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(12) United States Patent (10) Patent No.: US 8,268,567 B2 Clark (45) Date of Patent: Sep USOO8268567B2 (12) United States Patent (10) Patent No.: US 8,268,567 B2 Clark (45) Date of Patent: Sep. 18, 2012 (54) REACTION SENSING IN LIVING CELLS FOREIGN PATENT DOCUMENTS WO WO-99/45357 9, 1999 (75) Inventor: Heather Clark, Lexington, MA (US) WO WO/O1/O8660 2, 2001 WO WO 2004083902 * 9/2004 (73) Assignee: The Charles Stark Draper Laboratory, Cambridge, MA (US) OTHER PUBLICATIONS Bucket al. (Talanta 2004 vol. 63, p. 41-59).* European Search Report for European Patent Application No. 06851 (*) Notice: Subject to any disclaimer, the term of this 882.8, mailed Mar. 31, 2009. patent is extended or adjusted under 35 Barker et al., “Radiometric and Flourescense-Lifetime-Based U.S.C. 154(b) by 932 days. Biosensors Incorporating Cytochrome Cand the Detection of Extra and Intracellular Macrophage Nitric Oxide.” Anal Chem, vol. 71, No. (21) Appl. No.: 11/522,169 9, May 1, 1999, pp. 1767-1772. Bucket al., “Nanoscale Probes Encapsulated by Biologically Local ized Embedding (PEBBLEs) for Ion Sensing and Imaging in Live (22) Filed: Sep.15, 2006 Cells.” Talanta, vol. 63, No. 1, May 10, 2004, pp. 41-59. Buehlmann et al., "Carrier-Based Ion-Selective Electrodes and Bulk (65) Prior Publication Data Optodes. 2. Ionophores for Potentiometric and Optical Sensors.” Chem Rev, vol. 98, Jan. 1, 1998, pp. 1593-1687. US 2010/0227334 A1 Sep. 9, 2010 Dubach et al., “Flourescent Ion-Selective Nanosensors for Intracel lular Analysis with Improved Lifetime and Size.” Nano Lett, vol. 7. Related U.S. Application Data No. 6, Jun. 2007, pp. 1827-1831. Kohls et al., “Setup of a Fiber Optical Oxygen Multisensor-System (60) Provisional application No. 60/718.255, filed on Sep. and its Applications in Biotechnology.” Sensors Actuators B. vol. 70, 16, 2005. No. 1-3, Nov. 1, 2000. Sigworth et al., “Microchip Technology in Ion-Channel Research.” (51) Int. C. IEEE Trans Nanobiosci, vol. 4. No. 1, Mar. 1, 2005, pp. 121-127. GOIN33/53 (2006.01) International Search Report for PCT Application No. PCT/US2006/ U.S. Cl. ............................................. 435/7.1; 435/4 036040, mailed Aug. 29, 2008, 4 pages. (52) Written Opinion for PCT Application No. PCT/US2006/036040, (58) Field of Classification Search ........................ None mailed Aug. 29, 2008, 7 pages. See application file for complete search history. Brasuel et al. (Anal Chem 2001 vol. 73, p. 2221-2228). Examination Report mailed Mar. 11, 2010 in European Patent Appli (56) References Cited cation No. 0685 1882.8 (5 pages). McGraw-Hill Dictionary of Scientific and Technical Terms, Sixth U.S. PATENT DOCUMENTS Edition, p. 1633, year 2005. 4,272.484 6, 1981 Lubbers * cited by examiner 4,272.485 6, 1981 Lubbers 4,379,041 4, 1983 Petranek et al. Primary Examiner — Jacob Cheu 4,945,171 7/1990 Haugland et al. 5,057.431 10, 1991 Lubbers et al. (74) Attorney, Agent, or Firm — Foley & Lardner LLP 5,128,019 7/1992 Karphet al. 5,132,095 7, 1992 KOshiishi et al. (57) ABSTRACT 5,494,640 2f1996 Simon et al. 5,691,205 11/1997 Kawabata et al. Chemical reactions occurring within a living cell are mea 5,908,624 6, 1999 Scott et al. sured in a manner that does not affect the viability of the cell 6,143,558 11/2000 Kopelman et al. or the reaction under study. In one embodiment, one or more 6,210,551 4/2001 Osman et al. ................. 205,778 sensors are introduced into the cell and/or covalently associ 6,379,955 B1 4/2002 Kopelman et al. 6,699.465 B2 3, 2004 Scott ated with the exterior cell membrane. The sensor(s) emit an 2002fO155600 10, 2002 Kopelman et al. observable signal indicating a value of a parameter associated 2003. O157535 8, 2003 Berkovic .......................... 435/6 with the chemical reaction, e.g., the concentration of a reac 2003/0213691 11, 2003 Peper et al. tion product. Because cell viability is not compromised, the 2003/0217920 11, 2003 Peper et al. 2004/0058384 3, 2004 Bakker et al. cell may be stimulated (e.g., by Subjection to an agonist or 2004/0058458 3, 2004 Anker et al. antagonist, a pathogen, a pharmaceutical compound, or a 2004/0146944 T/2004 Fang et al. ..................... 435/72 potential toxin) so as to affect the reaction under study. 2005/OO 11760 1/2005 Bakker et al. 2006, OOO8924 1, 2006 Anker et al. 41 Claims, 10 Drawing Sheets U.S. Patent Sep. 18, 2012 Sheet 1 of 10 US 8,268,567 B2 FIG. I. 134 Agent Eccitation FE Introduction Control U9 Module Module U.S. Patent Sep. 18, 2012 Sheet 2 of 10 US 8,268,567 B2 20 N FIG. 24 22 24 s N-- 5. FIG. 2B 24 FIG. 2C 28 U.S. Patent Sep. 18, 2012 Sheet 3 of 10 US 8,268,567 B2 FIG. 2D 9. -24 testesters----------------- - - - - U.S. Patent Sep. 18, 2012 Sheet 4 of 10 US 8,268,567 B2 | ? 0I£ s £(9IAI -- s: 008 U.S. Patent Sep. 18, 2012 Sheet 5 of 10 US 8,268,567 B2 9(9.I.H. „~~~ U.S. Patent US 8,268,567 B2 9(9IAI 0 00°0 30 u23S3On-UU Og 3ZeudoN U.S. Patent Sep. 18, 2012 Sheet 7 of 10 US 8,268,567 B2 Z’9ICH U.S. Patent Sep. 18, 2012 Sheet 8 of 10 US 8,268,567 B2 s x N c s o S s : s o d w (OAg/09) eoueoseonjo ope U.S. Patent Sep. 18, 2012 Sheet 9 of 10 US 8,268,567 B2 s E 3 N c O 4. r N t (O1970 g) epueoseon Jo Ole U.S. Patent Sep. 18, 2012 Sheet 10 of 10 US 8,268,567 B2 s 5 E o l s o o o o N N c co s s c (OASIO/9) eoueoSejon-Jo ope US 8,268,567 B2 1. 2 REACTION SENSING IN LIVING CELLS In one embodiment, one or more sensors are introduced into the celland/or covalently associated with the exterior cell CROSS-REFERENCE TO RELATED CASES membrane. The sensor(s) produce an observable signal indi cating a value of a parameter associated with the chemical This application claims the benefit of U.S. Provisional reaction, e.g., the concentration of a reaction product. Patent Application No. 60/718.255, entitled “Optical Biosen Because cell viability is not compromised, the cell may be sor Array and Related Systems, Methods and Devices, filed stimulated (e.g., by Subjection to an agonist or antagonist, a on Sep. 16, 2005, the entire disclosure of which is hereby pathogen, a pharmaceutical compound, or a potential toxin) incorporated by reference as if set forth herein in its entirety. so as to affect the reaction under study. By “observable' is 10 meant visible or measurable using instrumentation. FIELD OF THE INVENTION In some embodiments, the sensor is used to monitor the activity of an ion channel through the cell membrane. In Such The invention relates to monitoring chemical reactions cases, the sensor may be introduced within the cell, may be occurring within living cells, and more particularly to meth attached to the exterior cell membrane, or both. For example, ods and apparatus for measuring such reactions without com 15 the external sensor may be attached to the cell membrane promising the viability of the cells. proximate to an ion channel of the cell, e.g., via an antibody specific to the ion channel (which typically comprises a pro BACKGROUND OF THE INVENTION tein). The sensor may be an ion sensor, and sensors internal and external to the cell may monitor differentions. Numerous techniques exist for monitoring the extent and In some embodiments, the sensor comprises an ion-selec time profile of a chemical reaction of interest occurring tive optode. The reaction may produce anionic product that is within a living cell. Many Such techniques are indirect, sequestered by the optode, or instead, the reaction may pro involving an assay for reaction products outside the cell that duce a non-ionic product that is ionized to facilitate monitor reflect the progress of reaction(s) taking place in the cyto ing using the optode. In this way, the invention can be used in plasm. A disadvantage of such assays is their Vulnerability to 25 numerous pharmaceutical applications involving Small mol influence by the spurious presence of reaction products in the ecules. cells environment that do not, in fact, emanate from the cell. In some embodiments, the optode comprises an ion-selec Intracellular techniques have also been developed; these tive ionophore, a source of triggering ions, and a signaling include, for example, genetically engineering cells to fluo agent responsive to the triggering ions. Typically the iono resce in the presence of a compound of interest, radioactive 30 phore is selective for the ionic reaction product, and upon binding assays, and colorimetric assays. These techniques, binding of a reaction-production, a triggering ion is released however, require potentially destructive modification or inva to interact with the signaling agent and thereby produce the sion of the cell. signal. The strength of the signal may be indicative of the Intracellular and extracellular assays are often employed to concentration of the triggering ion, which itself reflects the trackenzymatic activity by measuring the amount of reaction 35 concentration of the ionic reaction product. Different sensors, product through the use of coupling. Enzymatic coupling is a each responsive to differentions and producing differentiable complicated approach that links a non-quantifiable enzymatic signals, can be used depending on the reaction(s) being moni reaction of interest with an optically measurable (by means of tored.
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