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.

Proximity hybridization-mediated isothermal exponential amplification for ultrasensitive electrochemical protein detection

“…10,11 Different from the toehold-mediated strand displacement, 12,13 which has been extensively employed to design DNA assembly, a proximity hybridization-induced DNA assembly homogeneous assay has been proposed. 14,15 The method was based on the target-binding process to promote strand displacement and reinforce the hybridization of two short DNA sequences, which cannot form stable duplex at normal circumstances. Through converting the target binding event into detectable DNA assembly, proximity hybridization strategy can be combined with various signal amplification strategies, such as enzyme-free cyclic assembly of hairpins, 16 ligation-mediated strand recycling, 17 and binding-induced self-assembly of DNAzyme 18 to build ultrasensitive biosensors.…”

Aptamer-based fluorescent solid-phase thrombin assay using a silver-coated glass substrate and signal amplification by glucose oxidase

“…The thrombin-binding aptamer (TBA) is well known and has been applied to establish biosensors with methods of electrochemistry [7,8], fluorescence [9,10], colorimetry [11,12], surface enhanced Raman scattering [13] and surface plasmon resonance [14,15] successfully. Fluorescence aptasensors have remarkable advantage of simplicity and high sensitivity.…”

A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins

“…Thus, to make the detection of trace levels of the analyte possible, many researchers have devoted effort to developing amplification techniques. Signal amplification strategies, such as the polymerase chain reaction (PCR) [5][6][7], rolling circle amplification (RCA) [8][9][10][11], enzyme-assisted signal amplification [12][13][14] and DNA based technologies [15][16][17][18] have been employed to improve the sensitivity of protein detection. Meanwhile, a further development in DNA amplification is the hybridization chain reaction (HCR) [19], which is enzyme-free and needs initiator DNA to trigger a self-assembly process.…”