Quantitative Electrochemical DNA Microarray on a Monolith Electrode with Ten Attomolar Sensitivity, 100 % Specificity, and Zero Background

Abstract

Circulating microRNA are promising diagnostic and prognostic biomarkers of disease in quantitative blood tests. A label-free, PCR-free, electrochemical microarray technology on a monolith electrode is described, with 10 attomolar (aM) sensitivity and responsiveness to binding of <1 zeptomole of target to immobilized ssDNA probes with zero background. Specificity is 100% in a mixture with five nonspecific miRNA each with a 103-fold higher concentration. Direct measurement on plasma-derived miRNA without cDNA conversion and PCR demonstrated multiplexing and near-ideal quantitative correlation with an equivalent pure sample. The dynamic range is a target concentration ranging from 10–2 to 103 femtomolar (fM). This PCR-free novel technology can be applied as a test for cancer diagnosis/prognosis to detect 103 copies of a miRNA sequence in RNA extracted from 100 μL of plasma.

Electrochemical Characteristics of a DNA Modified Electrode as a Function of Percent Binding

“…8−10 Recently, the insulator−conductor transition has become an attractive principle for developing high-impact liquid biopsy applications for detection of cancer by profiling cell-free nucleic acids (cfNA). 11,12 The importance of electronic conduction in dsDNA for electrochemical devices was realized at the onset as the charge injection was accomplished via redox coupling. 13,14 The conduction is attributed to overlapping of π orbitals in the base pairs, 13,15 leading to short-range transport by nonresonant tunneling 16 and long-range conduction by hopping between adjacent bases.…”

“…23 Applying negative potential to the electrode lifts the tethered ssDNA probe, 26,27 which partly explains the significant enhancement in binding on application of periodic electrochemical potential. 12,28,29 Second, several studies have clearly demonstrated that electrostatic repulsion tends to reduce binding of the target as the probe coverage increases. 30−33 For example, measurement of surface charge by electrochemical impedance spectroscopy (EIS) indicates that DNA charge is not completely screened by the ions in the solution to cause electrostatic repulsion that limits binding at high probe coverage.…”

“…A 1 cm 2 Silicon chip (SiO 2 /Si) with five 0.8 × 8 mm and two 0.8 × 6 mm (control) Au electrodes were patterned by standard lithography (SI, Figure S1). There were four steps to make the DNA electrode in an array of seventy-two 50 μm diameter microwells (see SI, Section S2, Figure S2−S4 for details, and prior publication 12 ). The binding in step 3, was performed electrochemically (SI, Figure S4).…”

Utilizing the Organizational Power of DNA Scaffolds for New Nanophotonic Applications

“…This is due to the high sensitivity that can be achieved and the simple fluorescent readout that does not require additional assay steps . Such DNA biosensors have applications in sensing a myriad of DNA targets as well as RNA; of particular interest currently is the detection of circulating microRNAs which may prove to be a simple diagnostic biomarker for a myriad of infection and disease states . However, the small size and limited number of available bps in these microRNAs make them quite challenging for direct hybridization‐based sensing and they often require amplification steps and use of adaptors to extend their sequence length for FRET‐based interrogation.…”

Electrochemical Biosensor for MicroRNA Detection Based on Cascade Hybridization Chain Reaction

“…However, miRNAs detection remains a big challenge because of their low abundance, extremely short length and sequence homology . Many efforts have been made on miRNAs detection using various techniques such as northern blotting, quantitative fluorescence reverse transcription polymerase chain reaction, microchip assays,, surface enhanced Raman scattering, fluorescent, colorimetric, chemiluminescence, and electrochemical methods …”