Highly Selective And Sensitive Electrochemical Biosensor For The Detection Of Small Biological Molecules Based On DNAzymes

Biosensors, which were a new field based on biology, iatrology, physics, chemistry and electronic technology, have become the main analytical methods in clinical and biochemical analyses as well as in other areas such as environmental control, food quality control, etc. With their potential utility as specific, simple and direct detection techniques and reductions in the cost and time of analysis, biosensors continue to be a very active area of research. In recent years, as the new detection technologies constantly spring up and get perfected both in the research and practical application, the sensitivity and the specificity of the biosensor have been largely improved. However, with the research developing, the development of the sensitive, selective and inexpensive biosensors are of paramount importance for biomedical research and clinical diagnosis.Based on the above consideration and previously reported papers, this research mainly employs self-cleavage DNAzymes and nicking endonuclease N.BstNB I to cyclically amplify the electrochemical detection signal, so as to improve the sensitivity of the newly designed biosensor. In this study, we adopt T4DNA ligase and cofactor-dependent DNAzyme to consist the recognition element of the proposed biosensors. By taking advantage of the specifity of biological enzyme, the selectivity of the newly designed biosensors is improved dramatically.The main contents of this study are as follows:(1) The performance study of the electrochemical sensing system based on the dual strategy of ATP-dependent enzymatic ligation reaction and self-cleaving DNAzyme-based cyclic amplification. The dual strategy that combines the adenosine triphosphate (ATP)-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection is employed to construct the biosensor. By taking advantage of the highly specific cofactor dependence of the DNA ligation reaction, the proposed ligation-induced DNAzyme cascades demonstrate ultrahigh selectivity toward the target cofactor ATP, A catalytic and molecular beacons strategy is further adopted to amplify the electrochemical signal detection achieved by cycling and regenerating the8-17DNAzyme to realize enzymatic multiple turnover, thus one DNAzyme can catalyze the cleavage of several hairpin-structured substrates, which improves the sensitivity of the newly designed electrochemical sensing system. The detection limit of the proposed biosensor for ATP determination was calculated to be0.053nM (S/N=3).(2) The performance study of the electrochemical sensing system based on the dual strategy of specific DNAzyme and nicking endonuclease N.BstNB I based cyclic amplification. In the presence of target molecule L-histidine, the L-histidine dependent DNAzyme will be cut at the specific cleavage site (rA), then the cleaved substrate sequence will be released and hybridize with HP-Fc DNA. Due to the hybridization reaction, the produced DNA duplex containing the specific sequence will be specifically recognized by nicking endonuclease N.BstNB I. After the cleavage, the intact DNA sequence will be released from the damaged duplex and hybridize with another HP-Fc DNA, then the same cleavage process will conduct again. Therefore, the detection signal will be amplified because of the nicking endonuclease based cyclic amplication. The detection limit of the proposed biosensor for L-histidine determination was calculated to be360nM (S/N=3).(3) Study on the electrochemical sensing system based on the strategies of L-histidine-dependent DNAzyme, nicking endonuclease N.BstNB I based cyclic amplification, and methylene blue-labeled short-chain DNA probe. In order to improve the sensitivity of the biosensor for L-histidine detection, our research group proposed to introduce methylene blue-labeled short-chain DNA probe to transform the signal-off biosensor into signal-on mode sensing system. The detection limit was calculated to be0.21μM (S/N=3), and a linear relationship between peak current and L-histidine concentration in the range of5.0×10-7-1.0×10-3M was obtained with a correlation factor of0.995. Compared with previously reported methods, the sensitivity of the novel signal-on electrochemical biosensor based on DNAzyme and nicking endonuclease was improved remarkably.