Binary light-up fluorescent probe based on silver nanoclusters for MicroRNA detection

Silver nanoclusters (Ag NCs) are widely applied in the biosensing of metal ions, small organic molecules, nucleic acids, amino acids, and proteins due to their particular fluorescence and chemical properties. Organic matrices such as DNA are usually employed for Ag NC synthesis and stabilization. They make Ag NC/matrix complexes biocompatible and sensitive to the environment. It has recently been shown that Ag NCs based on DNA matrices are capable of self-assembly and rearrangement followed by a change in the fluorescence and absorbance characteristics. These attributes allow the development of sensors with target molecule detection visible even by the naked eye. Here we suggest a simple one-step turn-on highly specific microRNA-210 sensor based on a fluorescent Ag NC. The main feature of the sensor is the smart design of a binary matrix, which provides the appearance of a bright green fluorescence signal only after Ag NCs/DNA-matrix complexes are bonded to the target sequence. The microRNA detection assay requires no additional action because the process proceeds by itself. A comprehensive optimization of the binary probe structure and location was carried out. An approach to detection leading to minimal background signal was defined as follows. The approach involves the preliminary synthesis of non-fluorescent silver clusters using a single strand of the binary matrix containing a 5'-CCCGTTTT-3' part. It was shown that these "dark" structures can be stored for at least a month before analysis. The fluorescence intensity of the green Ag NCs increases in the presence of the microRNA-210 sequence, and that dependence on the target concentration tends to be linear in the range of 5-500 nM. The sensor demonstrates specificity to the miR-210 sequence, and the LOD (limit of detection) was established as 5 nM in serum samples.