Miniaturised Biosensor with Optimized Amperometric Detection

• US 20090178935A1
• Filed: 03/29/2007
• Published: 07/16/2009
• Est. Priority Date: 04/10/2006
• Status: Abandoned Application

• Current Assignee: Biomericux S.A., Biomerieux SA (Compagnie Merieux Alliance SAS), Diagnoswiss SA
• Original Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS), Diagnoswiss SA
• Inventors: Reymond, Frederic, Rossier, Joel Stephane, Morier, Patrick
• Application Number:
  US12/296,639
• Publication Number:
  US 20090178935A1
• Time in Patent Office: Days
• US Class Current: 205/777.500
• CPC Class Codes: G01N 27/3273 Devices therefor, e.g. test element readers, circuitry details not specific to biochemical electrodes G01N33/4875

Abstract

A method to optimize the amperometric detection in a microsystem consists in limiting the detection to times when the diffusion layer (18-20) of the analyte to detect remains smaller than the microchannel (7) height. The charge detected during the second part of the amperometric measurement (which corresponds to the integral of the measured current over the corresponding time period) can also be considered so as to remove the contribution of the capacitive current and, when applicable, of the current resulting from the reduction or oxidation of the analyte molecules present in a recess above the electrode at the beginning of the detection. A microfluidic amperometric sensor for performing the method comprises at least one microchannel (7) having at least one electrode (15-17), integrated in one wall of the microchannel, and having a characteristic length or radius which is smaller than half the microchannel height.

Claims

1. :

An amperometric detection method for determining the presence, the amount and/or the concentration of an analyte in a microfluidic sensor comprising the steps of;

a) providing a microfluidic sensor comprising at least one microstructure including at least one working electrode integrated with precise size and location in one wall portion of said microstructure, the height of said microstructure above said integrated working electrode being at least twice the characteristic length—

or the radius in the case of a circular electrode—

r of said integrated working electrode;

b) filling said microfluidic sensor with the sample to analyse;

c) applying the potential required to directly or indirectly detect said analyte by amperometry for a time period shorter than the ratio r²/D, where r is in metres and D is the diffusion coefficient of said analyte in m²/s, and measuring the resulting oxidation or reduction current at said integrated working electrode during this time period, so that only the analyte molecules submitted to a hemi-spherical or hemi-cylindrical diffusion regime above said at least one integrated working electrode are probed during the amperometric measurement;

and optionally

d) performing sequential amperometric measurements by repeating step c) after a relaxation time longer than half of the ratio r²/D.

5. :

An amperometric detection method according to claim 1, wherein a relaxation time separating sequential amperometric measurements is longer than about 1 second but shorter than about 1 minute.

6. :

An amperometric detection method according to claim 1, wherein an effective detection signal considered for determining the presence, concentration and/or amount of an analyte in said microfluidic sensor is restricted to only a portion of the current measured during step c), and optionally step d), said portion of the measured current being selected over a time period where the capacitive current can be considered constant with respect to the faradic current and where are detected only analyte molecules submitted to a hemi-spherical or hemi-cylindrical diffusion regime.

7. :

An amperometric detection method according to claim 6, wherein the current measured during the first part of the potential application in step c), and optionally step d), is eliminated and not considered as the effective detection signal, said first part of the potential application having a duration of at least 1 second, or, in the case where said at least one integrated working electrode has a recess of length L, a duration at least equal to the ratio L²/2D.

8. :

An amperometric detection method according to claim 1, wherein an effective detection signal is obtained by integrating the current measured during step c), and optionally step d) over only a portion of the potential application period, so as to obtain a detection signal corresponding to the value of the charge Q in Coulombs resulting from the detection of analyte molecules submitted only to a hemi-spherical or hemi-cylindrical diffusion regime, and wherein the presences the amount and/or the concentration of said analyte is determined from the value of this charge Q.

9. :

An amperometric detection method according to claim 8, wherein the detection signal is obtained by eliminating the current measured during at least the first second of potential application, and by considering the charge Q corresponding to the integration of the measured current over the remaining duration of the potential application.

11. :

An amperometric detection method according to claim 8, wherein the presence, the amount and/or the concentration of an analyte is determined from the time evolution of said charge Q over sequential amperometric measurements.

13. :

An amperometric detection method according to claim 1, wherein the detected analyte molecules are partially or totally regenerated during sequential amperometric measurements and/or during the relaxation time separating two sequential amperometric measurements.

14. :

An amperometric detection method according to claim 13, wherein the detected analyte molecules are partially or totally regenerated during the relaxation time separating two sequential amperometric measurements by inverting the potential applied to said at least one integrated working electrode to a value enabling to reduce, or respectively oxidise, the detected molecules back into analyte molecules that are then detectable during the next amperometric measurement.

15. :

An amperometric detection method according to claim 13, wherein the detected analyte molecules are partially or totally regenerated on at least one counter electrode integrated in at least one wall portion of said microstructure.

16. :

An amperometric detection method according to claim 13, wherein the presence, amount and/or concentration of an analyte is determined by considering the currents resulting from both said amperometric measurement(s) and said regeneration of the detected molecules.

69. :

Use of an amperometric microfluidic sensor in conjunction with an amperometric detection method according to claim 1 for performing chemical and/or biological reactions in solution and particularly in connection with synthesis, and/or for performing chemical and/or biological analysis particularly in connection with chemical and/or biological assays such as but not limited to protein assays, affinity assays, immunoassays, enzymatic assays, enzyme-linked immunosorbent assays, cellular assays, virus assays, pathogen assays, DNA assays, hybridization assays, oligonucleotide assays, physico-chemical characterisation assays, lipophilicity assays, solubility assays or permeability assays, said sensor comprising at least one microstructure including;

a. at least one working electrode integrated with precise size and location in one wall portion of said microstructure, the height of said microstructure above said integrated working electrode being at least twice the characteristic length—

or the radius in the case of a circular electrode—

r of said integrated working electrode, and, optionally, said microstructure exhibiting a recess of length L in metres above said at least one integrated electrode, and

b. at least one counter electrode or one pseudo-reference electrode integrated in one wall portion of said microstructure, characterised in that the distance between said at least one counter electrode or one pseudo-reference electrode and said at least one working electrode is smaller than twice the microstructure height.

2. :

An amperometric detection method for determining the presence, the amount and/or the concentration of an analyte in a microfluidic sensor comprising the steps of;

a) providing a microfluidic sensor comprising at least one microstructure including at least one working electrode integrated with precise size and location in one wall portion of said microstructure, the height of said microstructure above said integrated working electrode being at least twice the characteristic length—

or the radius in the case of a circular electrode—

r in metres of said integrated working electrode, and said microstructure exhibiting a recess of height L in metres above said at least one integrated working electrode;

b) filling said microfluidic sensor with the sample to analyse;

c) applying the potential required to directly or indirectly detect said analyte by amperometry for a time period shorter than the ratio (r+L)²/D, where D is the diffusion coefficient of said analyte in m²/s, and measuring the resulting oxidation or reduction current at said integrated working electrode during this time period, so that only the analyte molecules submitted to a hemi-spherical or hemi-cylindrical diffusion regime within said microstructure are probed during the amperometric measurement;

and optionally

d) performing sequential amperometric measurements by repeating step c) after a relaxation time longer than half of the ratio (r+L)²/D.

4. :

An amperometric detection method according to claim 2, wherein the potential is applied—

and the related current measured—

at said at least one integrated electrode during a time period of no more than about 2 seconds.

10. :

An amperometric detection method according to claim 4, wherein the detection signal is obtained by eliminating the current measured during a first potential application of duration at least equal to L²/2D, and by considering the charge Q corresponding to the integration of the measured current over the remaining duration of the potential application, and wherein the detection signal is obtained by considering the charge Q corresponding to the integration of the current measured over the time interval t=˜

1 s and t=˜

2 s.

3. (canceled)

12. (canceled)

17-19. -19. (canceled)

20. :

An amperometric microfluidic sensor comprising at least one microstructure including;

a. at least one working electrode integrated with precise size and location in one wall portion of said microstructure, the height of said microstructure above said integrated working electrode being at least twice the characteristic length—

or the radius in the case of a circular electrode—

r of said integrated working electrode, and, optionally, said microstructure exhibiting a recess of length L in metres above said at least one integrated electrode,

b. at least one counter electrode or one pseudo-reference electrode integrated in one wall portion of said microstructure, characterised in that the distance between said at least one counter electrode or one pseudo-reference electrode and said at least one working electrode is smaller than twice the microstructure height,

said amperometric microfluidic sensor being adapted to detect signals resulting only from the analyte molecules submitted to a hemi-spherical or hemi-cylindrical diffusion regime above said at least one integrated working electrode upon application of the potential required to directly or indirectly detect said analyte by amperometry for a time period shorter than the ratio (r+L)²/D where D is the diffusion coefficient of said analyte in m²/s, and measuring the resulting oxidation or reduction current at said integrated working electrode during this time period.

22. :

An amperometric microfluidic sensor according to claim 20, wherein the ratio of the microstructure height over the characteristic length—

or radius—

of said at least one integrated working electrode is comprised between about 2 and 5.

35. :

An amperometric microfluidic sensor according to claim 20, wherein said microstructure comprises a plurality of working electrodes integrated with precise size and location in one wall portion of said microstructure, the height of said microstructure above said integrated working electrode being at least twice the characteristic length—

or the radius in the case of a circular electrode—

r of said integrated working electrodes, and the distance between two adjacent integrated working electrodes being equal or larger than twice their characteristic length—

or radius in case of circular electrodes.

39. :

An amperometric microfluidic sensor according to claim 20, wherein said microstructure and/or said at least one integrated working electrode is/are fabricated by any one of physical or chemical etching, injection moulding, laser photoablation, polymer casting, UV-LIGA, embossing, silicon technology, assembly of a series of layers or any combination thereof.

44. :

An amperometric microfluidic sensor according to claim 20, wherein the reference or pseudo-reference electrode(s) is(are) placed in the reservoir(s) at the inlet and/or outlet of said microstructure, said microchannel or said array or network of microchannels.

45. :

An amperometric microfluidic sensor according to claim 20, wherein said microstructure and/or a reservoir surrounding said microstructure inlet or outlet comprise(s) at least one of a biological material and of a chemical compound or reagent.

46. :

An amperometric microfluidic sensor according to claim 45, wherein said biological material or chemical compound or reagent is dried and/or reversibly or irreversibly immobilised;

either directly within said reservoir and/or within at least one portion of said microstructure such as on a wall portion or on said integrated working electrode(s);

or on a support material like a membrane, a gel, a sol-gel or beads, placed either within said reservoir and/or within at least one portion of said microstructure.

73. :

An amperometric microfluidic sensor according to claim 20, wherein said at least one working electrode is facing said at least one counter-electrode or one pseudo-reference electrode, so that said integrated working electrode is located on one face of a microstructure support of said amperometric microfludic sensor while said at least one counter-electrode or pseudo-reference electrode is located on the opposite face of the microstructure support of said amperometric microfluidic sensor.

21. (canceled)

23-34. -34. (canceled)

36-38. -38. (canceled)

40-43. -43. (canceled)

47-51. -51. (canceled)

52. :

A method of fabricating an amperometric microfluidic sensor comprising

a. at least one microstructure including at least one working electrode integrated with precise size and location in one wall portion of said microstructure, the height of said microstructure above said integrated working electrode being at least twice the characteristic length—

or the radius in the case of a circular electrode—

r of said integrated working electrode, and, optionally, said microstructure exhibiting a recess of height L in metres above said at least one integrated electrode,

b. at least one counter electrode or one pseudo-reference electrode integrated in one wall portion of said microstructure, characterised in that the distance between said at least one counter electrode or one pseudo-reference electrode and said at least one working electrode is smaller than twice the microstructure height,

said amperometric microfluidic sensor being adapted to detect signals resulting only from the analyte molecules submitted to a hemi-spherical or hemi-cylindrical diffusion regime above said at least one integrated working electrode upon application of the potential required to directly or indirectly detect said analyte by amperometry for a time period shorter than the ratio (r+L)²/D, where D is the diffusion coefficient of said analyte in m²/s, and measuring the resulting oxidation or reduction current at said integrated working electrode during this time period.

55. :

A method of fabricating an amperometric microfluidic sensor according to claim 52, wherein said integrated working, counter and/or pseudo-reference electrode(s) is(are) supported on conducting pad(s) placed on the material serving as microstructure support, on the opposite side of said microstructure and wherein said integrated working, counter, reference and/or pseudo-reference electrode(s) is(are) manufactured by elimination of material of the microstructure support at the bottom of said microstructure so as to create recessed electrode(s) at the bottom of said microstructure.

53-54. -54. (canceled)

56-68. -68. (canceled)

70-72. -72. (canceled)