A PCR-free electrochemical method for messenger RNA detection in cancer tissue samples
Md Nazmul Islam1, Vinod Gopalan2, Md Hakimul Haque2
1School of Natural Sciences, Griffith University, Nathan Campus, QLD 4111, Australia; Queensland Micro-, and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia.
Abstract:
Despite having reliable and excellent diagnostic performances, the currently available messenger RNA (mRNA) detection methods mostly use enzymatic amplification steps of the target mRNA which is generally affected by the sample manipulations, amplification bias and longer assay time. This paper reports an amplification-free electrochemical approach for the sensitive and selective detection of mRNA using a screen-printed gold electrode (SPE-Au). The target mRNA is selectively isolated by magnetic separation and adsorbed directly onto an unmodified SPE-Au. The surface-attached mRNA is then measured by differential pulse voltammetry (DPV) in the presence of [Fe(CN)6]4-/3- redox system. This method circumvents the PCR amplification steps as well as simplifies the assay construction by avoiding multiple steps involved in conventional biosensing approaches of using recognition and transduction layers. Our method has demonstrated good sensitivity (LOD = 1.0pM) and reproducibility (% RSD = <5%, for n = 3) for detecting FAM134B mRNA in two cancer cell lines and a small cohort of clinical samples (number of samples = 26) collected from patients with oesophageal cancer. The analytical performance of our method is validated with a standard qRT-PCR analysis. We believe that our PCR-free approach holds a great promise for the analysis of tumor-specific mRNA in clinical samples.
