Novel label-free colorimetric and electrochemical detection for MiRNA-21 based on the complexation of molybdate with phosphate

MicroRNA-21 (miRNA-21) is a promising biomarker for the early diagnosis of cancer in blood and body fluids in abnormal expression. Therefore, it will be helpful to develop simple, portable, and on-site sensors. In this work, a smartphone colorimetric and electrochemical-based miRNA sensors were developed by combining two strategies: i) acid treatment of formed DNA and miRNA target on microplates and ii) detection of released phosphate groups based on molybdate reaction. For the first time, an unmodified DNA strand as a probe was immobilized on polystyrene 96-well microplates as ready-to-use, stable, and portable bio-platforms using a silanization process. In the presence of miRNA-21 as a target sequence, the capture probe was hybridized on each microplate. Each well was acid-treated on the way to release the phosphate groups from the backbone of the DNA strand. Moreover, the released phosphate groups were reacted with molybdate to produce a redox phosphomolybdate complex which, in the presence of ascorbic acid, generates a blue color with intensity proportional to phosphate concentration and thus to the concentration of miRNA-21. The color intensity was measured by a smartphone. In addition, the phosphomolybdate complex has been assessed by square wave voltammetry (SWV). The smartphone and electrochemical-based assays showed a good linear range between 1 pM and 50 nM and 10 fM - 50 nM, with a detection limit of 600 fM and 8 fM, respectively. The proposed colorimetric method was successfully used to analyze microRNA-21 in (spiked) human serum samples.