Method for analyzing biological substances in a conductive liquid medium

• US 6,150,106 A
• Filed: 08/21/1998
• Issued: 11/21/2000
• Est. Priority Date: 07/07/1994
• Status: Expired due to Term

• Current Assignee: Centre National De La Recherche Scientifique, L'Ecole Centrale De Lyon, The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: Centre National De La Recherche Scientifique, L'Ecole Centrale De Lyon
• Inventors: Souteyrand, Eliane, Martin, Jean-Rene, Mikkelsen, Susan Rosalie, Lawrence, Marcus Francois
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/137,964
• Time in Patent Office: 823 Days
• Field of Search: 435/6, 435/7.1, 195/103.5, 204/403, 204/182.8
• US Class Current: 435/6.11
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

The present invention relates to a procedure for the qualitative and/or quantitative analysis of biological substances, which are preferably biological substances, that are present in a conductive liquid medium, with the aid of at least one affinity sensor that includes at least one structure that includes at least one semiconductor material, which is coated on one of its surface with at least one layer of an isolating material, which in turn is affixed adhesively to at least one sensitive membrane, which is in contact with the conductive medium and which includes ligands that are complementary to the biological substances in question and which are capable of, and suitable for, forming pairs specifically with the latter biological substances, with the said procedure being characterized by the fact that it consists essentially of applying a voltage between the semiconductor and the conductive medium; of gathering the variations in the electrical signals induced by a charge- effect phenomenon directly and essentially linked to the specific pairings of the biological substances with their complementary ligands included in the sensitive membrane; and interpreting the collected signals in order to identify and/or quantify the substances in question.

Claims

1. A method for detecting biological substances present in a conductive liquid medium (LC), comprising:

providing at least one affinity sensor which includes at least one structure having at least one semiconductor material coated on one surface with at least one isolating layer and at least one sensitive membrane affixed to said isolating layer, said sensitive membrane in fluid contact with a conductive liquid medium (LC) and which includes at least one ligand that is complementary to and capable of forming a hybridizing pair with said biological substance;

applying a voltage between the semiconductor and the conductive liquid medium;

detecting variations in electrical signals resulting from a pairing of said biological substance in said conductive liquid medium with said ligand coupled on said sensitive membrane; and

interpreting the variations in electrical signals detected in said detecting step in order to detect said biological substance.

2. The method of claim 1, wherein:

said sensor further comprises at least one working electrode consisting of the semiconductor/isolating layer/sensitive membrane structure, at least one reference electrode that forms a reference system and at least one counter-electrode;

said applying step comprises applying a polarization voltage V_pol to said working electrode, said polarization voltage being continuous in relation to said reference electrode immersed in said conductive liquid medium, superimposing on said voltage V_pol, a sinusoidal electrical modulation; and

said detecting step comprises measuring a voltage between the working electrode and the reference electrode, and a current passing through the working electrode, calculating a phase impedance Zp or the quadrature impedance Zq, plotting the Zp or Zq curve as a function of V_pol, and monitoring a shift of said curve, with said shift corresponding to said variations in the electrical signals, as induced by said charge effect resulting from said pairing of said biological substance with said ligand.

3. The method of claim 1 or 2, wherein said pairing comprises a reaction selected from the group consisting of nucleotide hybridization, and immunological coupling.

4. The method of claim 2, wherein said shift in said Zp or Zq curve is monitored by monitoring a flat-band potential (V_bp) of said semiconductor material, which is deducted from the said curve.

5. The method of claim 2, wherein said polarization voltage varies within a depletion range of said semiconductor material.

6. The method of claim 2, wherein said polarization voltage varies within a depletion range of said semiconductor material.

7. The method of claim 2, wherein said polarization voltage is from -3 to +3 V.

8. The method of claim 2, wherein said polarization voltage is from -1.5 to +1.5 V.

9. The method of claim 1, wherein:

said affinity sensor includes at least one transducer element formed by at least one field-effect transistor having a gate in contact with the conductive liquid medium, said gate formed by said sensitive membrane, with said transistor being associated with at least one reference electrode that is also immersed in said conductive liquid medium; and

said electrical signals induced by said pairing of said biological substance with said ligand on said sensitive membrane comprise variations in current or voltage.

10. The method of claim 9, wherein:

a potential (V_ds) between a drain and a source of said polarization voltage (V_pol) is assigned a constant value;

a drain current is kept constant, at an index value, with a potential between the reference and the source, V_rs, being adjusted; and

variations in V_rs are measured in response to said pairings between the biological substances and said ligands on said sensitive membrane.

11. The method of claim 1, wherein said affinity sensor further comprises at least one semiconductor/isolating layer structure which does not include a sensitive membrane and in which the isolating layer and the semiconductor form a field-effect transistor (FET) used as a reference (REFET).