Fluorescence microarrays for enzyme-free DNA detection based on web hybrid chain reaction

The diagnosis of pathogenic microorganism infection mainly relies on molecular detection of nucleic acids or proteins, and the nucleic acid detection is the gold standard for the pathogen detection. The viral genome mutates quickly, and the genome of the host cell usually binds to multiple sites in the viral genome, leading to patient infections. Therefore, rapid nucleic acid detection methods with high specificity are essential for controlling the spread of viruses and maintaining human health. In this study, Cy3-labeled DNA probes were obtained based on a convenient and enzyme-free web hybridization chain reaction (wHCR) and used for DNA diagnosis of hepatitis B virus (HBV) in an isothermal platform. In this microarray platform, the target DNA is directly captured on the microarray and identified by the aggregated DNA probes to amplify the fluorescence signal. After fluorescence scanning analysis, the detection limit of HBV DNA fragments on this DNA microarray is 84 fM. In addition, this method not only specifically distinguishes single-base mismatched sequences, but also obtains the quantitative detection of HBV DNA in serum samples. Compared with the enzymatic amplification reaction in which the target nucleic acids are used as the amplification template, this method effectively avoids cross-contamination. Furthermore, compared to enzymatic reactions, the requirements for reaction conditions such as temperature and pH are relatively lenient. This method is user-friendly and suitable for molecular detection of infectious diseases under limited resource conditions.