Development of Enzyme-Free DNA Amplifier Based on Chain Reaction Principle

Enzyme-free DNA amplifiers can amplify the signal of nucleic acid molecules. They can be applied to DNA molecular operation and nucleic acid detection. The reaction speed is the core index to evaluate DNA amplifiers. In this study, we designed a DNA amplifier based on an enzyme-free chain reaction. This DNA amplifier can release one more signal molecule in each round of reaction and trigger the next round, which significantly improved reaction speed. Moreover, because the amplifier used a stable DNA structure, the reaction can occur at room temperature. To integrate the amplifier into other DNA molecular operations, we performed the amplification reaction in a microfluidic chip module. The results showed that the amplifier can realize real-time signal feedback at a proper input molecule concentration and reach the endpoint in 40 s, even at a low relative concentration. To apply the amplifier for nucleic acid detection, we also used a conventional fluorescent polymerase chain reaction instrument for the reaction. The results showed that the amplifier specifically detected trace DNA single-stranded molecules. To solve the leakage problem of existing amplifiers, we designed a DNA molecule as the chain reaction's inhibitor, which was crucial in controlling the reaction speed and preventing leakage. STATEMENT OF SIGNIFICANCE: Traditional amplifier strategies of enzyme-free DNA amplifiers relied on a constant number of cycling molecules to catalyze the amplifier molecules' changing structure and release fluorescent signals, which lead low reaction speed. Based on an enzyme-free chain reaction, we designed a DNA amplifier which can release one more cycling molecule in each loop and trigger the next loop and significantly improve reaction speed in this study. Our analysis on microfluidic chip module and PCR instrument verifies high sensitivity and selectivity. And this strategy of DNA amplifier realizes the control of reaction and prevents leakage. We believe that this automated amplification strategy could have great applications in vivo signal detection, imaging, and signal molecule translation.