Construction Of DNA Biosensors On Silicon Chip And Magnetic Beads And Their Applications In Fluorescence Analysis

In recent years, DNA detection technology has become more and more important with the development of science and technology. Traditional gel electrophoresis is laborious and time-consuming. DNA sensors based on base pairing emerged at a historic moment. Among these, DNA fluorescent sensors have drawn considerable attention due to their high sensitivity, low cost and convenience.Based on these, we have constructed four DNA fluorescent sensors. Firstly, morpholino probe was immobilized on the surface of a silicon chip and then hybridized with DNA in the ensuing step. The fluorescence label was introduced by strongly binding rhodamine B with DNA by means of phosphate-zirconium-carboxylate coordination. It was applied for DNA detection through fluorescence imaging analysis.Secondly, morpholino probe was modified on the surface of magnetic microspheres, and then hybridized with target DNA. Biotin-labeled signal probe DNA was designed to hybridize with target DNA, and the biotin was used to bind with streptavidin-alkaline phosphatase. L-Ascorbic acid-2-phosphate was hydrolyzed by alkaline phosphatase to generate L-ascorbic acid, which could reduce resazurin to resorufin, resulting in a turn-on fluorescence signal. It was used to detect DNA through fluorescence spectra analysis.Thirdly, target DNA was hybridized to morpholino that was also immobilized on the surface of magnetic microspheres.1-pyrenebutanoic acid was incorporated to the backbone of target DNA through phosphate-zirconium-carboxylate coordination. Then fluorescent hypericin was linked to 1-pyrenebutanoic acid via π-π stacking interaction.Fourthly, target DNA was hybridized with the capture DNA that was immobilized on the surface of magnetic microspheres. Terminal deoxynucleotidyl transferase was employed to generate the polyT at the 3’end of target DNA for the formation of CuNPs. The formation of CuNPs was due to binding interactions between the DNA template and Cu2+ ions, and the DNA-complexed Cu2+ was reduced to Cu+, which was followed by the disproportionation of Cu+ into Cu2+ and Cu0. The formed Cu0 was then clustered on the DNA template, thus producing stable fluorescent nanoparticles.