Digital protein sensing chip and methods for detection of low concentrations of molecules

Abstract

A sensing device is provided that includes a tunnel junction created by forming a hole in a layered tunnel junction (for example). A chemically, well-defined surface may be formed by coupling affinity reagents to the electrodes, which, by these means, the surface may be configured to be selective for a particular analyte.

1 . A device for detecting the binding of a molecule to a cognate ligand, comprising:
 a first planar electrode having a length, a width and a thickness;   an insulating layer having a length, a width and a thickness;   a second planar electrode having a length, a width and a thickness;   an opening between the electrodes configured to expose the gap between them and establishing an electrodes-insulating layer junction, wherein the opening is spaced away from the perimeter of each electrode;   one or more recognition molecules comprising a cognate ligand;   a chemical group configured to couple the cognate ligand to at least one of the electrodes; and   a reference electrode in communication with at least one of the electrodes.   
     
     
         2 . The device of  claim 1 , wherein the reference electrode is configured with a substantially constant potential difference with respect to at least one of the electrodes. 
     
     
         3 . The device of  claim 1 , wherein the cognate ligand comprises at least one of an antibody, a Fab fragment, an aptamer and a peptide configured to bind to one or more protein targets. 
     
     
         4 . The device of  claim 1 , wherein the device is configured to detect single molecular binding events corresponding to low concentration sample solutions. 
     
     
         5 . The device of  claim 1 , wherein the device is configured for sequencing a peptide chain. 
     
     
         6 . A system for detecting the binding of a molecule to a cognate ligand, comprising:
 the device of  claim 1 ; and   purification means for purifying a patient serum sample for obtaining one or more target proteins.   
     
     
         7 . The system of  claim 6 , wherein the purification means comprises a lab-on-a-chip. 
     
     
         8 . The system of  claim 6 , further comprising a pair of fluid chambers, the device formed between the pair of fluid chambers. 
     
     
         9 . The system of  claim 8 , wherein the pair of fluid chamber are in fluid communication via the opening in the device. 
     
     
         10 . A method for detecting at least one binding event between a recognition molecule and a target molecule, comprising:
 providing the device of  claim 1 ;   providing a substantially fixed bias between the electrodes; flowing a sample solution adjacent the junction;   recording current signals generated as a result of one or more binding events between one or more molecules in the sample and corresponding recognition molecules; and   determining at least one of a number and type of molecules present in a sample solution based on the characteristics of the signal generated as each type of molecule binds.   
     
     
         11 . The method according to  claim 10 , further comprising recording at least one of the number and type of molecules determined. 
     
     
         12 . The method according to  claim 10 , wherein the characteristics comprise at least one of baseline current, peak current above a baseline, peak width, peak shape as encoded by Fourier, wavelet or Cepstrum component amplitudes, and flatness of the peak top expressed as root mean square signal variation. 
     
     
         13 . The method according to  claim 10 , further comprising at least one of recording and counting signals generated by single molecule binding events of a plurality of protein variants contained in the sample solution. 
     
     
         14 . The method according to  claim 10 , further comprising providing the target molecule, including:
 providing a sample to a first channel of a processing device, the sample including cells containing the target molecule, the first channel including antibodies for capturing the cells in the sample;   lysing the captured cells to generate a lysate including the target molecule;   eluting the lysate into a second channel including antibodies for binding the target molecule;   generating fragments of the target molecule from the bound target molecule; and   providing the fragments of the target molecule as the target molecule to the device.   
     
     
         15 . The method according to  claim 14 , wherein the sample includes blood cells, wherein the target molecule includes a protein, and wherein the fragments of the target molecule includes peptides of the protein. 
     
     
         16 . The method according to  claim 14 , the generating the fragments further including capturing a portion of the fragment of the target molecule, the providing the fragments including providing the portion of the fragment of the target molecule to the device. 
     
     
         17 . The method according to  claim 14 , herein the fragments of the target molecule includes peptides of the protein, further comprising, prior to providing the fragments, modifying the fragments to ligate a charged peptide to the N terminus of the peptides of the protein, the providing including providing the modified fragments as the target molecule to the device. 
     
     
         18 - 28 . (canceled)