Electrochemical biosensors, applications and methods of making biosensors

• US 7,892,414 B1
• Filed: 11/15/2005
• Issued: 02/22/2011
• Est. Priority Date: 11/19/2004
• Status: Expired due to Fees

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Sumner, James J.
• Primary Examiner(s): Neckel; Alexa D
• Assistant Examiner(s): DIETERLE, JENNIFER M
• Application Number:
  US11/274,709
• Time in Patent Office: 1,925 Days
• Field of Search: 204/403, 204/400, 204/403.01, 205/198, 205/778, 205/777.5, 435/14, 422/68.1, 422/82.02, 436/806
• US Class Current: 205/778
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 2400/28 Chitin, chitosan
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property

Abstract

Electrochemical biosensors, methods of making biosensors and methods of detecting a target analyte in a sample to be analyzed are provided. A preferred embodiment device comprises a non-conductive polymer deposited on the electrode to form a matrix material having a plurality of pores; a capture biomolecule immobilized on the electrode such that the binding of the target analyte to the capture biomolecule effectively blocks a sufficient number of pores in the matrix material to produce a measurable decrease in the rate of a redox reaction occurring at the electrode; a current source for driving a redox reaction at the electrode; a redox species in contact with the electrode; and a detector element operatively connected to the electrode to detect the measureable decrease of a change in the rate of a redox reaction occurring due to the blockage of pores in the matrix material by the target analyte.

Claims

1. A biosensor comprising:

a) an electrode;

b) a non-conductive polymer deposited on the electrode to form a matrix material having a plurality of pores therethrough;

c) a capture biomolecule immobilized on the matrix material capable of binding a target analyte in a sample to be analyzed;

wherein the binding of the target analyte to the capture biomolecule effectively blocks a sufficient number of pores in the matrix material to produce a measurable decrease in the rate of a redox reaction occurring at the electrode;

d) a current source, wherein the current source provides a flow of electrons to drive a redox reaction at the electrode between the electrode and a redox species in contact with the electrode; and

e) a detector element coupled to the electrode to detect the measureable decrease a change in the rate of a redox reaction occurring at the electrode due to the number of pores in the matrix material blocked by the binding of the target analyte.

2. The biosensor of claim 1, wherein the matrix material comprises chitosan and the electrochemical deposition of chitosan is performed on the electrode, and wherein the capture biomolecule is unlabeled, and wherein the pores of the matrix material are large enough to permit a flow of a redox species to the electrode, and wherein the pores are small enough to substantially prevent the flow of any contaminating molecules present in the sample to be analyzed to the electrode.

3. The biosensor of claim 1, wherein the binding of the target analyte to the capture biomolecule results in the target analyte blocking a significant percentage of the pores leading to the electrode resulting in an increase in the impedence.

4. The biosensor of claim 1, wherein electrode is immersed in an electrolyte comprising redox molecules and wherein the binding of the target analyte to the capture biomolecule significantly affects the ability for the redox molecules to reach the electrode, thereby decreasing the rate of redox detected by the detector element.

5. The biosensor of claim 1, wherein the capture biomolecule is a nucleic acid sequence.

6. The biosensor of claim 1, wherein the capture biomolecule is a protein or peptide.

7. The biosensor of claim 1, wherein the signal element further quantifies the amount of target analyte present in the sample based on the amount of change in the rate of the redox reaction occurring at the electrode.

8. The biosensor of claim 1, wherein the packing density of the capture biomolecule on the matrix is in a range of about 1×

10¹¹ to about 1×

10¹³ per square centimeters.

9. The biosensor of claim 1, wherein the redox species is contained in an aqueous solution in contact with the electrode.

10. The biosensor of claim 9, wherein the redox species is potassium ferricyanide.

11. The biosensor of claim 1, wherein the nonconductive polymer comprises a chitosan film that is electrochemically deposited on electrode surface, and the capture biomolecule comprises an unlabeled immobilized capture strand of oligonucleotide which is immobilized to the surface of the chitosan film.

12. The biosensor of claim 11, further comprising glutaraldehyde having a concentration in the range of 0.05 to approximately 0.1% so that there are enough bonding sites for the capture biomolecule in order to obtain sufficient packing density to yield the blocking effect, yet not significantly in excess of 0.1% glutaraldehyde so that cross-linking of the matrix material and closing of the pores occurs, and potassium ferricyanide;

and wherein the concentration of chitosan is within the range of about 0.01% to 1%.

13. The biosensor of claim 1, wherein the capture biomolecule is a first element of a binding pair and the target analyte is a second element of the binding pair;

and wherein neither the capture biomolecule nor the target analyte interacts with the redox molecules to generate redox products.

14. The biosensor of claim 13, wherein the binding pair is selected from:

antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin and complementary nucleic acid sequences.

15. The biosensor of claim 13, wherein the binding pair comprises complementary nucleic acid sequences and wherein the nucleic acid sequence of the capture biomolecule is complementary to at least a portion of the sequence of a target analyte.

16. The biosensor of claim 1, wherein the matrix material is a non-conductive polymer having a functional group capable of binding a capture biomolecule or a linking material and wherein the matrix material is deposited on the electrode by electrochemical deposition to thereby form a lattice;

and wherein the pores in the lattice are chemically constricted by a cross-linking material.

17. The biosensor of claim 16, wherein the non-conductive polymer is substantially soluble at a generally acidic pH or a generally basic pH and is substantially insoluble at a generally neutral pH.

18. The biosensor of claim 17, wherein the non-conductive polymer is selected from:

chitosan, poly-L-Iysine, and combinations thereof.

19. A biosensor comprising:

a) an electrode;

b) a matrix material deposited on the electrode, wherein the matrix material comprises a plurality of pores therethrough;

c) a capture biomolecule immobilized on the electrode capable of binding a target analyte in a sample to be analyzed, wherein the binding of the target analyte to the capture biomolecule effectively blocks a sufficient number of pores in the matrix material to produce a measurable decrease in the rate of a redox reaction occurring at the electrode; and

d) a detector element coupled to the electrode to detect the measureable decrease in the rate of a redox reaction occurring at the electrode in order to detect the presence of the target analyte due to the number of pores in the matrix material blocked by the target analyte.

20. A method of making a biosensor comprising:

a) providing an electrode;

b) immersing the electrode in a solution of a non-conductive polymer material;

c) running a current to the electrode sufficient to cause the non-conductive polymer material to deposit on the surface of the electrode, thereby forming a matrix material having a plurality of pores therethrough;

d) immobilizing an unlabelled capture biomolecule capable of binding a target analyte onto the matrix material, wherein the binding of the target analyte by the capture biomolecule effectively blocks a sufficient number of pores in the matrix material to produce a measurable decrease in the rate of a redox reaction occurring at the electrode; and

e) coupling the electrode to a signal element to detect and report a change in the rate of a redox reaction occurring at the electrode.

21. A method of detecting a target analyte in a sample to be analyzed comprising:

a) immersing an electrode in the sample to be analyzed, wherein the electrode comprises;

i) a non-conductive polymer deposited on the electrode to form a matrix material having a plurality of pores therethrough, and wherein the matrix material is of sufficient thickness to produce a detectable change in the rate of a redox reaction occurring at the electrode; and

ii) an unlabelled capture biomolecule immobilized on the electrode capable of binding the target analyte contained in the sample to be analyzed, wherein the binding of the target analyte to the capture biomolecule effectively blocks a sufficient number of pores in the matrix material to produce a measurable decrease in the rate of a redox reaction occurring at the electrode,

b) providing a flow of electrons to drive a redox reaction at the electrode between the electrode and a redox species in contact with the electrode; and

c) detecting and reporting a change in the rate of a redox reaction occurring at the electrode that signals the binding of the target analyte to the capture biomolecule.