Device and methods for detecting the response of a plurality of cells to at least one analyte of interest

• US 7,713,733 B2
• Filed: 08/06/2002
• Issued: 05/11/2010
• Est. Priority Date: 08/06/2001
• Status: Active Grant

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Gilligan, Jonathan M., Wikswo, John P., Cliffel, David, Baudenbacher, Franz J., Balcarcel, R. Robert, Eklund, Sven
• Primary Examiner(s): Beisner; William H
• Assistant Examiner(s): BOWERS, NATHAN ANDREW
• Application Number:
  US10/483,466
• Publication Number:
  US 20050014129A1
• Time in Patent Office: 2,835 Days
• Field of Search: 205/777.5, 205/778, 205/779, 435/305.1, 435/305.2, 435/288.5, 435/288.3, 435/305.3, 435/244, 435/288.4, 435/30
• US Class Current: 435/305.1
• CPC Class Codes: C12M 41/32 of substances in solution
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5038 involving detection of metabolites per se
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: Y10T 436/115831 Condition or time responsive

Abstract

An apparatus and methods for detecting at least one analyte of interest either produced or consumed by a plurality of cell. In one embodiment of the present invention, the method includes the steps of providing a housing defining a chamber, placing a plurality of cells in the chamber, and simultaneously detecting at least two analytes of interest either produced or consumed by the plurality of cells in the chamber.

Claims

1. A device for detecting at least two analytes of interest either produced or consumed by a plurality of cells, comprising:

(a) a body portion having a first end, an opposite second end, a central axis running through the center of the body portion from the first end to the second end, and a planar axis perpendicular to the central axis, wherein the body portion is configured to have a recess proximate to the second end;

(b) a substrate for covering the recess of the body portion to define a chamber proximate to the second end, for receiving a plurality of cells therein, and further defining an optical window for allowing optical communication;

(c) an insert placed into the chamber, containing the plurality of cells;

(d) an inlet channel formed in the body portion and in fluid communication with the chamber for introducing a medium into the chamber;

(e) an outlet channel formed in the body portion and in fluid communication with the chamber for introducing a medium away from the chamber;

(f) means for simultaneously detecting at least two analytes of interest either produced or consumed by the plurality of cells in the chamber;

(g) a membrane positioned over the optical window of the substrate and between the substrate and the second end of the body portion, wherein the membrane is at least partially transparent; and

(h) a controller in communication with the simultaneously detecting means and comprising a means for storing, processing, and analyzing at least one detected signal, wherein the inlet channel and the outlet channel are formed substantially in parallel to the central axis of the body portion, wherein the substrate is formed substantially perpendicular to the central axis of the body portion and substantially in parallel to the planar axis of the body portion, and wherein the simultaneously detecting means comprises a first electrode to detect one of the at least two analytes of interest and a second electrode to detect another of the at least two analytes of interest, the first electrode and the second electrode positioned apart from each other, and wherein the controller is programmed to cause the simultaneously detecting means to cause the first electrode to detect the one of the at least two analytes of interest and generate a first corresponding signal and to cause the second electrode to detect the other of the at least two analytes of interest and generate a second corresponding signal, and wherein the controller is further programmed to measure at least one physical quantity associated with physiological activities of the at least one cell based on the detected signal from each of the first electrode and second electrode.

2. The device of claim 1, wherein the first electrode and the second electrode have different electrochemical characteristics.

3. The device of claim 2, wherein the first electrode comprises a gold electrode and the second electrode comprises a platinum electrode.

4. The device of claim 1, further comprising a potentiostat electrically coupled to the first electrode and the second electrode, and in communication with the controller, for detecting a voltage as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber.

5. The device of claim 1, further comprising a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode, and in communication with the controller, for detecting a current as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber.

6. The device of claim 1, further comprising an optical detector in communication with the controller to detect one of the at least two analytes of interest.

7. The device of claim 6, wherein the optical detector comprises an optical fiber.

8. The device of claim 1, wherein the controller is further programmed to analyze the at least two analytes of interest based on the measured at least one physical quantity.

9. The device of claim 8, wherein in operation, the at least two analytes of interest are produced or consumed by the plurality of cells in response to being exposed to an unknown agent and the controller is further programmed to identify the unknown agent based on the analysis.

10. The device of claim 1, wherein the at least one physical quantity comprises at least one of heat production, oxygen consumption, uncoupling ratio between heat production and oxygen consumption, free radical synthesis, fraction of oxygen diverted to free radical synthesis, reduced nicotinamide adenine dinucleotide phosphate (“

NAD(P)H”

), acid production, glucose uptake, lactate release, gluconeogenesis, transmembrane potential, intracellular messengers, membrane conductance, transmembrane pump and transporter rates, messenger RNA expression, neurotransmitter secretion, intracellular glycolitic stores, transmembrane action potential amplitude and firing rate, heat-shock protein expression, intracellular calcium, and calcium spark rate.

11. A device for detecting a plurality of analytes of interest either produced or consumed by a plurality of cells, comprising:

(a) a body portion having a first end, an opposite second end, a central axis running through the center of the body portion from the first end to the second end, and a planar axis perpendicular to the central axis, wherein the body portion is configured to have a recess proximate to the second end;

(b) a substrate for covering the recess of the body portion to define a chamber proximate to the second end, for receiving a plurality of cells therein, and further defining an optical window for allowing optical communication;

(c) an insert placed into the chamber, containing the plurality of cells;

(d) an inlet channel formed in the body portion and in fluid communication with the chamber for introducing a medium into the chamber;

(e) an outlet channel formed in the body portion and in fluid communication with the chamber for introducing a medium away from the chamber;

(f) means for simultaneously detecting a plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber;

(g) a membrane positioned over the optical window of the substrate and between the substrate and the second end of the body portion, wherein the membrane is at least partially transparent; and

(h) a controller in communication with the simultaneously detecting means and comprising a means for storing, processing, and analyzing at least one detected signal, wherein the inlet channel and the outlet channel are formed substantially in parallel to the central axis of the body portion, wherein the substrate is formed substantially perpendicular to the central axis and substantially in parallel to the planar axis of the body portion, and wherein the simultaneously detecting means comprises a plurality of electrodes to detect the plurality of analytes of interest, respectively, wherein the controller is programmed to cause the plurality of electrodes to detect the respective plurality of analytes of interest and generate signals corresponding to each of the plurality of electrodes, measure at least one physical quantity associated with physiological activities of the plurality of cells based on the detected signals from the plurality of electrodes, and analyze the plurality of analytes of interest based on the at least one physical quantity.

12. The device of claim 11, wherein the plurality of electrodes each has different electrochemical characteristics.

13. The device of claim 11, wherein the plurality of electrodes comprise a gold electrode.

14. The device of claim 11, wherein the plurality of electrodes comprise a platinum electrode.

15. The device of claim 11, further comprising a reference electrode, and an amperemeter electrically coupled to the plurality of electrodes, respectively, and in communication with the controller, for detecting a current as a function of the plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber.

16. The device of claim 11, further comprising a potentiostat electrically coupled to the plurality of electrodes, respectively, and in communication with the controller, for detecting a voltage as a function of the plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber.

17. The device of claim 11, further comprising an optical detector to detect the at least one of the plurality of analytes of interest.

18. The device of claim 17, wherein the optical detector comprises an optical fiber.

19. A device for detecting at least two analytes of interest either produced or consumed by at least one cell, comprising:

(a) a body portion having a first end, an opposite second end, a central axis running through the center of the body portion from the first end to the second end, and a planar axis perpendicular to the central axis, wherein the body portion is configured to have a recess proximate to the second end;

(b) a substrate for covering the recess of the body portion to define a chamber proximate to the second end, for receiving a plurality of cells therein, and further defining an optical window for allowing optical communication;

(c) an insert placed into the chamber, containing the plurality of cells;

(d) an inlet channel formed in the body portion and in fluid communication with the chamber, wherein the inlet channel is formed substantially in parallel to the central axis of the body portion, and wherein the substrate is formed substantially perpendicular to the central axis and substantially in parallel to the planar axis of the body portion;

(e) a first electrode having a first electrochemical characteristic;

(f) a second electrode positioned away from the first electrode and having a second electrochemical characteristic that is different from the first electrochemical characteristic;

(g) a membrane positioned over the optical window of the substrate and between the substrate and the second end of the body portion, wherein the membrane is at least partially transparent, and

(h) a controller in communication with the first electrode and the second electrode, and comprising a means for storing, processing, and analyzing at least one detected signal,

wherein the controller is programmed to cause the first electrode to detect a first analyte of interest either produced or consumed by at least one cell and to cause the second electrode to detect a second analyte of interest either consumed or produced by at least one cell, respectively and simultaneously, wherein the second analyte of interest either produced or consumed by at least one cell is different from the first analyte of interest, and wherein the controller is further programmed to measure at least one physical quantity associated with physiological activities of the at least one cell based on the detected signal from each of the first electrode and second electrode, and analyze the first analyte of interest and the second analyte of interest based on the measured at least one physical quantity.

20. The device of claim 19, further comprising a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber.

21. The device of claim 19, further comprising a potentiostat electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber.

22. The device of claim 19, further comprising additional electrodes, each having a different electrochemical characteristic and being positioned away from the first and second electrodes.

23. The device of claim 19, further comprising an outlet in fluid communication with the chamber for introducing a medium away from the chamber.

24. The device of claim 19, further comprising an optical detector to detect optical signatures of intracellular physiological processes of the cell.

25. The device of claim 19, further comprising an optical detector to detect at least one of the analytes of interest.

26. The device of claim 25, wherein the optical detector comprises an optical fiber having a first end, a second end and a body portion defined therebetween.

27. The device of claim 26, wherein the first end of the optical fiber reaches in the chamber and is capable of detecting an optical signal related to the two analytes of interest either produced or consumed by at least one cell.

28. The device of claim 25, wherein the optical detector further comprises:

(a) a cover slip member having a first surface and a second surface, wherein the first surface of the cover slip is underneath the chamber and the second surface of the cover slip is optically coupled to the first end of the optical fiber; and

(b) a light source optically coupled to the second end of the optical fiber.

29. The device of claim 28, further comprising:

(a) a beam splitter optically coupled to the optical fiber and positioned between the light source and the cover slip for directing optical signals transmitted through the optical fiber corresponding to the optical response from a first direction to a second direction; and

(b) an analyzer for receiving the optical signals directed by the beam splitter.

30. The device of claim 19, wherein the membrane comprises a Si/SiN membrane.

31. The device of claim 19, wherein the first electrode is formed with a first diameter, and the second electrode is formed with a second diameter that is different from the first diameter.

32. The device of claim 19, wherein the first electrode is formed with a first conductive material, and the second electrode is formed with a second conductive material that is different from the first conductive material.

33. A device for detecting at least two analytes of interest either produced or consumed by at least one cell exposed to an unknown agent, comprising:

(a) a body portion having a first end, an opposite second end, a central axis running through the center of the body portion from the first end to the second end, and a planar axis perpendicular to the central axis, wherein the body portion is configured to have a recess proximate to the second end;

(b) a substrate for covering the recess of the body portion to define a chamber proximate to the second end, for receiving a plurality of cells therein, and further defining an optical window for allowing optical communication;

(c) an insert placed into the chamber, containing the plurality of cells;

(d) an inlet channel formed in the body portion and in fluid communication with the chamber, wherein the inlet channel is formed substantially in parallel to the central axis of the body portion, and wherein the substrate is formed substantially perpendicular to the central axis and substantially in parallel to the planar axis of the body portion;

(e) a first electrode having a first electrochemical characteristic;

(f) a second electrode positioned away from the first electrode and having a second electrochemical characteristic that is different from the first electrochemical characteristic;

(g) a membrane positioned over the optical window of the substrate and between the substrate and the second end of the body portion, wherein the membrane is at least partially transparent; and

(h) a controller in communication with the first electrode and the second electrode and comprising a means for storing, processing, and analyzing at least one detected signal, programmed to cause the first electrode to detect a first analyte of interest either produced or consumed by at least one cell and to generate a first corresponding signal, and to cause the second electrode to detect a second analyte of interest either produced or consumed by at least one cell and to generate a second corresponding signal, respectively and simultaneously, wherein the second analyte of interest is different from the first analyte of interest, and wherein the controller is further programmed to measure at least one physical quantity associated with physiological activities of the at least one cell based on the first signal and the second signal, analyze the at least two analytes of interest based on the measured at least one physical quantity, and identify the unknown agent based on the analysis.

34. The device of claim 33, further comprising a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber, wherein the reference electrode and the amperemeter are in communication with the controller, wherein the controller is further programmed to cause the reference electrode and amperemeter to detect the current and to analyze the at least two analytes of interest based on the detected current.

35. The device of claim 33, further comprising a potentiostat electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber, wherein the potentiostat is in communication with the controller and the controller is further programmed to cause the potentiostat to detect the voltage and to analyze the at least two analytes of interest based on the detected voltage.

36. The device of claim 33, further comprising an optical detector for detecting an optical signal as a function of at least one of the analytes of interest, wherein the optical detector is in communication with the controller and the controller is further programmed to cause the optical detector to detect the optical signal and to analyze the at least two analytes of interest based on the detected optical signal.