Amplification-Free Point-of-Care microRNA Detection in Urine Using Molecular Beacon Based Electrochemiluminescence

Abstract

The burgeoning field of miRNA detection using electrochemiluminescence (ECL) has garnered a great deal of attention in recent times.1 ECL presents many advantages over alternative detection strategies as ECL analyses generally exhibit low-background signals, high sensitivity, are reproducible and scale-able.2 When combined with magnetic-beads for the immobilisation of biological recognition elements, ECL biosensors offer exceptional sensitivity owing to the ability to disperse the beads in large sample volumes to capture the biological target of interest via mixing then concentrate prior to analysis via magnetic extraction.3, 4 Herein, we present a novel biosensor for the sensitive detection of miRNA-21 comprising a molecular beacon ECL probe immobilised on magnetic beads. We obtained a limit of detection of 500 attomoles for miRNA-21 without enzymatic amplification or the addition of detection probes or intercalating labels, negating the need for lengthy and stringent washing procedures to reduce the background signal. Furthermore, we demonstrate the immense potential for point-of-care applications by developing a fully integrated miRNA detection platform. The platform comprises a mini-potentiostat for ECL generation, integrated silicon photodiode for ECL detection and a custom 3D-printed cell holder to readily interface a disposable screen-printed electrode with the potentiostat and photodiode.We applied our magnetic miRNA extraction technology to the detection of miRNA-21 in human urine, a promising biomarker for the early diagnosis of chronic kidney disease.5 Urine is an ideal sample matrix as it is non-invasive to obtain in large volumes, does not require a phlebotomist to acquire and miRNAs in urine are stable in exosomes. When combined with point-of-care miRNA extraction methods, the molecular beacon based ECL system we have developed presents a promising tool for point-of-care diagnosis and management of numerous diseases. 1. P. K. Kalambate, N. S. Gadhari, X. Li, Z. Rao, S. T. Navale, Y. Shen, V. R. Patil and Y. Huang, TrAC, Trends Anal. Chem., 2019, 115643.2. Y. Cheng, L. Dong, J. Zhang, Y. Zhao and Z. Li, Analyst, 2018, 143, 1758-1774.3. M. H. Shamsi, K. Choi, A. H. Ng, M. D. Chamberlain and A. R. Wheeler, Biosens. Bioelectron., 2016, 77, 845-852.4. W. Liu, X. Zhou and D. Xing, Biosens. Bioelectron., 2014, 58, 388-394.5. M. L. Alvarez, M. Khosroheidari, R. Kanchi Ravi and J. K. DiStefano, Kidney Int., 2012, 82, 1024-1032.