Rapid and Sensitive Detection of miRNA Based on AC Electrokinetic Capacitive Sensing for Point-of-Care Applications.

Nan Wan,Xiaogang Lin,Yu Jiang,Jayne Wu,Jiamei Huang,Shigetoshi Eda,Rania Oueslati

doi:10.3390/s21123985

Abstract

A sensitive and efficient method for microRNAs (miRNAs) detection is strongly desired by clinicians and, in recent years, the search for such a method has drawn much attention. There has been significant interest in using miRNA as biomarkers for multiple diseases and conditions in clinical diagnostics. Presently, most miRNA detection methods suffer from drawbacks, e.g., low sensitivity, long assay time, expensive equipment, trained personnel, or unsuitability for point-of-care. New methodologies are needed to overcome these limitations to allow rapid, sensitive, low-cost, easy-to-use, and portable methods for miRNA detection at the point of care. In this work, to overcome these shortcomings, we integrated capacitive sensing and alternating current electrokinetic effects to detect specific miRNA-16b molecules, as a model, with the limit of detection reaching 1.0 femto molar (fM) levels. The specificity of the sensor was verified by testing miRNA-25, which has the same length as miRNA-16b. The sensor we developed demonstrated significant improvements in sensitivity, response time and cost over other miRNA detection methods, and has application potential at point-of-care.

Highlights

MicroRNAs are noncoding small RNAs of 18–25 nucleotides that regulate the expression of multiple genes
The first experiment was to confirm whether heat and fast annealing led to improvement, before other optimizations were employed
The melting temperature (Tm) of miRNAs are around 45 ◦ C, so the samples were heated to 95 ◦ C put on ice to cool

Summary

Introduction

MicroRNAs (miRNAs) are noncoding small RNAs of 18–25 nucleotides that regulate the expression of multiple genes. As the presence of distinct miRNAs indicates specific medical conditions—and because miRNAs can stably exist in various body fluids—miRNAs have been investigated as biomarkers for disease diagnosis [1]. Extracellular miRNAs circulate in the peripheral blood [4,5,6,7], and widely exist in other body fluids such as saliva, urine, tears, amniotic fluid and breast milk [7,8,9]. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), as the gold standard for RNA quantification [10], requires unwieldy and expensive thermal cycling equipment for amplification and quantification, rendering it unsuitable for point-of-care (POC) detection of circulating miRNAs diagnostics. Due to the short lengths of miRNAs, RT-PCR of miRNA is rather costly and technically demanding to perform, even compared with regular RT-PCR applications

Methods

Results

Conclusion