Target amplification-free detection of urinary microRNA for diabetic nephropathy diagnosis with electrocatalytic reaction

Diabetic nephropathy (DN) is a serious diabetic complication, usually developed from type II diabetes mellitus (T2DM) and known as type II DN (T2DN). New emerging biomarkers for T2DN are microRNAs (miRNAs) which have been studied for the noninvasive early-stage detection of the disease. In this work, a nucleic acid amplification-free miRNA-124 sensor based on target-induced strand displacement on magnetic beads, and by using methylene blue-loaded silica particles as a label was developed. Measurement methods can be either visual observation, spectrophotometry, or electrochemistry. After incubation and separation of the magnetic particles, a blue-violet solution (564 nm) appeared, depending on the concentration of miRNA displaced. For electrochemical detection, methylene blue on the silica served as a redox mediator for the coupled reaction with ferricyanide in the solution phase. At the electrode surface, ferricyanide was re-reduced to ferrocyanide, and was thus available for further reaction with methylene blue, forming an amplification cycle. After optimization, the total assay time was 60 min, and limits of detection were 1 pM, 6 fM, and 0.65 fM, by the naked eye, spectrophotometry and electrochemistry, respectively. The miRNAs in 42 suspected urine samples from patients suffering from either diabetic nephropathy, diabetes mellitus, or chronic kidney disease were validated by comparing with the droplet digital polymerase chain reaction (ddPCR).