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8-21-2012 Nanostructuring on Multilayered Graphene Petal Nanosheets for Electrochemical Biosensing Jonathan C. Claussen Birck Nanotechnology Center, Purdue University, [email protected]

Anurag Kumar Birck Nanotechnology Center, Purdue University, [email protected]

David B. Jaroch Birck Nanotechnology Center, Purdue University, [email protected]

M. Haseeb Khawaja Birck Nanotechnology Center, Purdue University

Allison Hibbard Birck Nanotechnology Center, Purdue University

See next page for additional authors

Follow this and additional works at: http://docs.lib.purdue.edu/nanopub Part of the Nanoscience and Nanotechnology Commons

Claussen, Jonathan C.; Kumar, Anurag; Jaroch, David B.; Khawaja, M. Haseeb; Hibbard, Allison; Porterfield, D. Marshall; and Fisher, Timothy S., "Nanostructuring Platinum Nanoparticles on Multilayered Graphene Petal Nanosheets for Electrochemical Biosensing" (2012). Birck and NCN Publications. Paper 1153. http://dx.doi.org/10.1002/adfm.201200551

This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Authors Jonathan C. Claussen, Anurag Kumar, David B. Jaroch, M. Haseeb Khawaja, Allison Hibbard, D. Marshall Porterfield, and Timothy S. Fisher

This article is available at Purdue e-Pubs: http://docs.lib.purdue.edu/nanopub/1153 www.afm-journal.de www.MaterialsViews.com FULL PAPER Nanostructuring Platinum Nanoparticles on Multilayered Graphene Petal Nanosheets for Electrochemical Biosensing

Jonathan C. Claussen , Anurag Kumar , David B. Jaroch , M. Haseeb Khawaja, Allison B. Hibbard , D. Marshall Porterfi eld , and Timothy S. Fisher *

unique properties including more favo- Hybridization of nanoscale metals and carbon nanotubes into composite rable Faradic-to-capacitive current ratios, has produced some of the best-performing sensors to date. higher current densities, and faster mass The challenge remains to develop scalable nanofabrication methods that transport by convergent diffusion than their larger micro/macro electrode coun- are amenable to the development of sensors with broad sensing ranges. A terparts.[ 6 , 7 ] In order to increase biosensor scalable nanostructured biosensor based on multilayered graphene petal current output to measurable levels, large nanosheets (MGPNs), Pt nanoparticles, and a biorecognition element arrays of (i.e. , nanoelec- ( oxidase) is presented. The combination of zero-dimensional nano- trode arrays [NEAs]), have been immobi- [8,9] particles on a two-dimensional support that is arrayed in the third dimension lized on electrode surfaces. These NEA biosensors, fabricated with various nano- creates a sensor platform with exceptional characteristics. The versatility of structures (e.g. , , nanotubes, the biosensor platform is demonstrated by altering biosensor performance and nanocrystals) have shown promising (i.e., sensitivity, detection limit, and linear sensing range) through changing results, displaying high sensitivities and the size, density, and morphology of electrodeposited Pt nanoparticles on fast response times; however, the concom- the MGPNs. This work enables a robust sensor design that demonstrates itance of a scalable fabrication technique, robust biofunctionalization scheme, and exceptional performance with enhanced glucose sensitivity (0.3 μM detection > enhanced performance (i.e. , low detec- limit, 0.01–50 mM linear sensing range), a long stable shelf-life ( 1 month), tion limit and wide linear sensing range) and a high selectivity over electroactive, interfering species commonly found remains to be seen. in human serum samples. To bridge the gap between fundamental research and commercial biosensor appli- cations, fundamental nanoelectrode array 1. Introduction fabrication challenges still need to be addressed including pro- tocols for effi cient electrode development and immobi- Nanostructures have recently been utilized in a variety of bio- lization. Existing nanoelectrode fabrication protocols typically sensing applications due to their enhanced surface area, precise require lithography and chemical processing steps that are biomolecule-electrode connections, and enhanced delivery of expensive, limited to serial processing, and not suitable for a amplication agents.[ 1–5 ] For electrochemical sensing, conduc- wide variety of materials.[ 10 ] Templated development of nano- tive nanostructures immobilized on electrodes enhance elec- electrode arrays with anodic alumina or polycarbonate generally trocatalytic behavior due to quantum confi nement and exhibit eliminates the need for lithography, but still requires multi-step fabrication protocols ( e.g., physical vapor deposition, anodization, and electrodeposition) to develop the template and subsequent nanostructures.[ 11 , 12 ] Similarly, biosensors comprised of ordered A. Kumar , M. H. Khawaja , A. B. Hibbard , Prof. T. S. Fisher and semi-ordered arrays of carbon nanotubes (CNTs) typically Birck Nanotechnology Center require template development or lithography to geometrically Department of Mechanical Engineering distribute metal catalysts and/or chemical functionalization steps Purdue University to prepare CNTs for conjugation with biorecognition agents.[ 13–15 ] 1205 W. State St., West Lafayette, IN 47907-2057 USA However, recently developed graphene petal nanosheets with E-mail: tsfi [email protected] highly reactive edge planes can be grown directly on a variety of Dr. J. C. Claussen , Prof. D. M. Porterfi eld Birck Nanotechnology Center surfaces without the need for metal catalysts—creating a nano- Department of Agricultural and Biological Engineering structured surface well suited for integration into numerous Purdue University, USA electrochemical sensing applications.[ 16–18 ] D. B. Jaroch , Prof. D. M. Porterfi eld Various biofunctionalization techniques have been devel- Birck Nanotechnology Center oped to immobilize biorecognition agents onto electrode sur- Weldon School of Biomedical Engineering Purdue University, USA faces including covalent binding through self-assembled mono- layers (SAMs), non-covalent membranes, and electrodeposition DOI: 10.1002/adfm.201200551 with conductive polymers. Self-assembled monolayers provide

Adv. Funct. Mater. 2012, 22, 3399–3405 © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com 3399 3400 FULL PAPER with aqueouselectrolytes. wileyonlinelibrary.com strate usingamicrowaveplasma chemicalvapordeposition An arrayofMGPNsweregrownacrossaTi-coated siliconsub- 2.1. MultilayeredGraphenePetalNanosheet(MGPN)Electrodes Results 2. in 1962), biosensing paradigmsinceitsinceptionfromClarkandLyons cose oxidase(GOx)(perhapsthemostwidelystudiedenzymatic against thebroadestcollectionofbiosensors,enzymeglu- the electrodesurface.Inordertobenchmarkperformance tive polymerPoly(3,4-ethylenedioxythiophene) (PEDOT)onto is usedtoelectrodepositenzymewiththeelectricallyconduc- sensing range.Arobustsensorbiofunctionalizationprotocol manipulated tomaximizethebiosensorsensitivityanddynamic chemical performance.ThesizeanddensityofthePtNPsare along thegraphenepetaledgesandplanestoenhanceelectro- tion processisusedtogrowplatinumnanoparticles(PtNPs) reported foruseinglucose PEDOT wellsuitedforenzymeimmobilizationmatrixesas bility, owingtoinherentdioxyethylenebridginggroups,makes cataltyic behaviorinfuelcells, electrodeposition—nanoparticles thatarewellknowfortheir a highlyconductivetemplateforsubsequentPtnanoparticle a chemicalvapordepositiontechnique.TheMGPNsactas graphene petalnanosheets(MGPNs)onasiliconwaferthrough catalyst drivencarbonnanotubearrayswegrowmultilayered etch backfabricationtechniques,poroustemplates,ormetal electrode arraybiosensorscurrentlyface.Inlieuoflithography/ tured biosensortoaddressmanyofthelimitationsthatnano- biosensing applications. ance ofglucosebiosensorspreviouslyreportedintheliterature. linear sensingrangethatgreatlyimprovesupontheperform- in humanserumsamples,andalowdetectionlimitwide ascorbic acid,uricandacetaminophen)typicallyfound imal interferencefromendogenouselectroactivespecies( with strongglucosesensitivityevenafter5weeksofuse,min- MGPN glucosebiosensorperformanceprovestobeexemplary sequent amperometricglucosesensing.TheoptimizedPtNP- due toitsbiocompatibility, stability, andhighconductivity. polymeric materialthatcanbeutilizedinbiosensorinterfaces ethylenedioxythiophene) (PEDOT)isanelectricallyconductive ibility inbiologicalsensingapplications. they aresubjecttodelaminationandshowlimitedreproduc- branes canberapidlyassembledonelectrodesurfaces,but ductive surfaces. ronments andcanbecontrollablyelectrodepositedontocon- and Poly(styrene-sulfonate) (PSS)aresolubleinaqueousenvi- Mixtures ofthemonomer3,4-ethylenedioxythiophene(EDOT) ical sensors, diminish biosensorsensitivity. barrier thatcanimpedeheterogeneouschargetransportand and electrodesurface;buttheyalsocreateanelectron-transfer a strongandstablecovalentlinktothebiorecognitionagent www.afm-journal.de In thispaperwepresentafundamentallynewnanostruc- [ 34 , 35 [ ] 30 isencapsulatedwithinthePEDOTmatrixforsub- ] andbiosensors. [ 24 ] Furthermore PEDOTdisplayshighstability [ [ 23 25 ] ] Thishighelectrochemicalsta- andcholesterol [ 27 [ 31–33 , 28 [ 12 ] hydrogenstorage, , 19 ] Aone-stepelectrodeposi- , 20 ] Nncvln mem- Non-covalent [ 21 ] © Howeverpoly(3,4- 2012WILEY-VCHVerlag GmbH&Co. KGaA, Weinheim [ 26 ] amperometric [ 29 ] chem- [ 22 i.e. , 23 , ]

a magnifi ed view. vapor deposition(MPCVD)onaTicoatedsiliconsubstrate.Insetshows scopy imagesofaMGPNelectrodegrownbymicrowaveplasmachemical 1. Figure tance ofapproximately500nmfromthesurface( grow acrossthesurfaceofelectrode—protrudingadis- technique (seeExperimentalMethods). Thepetalsuniformly phology areelectrodepositedonto theMGPNs( tubes. planes, surface moreelectroactivethantheuntreated,superfi cial graphenelayers.Thegenerateddefectsrenderthe etch generatingdefectsandoxygenatedspeciesonthesuperfi the abilityofelectrolytetoimpregnatecarbonsurface. electrode naturefromhydrophobictohydrophilic—enhancing In anefforttoincreaseelectro-reactivitytowardsH 2.2. PlatinumNanoparticle(PtNP)Modifi morphologies correspondingto5–25graphenelayers. porting Information)areconsistentwithpreviousreported measured bytunnelingelectronmicroscopy(Figure S1inSup- force microscope)andtheaverage12layergraphenedepth 1–10 nmthicknessofthepetals(measuredbyaVeeco atomic tion, deposition onHOPG, trochemical performanceandsubsequentmetallicnanoparticle vious studiesthatdemonstrateplasmaetchingimproveselec- electrochemical characterizationexperimentscorroboratepre- (Figure S2inSupporting Information).Thesespectroscopy and create fi (see ExperimentalSection).Five distinctcurrentsareusedto nique withasimilar3-electrode set-upasmentionedpreviously nanoparticles areelectrodeposited throughacurrentpulsetech- metry, andamperometrichydrogenperoxide(H terized withRamanspectroscopy, ferricyanidecyclicvoltam- plasma etch.Theeffectsofthisetchingprocesswerecharac- electrodes wereexposeduniformlytoa30secondoxygen trodes andimprovesubsequentPtNPdeposition,theMGPN an efforttoincreasetheelectroactivenatureofMGPNelec- [ 13 [ 37 , 33 [ ve Pt-MGPNelectrodeswithPt nanoparticlesofunique 38 ] Such improvementscanbeattributedtotheO , Field emissionscanningelectronmicroscopy(FESEM)micro- , 41 39 ] ] Ptnanoparticlesofvaryingsize,density, andmor- whilenewlyformedoxygenatedspeciesalter the [ 36 ] carbonnanowalls, Adv. Funct. ed MGPNElectrodes www.MaterialsViews.com

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lm. gluconicacid sensitivity of the optimized sensitivity 2 − O 2 D supplementary control experi- supplementary was electrodepositedment, Pt highly ordered pyro- onto planar (HOPG) at thelytic graphite (2.5 mA currentsame conditions as the opti- pulses, 250 cycles) Themized Pt-MGPN electrode. H Pt-MGPN was nearly 5 timesPt-MGPN was nearly elec- as great as the Pt-HOPG enhancedtrode—illustrating the over con- sensitivity of MGPNs sub- ventional carbon-based S3 in Supporting strates (Figure Information). 2.3. Glucose Sensing In order to convert the PtNP- MGPN electrodes into enzy- matic biosensors, the enzyme GOx is mixed with the con- mer PEDOT and ductive poly GOx

−→ − O 2 2e H + testing where values continue to increase 2 + 2 A (orange), O 2 O 2 μ sensitivity of O 2 + testing. Amperometric glucose calibration plots O + 2 2 + O 2 2H → 2 glucose O During electrochemical glucose sensing, where glucose is the During electrochemical glucose sensing, where glucose is Amperometric glucose sensing is carried out in the same Amperometric − 2 urable current signal ( Equation 1 & 2 ). A schematic portraying ). A schematic portraying producing a meas- sequently oxidized at the electrode surface, 2 & Equation 1 urable current signal ( biosensors as well the biofunctionalized PtNP-MGPN glucose as the enzymatic function of GOx is illustrated in

for all 5 PtNP-MGPN biosensors are created by adding succes- sive aliquots of increasing concentrations of glucose and meas- uring the corresponding steady-state signal response, typically ). The glucose sensitivity for 3 achieved within 5 seconds (Figure the Pt-MGPN biosensors and linear sensing range of the PtNP- MGPN glucose biosensors follow similar trends found in the amperometric H PEDOT layer was electrodeposited under the same conditions PEDOT layer was electrodeposited under pulses of 1.05 mA for all 5 Pt-MGPN samples [constant current (Pt-MGPN) and that are applied between the working electrode aberrations in grain auxiliary electrode (Pt gauze) for 500 cycles] samples are con- size and average thickness between electrode of the PEDOT sidered to be negligible. Thus the conductivity on each MGPN and amount of GOx enzyme electrodeposited electrode is considered equivalent. (H substrate of GOx, generated peroxide D H for higher Pt electrodeposition current pulses until a maximum sensitivity and linear sensing range is reached for the PtNP- MGPN biosensor with 2.50 mA current pulses ( subsequently electrodeposited onto the electrode surface (see subsequently electrodeposited onto the size can alter the Experimental Section). The PEDOT grain conductivity of the deposited PEDOT fi 3-electrode set-up and working potential (500 mV) as the amper- ometric H

2 2 2 − − O 2 10) ± cm cm A ini- 1 1 − μ − 10) nm with 25) nm) while ± ± 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2012 WILEY-VCH Verlag © 10 nm in width) extending 10 nm in width) extending A current pulses. The H 5) nm and a nearly smooth 5) nm

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31 2 [ − (MGPN electrode after oxygen plasma (MGPN electrode ower-like morphology become more ower-like 2 − cm calibration plots were performed in the 1 2 − ower-like morphology) began to form on ower-like cm sensitivity of the MGPN electrode is greatly O 1 2 , 3399–3405 2 − 50) nm with a rose-like morphology while O 22 sensitivity decreases to 12.9 mA mM ± 2 , 2 O 2 5) nm at 625 and glucose sensing. and glucose 2012

2 ± O 2 Characterization of the platinum modifi ed multilayered graphene petal nanosheet ed nanoparticle modifi Characterization of the platinum ower-like morphology. The ridgeline nanoparticles The ridgeline nanoparticles morphology. ower-like A (red), (c) 1.25 mA (green), (d) 2.5 mA (blue), and (e) 5.0 mA (purple) were used to electrodeposit A (red), (c) 1.25 10 nm in width) extentding from petal face nanoparti- 20 nm, caulifl μ 20 nm) with caulifl < < 20 nm). At 5.0 mA the Pt ridgelines have now expanded 5.0 mA the 20 nm). At < < Amperometric H in width to (300 cles ( the petal face nanoparticles have grown to (35 all visible petal faces contain an array of needle-like Pt nanopar- all visible petal faces contain an array of needle-like ticles ( Thus by changing the a needle-like to rose-like morphology. and pulse deposition current the Pt nanoparticle size, density, morphology can be altered. nm with needle-like morphology while petal face nanoparti- nm with needle-like morphology while cles ( (100 from the ridgeline nanoparticles (width of apparent. At 2.5 mA the petal tips are almost entirely coated 2.5 mA the petal tips are almost entirely apparent. At with needle-like Pt nanoparticles ( each petal face. Ridgeline nanoparticles begin to coalesce at each petal face. Ridgeline nanoparticles widths of (100 current pulses of 1.25 mA with average ticles ( to caulifl grow to (86 tiate nanoparticle growth along the MGPN ridge lines with an tiate nanoparticle growth along the MGPN average nanoparticle width of (46 size, density, and morphology. Current pulses of 312 and morphology. size, density, same manner as mentioned previously with a working potential The H of 500 mV. (b) 625 the MGPNs. (f)Pt nanoparticles of distinct size and density onto the H Bar graph displaying enhanced with the introduction of Pt as the sensitivity increases from 0.595 mA mM after Pt electrodeposition with 312 etch) to 9.71 mA mM sensitivity increases as a function of the Pt electrodeposition cur- rent pulse until a maximum sensitivity of 13.7 mA mM is reached for the Pt-MGPN biosensor with 2.50 mA current pulses. The H for the Pt-MGPN biosensor with 5.0 mA current pulses. As a characteristics have a signifi sequent H Adv. Funct. Mater. Funct. Adv. (PtNP-MGPN) electrodes before enzyme immobilization. (a-e) Field emission scanning electron microscopy (PtNP-MGPN) electrodes before enzyme immobilization. on MGPNs. Current pulses (500 ms) of (a) 312 (FESEM) micrographs of PtNPs electrodeposited Figure 2 . the MGPN electrode (before and after the oxygen plasma etch) and the PtNP-MGPN electrodes. Errors bars the MGPN electrode (before and after the oxygen show standard deviation for 3 different experiments. www.MaterialsViews.com 3402 FULL PAPER ( materials includinggraphene,carbonnanotubes,andPtNPs listed andcomparedtoglucosebiosensorscomprisedofsimilar linear sensingrangeoftheoptimizedPt-MGPNbiosensor is glucose detectionlimit(S/N enables sensingwithinthephysiological rangeforbloodglucose the broadlinearsensingrange of thePt-GPNbiosensornotonly structured biosensorsreported intheliterature.Furthermore, MGPN biosensoriswiderrespectively thananyothernano- suming O steps of10–50 glucose concentrationrangeof0.01mMto26.65byincremental glucoseconcentration analysis andcoeffi cient ofdetermination(R by 5mMaliquotsand(c)correspondinglinearglucosesensingrange withlinearregression (purple)] portraysthedynamiccurrentresponseforaglucoseconcentration rangeof5–60mM current pulsesof312 wileyonlinelibrary.com on asinglePtNP.GlucosebindswithintheGOxenzymaticpocketproducingH PtNP-MGPN glucosebiosensorwithadjacentmagnifi ed viewportrayalofGOximmobilized 3. Figure determination (R step of10mMglucoseand(e)correspondinglinearsensing range andcoeffi cient of www.afm-journal.de Table To ourknowledge,the sensingrangeoftheoptimizedPt-

1 . ) (a) Tiltedcross-sectionalschematicillustratingtheGOx/PEDOTbiofunctionalized 2

. (b)GlucosecalibrationplotsofthePt-MGPNbiosensors[Ptelectrodeposition μ 2 M by10 ) asillustrated. μ A (orange),625 μ M, 100-500 = 3,signal-to-noiseratioof3)and μ μ A (red),1.25mA(green),2.5(blue),and5.0 M by100 2 ) asillustrated.(d)Glucosecalibrationplotfor a © 2012WILEY-VCHVerlag GmbH&Co. KGaA, Weinheim μ M, 1–5mMby1mM,followedone MGPNs outperformconventional planarPtnanoparticle/ and/or multi-steplithography steps.ThesePtnanoparticle/ cally includesanodicalumina orpolycarbonatetemplates nates thecomplexityoftraditional NEAdesignsthattypi- present ananoelectrodearray fabricationprotocolthatelimi- to enhancetheelectro-reactivity ofthepetalsand,ineffect, using theMGPNsastemplatesforPtnanoparticlegrowth edge planes. tips thatexhibitthefastETratestypicallyfoundingraphene electrochemical propertiesstemmingfromtheexposedpetal 2 O 2 wie con- while [ 18 ] Inthiswork,we introducetheconceptof 20.8 mM(20mg/dL–350mg/dL)]patients; dL–135 mg/dL)]anddiabetic[1.1mM of healthy[3.6mMand7.5(65mg/ Fgr 4c). (Figure ence fromendogenouselectroactivespecies AP, UA,andAAexhibitsminimalinterfer- tored aftertheadditionof100μMaliquots centration of5mMelectrochemicallymoni- in humanserumsamples.Aglucosecon- and ascorbicacid(AA)),commonlyfound cies (uricacid(UA),acetaminophen(AP), glucose withinthreeknownelectroactivespe- tion of5mA)hasalsobeentestedbysensing MGPN glucosebiosensor(Ptelectrodeposi- glucose selectivityoftheoptimizedPtNP- cyclic testingandstorage(Figure 4 b). The of theenzymeimmobilizationprotocolwith testing—demonstrating therobustnature 75% ofitssensitivityevenafter5weeks Pt-MGPN biosensorretainsmorethan glucose monitoringsystems.Theoptimized off-the-shelf storagetypicalofhome blood petri dishwithnorefrigeration—mimicking testing, thesensorsremainwithinacapped ments overa5-weekperiod.Betweenweekly by performingglucosebiosensingmeasure- trodeposition techniquehasbeenvalidated The durabilityoftheGOx/PEDOTelec- and Selectivity 2.4. BiosensorLifetimePerformance be monitoredsimultaneously(Figure cose levelsfromdiversehumanserumscould non-invasive sensingprotocolsinwhichglu- sensing rangeopenspossibilitiesfornew research hasbeguntouncoverfavorable with MGPNsareavailable,butinitial application. Few priorreportsofsensing (MGPNs) inanelectrochemicalbiosensing multilayered graphenepetalnanosheets We haveshownthefi Conclusions 3. and urine it enablesglucosesensinginsaliva, [ 57 ] aswell.Thusthiswidelinear Adv. Funct. rst exampleofusing www.MaterialsViews.com

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] ] 41 46 [ [ Ref. wileyonlinelibrary.com www.afm-journal.de [mM] Sensing Range Sensing Range The work presented in ] 58 , This optimization process ] 3 [ M] 1 1–20 [ 44 ] 3 .012–3.8 [ 47 ] 5 .01–10 [ 51 ] 3 .002–.040 [ 45 ] 10 .02–1.02 80 up to 30 [ 15 ] 30 .03–2.43 [ 48 ] 20 0.05–22 [ 49 ] 59 1.5 0.003–9 [ 52 ] 0.5 0.005–5 2.7 .005–2.4 [ 53 ] 0.3 0.01–50 this work 5.7 0.006–1.5 [ 50 ] μ , [ Limit 42 , 31 [ , particles covering the graphene petal i.e. Carbon Carbon Graphite Graphite Graphite Graphene/ Nanotubes Nanotubes Nanotubes Nanotubes Nanotubes Nanotubes Nanotubes Nanoparticles Nanoparticles Nanoparticles Carbon Carbon 2 Electrochemical biosensor performance comparison of glucose comparison biosensor performance Electrochemical The link between electrode nanostructuring and enzymatic Biosensor Base Material Detection GOx-CS-Nafi on/AuNPsGOx-CS-Nafi Metallic GOx-CS-IL/AuNPs Metallic GOx-PtNPs-CNTs/TiO GOx-PtNPs-CNTs/TiO GOx/aligned-SWCNTs Carbon GOx/AuNWs-CS Metallic GOx-Fc-MWCNTsGOx-HRP-Ppy-SWCNT Carbon Carbon on/AuNPs- GOx-Nafi MWCNTs GOx-PtNPs-SWCNTs Carbon GOx-PEDOT/PtNP/ MGPNs GOx-Nafi on-Pt-xGnPsGOx-Nafi Graphene/ GOx-CNx-MWCNTs Carbon Ppy-GOx-Gn Graphene/ this manuscript further elucidates the subject, by demonstrating and density can be con- how Pt nanoparticle size, morphology, trolled to improve the linear sensing range and the detection limit of the enzymatic biosensors. MGPN electrodes decorated with densely-packed ( biosensing sensitivity remains relatively unexplored in the liter- ature. Soleymani and co-workers have begun to piece together the relationship between biodetection sensitivity and controlled nanostructuring through their work with palladium microelec- trodes and nucleic acid detection. ridgelines and peppering the graphene petal faces), needle-like Pt nanoparticles yieled the best results in terms of hydrogen peroxide and glucose sensing—corroborating similar reports connecting tightly-packed densities and needle-like morpholo- gies of Pt nanoparticles immbolized on carbon substrates to higher electroreactive ability. T a b l e and carbon nanotubes, based upon, graphene/graphite, biosensors 1 . metallic nanoparticles. (GOx)–glucose oxidase, (xGnPs)–exfoliated graphite nanoplatelets, (Gn)–graphene, (GOx)–glucose (SWCNT)–single- (Ppy)–polypyrrole, (MWCNT)–multi-walled carbon nanotubes, (AuNPs)–gold nano- walled , (PtNPs)–platinum nanoparticles, doped multi-walled nanowires, (CNx-MWCNTs)–nitrogen particles, (AuNWs)–Au peroxidase, (HRP)–horseradish carbon nanotubes, (Fc)–ferrocenecarboxaldehyde, (CS)–chitosan, (IL)–ionic liquid A M μ μ A (orange), 625 A (orange), μ 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2012 WILEY-VCH Verlag © 5:1 respectively), thus 5:1 respectively), thus ∼ sensitivity ( 2 O 2 , 3399–3405 22 , 2012

(a) Glucose sensing ranges of the Pt-MGPN glucose biosen- (a) Glucose sensing ranges of the Pt-MGPN Adv. Funct. Mater. Funct. Adv. aliquots of uric acid (UA), acetaminophen (AP), and ascorbic acid (AA) and successful detection of glucose (5 mM) within the backdrop of said electroactive, interfering species for the Pt-MGPN glucose biosensor (Pt electrodeposition of 2.5 mA). Figure 4 . sors [Pt electrodeposition current pulses of 312 (red), 1.25 mA (green), 2.5 mA (blue), and 5.0 mA (purple)] as compared to glucose levels found in urine, blood, tears, and saliva. (b) Biosensor lifetime measurements where the glucose sensitivity for each distinct Pt-MGPN glucose biosensor was monitored over a period of 5 weeks. (c) Selectivity test demonstrating minimal interference from 100 www.MaterialsViews.com HOPG in terms of H demonstrating the impact nanostructured, three dimension- ally arrayed MGPNs fused with Pt nanoparticles can exhibit in electrochemical sensing. 3404 FULL PAPER wileyonlinelibrary.com subsequently driedunderagentlestreamofN cleanedwithacetone,,andisopropylalcohol surface duringcuttingoperation.Afterwaferdicing,theelectrodesare spun andhardbaked(10minat120 similar protocolsestablishedinour previouswork. deposition (MPCVD)withaSEKI AX5200S MPCVDreactorfollowing of theMGPNsiscarriedoutby microwave plasmachemicalvapor photoresist anddebrisbeforeMGPN Synthesis. sized electrodes(0.35cm diced withadiamond-bladedicingsaw(DiscoDAD-2H/6)intoequally- (500 nm)]atabasepressureof5.0 evaporated ontoanoxidizedsiliconwafer[P silicon electrodesareelevated17 mmabovea5.1cmdiameter 4. ExperimentalSection partially negative. transforming thePEDOTpolymerchainchargefrompositiveto work canover-oxidize carbonatomsonthepolymerbackbone— PEDOT athighconcentrations( by theelectrodepositedPEDOTlayer. Theelectrodepositionof results oftheselectivityexperimentscanbeexplainedinpart tive speciescommonlyfoundinhumanserumsamples.The interference foroveronemonthfromendogenouselectroac- modifi blood. centration levelsfoundinurine,tears,andsalivaadditionto widening theglucosesensingrangeintophysiologicalcon- yields outstandingresultsintheglucosesensingparadigmby generation bio-ethanolfueltechnologies. agriculture, foodsafety, andsecurity. monitoring ofnumerousanalytesassociatedwithmedicine, fundamental physiologyresearch,itwillenablemultiplexed PtNP-MGPN biosensortechnologywillnotonlyimprove for multi-plexedbiosensingpurposes.Thuswebelievethe can allbeelectrodepositedontodistinctmicroelectrodes platforms wheretheMGPNs,PtNPs,andrespectiveenzymes these fabricationprotocolsareamenabletolab-on-a-chip disorders, in-fi oxidasefortheadvancementofbasicresearchand suchasglutamateoxidase,lactateand quite versatileastheGOxcanbeinterchangedwithother protocols. Thishigh-sensitivitybiosensingplatformcanbe tion longevitythatisnotaccessibleinmanysensordesign development, sensitivitytuningtechniques,andbiorecogni- rication protocolspresentedhereinfacilitaterapidsensor broader contextthefi should haveatremendousimpactonbiosensing,andin integrated intoawidearrayofelectronicdevices.Thiswork are allscalablefabricationtechniquesthatcanbepotentially enzyme encapsulatedwithintheconductivepolymerPEDOT trodeposition ofPtnanoparticles,andelectrodeposition electrochemical biosensing. ascorbic anduricacid)duetoelectrostaticrepulsionduring to repelnegatively-chargedelectrochemicalinterferents( www.afm-journal.de Multilayeredgraphenepetalnanosheet (MGPN)synthesis Baseelectrodefabrication The electrodepositionofGOxwithPEDOTontothePtNP The bottom-upgrowthofMGPNsonasiliconwafer, elec- eld sensingofbiomarkersassociatedwithneurological ed MGPNsenablesrobustglucosesensingwithminimal [ 60 ] patienttrauma, [ 60 , 61 ] ThustheelectrodepositedPEDOTtends eld ofelectrochemicalsensingasfab- 2 ) afterathinfi lm ofAZ1518photoresistis : Athin fi lm ofTi(100nm)ise-beam

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a platingsolutionconsistingofH electrode, andAg/AgClasthereferenceelectrodeweredippedwithin the MGPNsactedasworkingelectrode,Ptgauzeauxiliary set-up (BASiEpsilonThree-ElectrodeCell Stand-potentiostat)where the 100Wpowersetting. over theMGPNelectrodefor30secondsbysettingRFgeneratorto (GOx) (SigmaAldrichG7141)dissolvedinH the mixturewhilesolutionisagitated.Theenzymeglucoseoxidase 3,4-ethylenedioxythiophene (0.03M,SigmaAldrich483028)isaddedto deposited ontheMGPNs. 250 tomanipulatethesize,density, andmorphologyofPtnanoparticles 312 uA,6251.25mA,2.5mAand5.0wereutilizedincyclesof nanoparticles ontotheMGPNelectrodes.Currentpulses(500ms)of submergible sensorregionof0.35cm is dicedandlacquercoatedinanidenticalmanner, eachdipstickhasa testing donotchangethesubmergedsurfacearea.Sinceeachdipstick midpoint ofthelacquerlinesosmallvariationsinfl In thetestingvial,nanostructureddipsticksaresubmergedto wired tothepotentiostatviaanalligatorclipandcableconnectingwire. is submergedinthetestingsolutionandotherendelectrically lacquer line(0.2cmwide)separatesthedipstickinhalf,whereoneend of theelectrodeismaskedbyanacidresistantlacquer. Thehorizontal as smallelectrochemicaldipstickswerethesensorregion(0.35cm analyte werepipettedintothetestingvial.ThePt-MGPNbiosensorsact bar whilesuccessiveincreasingconcentrationaliquotsofsaidtarget under constantstirring(500rpm)witha0.5cm(length)magneticstir buffered saline(PBS,0.1MpH7.4)ataworkingpotentialof500mV electrode (Ptgauze)for500cycles. that areappliedbetweentheworkingelectrode(Pt-MGPN)andauxiliary onto eachPt-MGPNelectrodeviaconstantcurrentpulsesof1.05mA to themixture.ThesubsequentPEDOT/GOxsolutioniselectrodeposited 206083) andNa required beforeimageanalysis. microscopy (FESEM)micrographs. Noadditionalprocessingstepswere power settingof5.0kVtoobtain all fi eld emission scanningelectron laser powerwas2mWanda50Xobjective lenswasused. room temperatureusingalaserexcitation at488nmwavelength.The T64000 systembyHoribaScientifi c. Allthespectrawerecollectedat electrode (Figure 1 ). to permitmonolithicMGPNgrowthacrosstheentiresurfaceof of 10SCCM.Thehydrogenplasmadecomposesthemethanegas growth, ispumpedintothechamberfor10minutesatafl (H Ag/AgCl asthereferenceelectrode.Amperometrichydrogenperoxide acted astheworkingelectrode,aPtwireauxiliaryand Cell Stand-potentiostat)wheretheMGPNorPtNP-MGPNelectrodes was performedina3electrodeset-up(BASiEpsilonThree-Electrode O species thatmayhavebeenintroducedintothechamberduringloading. to abasepressureof0.1mTorr toeliminate/minimizecontaminating placed insidethevacuumchamberofreactorandpumpeddown within aPlasmaTech ReactiveIonEtch(RIE).TheMGPNelectrodewas created byfi rst mixingpoly(styrenesulfonate)(0.1M)inH onto thePtNP-MGPNelectrodes.TheGOx/PEDOTsolutionis Poly(3,4-ethylenedioxythiophene) (PEDOT)beforeitiselectrodeposited generator, whilemethane(CH is generatedoverthesampleviaa5kWSEKIAX5200Smicrowave frequency powersupplyatapressureof30Torr. Ahydrogenplasma and heatedto700 molybdenum puck,placedinsidetheMPCVDreactorchamber of theMGPNs,MGPNelectrodewasexposedtoanO chamber pressurewasadjustedto60mTorr. AO 2 wasintroducedintothechamberatafl 2 SensorImaging RamanSpectroscopy Ptnanoparticleelectrodeposition Glucoseoxidase(GOx)immobilization Electrochemicalmeasurements/set-up Oxygenplasmaetch O 2 ) andglucosesensingexperimentswereperformedinphosphate 2 : AS-4800 Hitachi microscopewasutilizedata SO ° 4 C inahydrogenambientby3.5kWradio- : Inanefforttoimprovetheelectroactivenature (0.5M,Fluka71960)toelectrodepositPt : Ramanspectroscopywasperformedusinga 4 ) gas,theactingprecursorforMGPN 2 : A3electrodeelectrochemical PtCl 2 Adv. Funct. . : Glucoseoxidaseismixedwith : Allelectrochemicaltesting 6 ·6H ow rateof50SCCMandthe 2 2 O (2 mg/ml) isnextadded O (4mM,SigmaAldrich www.MaterialsViews.com

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