Polymerase chain reaction-based ultrasensitive detection of HBV DNA via G-quadruplex selective iridium(III) complex...

Polymerase chain reaction (PCR) is the gold standard for low-abundant DNA detection. Here, to expand the application of PCR with novel detecting methods, we developed a label-free fluorescent sensor for ultrasensitive and one-step detection of hepatitis B virus (HBV) DNA using the G-quadruplex selective iridium(III) complex luminescent probe. By using HBV DNA as the template with two hairpin structure primers that contained oxyethylene glycol tethers, PCR amplification occurred and generated numbers of specific PCR products with free G-quadruplex sequences at both ends. Such free G-quadruplex sequences can change into G-quadruplex structure with the help of K⁺, resulting in a strong luminescence intensity upon their binding with the G-quadruplex selective iridium(III) complex. The luminescence intensity increase was proportional to the concentration of PCR products, and indirectly related with HBV DNA concentration. Moreover, the utilization of the iridium(III) complex effectively improved the specificity of the sensor, while PCR paved the way for the ultrasensitive detection of DNA in the linear range of 3.0 fM to 800 pM, with a detection limit of 1.6 fM. Notably, this assay was successfully used to detect HBV DNA in normal and patient serum samples, indicating a potential application for biomolecular analysis.