| Summary: | An impedance-based biosensor for the ultrasensitive, selective, and label-free detection of a blood miRNA associated to Alzheimer disease (AD), miRNA-206, was developed. The principle was grounded in the changes in the charge transfer resistance (RCT) as an effect of intramolecular forces between miRNAs and ferro/ferricyanide in a well-structured transducer platform. A compact well-ordered mixed monolayer made of co-immobilized miRNA capture to 6-mercapto-1-hexanol (MCH) in a 1:4 M ratio (at 37 °C), uplifted the performance of the sensor through effectively assisting the orientation of the oligonucleotides. In this work, the remarkable response of the sensor was generated through new insights into the use of different moieties of miRNA capture to MCH, aiming to control interfacial constants, surface densities, and hybridization efficiency.A very low limit of detection, 0.15 aM, is achieved and the sensor has a wide linear dynamic range (from 1 aM to 1 μM), high selectivity to mismatches, low non-specific binding of proteins (BSA) and good stability (<10 % change in response after 14 days storage). Importantly, the sensor successfully measured miRNA-206 concentrations in real plasma samples (>95 % recovery), correlating directly with qPCR results. Nanomolar concentrations of miRNA-206 were found in the plasma of confirmed AD patients, while healthy controls, had a concentration of pM or lower. The biosensor's ability to quantitatively detect miRNA-206 in plasma without target amplification, e.g., using PCR, is significant, opening the possibility of developing a point-of-care diagnostic device for AD screening, contributing to clinical trials and patient care.
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