pH comprises another polymeric material. of the contents of the fermentation reactor over the 10-day 6. The sensor of claim 1, wherein the polymeric material period. The redox-active moiety-based pH sensor tracked the has the formula C7HFOS*C.F. glass electrode sensor without any appreciable drift and with 30 7. The sensor of claim 1, wherein the layer of the polymeric out exhibiting any appreciable electronic background noise material comprises a porous plastic. throughout the course of the 10-day period. In contrast, the 8. The sensor of claim 1, wherein the first or second solid glass electrode sensor exhibited electronic noise during the state electrode comprises carbon. 10-day period. 9. The sensor of claim 1, wherein the first or second solid Electrochemical sensor systems, devices and methods pro 35 state electrode comprises silicon. vided herein can be combined with or modified by other 10. The sensor of claim 1, wherein the layer of the poly systems, devices and methods. For example, electrochemical meric material comprises pores. sensors described herein, including methods for forming Such 11. The sensor of claim 10, wherein said pores have a pore sensors, can be combined with or modified by Systems and size between about 0.1 micrometers and 1 micrometer. methods described in U.S. patent application Ser. No. 12/049, 40 12. The sensor of claim 1, wherein the analyte is hydrogen 230 to Kahnet al. (“SILICONELECTROCHEMICAL SEN 1O. SORS) and PCT/US2008/066165 to Kahn et al. (“SEMI 13. The sensor of claim 1, wherein the layer of the poly CONDUCTOR ELECTROCHEMICAL SENSORS), meric material is an ion selective layer. which applications are entirely incorporated herein by refer 14. A solid State sensor for detecting the presence or CCC. 45 absence of an analyte, comprising (i) a solid State electrode In some embodiments, sensors have been described as having a Surface having immobilized thereon a layer of a being used to detect the presence or absence of an analyte redox-active moiety, (ii) a layer of a polymeric material and (e.g., H+). It will be appreciated, however, that detecting the (iii) a protective layer adjacent to the layer of the polymeric presence or absence of an analyte can include detecting (or material, wherein the layer of the polymeric material is measuring) the concentration of an analyte. For example, 50 porous and covers the layer of the redox-active moiety, detecting the presence or absence of H+ in a liquid sample wherein the redox-active moiety has an oxidation potential using any of the sensors described herein can include detect and/or a reduction potential that is directly sensitive to the ing (or measuring) the concentration of H+. presence of the analyte, and wherein the redox-active moiety It should be understood from the foregoing that, while is immobilized on the Surface through a covalent interaction particular implementations have been illustrated and 55 with the solid state electrode. described, various modifications can be made thereto and are 15. The solid state sensor of claim 14, further comprising a contemplated herein. It is also not intended that the invention light blocking layer adjacent to the layer of the polymeric be limited by the specific examples provided within the speci material. fication. While the invention has been described with refer 16. The solid state sensor of claim 15, wherein the light ence to the aforementioned specification, the descriptions and 60 blocking layer transmits less than 10% of light incident on the illustrations of the preferable embodiments herein are not light blocking layer. meant to be construed in a limiting sense. Furthermore, it 17. The solid state sensor of claim 16, wherein the light shall be understood that all aspects of the invention are not blocking layer transmits less than 5% of light incident on the limited to the specific depictions, configurations or relative light blocking layer. proportions set forth herein which depend upon a variety of 65 18. The solid state sensor of claim 17, wherein the light conditions and variables. Various modifications in form and blocking layer transmits less than 1% of light incident on the detail of the embodiments of the invention will be apparent to light blocking layer. US 8,758,584 B2 89 90 19. The solid state sensor of claim 14, wherein the analyte is hydrogen ion. 20. The solid state sensor of claim 14, wherein the solid state electrode comprises silicon. 21. The solid state sensor of claim 14, wherein the layer of 5 the polymeric material is an ion selective layer. 22. The solid state sensor of claim 1, wherein the solid state electrode is non-metallic. 23. The solid state sensor of claim 14, wherein the layer of the polymeric material selectively screens off anions and only 10 allows the passage of cations. k k k k k