Protein and peptide sensors using electrical detection methods

Abstract

The present invention provides an apparatus and methods for the electrical detection of molecular interactions between a probe molecule and a protein or peptide target molecule, but without requiring the use of electrochemical or other reporters to obtain measurable signals. The methods can be used for electrical detection of molecular interactions between probe molecules bound to defined regions of an array and protein or peptide target molecules which are permitted to interact with the probe molecules.

1 . An apparatus for the electrical detection of molecular interactions between an immobilized probe molecule and a protein or peptide target molecule. comprising: 
 (a) a supporting substrate;    (b) one or a plurality of microelectrodes in contact with the supporting substrate;    (c) one or a plurality of linking moieties in contact with the microelectrodes and to which probe molecules have been immobilized;    (d) at least one counter-electrode in electrochemical contact with the microelectrodes;    (e) a means for producing an electrical signal at each microelectrode;    (f) a means for detecting changes in the electrical signal at each microelectrode; and    (g) an electrolyte solution in contact with the microelectrodes. the linking moieties, and the counter-electrode, wherein molecular interactions between the immobilized probe molecules and protein or peptide target molecule are detected as a difference in the electrical signal at each microclectrode in the presence and absence of the protein or peptide target molecule.    
     
     
         2 . The apparatus of  claim 1 , w herein the linking moieties comprise polyacrylamide gel, agarose gel, polyethylene glycol, cellulose gel, sol gel, or combinations thereof.  
     
     
         3 . The apparatus of  claim 2 , wherein the linking moieties comprise polyacrylamide gel.  
     
     
         4 . The apparatus of  claim 1 . wherein the linking moieties comprise a conjugated polymer or copolymer film.  
     
     
         5 . The apparatus of  claim 4 , wherein the conjugated polymer or copolymer film is polypyrrole, polythiphene, polyaniline, polyfuran, polypyridine, polycarbazole, polyphenylene, poly(phenylenvinylene), polyfluorene, or polyindole, or their derivatives copolymers, or combinations thereof.  
     
     
         6 . The apparatus of  claim 1 , wherein the linking moieties comprise a neutral pyrrole matrix.  
     
     
         7 . The apparatus of  claim 1 , wherein the supporting substrate comprises ceramic, glass, silicon, silicon nitride, fabric, rubber, plastic, printed circuit board, or combinations thereof.  
     
     
         8 . The apparatus of  claim 1 , wherein the microelectrodes comprise a conductive material and an insulating material.  
     
     
         9 . The apparatus of  claim 8 , wherein the conductive material is solid or porous gold, silver, platinum, titanium, copper, chromium, or aluminum, or metal oxide, metal nitride, metal carbide, carbon, graphite, conductive plastic, metal impregnated polymers or combinations thereof.  
     
     
         10 . The apparatus of  claim 9 , wherein the conductive material is platinum.  
     
     
         11 . The apparatus of  claim 9 , wherein the conductive material is gold.  
     
     
         12 . The apparatus of  claim 8 , wherein the insulating material is glass, silicon, silicon nitride, plastic, rubber, fabric, ceramic, printed circuit board, or combinations thereof.  
     
     
         13 . The apparatus of  claim 12 , wherein the insulating material is silicon.  
     
     
         14 . The apparatus of  claim 12 , wherein the insulating material is glass.  
     
     
         15 . The apparatus of  claim 8 , wherein the conductive material is embedded the supporting substrate and the supporting substrate comprises the insulating material.  
     
     
         16 . The apparatus of  claim 1 , further comprising at least one reference electrode.  
     
     
         17 . The apparatus of  claim 16 . wherein the reference electrode comprises a conductive material and an insulating material.  
     
     
         18 . The apparatus of  claim 17 , wherein the conductive material is solid or porous gold, silver, platinum, titanium, copper, chromium, or aluminum, or metal oxide, metal nitride, metal carbide, carbon, graphite, conductive plastic, metal impregnated polymers or combinations thereof.  
     
     
         19 . The apparatus of  claim 17 . wherein the conductive material is silver/silver chloride.  
     
     
         20 . The apparatus of  claim 17 , wherein the insulating material is glass, silicon, silicon nitride, plastic, rubber, fabric. ceramic. printed circuit board, or combinations thereof.  
     
     
         21 . The apparatus of  claim 1 , wherein the supporting substrate further comprises a plurality of wells, each of which encompasses at least one microelectrode in contact with a linker moiety and at least one counter-electrode.  
     
     
         22 . The apparatus of  claim 1 . wherein the probe molecules are oligonucleotides or nucleic acids.  
     
     
         23 . The apparatus of  claim 22 , wherein the probe molecules are aptamers.  
     
     
         24 . The apparatus of  claim 1 , wherein the probe molecules are proteins or peptides.  
     
     
         25 . The apparatus of  claim 24 , wherein the probe molecules are antibodies.  
     
     
         26 . The apparatus of  claim 25 , wherein the antibodies are polyclonal antisera, polyclonal antibodies, or F(ab), F(ab)′, F(ab) 2 , or F v  fragments thereof.  
     
     
         27 . The apparatus of  claim 25 , wherein the antibodies are monoclonal antibodies, or F(ab), F(ab)′, F(ab) 2 , or F v  fragments thereof.  
     
     
         28 . The apparatus of  claim 25 , wherein the antibodies are F(ab) fragments or single-chain F v  fragments produced by in vitro libraries.  
     
     
         29 . The apparatus of  claim 1 , wherein the probe molecules comprise a natural products library, a peptide library. a phage display library, or a combinatorial library.  
     
     
         30 . The apparatus of  claim 1 , wherein the linking moieties further comprise streptavidin and the probe molecules are biotinylated.  
     
     
         31 . The apparatus of  claim 1 , wherein molecular interactions between probe molecules and protein or peptide target molecules are detected by using an electrical detection method selected from the group consisting of impedance spectroscopy, cyclic voltammetry, AC voltammetry, pulse voltammetry, square wave voltammetry, AC voltammetry, hydrodynamic modulation voltammetry, conductance, potential step method, potentiometric measurements, amperometric measurements. current step method, other steady-state or transient measurement methods, and combinations thereof.  
     
     
         32 . A method for the electrical detection of molecular interactions between an immobilized probe molecule and a protein or peptide target molecule. comprising: 
 (a) detecting a first electrical signal in one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized;    (b) exposing the one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized to a sample mixture containing protein or peptide target molecules;    (c) detecting a second electrical signal in one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized;    (d) comparing the first electrical signal with the second electrical signal, and    (e) determining whether the first electrical signal is different from the second electrical signal.    
     
     
         33 . The method of  claim 32 . wherein molecular interactions between probe molecules and protein or peptide target molecules are detected by using an electrical detection method selected from the group consisting of impedance spectroscopy, cyclic voltammetrv, AC voltammetry, pulse voltammetry. square wave voltammetry, AC voltammetry, hydrodynamic modulation voltammetry, conductance, potential step method, potentiometric measurements, amperometric measurements, current step method, other steady-state or transient measurement methods, and combinations thereof.  
     
     
         34 . The method of  claim 32 , wherein the electrical detection method is AC impedance that is measured over a range of frequencies.  
     
     
         35 . The method of  claim 32 , wherein the electrical detection method is AC impedance that is measured by transient methods with AC signal perturbation superimposed upon a DC potential applied to an electrochemical cell.  
     
     
         36 . The method of  claim 32 , wherein the electrical detection method is AC impedance that is measured by impedance analyzer. lock-in amplifier, AC bridge, AC voltammetry, or combinations thereof.  
     
     
         37 . The method of  claim 32 , wherein the linker moieties comprise polyacrylamide gel, agarose gel. polyethylene glycol. cellulose gel. sol gel, or combinations thereof and the protein or peptide target molecules are labeled with an electrochemically active reporter molecule prior to exposing the one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized to the sample mixture containing protein or peptide target molecules.  
     
     
         38 . The method of  claim 37 , wherein the electrochemically active reporter molecule comprises a transition metal complex.  
     
     
         39 . The method of  claim 37 , wherein the transition metal ion is ruthenium, cobalt, iron. zinc, copper, magnesium, nickel, or osmium.  
     
     
         40 . The method of  claim 37 , wherein the electrochemically active reporter labeled target molecules are labeled with electrochemical reporter groups selected from the group consisting of 1,4-benzoquinone, ferrocene, tetracyanoqublodimethane, N,N,N′,N′-tetramethyl-p-phenylenediamine, and tetrathiafulvalene.  
     
     
         41 . The method of  claim 37 , wherein the electrochemically active reporter labeled target molecules are labeled with electrochemical reporter groups selected from the group consisting of 9-aminoacridine, acridine orange, aclarubicin, daunomycin, doxorubicin, pirarubicin, ethidium bromide, ethidium monoazide, chlortetracycline, tetracycline, minocycline, Hoechst 33258, Hoechst 33342, 7-aminoactinomycin D, Chromomycin A 3 , mithramycin A, Vinblastine, Rifampicin, Os(bipyridine) 2 (dipyridophenazine) 2   + , Co(bipyridine) 3   3+ , and Fe-bleomycin.  
     
     
         42 . The method of  claim 32 , further comprising: 
 (f) exposing the one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized to an electrochemically labeled target binding molecule;    (g) detecting a third electrical signal in one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized.    (h) comparing the second electrical signal with the third electrical signal, and    (i) determining whether the second electrical signal is different from the third electrical signal.    
     
     
         43 . The method of  claim 42 . wherein the target binding molecules are oligonucleotides or nucleic acids.  
     
     
         44 . The method of  claim 43 , wherein the target binding molecules are aptamers.  
     
     
         45 . The method of  claim 42 . wherein the target binding molecules are proteins or peptides.  
     
     
         46 . The method of  claim 45 . wherein the target binding molecules are antibodies.  
     
     
         47 . The method of  claim 46 , wherein the antibodies are polyclonal antisera, polyclonal antibodies, or F(ab), F(ab)′, F(ab) 2 , or F v  fragments thereof.  
     
     
         48 . The method of  claim 46 , wherein the antibodies are monoclonal antibodies, or F(ab), F(ab)′, F(ab) 2 , or F v  fragments thereof.  
     
     
         49 . The method of  claim 46 , wherein the antibodies are F(ab) fragments or single-chain F v  fragments produced by in vitro libraries.  
     
     
         50 . The method of  claim 42 , wherein the target binding molecules are a natural products library, a peptide library, a phage display library, or a combinatorial library.  
     
     
         51 . A method for the electrical detection of molecular interactions between an immobilized probe molecule and a protein or peptide target molecule, comprising: 
 (a) detecting a first electrical signal in one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized;    (b) exposing the one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized to a sample mixture containing a first protein or peptide target molecule and to a second protein or peptide target molecule, wherein the second protein or peptide target molecule is labeled with an electrochemical reporter;    (c) detecting a second electrical signal in one or a plurality of microelectrodes in contact with linker moieties to which probe molecules have been immobilized;    (d) comparing the first electrical signal with the second electrical signal; and    (e) determining whether the first electrical signal is different from the second electrical signal.