Highly sensitive and label-free electrochemical detection of microRNAs based on triple signal amplification of multifunctional gold nanoparticles, enzymes and redox-cycling reaction

• We reported a label-free and triple signal amplification strategy for miRNAs detection.
• Aminophenylboronic acid/biotin-modified AuNPs were used for the recognition of miRNAs and the coupling of streptavidin-conjugated alkaline phosphatase.
• Tris(carboxyethyl)phosphine was used as the reducing reagent for the p-aminophenol redox cycling.
• A detection limit of 3 fM for miRNA-21 was achieved.

MicroRNAs (miRNAs) are believed to be important for cancer diagnosis and prognosis, serving as reliable molecular biomarkers. In this work, we presented a label-free and highly sensitive electrochemical genosensor for miRNAs detection with the triple signal amplification of gold nanoparticles (AuNPs), alkaline phosphatase (ALP) and p-aminophenol (p-AP) redox cycling. The label-free strategy is based on the difference in the structures of RNA and DNA. Specifically, miRNAs were first captured by the pre-immobilized DNA probes on a gold electrode. Next, the cis-diol group of ribose sugar at the end of the miRNAs chain allowed 3-aminophenylboronic acid (APBA)/biotin-modified multifunctional AuNPs (denoted as APBA–biotin–AuNPs) to be attached through the formation of a boronate ester covalent bond, which facilitated the capture of streptavidin-conjugated alkaline phosphatase (SA–ALP) via the biotin–streptavidin interaction. After the addition of the 4-aminophenylphosphate (p-APP) substrate, the enzymatic conversion from p-APP to p-AP occurred. The resulting p-AP could be cycled by a chemical reducing reagent after its electro-oxidization on the electrode (known as p-AP redox cycling), thus enabling an increase in the anodic current. As a result, the current increased linearly with the miRNAs concentration over a range of 10 fM–5 pM, and a detection limit of 3 fM was achieved. We believe that this work will be valuable for the design of new types of label-free and sensitive electrochemical biosensors.