title: Quantitation of femtomolar protein levels via direct readout with the electrochemical proximity assay.
abstract: We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout.
authors.affiliation: Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
authors.forename: Jiaming
authors.initials: J
authors.lastname: Hu
authors.forename: Tanyu
authors.initials: T
authors.lastname: Wang
authors.forename: Joonyul
authors.initials: J
authors.lastname: Kim
authors.forename: Curtis
authors.initials: C
authors.lastname: Shannon
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Easley
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry; D002417:Cattle; D004247:DNA / Q000737:chemistry; D055664:Electrochemical Techniques; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D006801:Humans; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013917:Thrombin / Q000032:analysis* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D012710:Serum Albumin, Bovine; D006046:Gold; D004247:DNA; D013917:Thrombin