The Construction Of Biosensor Based On Metal DNAzyme And Its Application In Hg²⁺ Detection

Environmental safety has always been a hot issue that people are concerned about.Mercury is one of the most widely used harmful heavy metals,which can seriously damage living organisms at extremely low concentrations.As one of the most common heavy metal ions,mercury ion is a cumulative toxin with low biodegradability and high mobility rate.It is difficult to degrade in the cycle of biological matter and energy.Even at a very low concentration,it can threaten human health through the biological enrichment of the food chain and may even damage the brain and kidneys.Water-soluble mercury ions are one of the most common and stable forms of mercury pollution.In recent years,anthropogenic mercury and its impact on ecosystem functions and human health have received widespread attention.In particular,the discharge of mercury-containing industrial wastewater has made it widely distributed in the water environment.According to the regulations of the World Health Organization（WHO）,the maximum allowable concentration of Hg²⁺in drinking water is 2 ppb（10 n M）.At present,some traditional detection methods such as inductively coupled plasma mass spectrometry,atomic emission spectrometry and atomic absorption spectrometry are widely used in the detection of Hg²⁺in water bodies.However,these technical methods involve complicated sample preparation process,complicated operating procedures,and expensive equipment.Therefore,there is an urgent need to develop a simple,efficient,and sensitive method to realize the trace detection of Hg²⁺in the water environment.Here,a biosensor based on metal DNAzyme combined with a variety of isothermal signal amplification technologies was constructed to detect trace amounts of Hg²⁺in the water environment.In this study,three simple,efficient and sensitive biosensors with high specificity and selectivity for Hg²⁺were constructed by using T-Hg²⁺-T structure combined with metal DNAzyme and isothermal signal amplification technology,and were successfully applied to the detection of water samples.The main research results are as follows:By combining the T-Hg²⁺-T structure with the metal DNAzyme walker,a simple and powerful colorimetric sensing strategy has been developed to detect Hg²⁺.This strategy relies on the target Hg²⁺to form a T-Hg²⁺-T structure to trigger the conformational changes of the metal DNAzyme walker,and the metal DNAzyme cleavage cycle and walking can achieve signal amplification.As far as we know,this work is the first time that the T-Hg²⁺-T structure is combined with a metal DNAzyme walker for Hg²⁺colorimetric detection.It is worth noting that both DNAzyme walker and hairpin（HAP）are modified on the surface of Au NPs by poly A.HAP not only contains the substrate sequence of metal DNAzyme,but also contains a G-rich sequence.When there is no target Hg²⁺,after adding Mg²⁺,the metal DNAzyme walker will hybridize with HAP and cleave until the HAP on the surface of Au NPs is completely cleaved.Finally,after adding hemin,many G-quadruplex DNAzymes are formed on the surface of Au NPs,and then a highly catalytic nano-DNAzyme can catalyze the color development of 3,3’,5,5’-tetramethylbenzidine（TMB）,and realize the detection of Hg²⁺.This strategy has a good correlation in the range of Hg²⁺concentration of 1-10⁴ n M,and its detection limit is as low as 0.16 n M.In addition,the biosensor has the advantages of high specificity,simplified operation and short analysis time.Therefore,the biosensor can be used as a universal platform for detecting Hg²⁺.In addition,by redesigning the corresponding binding molecules,the colorimetric sensing strategy can be easily extended to the detection of other targets.Combining DNAzyme and primer exchange cascade amplification reaction,a metal DNAzyme-mediated primer exchange cascade amplification reaction is proposed for the ultra-sensitive detection of Hg²⁺biosensing platform.This platform relies on the T-Hg²⁺-T structure to activate the activity of the metal DNAzyme,which in turn triggers the primer exchange cascade amplification reaction to achieve exponential signal amplification.The strategy has ingeniously designed a hairpin H2 whose stem contains a repetitive sequence and a stop site.It will hybridize with the primer and under the action of polymerase,the primer would extend using the H2 stem as a template.When it reaches the stop site,it stops extending.At this time,H2 can fold itself and release the extended primer.Due to the presence of the repetitive sequence,the released primer will hybridize and extend with H2,and an endless primer exchange cascade amplification reaction occurs.Based on this amplification strategy,the built sensor has superior sensitivity and specificity for Hg²⁺detection,with detection limit as low as 0.99 f M.By improving the performance of the sensor,the sensor shows higher reproducibility.In addition,this strategy shows a broader application prospect in actual sample detection.By combining the proximity of the metal DNAzyme mediated by T-Hg²⁺-T cleavage and the catalytic hairpin self-assembly（CHA）,a T-Hg²⁺-T-mediated metal DNAzyme double signal amplification for detecting Hg²⁺fluorescence sensing platform.Under the action of the target Hg²⁺,the split DNAzyme approaches,and then hybridizes with H1 and undergoes a cleavage reaction.The release of primers triggers the catalytic hairpin self-assembly（CHA）reaction to form a metal DNAzyme three-channel structure,achieving double signal amplification of metal DNAzyme.A large number of signal output chains are generated,and fluorescent signals are generated by intercalating thioflavin T（Th T）.The cleavage reaction of CHA and metal DNAzyme does not require the participation of enzymes and requires little environmental conditions.The experiment optimized the performance of this strategy,and obtained the standard curve for detecting Hg²⁺under the optimal conditions,which can realize the quantitative detection of Hg²⁺.In addition,by redesigning the corresponding binding molecules,the proposed strategy can be easily extended to other target detections.