Method and apparatus for detecting physicochemical changes emitted by biological sample

• US 20030022387A1
• Filed: 07/29/2002
• Published: 01/30/2003
• Est. Priority Date: 07/30/2001
• Status: Active Grant

• Current Assignee: Matsushita Electric Industrial Company Limited (Panasonic Holdings Corporation)
• Original Assignee: Matsushita Electric Industrial Company Limited (Panasonic Holdings Corporation)
• Inventors: Oka, Hiroaki, Ozaki, Nobuhiko, Sugihara, Hirokazu
• Granted Patent: US 7,326,384 B2
• Time in Patent Office: Days
• US Class Current: 436/149
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10

Abstract

A device for detecting a physicochemical change in a biological sample comprising at least one measuring electrode provided by causing a conductive material to enter into a porous film, and a conductor connected to the measuring electrode.

Claims

1. A device for detecting a physicochemical change in a biological sample, comprising:

at least one measuring electrode provided by causing a conductive material to enter into a porous film; and

a conductor connected to the measuring electrode.

2. A device according to claim 1, further comprising:

a support substrate for supporting the porous film;

a cell isolation portion provided on a surface of the porous film, for defining at least one chamber for accommodating a biological sample; and

a suction portion to be coupled in fluid communication with the biological sample in the chamber through the porous film,

wherein the measuring electrode is provided on a rear side of the porous film under the chamber.

3. A device according to claim 1, wherein the measuring electrode is provided by sputtering a conductive material onto the porous film, and the conductor is provided by sputtering or printing a conductive material onto a mask layer provided on the porous film.

4. A device according to claim 1, wherein each measuring electrode and the conductor connected thereto are insulated from the other measuring electrodes and the conductors thereto, so that the measuring electrodes can perform detection independently from each other.

5. A device according to claim 4, wherein each measuring electrode and the conductor connected thereto are insulated from the other measuring electrodes and the conductors thereto by an insulating resin injected to the porous film.

6. A device according to claim 4, wherein each measuring electrode and the conductor connected thereto are insulated from the other measuring electrodes and the conductors thereto by destroying a porous structure of surroundings of each measuring electrode and the conductor connected thereto with heat treatment, a laser or pressure.

7. A device according to claim 4, wherein each measuring electrode and the conductor connected thereto are insulated from the other measuring electrodes and the conductors thereto by adhesive for adhering the cell isolation portion provided on the porous film to the porous film.

8. A device according to claim 1, further comprising:

a support substrate for supporting the porous film;

a cell isolation portion provided on a surface of the porous film, for defining at least one chamber for accommodating the biological sample; and

at least one reference electrode,

wherein the measuring electrode is provided on a rear side of the porous film under the chamber, a reference electrode is provided on an inner wall of the chamber, and a conductor connected to the reference electrode is provided on an upper side of the cell isolation portion by patterning.

9. A device according to claim 2, wherein the cell isolation portion is made of an insulating material.

10. A method for measuring a physicochemical change in a biological sample of interest, comprising the steps of:

providing at least one reaction system for measuring a physiochemical characteristic of the biological sample of interest;

placing the biological sample of interest in the reaction system; and

detecting a change in the physiochemical characteristic of the biological sample of interest,

wherein the reaction system comprises a device according to claim 8, an arrangement and an environment or a characteristic of the reference electrode and the measuring electrode are adapted to characterize a change in the physiochemical characteristic of the biological sample of interest.

11. A method according to claim 10, wherein the arrangement and the environment or the characteristic are volumes of domains, the reference electrode and the measuring electrode being provided in the respective domains, and the volume of the domain provided with the measuring electrode is smaller than the volume of the domain provided with the reference electrode.

12. A method according to claim 10, wherein the arrangement and environment or characteristic are impedances of the reference electrode and the measuring electrode, and the impedance of the measuring electrode is lower than the impedance of the reference electrode.

13. A method according to claim 10, wherein the arrangement and environment or characteristic are frequency characteristics of the reference electrode and the measuring electrode, the impedance of the measuring electrode at about 10 Hz to about 10 kHz is smaller than the impedance of the reference electrode at about 10 Hz to about 10 kHz.

14. A method according to claim 10, wherein the arrangement and environment or characteristic are the amounts of electrolyte immersing the reference electrode and the measuring electrode, and the amount of the electrolyte immersing the reference electrode is greater than the amount of the electrolyte immersing the measuring electrode.

15. A method according to claim 14, wherein the amount of the electrolyte immersing the reference electrode is about 5 or more times as great as the amount of the electrolyte immersing the measuring electrode.

16. A method according to claim 12, wherein the impedance of the reference electrode is about 5 or more times as great as the impedance of the measuring electrode.

17. A method according to claim 10, wherein the reaction system has an arrangement and an environment or a characteristic selected from the group consisting of the impedance of the reference electrode:

the impedance of the measuring electrode=5;

1 and the volume of the electrolyte immersing the reference electrode;

the volume of the electrolyte immersing the measuring electrode=5;

1.

18. A method according to claim 10, wherein the step of detecting the physicochemical characteristic of the biological sample of interest comprises the steps of:

(a) recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

(b) sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

(c) calculating an average of each of the standard deviations; and

(d) detecting a change in the physiochemical characteristic of the biological sample based on the average.

19. A method according to claim 10, wherein the step of detecting the physicochemical characteristic of the biological sample of interest comprises the steps of:

(a) recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

(b) sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

(c) dividing the standard deviations into a plurality of classes having a predetermined size of standard deviation as a unit, and obtaining a distribution indicating physicochemical characteristics of the plurality of groups of extracted data belonging to the classes;

(d) approximating the distribution to a normal distribution;

(e) calculating an average and a half-width of the resultant normal distribution;

(f) optionally repeating (b) to (e); and

(g) detecting a change in the physicochemical characteristic of the biological sample based on the average and half-width.

20. A method according to claim 19, wherein the steps (b) to (e) are repeated and the number of the time-series signal values to be sampled is changed in each repetition.

21. A method according to claim 19, wherein before the step (b), the method further comprises adding up the time-series signal values emitted by a plurality of biological samples of interest provided in the reaction systems.

22. A method according to claim 21, wherein before the step (a), the method further comprises simultaneously stimulating the plurality of the biological samples of interest.

23. A method according to claim 10, wherein the step of detecting a change in the physicochemical characteristic of the biological sample of interest, comprises:

(a) recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

(b) sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

(c) dividing the standard deviations into a plurality of classes having a predetermined size of standard deviation as a unit, and obtaining a distribution indicating physicochemical characteristics of the plurality of groups of extracted data belonging to the classes;

(d) approximating the distribution by curvilinear approximating analysis selected from the group consisting of exponential decreasing analysis, exponential increasing analysis, Gaussian distribution, Lorentz distribution, a analysis, multiple peak analysis, and nonlinear analysis; and

(e) detecting a change in the physicochemical characteristic of the biological sample based on gradients before and after a peak on the approximated curve obtained by the step (d).

24. A method according to claim 18, wherein the sampling in the step (b) is carried out in a time-series manner.

25. A method according to claim 18, wherein the sampling in the step (b) is carried out at random.

26. A method according to claim 18, wherein the sampling in the step (b) is carried out a plurality of times from initial data a in a time-series manner and a plurality of times from data b recorded at a predetermined time after the initial data a in a time-series manner.

27. A method according to claim 19, wherein the sampling in the step (b) is carried out a plurality of times from initial data a in a time-series manner and a plurality of times from data b recorded at a predetermined time after the initial data a in a time-series manner.

28. A method according to claim 10, wherein the step of detecting a change in the physicochemical characteristic of the biological sample of interest, comprises:

(a) recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

(b) sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

(c) sampling the resultant standard deviations to obtain a plurality of groups of extracted standard deviations consisting of a plurality of values, and calculating an average of each of the plurality of groups of extracted standard deviations; and

(e) obtaining an index value detecting a change in the physicochemical characteristic of the biological sample based on a time of occurrence of the time-series signal value when the average reaches a predetermined threshold.

29. A method according to claim 18, wherein the biological sample of interest is a cell, and the physiochemical signal is a signal associated with activation of an ion channel or receptor of the cell, or actuation of an intracellular signal transduction system.

30. A method according to claim 19, wherein the step (a) is carried out in the presence of a standard chemical substance having a known action on the biological sample, and in the presence of a subject chemical substance, respectively, and the step (f) is carried out with concentrations of the standard chemical substance and the subject chemical substance being changed, respectively, and

the method further comprises comparing changes in the physicochemical characteristic in the presence of the standard chemical substance obtained in the step (g) with a change in the physicochemical characteristic in the presence of the subject chemical substance obtained in the step (g), and characterizing an action of the subject chemical substance on the biological sample.

31. An apparatus for measuring an action of a subject chemical substance on a biological sample, comprising:

a reaction system for measuring a physicochemical characteristic of the biological sample, comprising a device according to claim 8;

means for recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

means for sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

means for calculating an average of each of the standard deviations; and

means for detecting a change in the physiochemical characteristic of the biological sample based on the average.

32. An apparatus for measuring an action of a subject chemical substance on a biological sample, comprising:

a reaction system for measuring a physicochemical characteristic of the biological sample, comprising a device according to claim 8;

means for recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

means for sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

means for dividing the standard deviations into a plurality of classes having a predetermined size of standard deviation as a unit, and obtaining a distribution indicating physicochemical characteristics of the plurality of groups of extracted data belonging to the classes;

means for approximating the distribution to a normal distribution;

means for calculating an average and a half-width of the resultant normal distribution; and

means for detecting a change in the physicochemical characteristic of the biological sample based on the average and half-width.

33. An apparatus for measuring an action of a subject chemical substance on a biological sample, comprising:

a reaction system for measuring a physicochemical characteristic of the biological sample, comprising a device according to claim 8;

means for recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

means for sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

means for dividing the standard deviations into a plurality of classes having a predetermined size of standard deviation as a unit, and obtaining a distribution indicating physicochemical characteristics of the plurality of groups of extracted data belonging to the classes;

means for approximating the distribution by curvilinear approximating analysis selected from the group consisting of exponential decreasing analysis, exponential increasing analysis, Gaussian distribution, Lorentz distribution, σ

analysis, multiple peak analysis, and nonlinear analysis; and

means for detecting a change in the physicochemical characteristic of the biological sample based on gradients before and after a peak on the obtained approximated curve.

34. An apparatus for measuring an action of a subject chemical substance on a biological sample, comprising:

a reaction system for measuring a physicochemical characteristic of the biological sample, comprising a device according to claim 8;

means for recording a physiochemical signal emitted from the biological sample of interest as a time-series signal value;

means for sampling the time-series signal value to obtain a plurality of groups of extracted data consisting of a plurality of values, and calculating a standard deviation of each of the plurality of groups;

means for sampling the resultant standard deviations to obtain a plurality of groups of extracted standard deviations consisting of a plurality of values, and calculating an average of each of the plurality of groups of extracted standard deviations; and

means for obtaining an index value detecting a change in the physicochemical characteristic of the biological sample based on a time of occurrence of the time-series signal value when the average reaches a predetermined threshold.

35. An apparatus for screening drugs at high speed, comprising:

at least one reaction system for measuring a physicochemical characteristic of a biological sample of interest, comprising a device according to claim 8;

and

means for detecting a change in the physicochemical characteristic of the biological sample of interest,

wherein the reaction system comprises a reference electrode and a measuring electrode, and an arrangement and an environment or a characteristic of the reference electrode and the measuring electrode are adapted to characterize a change in the physiochemical characteristic of the biological sample of interest.