Quantitative Detection Mechanism and Method of Fluorescence Signal Amplification Based on DNA Walker

The polymerase chain reaction (PCR) is widely used for amplifying the bio-sequence in deoxyribonucleic acid (DNA) quantitative detection to overcome the limited sensitivity of biochip scanners, but the PCR is not suitable for medical on-site testing due to its complex, expensive, and bulky heating and cooling equipment. This study proposes a new approach based on cyclic cleaving by DNA walkers, which exhibits a proportional relationship between the reaction product and the initial concentration of DNA walker when the detecting probe sequence is excessive. With that, the fluorescence probe of Cy3-DRNA/MCH/Au is constructed and bonded on the Au-surface of indium tin oxide (ITO) glass. Via the charge coupled device (CCD) sensor, the grayscale intensity of each pixel is linearly converted to correspond to the Cy3 concentration on the ITO-Au surface of the detection probe. And the reaction products cleaved by DNA walker are proportional to the accumulation of all intensity differences for each pixel in the probe. Experimental results show that the biochemical cleavage reaction maintains good linear characteristics with the reaction time from 0 to 40 min, and the linear fitting correlation coefficient R2 is 0.99147 at the biochemistry cleaving time of 25 min. This new approach provides a novel and promising technique for biological signal amplification and quantitative detection based on DNA walker, which is expected to have a wide range of applications in biology, medicine, food, and the environment.