Method for identifying and/or analyzing biological substances, present in a conductive liquid, device and affinity sensor used for implementing said method

• US 6,475,728 B1
• Filed: 03/31/2000
• Issued: 11/05/2002
• Est. Priority Date: 06/11/1997
• Status: Expired due to Fees

• Current Assignee: Centre National De La Recherche Scientifique, École centrale de Lyon
• Original Assignee: Centre National De La Recherche Scientifique, École centrale de Lyon
• Inventors: Souteyrand, Eliane, Martin, Jean-René
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): KIM, YOUNG J
• Application Number:
  US09/424,877
• Time in Patent Office: 949 Days
• Field of Search: 435/4, 435/6, 435/34, 435/39, 204/1, 204/403, 204/412, 324/71.5
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 27/305 optically transparent or photoresponsive electrodes
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

An apparatus and method for identifying and/or quantifying a charged biological substance in a conductive liquid medium.

Claims

1. A apparatus for identifying and/or quantifying a charged biological substance in a conductive liquid medium, comprising:

a chamber for the conductive liquid medium;

an affinity sensor disposed in said chamber;

said affinity sensor comprising

a semiconductor having an upper and a lower surface;

said semiconductor having an ohmic contact disposed on its lower surface;

an insulator layer disposed on the upper surface of said semiconductor;

a plurality of probes in contact with said conductive liquid medium;

said

plurality of probes being disposed on the surface of said insulator layer;

said

plurality of probes comprising a recognition biological substance which is able to specifically hybridize with a charged biological substance present in the conductive liquid medium, whereby specific pairings of charged biological substances with their complementary recognition biological substances are formed;

said specific pairings modifying the surface electric charge of the affinity sensor;

means for biasing the semiconductor with respect to the conductive liquid medium;

means for illuminating the semiconductor;

means for measuring a photopotential V_ph at the terminals of the affinity sensor;

means for converting the detected signals into V_fb variations which are induced by a charge-effect phenomenon associated with said pairings modifying the surface electric charge of the affinity sensor; and

means for calculating and interpreting the Δ

V_fb variations in terms of the identification and/or quantitative determination of the charged biological substances.

2. An apparatus according to claim 1, wherein said semiconductor is a silicon semiconductor.

3. An apparatus according to claim 1, wherein said insulator layer is a silica or silicon nitride insulator layer.

4. An apparatus according to claim 1, wherein said recognition biological substance is a polynucleotide.

5. An apparatus according to claim 1, further comprising an auxiliary electrode.

6. An apparatus according to claim 5, wherein said auxiliary electrode is disposed on the insulator layer.

7. An apparatus according to claim 1, comprising a means. for evaluating the variation in V_ph signal with respect to a reference U, and a means for correcting the variation in V_ph signal by acting on the biasing means.

8. An apparatus according to claim 1, further comprising a reference structure not functionalized by a probe or probes.

9. An apparatus according to claim 1, wherein the probes are of different types, and wherein probes of the same type are grouped in the same region on the insulator layer to allow separate and/or successive illumination.

10. An affinity sensor according to claim 1.

11. A method of identifying and/or quantifying a charged biological substance in a conductive liquid medium using an apparatus comprising:

a chamber for the conductive liquid medium;

a affinity sensor disposed in said chamber;

said affinity sensor comprising

a semiconductor having an upper and a lower surface;

said semiconductor having an ohmic contact disposed on its lower surface;

an insulator layer disposed on the upper surface of said semiconductor;

a plurality of probes in contact with said conductive liquid medium;

said

plurality of probes being disposed on the surface of said insulator layer;

said

plurality of probes comprising a recognition biological substance which is able to specifically hybridize with a charged biological substance present in the conductive liquid medium, whereby specific pairings of charged biological substances with their complementary recognition biological substances are formed;

means for biasing the semiconductor with respect to the conductive liquid medium;

means for illuminating the semiconductor;

means for measuring a photopotential V_ph at the terminals of the affinity sensor;

means for converting the detected signals into V_fb variations which are induced by a charge-effect phenomenon associated with said pairings modifying the surface electric charge of the affinity sensor; and

means for calculating and interpreting the Δ

V_fb variations in terms of the identification and/or quantitative determination of the charged biological substances;

wherein the method comprises

providing an unlabeled recognition biological substance as a probe;

contacting the charged biological substance with the unlabeled recognition biological substance, whereby specific pairings of charged biological substances with their complementary recognition biological substances are formed;

said specific pairings modifying the surface electric charge of the affinity sensor;

biasing the semiconductor to ensure that the Fermi level corresponds approximately to, or exceeds, the intrinsic Fermi level at the surface of the semiconductor;

exposing the semiconductor to periodic illumination comprising photons having an energy greater than or equal to the band gap energy of the semiconductor;

measuring the photopotential V_ph at the terminals of the affinity sensor;

determining the complex impedance of the affinity sensor from the photopotential V_ph, determining the variations Δ

V_fb of the semiconductor which are induced by a charge-effect phenomenon associated with said specific pairings of the charged biological substances with their complementary recognition biological substances;

to the exclusion;

(i) of the variations resulting from possible charge and/or charge-transfer effects caused by enzyme-catalyzed chemical reactions in which some of the substances to be detected are consumed; and

(ii) and of the variations in the photoresponse which are associated with the appearance in the conductive medium of at least one tracer product capable of being revealed through pH variations or redox potential variations and/or through radiation-absorbing or radiation emitting labels; and

identifying and/or quantifying the charged biological substances in the conductive liquid medium by interpreting the received signals.

12. A method according to claim 11, wherein the specific pairings of charged biological substance comprise an enzyme-substrate complex.

13. A method according to claim 11, wherein the specific pairings of charged biological substance comprise an immunological complex.

14. A method according to claim 11, wherein the charged biological substance is a polynucleotide and the recognition biological substance is a polynucleotide.

15. A method according to claim 11, wherein the semiconductor is biased by imposing a bias voltage V_b by sweeping between a negative limiting voltage and a positive limiting voltage;

and

wherein Δ

V_fb is measured by detecting the photopotential V_ph at the terminals of the semiconductor;

optionally calculating the in-phase component Z_op and/or the quadrature component Z_oq of the optoelectrochemical impedance for each value of V_b;

constructing Z_op and/or Z_oq and/or V_ph curve as a function of V_b; and

monitoring the shift in the curve parallel to the x-axis, said shift corresponding to the desired variations in V_fb.

16. A method according to claim 11, wherein the periodic illumination is approximately sinusoidal weak illumination.

17. A method according to claim 11, wherein the periodic illumination is strong illumination.

18. A method according to claim 11, wherein the V_fb variations are obtained by detecting V_ph and constructing a V_ph=f(V_b)curve;

and monitoring a shift in the curve parallel to the x-axis, said shift corresponding to the desired Δ

V_fb values.

19. A method according to claim 11, wherein the Fermi level of the semiconductor is set approximately to the intrinsic Fermi level at the surface of the semiconductor by imposing a bias voltage V_b corresponding approximately to the abscissa of the point of inflection of the V_ph=f(V_b) or Z_oq=f(V_b) curve, wherein the ordinate of the point of inflection corresponds approximately to V_ph, ½

max and Z_oq, ½

max; and

wherein the variations in Δ

V_fb in V_fb are obtained (i) by measuring the change in V_ph and/or Z_oq; and

/or (ii) by taking into account the variations in the bias voltage Δ

V_b made necessary to keep V_ph, ½

max or Z_oq, ½

max constant, this Δ

V_b adjustment being the reflection of Δ

V_fb.

20. A method according to claim 11, which comprises detecting the photopotential signal V_ph;

optionally amplifying the signal V_ph;

optionally rectifying the signal to give a DC signal V′

_ph;

comparing the DC signal V′

_ph with a reference signal U corresponding approximately to the value of V′

_ph, ½

max;

obtaining the difference between V′

_ph and U, namely Δ

(V′

_ph−

U);

optionally amplifying Δ

(V′

_ph−

U);

applying the optionally amplified Δ

(V′

_ph−

U) between the semiconductor and the conductive liquid medium as a complement of V_b; and

recording Δ

(V′

_ph−

U).

21. A method according to claim 11, wherein the wavelength λ

of the illumination is greater than or equal to a threshold λ

₀, where ${\mathrm{\lambda <br><br>}}_0=\frac{1240}{E}<br><br>\text{\hspace{1em}<br><br>}<br><br>\mathrm{nm}.$

22. A method according to claim 21, wherein the wavelength is between 100 and 3000 nm.

23. A method according to claim 11, further comprising using a reference structure comprising a semiconductor and an insulator layer wherein said insulator layer is not functionalized by a probe;

and monitoring the difference between the V_bp variations measured by the two sensors, and the variation in this difference.

24. A method according to claim 11, wherein the insulator layer is functionalized by different types of polynucleotides as recognition biological substance, and wherein the conductive liquid medium comprises different types of polynucleotides as charged biological substance;

and wherein said probes of the same type are grouped in the same region on the insulator layer to allow separate and/or successive illumination.

25. A method according to claim 11, wherein the charged biological substance is selected from the group consisting of nucleotides, oligonucleotides, polynucleotides, nucleic acids and mixtures thereof.

26. A method according to claim 24, wherein at least one affinity sensor is used without a compartment at the surface of the affinity sensor.