Functionalized Nanopipette Biosensor

• US 20100072080A1
• Filed: 05/04/2009
• Published: 03/25/2010
• Est. Priority Date: 05/05/2008
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Umehara, Senkei, Webb, Chris D., Karhanek, Miloslav, Pourmand, Nader
• Granted Patent: US 8,940,142 B2
• Time in Patent Office: Days
• US Class Current: 205/792
• CPC Class Codes: G01N 27/4035 Combination of a single ion-sensing electrode and a single reference electrode G01N27/406 and G01N27/413 take precedence
• CPC Class Codes: G01N 27/42 Measuring deposition or liberation of materials from an electrolyte; Coulometry, i.e. measuring coulomb-equivalent of material in an electrolyte
• CPC Class Codes: G01N 33/48707 by electrical means G01N33/49, G01N33/493 take precedence

Abstract

Disclosed are methods and devices for biomolecular detection, comprising a nanopipette, exemplified as a hollow inert, non-biological structure with a conical tip opening of nanoscale dimensions, suitable for holding an electrolyte solution which may contain an analyte such as a protein biomolecule to be detected as it is passed through the tip opening. Biomolecules are detected by specific reaction withy peptide ligands chemically immobilized in the vicinity of the tip. Analytes which bind to the ligands cause a detectible change in ionic current. A sensitive detection circuit, using a feedback amplifier circuit, and alternating voltages is further disclosed. Detection of Il-10 at a concentration of 4 ng/nl is also disclosed, as is detection of VEGF.

Claims

1. A device for specific detection of an analyte in a sample containing electrolyte, comprising:

(a) a nanopipette having a tip with a nanoscale opening between an interior of the nanopipette and an external area for contacting the sample, said nanopipette defining an internal volume communicating with the sample and said nanoscale opening constructed so that ionic current may be detectibly reduced when the tip is blocked by specific binding of analyte in the sample to the peptide binding molecule;

(b) said tip having chemically attached thereto a peptide binding molecule specifically binding to the analyte in the sample;

(c) a first electrode, arranged to be in contact with electrolyte in the interior of the nanopipette, and further arranged to be connected to an amplifier input in a current detecting circuit; and

(d) a second electrode, arranged to be in contact with the electrolyte, exterior of the nanopipette, and further arranged for connection to the current detecting circuit, whereby electrolyte in the interior of the nanopipette and electrolyte in the bath permits ionic current to flow between the electrodes and through the nanoscale opening in the tip.

2. The nanopipette device of claim 1 further comprising a current detector circuit that acts as a voltage clamp in response to an alternating voltage signal from a signal generator.

3. The nanopipette device of claim 2 where the signal generator is an analog to digital converter and the signal is sinusoidal.

4. The nanopipette device of claim 2 where the first electrode is attached to a differential amplifier and the second electrode is attached to a reference electrode.

5. The nanopipette device of claim 1 further comprising a plurality of nanopipettes in a single external bath, different nanopipettes having different functionalities.

6. The nanopipette device of claim 1 wherein the first electrode and second electrode are separated by filter material preventing analyte flow between the electrodes but permitting ionic current between the electrodes.

7. The nanopipette device of claim 1 wherein the nanopipette is quartz.

8. The nanopipette device of claim 1 further comprising a layer of carboxylated polymer bonded to an amine-containing layer bonded to the nanopipette.

9. The nanopipette device of claim 8 wherein the carboxylated polymer is polyacrylic acid and the amine-containing layer is poly-1-lysine.

10. The nanopipette of claim 1 wherein the peptide binding molecule is chemically attached to the nanopipette through binding to a protein comprising protein A.

11. The nanopipette device of claim 10 comprising more than one nanopipette arranged to contact the sample.

12. The device of claim 11 further comprising a filter in the electrolyte for preventing analyte movement between the first electrode and the second electrode.

13. A method of making a nanopipette device, comprising the steps of:

(a) forming a nanoscale opening in a tip portion of a quartz tube;

(b) attaching a polyamine layer to the tip portion;

(c) attaching a carboxyl layer to amines formed in step (b);

(d) attaching a coupling protein to the carboxyl layer from step (c); and

(e) attaching a peptide ligand to the coupling protein.

14. The method of claim 13 further comprising the step of drawing the tube to create a nanoscale opening is between 10 and 100 nm in diameter.

15. The method of claim 13 where the carboxyl layer is formed by applying to the polyamine layer polyacrylic acid.

16. The method of claim 13 where the coupling protein is protein A/G and the peptide ligand is an antibody.

17. The method of claim 13 wherein step of attaching a peptide ligand comprises the step of dipping the tip in a solution of peptide ligand.

18. A method of detecting an analyte in a sample, comprising the steps of:

(a) contacting the sample with a nanopipette having a tip with a nanoscale opening between an interior of the nanopipette and an external bath, the interior of the nanopipette communicating with the tip opening, for containing analyte material and allowing it to pass through the opening, said tip further having directly chemically attached thereto a peptide binding molecule specifically binding to the analyte;

(b) applying an alternating voltage to a first electrode contacting analyte material in the interior of the nanopipette, said electrode being connecting to a current detecting circuit;

(c) measuring ionic current flow between the first electrode and a second electrode, also for connection to the current detecting circuit, arranged to be in contact with an electrolyte in the bath, whereby electrolyte in the interior of the nanopipette and electrolyte in the bath permits ionic current to flow between the electrodes and through the tip, said ionic current being detectibly reduced when the tip is blocked by analyte.

19. The method of claim 18 wherein the alternating voltage is sinusoidal.

20. The method of claim 19 wherein the voltage alternates at between 0.5 and 10 Hz.

21. The method of claim 20 wherein the alternating voltage produces an alternating ionic current that is reduced in both negative amplitude and positive current amplitude upon binding of an analyte.

22. The method of claim 21 wherein the reduction is between 3% and 20%.