Ratiometric biosensor array for multiplexed detection of microRNAs based on electrochemiluminescence coupled with cyclic voltammetry

Abstract A novel multiplexed ratiometric biosensor array was fabricated on a homemade screen-printed carbon electrode (SPCE) for near-simultaneous detection of microRNA (miRNA)-21 and miRNA-141 based on electrochemiluminescence (ECL) coupled with cyclic voltammetry (CV) method. In the detection system, the ECL signal tags (Ru-SiO 2 @PLL-Au) were fabricated using poly- l -lysine (PLL) as bridging agent and co-reactant to connect Ru-SiO 2 (Ru(bpy) 3 2+ -doped silica) and gold nanoparticles (Au NPs), which were respectively modified on two spatial resolved working electrodes (WE1 and WE2) of SPCE. Then the ferrocene (Fc)-labeled hairpin DNA (Fc-HDNA1 and Fc-HDNA2) as CV signal tags and ECL quenching material were immobilized on Ru-SiO 2 @PLL-Au. Upon miRNA-21 and miRNA-141 adding, the target miRNAs could hybridize with corresponding Fc-HDNA, which could lead to Fc away from Ru-SiO 2 @PLL-Au. Such conformational changes could recover the ECL of Ru-SiO 2 @PLL-Au and decreased the CV current of Fc, respectively. This “signal-on” of ECL and “signal-off” of CV were employed for dual-signal ratiometric readout. With the help of a multiplexed switch, two dual-signals from WE1 and WE2 were used for multiplexed detection of miRNA-21 and miRNA-141 down to 6.3 and 8.6 fM, respectively. This approach was used in real sample analysis and has significant potential for miRNA biomarkers detection in a clinical laboratory setting.