Veterans Affairs

One-step, label-free system for detection and direct quantification of microRNAs

Electrochemical sensor for enhanced detection of circulating biomarkers could advance diseases diagnosis and treatment

Medical & Biotechnology

The Department of Veterans Affairs is licensing out a novel medical diagnosis technology to businesses that would develop it into an available product.

System for current amplification when a sensing unit binds a target miRNA. Selected reductant in this example is tris-(2-carboxyethyl) phosphine hydrochloride (TCEP).

MicroRNAs (miRNAs) are a class of single-strand, short, endogenous non-protein-coding RNAs with approximately 19-23 nucleotides.

Recent studies have shown that miRNAs play important roles in a wide range of physiological processes, and their aberrant expressions are associated with various diseases.

Unlike most RNAs that are prone to degradation, research suggests that a stable population of miRNAs exist in circulation. Given this, miRNA expression and quantitative profiles can be employed as biomarkers for the onset and progression of disease states. However, the detection of miRNAs faces major challenges due to their unique characteristics, including low abundance and sequence similarity among family members. Sensors featuring fast, low-cost detection with suitable sensitivity and selectivity for broad applications remain to be established.

Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray are the mainstream techniques for identification and quantitation of circulating miRNAs in plasma or serum. Although microarray is a high throughput analysis platform, the sensitivity is not suitable for low-level miRNA quantitation. And, while qRT-PCR is almost the unanimous choice for quantifying miRNAs due to its high sensitivity, the methodology suffers from a time-consuming multi-step analysis process which requires highly skilled personnel working in a lab environment. Furthermore, most qRT-PCR analysis determines relative miRNA abundance (with respect to an often-not-validated reference miRNA) in biological samples which makes quantitative, point-of-care miRNA analysis impossible.

Building on advancements in electrochemical sensors and incorporation of nanoelectrodes in sensor fabrication, VA funded scientists have developed a one-step, label-free miRNA sensor based on a redox current reporter and a nucleic acid sequence complementary to that of the target. The sensing unit is bound to an electroconductive substrate and includes a signal amplification mechanism that does not rely upon a redox enzyme. In this manner, it overcomes a fundamental limitation of microelectrode DNA sensors that fail to generate detectable current in the presence of only small amounts of a target nucleic acid. By employing a reductant in the buffer solution bathing the sensing units, the redox current reporters are cyclically oxidized at the electrode and reduced by the reductant, thereby amplifying the signal in situ during the detection period.

The system can include multiple detection modes based on cyclic voltammetry and pulse voltammetry techniques (such as square wave voltammetry and differential pulse voltammetry). The parallel detection modes combine the high sensitivity of pulse voltammetry for detection and the previously inaccessible diagnostic power of cyclic voltammetry for method validation and optimization.

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