Functionalized nanopipette biosensor

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 with 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/ml is also disclosed, as is detection of VEGF.

What is claimed is: 
     
         1 . A nanopipette device for specific detection of one or more analytes in a sample containing electrolyte, comprising:
 (a) a quartz or glass capillary nanopipette having a tip with a nanoscale opening between an interior of the nanopipette and an external area for contacting the sample, said nanopipette having a hollow structure defining an internal volume communicating with the sample and said nanoscale opening in the tip of between 10 and 100 nm in diameter;   (b) peptide that specifically binds to a predetermined analyte in the sample, said peptide chemically attached to an internal service of said tip through a polymeric coating, whereby binding of an analyte to the peptide reduces size of said nanoscale opening;   (c) a first electrode, arranged to be in contact with electrolyte in the interior of the nanopipette, and connected to an input of an amplifier comprised in a current detecting circuit that detects ionic current through the nanoscale opening;   (d) a second electrode, arranged to be in contact with electrolyte exterior of the nanopipette, and further connected to said current detecting circuit, whereby electrolyte in the interior of the nanopipette and electrolyte in a bath permits ionic current to flow between the first electrode and the second electrode and through the nanoscale opening in the tip; and.   (e) said amplifier configured to apply an alternating voltage to the first electrode thereby producing positive and negative ionic currents, and said current detecting circuit configured to detect both of said positive and negative ionic currents.   
     
     
         2 . The nanopipette device of  claim 1  wherein said current detector circuit is configured as a voltage clamp in response to an alternating voltage signal from a signal generator. 
     
     
         3 . The nanopipette device of  claim 2  wherein the signal generator comprises an analog to digital converter and the alternating voltage signal is sinusoidal. 
     
     
         4 . The nanopipette device of  claim 2  wherein 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, said plurality of nanopipettes each having therein an electrode connected to an input of an amplifier comprised in a current detecting circuit that detects ionic current through a nanoscale opening in a member of said plurality of nanopipettes. 
     
     
         6 . The nanopipette device of  claim 1  wherein the first electrode and second electrode are separated from each other by filter material between the first electrode and the second electrode, thereby 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  wherein said polymeric coating comprises a layer of carboxylated polymer bonded to said polymeric coating wherein said polymeric coating comprises an amine-containing layer bonded to surface of the hollow structure of the nanopipette. 
     
     
         9 . The nanopipette device of  claim 8  wherein the carboxylated polymer is polyacrylic acid and the amine-containing layer is poly-l-lysine. 
     
     
         10 . The nanopipette device of  claim 1  wherein the peptide is chemically attached to the nanopipette through binding to a protein comprising protein A, covalently linked to the polymeric coating on the surface of the hollow structure of the nanopipette. 
     
     
         11 . The nanopipette device of  claim 10  comprising a second nanopipette arranged to contact the sample and comprising a second peptide chemically attached to said tip, and further comprising an electrode within the second nanopipette and arranged to be connected to a second amplifier input in said current detecting circuit. 
     
     
         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 . The nanopipette device of  claim 1  wherein the second peptide analyte binding molecule is an antibody polypeptide. 
     
     
         14 . The nanopipette device of  claim 1  wherein the peptide is an antibody polypeptide. 
     
     
         15 . The nanopipette device of  claim 1  wherein the peptide is attached to the inside of the nanopipette. 
     
     
         16 . The nanopipette device of  claim 1  wherein the electrolyte in the bath is an electrolyte gel separating the first and second electrodes. 
     
     
         17 . The nanopipette device of  claim 16  wherein the electrolyte gel is agar gel. 
     
     
         18 . The nanopipette device of  claim 1  wherein the peptide is attached to the nanopipette tip by an NHS (N-hydroxysuccinimide) coupling agent. 
     
     
         19 . The nanopipette device of  claim 1  wherein said polymeric coating comprises a poly-L-lysine layer. 
     
     
         20 . 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.   
     
     
         21 . The method of  claim 20  wherein the alternating voltage is sinusoidal. 
     
     
         22 . The method of  claim 21  wherein the alternating voltage alternates at between 0.5 and 10 Hz. 
     
     
         23 . 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. 
     
     
         24 . The method of  claim 23  wherein the reduction is between 3% and 20%.