Design And Application Of Signal Amplification Biosensor Based On DNAzyme

Enzymes are a group of proteins with catalytic activity which can maintain the normal physiological activities of cells.And the special biological activities of enzymes play an important role in human physiology and metabolic processes.Studies have shown that abnormal expression and function of some enzymes are closely related to the occurrence of diseases that threaten human health.Currently,a variety of methods reported to detecting enzymes have some limitations including radioactive materials utilization,specialized equipment requirement,low sensitivity and analysis in vitro.Therefore,the establishment of a new analytical method to achieve accurate and highly sensitive characterization of the expression and function of target enzymes in complex biological systems has shown important application value in disease diagnosis,drug screening and precise treatment.In this study,based on the advantages of DNAzyme,tetrahedral DNA nanostructure and reduced graphene oxide,we have constructed two signal amplification biosensors for sensitivity detection of apurininc/pyrimidine endonuclease 1（APE1）and ribonuclease A（RNase A）in living cells.The detailed contents are descried as follows.1.We developed a DNAzyme modified tetrahedral DNA nanostructure（DZ-TDN）and molecular beacon（MB）incorporated nano-biosensor for APE1 detection and living cell imaging.APE1 is an emerging target for sensitizing tumor cells to radiation and chemotherapy due to its critical function in DNA repair and reduction-oxidation regulation.Based on the specific recognition and hydrolysis of APE1 to AP sites,the nano-biosensor consists of DNAzyme modified tetrahedral DNA nanostructure（DZ-TDN）,a blocker containing AP sites,molecular beacon（MB）containing r A base.At normal status,DNAzyme activity was plugged by performing a duplex with a blocker strand containing AP site.The digestion of APE1 on AP site can restore DNAzyme activity,which consequently trigger MB cleavage recycle for high signal amplification.The method showed a linear response ranged for APE1 from 0.01 to 1U·m L^-1with a limit of detection（LOD）of 0.01 U·m L^-1.Moreover,the strategy was successfully used for effectors screening and intracellular imaging of APE1.2.We developed a nano-biosensor based on DNAzyme and rGO for RNase A detection and living cell imaging.As an endonuclease,RNase A plays an important role in anti-virus,anti-tumors and so on.Based on the specific cleavage activity of RNase A to RNA bases,the nano-biosensor consists of a hairpin probe containing rU and DNAzyme,a FAM-labeled single probe embedded with Ru base and rGO nanomaterial.In the absence of RNase A,the fluorescence of the signal probe is quenched by rGO due to its strong adsorption.Upon the introduction of RNase A,the recognition and hydrolysis of RNase A to the hairpin probe can release the active DNAzyme.The released DNAzyme can trigger the recycle cleavage of the single probe to produce the FAM-labeled shorts fragments with the help of cofactor,which cannot be adsorbed by rGO.The proposed strategy can detect RNase A with a detection limit of 0.1 pg·μL^-1.Moreover,this method was applied to screen the natural compounds targeted RNase A.The results showed that B6 can activate RNase A activity in a dose-dependent manner.Cell experiments indicated that the drug can regulate RNase A activity with difference in different cell lines.Finally,the method was successfully used for intracellular imaging of RNase A.In summary,the signal amplification nano-biosensor based on DNAzyme successfully achieved accurate detection and intracellular imaging of enzymes.It holds great significance for drug screening,cancer diagnosis and prognosis evaluation.