Femtomolar direct voltammetric determination of circulating miRNAs in sera of cancer patients using an enzymeless biosensor

Abstract

A disposable enzyme−free biosensing platform for the sensitive and selective voltammetric determination of miRNAs is reported. The bioplatform implies a sandwich−type hybridization configuration involving the use of two synthetic DNA probes that hybridize contiguously with the target miRNA−21. A thiolated capture probe was immobilized through thiol chemistry on disposable carbon electrodes modified with a hybrid nanomaterial composed of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs). A biotinylated detection probe was conjugated with ferrocene-capped AuNPs modified with streptavidin (Fc−AuNPs−Strep) which were used as labeling nanocarriers. The extent of the hybridization event was followed by differential pulse voltammetric measurement of the Fc oxidation peak. Under the optimized conditions, the developed biosensor provides attractive characteristics for the determination of the synthetic target miRNA, with a linear range between 10 fM and 2 pM and a limit of detection (LOD) of 5 fM, fully discrimination towards a highly homologous miRNA (with just one mismatched base) and a storage stability of at least two months. The biosensor was able to determine accurately the target miRNA directly in scarcely diluted serum from breast cancer (BC) patients with no need for a previous total RNA (RNAt) extraction and in a very small amount of RNAt extracted from breast adenocarcinoma cells without the need for amplification or reverse transcription to complementary DNA.