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 U ml−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. Researchers in China have developed an ultrasensitive DNA-based biosensing system with many possible medical and environmental applications. The team led by Yongxi Zhao of Xi'an Jiaotong University and Chunhai Fan of the Chinese Academy of Sciences proved the versatility of their one-step ‘DNA machine’ using lead ions and a DNA methyltransferase enzyme as example analytes. The system can be tailored so that a chosen analyte activates a rapid DNA amplification cascade that changes a quenched fluorescent group into an actively fluorescent state. The intensity of the fluorescence reveals the concentration of the analyte. The procedure is simpler, more sensitive and quicker than existing alternatives, and crucially does not require the cycled temperature changes generally needed for DNA amplification. The researchers now plan to adapt the process to detect many other chemicals, including biomarkers of cancer. We constructed a novel and universal biosensing platform based on polymerase-nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). It tactfully integrates two signal amplification modules including strand displacement amplification (SDA) and nicking enzyme signal amplification (NESA) into a one-step biosensing system via a bifunctional DNA probe with stem-loop structure. ESQM can be activated to afford a high amplified signal in the presence of target. The ultrasensitive detection of Pb2+ (30 fM) even in real water sample was achieved within 40 min, and the practicability of ESQM in DNA methyltransferase activity analysis demonstrated the universality of this biosensing platform.