Enhanced electrochemical detection using nanoparticles and precipitation

Abstract

The invention described herein relates generally to methods, sensors, devices and kits for electrochemical detection of a target analyte in a sample. In certain aspects, the methods, sensors, devices and kits described herein can be used to detect low concentrations of at least one target analyte using small sample volumes. In some embodiments, methods, sensors and kits for detecting a microbe, microbe fragment or released endotoxin in a test sample, including bodily fluids such as blood and tissues of a subject, food, water, and environmental surfaces, are also provided herein.

What is claimed is: 
     
         1 . A method comprising:
 (a) introducing simultaneously a reporter enzyme substrate, an electroactive mediator and a precipitating agent into an electrochemical sensor, wherein the electrochemical sensor comprises an electrode and a reporter enzyme conjugate, and wherein a reaction of the electroactive mediator precipitating composition with the reporter enzyme conjugate forms an electroactive precipitate locally adsorbed at a surface of the electrode;   (b) applying a voltage to the electrochemical sensor, wherein the voltage corresponds to the standard redox potential of the electroactive precipitate; and   (c) measuring a current generated from the electrode of the electrochemical sensor to detect a target analyte.   
     
     
         2 . The method of  claim 1 , wherein the electrochemical sensor comprises a plurality of electrodes. 
     
     
         3 . The method of  claim 2 , wherein at least two electrodes in the plurality of electrodes are adapted to detect different target analytes. 
     
     
         4 . The method of  claim 1 , wherein the electroactive mediator is selected from the group consisting of 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), 2,2′-Azinobis [3 -ethylbenzothiazoline-6-sulfonic acid] (ABTS), p-Nitrophenyl Phosphate (PNPP), 3,3′-diaminobenzidine (DAB), 4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), nitro blue tetrazolium (NBT), methylene blue, hydroquinone, ferrocene derivatives, and any combination thereof. 
     
     
         5 . The method of  claim 3 , wherein the electroactive mediator is TMB. 
     
     
         6 . The method of  claim 1 , wherein the reporter enzyme substrate is selected from the group consisting, of hydrogen peroxide, carbamide peroxide, nucleotides, oligonucleotides, RNA, DNA, phosphorylated peptides, phosphorylated proteins, phosphorylated small molecules, glucose, phenols, tyrosine, dopamine, catechol, urea, and any combination thereof. 
     
     
         7 . The method of  claim 6 , wherein the reporter enzyme substrate is hydrogen peroxide. 
     
     
         8 . The method of  claim 1 , wherein the precipitating agent is selected from the group consisting of a water-soluble polymer, a pyrrolidinone polymer, a polyaniline, a polypyrrole, a polythiophene, alginic acid, methyl vinyl ether/maleic anhydride copolymer, dextran sulfate, carrageenan, and any combination thereof. 
     
     
         9 . The method of  claim 8 , wherein the precipitating agent is a pyrrolidinone polymer. 
     
     
         10 . The method of  claim 1 , wherein the reporter enzyme conjugate comprises an enzyme conjugated with an antibody, an antibody fragment, a carbohydrate-binding protein, a peptide, a polypeptide, an aptamer, a cell-binding molecule, a lipid-binding molecule, a polynucleotide, a lipid, a carbohydrate, or any combinations thereof. 
     
     
         11 . The method of  claim 1 , wherein the reporter enzyme conjugate comprises an enzyme selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GOx), tyrosinase, urease, a DNAzyme, an aptazyme, and any combinations thereof. 
     
     
         12 . The method of  claim 11 , wherein the enzyme is HRP. 
     
     
         13 . The method of  claim 1 , wherein the electrochemical sensor comprises one or more microfluidic flow cells. 
     
     
         14 . The method of  claim 1 , wherein the electrochemical sensor comprises one or more open wells. 
     
     
         15 . The method of  claim 1 , wherein the electrode is a planar or 3-dimensional electrode. 
     
     
         16 . The method of  claim 1 , wherein the electrochemical sensor further comprises a counter electrode and a reference electrode. 
     
     
         17 . The method of  claim 1 , wherein the electrochemical sensor further comprises a positive control electrode and/or a negative control electrode. 
     
     
         18 . The method of  claim 1 , wherein the generated current corresponds to a reduction or oxidation current derived from reduction of the fully or partially oxidized electroactive mediator. 
     
     
         19 . A kit for electrochemical multiplex detection of a plurality of target analytes in a sample comprising:
 (a) an electrochemical sensor comprising a fluid-contact surface and a plurality of electrodes immobilized on at least a portion of the fluid-contact surface, wherein the electrodes are adapted to detect different target analytes;   (b) a plurality of reporter enzyme conjugates; and   (c) an electroactive mediator precipitating composition comprising a reporter enzyme substrate, an electroactive mediator and a precipitating agent, wherein a reaction of the reporter enzyme substrate and the electroactive mediator with a reporter enzyme conjugate in the plurality of reporter enzyme conjugates forms an electroactive precipitate locally adsorbed at a surface of at least one of the electrodes.   
     
     
         20 . An electrochemical sensor comprising:
 (a) a fluid-contact surface and a plurality of electrodes immobilized on at least a portion of the fluid-contact surface, wherein the electrodes are adapted to detect different target analytes;   (b) a plurality of reporter enzyme conjugates; and   (c) an electroactive precipitate locally adsorbed at the surfaces of at least one of the electrodes, wherein the electroactive precipitate is formed from a reaction of an electroactive mediator precipitating composition comprising a reporter enzyme substrate, an electroactive mediator and a precipitating agent, with a reporter enzyme conjugate in the plurality of reporter enzyme conjugates.