Polymerase/nicking enzyme synergetic isothermal quadratic DNA machine and its application for one-step amplified biosensing of lead (II) ions at femtomole level and DNA methyltransferase

Herein, we propose a novel and universal biosensing platform based on a polymerase-nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). This platform tactfully integrates two signal amplification modules, strand displacement amplification (SDA) and nicking enzyme signal amplification (NESA), into a one-step system. A bifunctional DNA probe with a stem-loop structure was designed to be partly complementary to the SDA product and digested substrate of NESA for bridging SDA and NESA. ESQM can be performed by using only the enzymes and buffer involved in the SDA module. In the presence of a target, this DNA machine is activated to afford a high quadratic amplified signal. Using Pb²⁺ and DNA adenine methylation (Dam) methyltransferase (MTase) as analytes, a sensitive biosensing platform is demonstrated. Low detection limits of 30 fM Pb²⁺ and 0.05 Uml^-1 Dam MTase were achieved within a short assay time (40 min), which were each superior to those of most previously reported methods. This DNA machine exhibited high selectivity for Pb²⁺. Furthermore, the successful detection of complex environmental water samples demonstrated the applicability of the proposed strategy in real samples, holding great potential for its application in environmental monitoring, biomedical research and clinical diagnosis.