Construction Of Electrochemical Biosensor Based On Multiple Signal Amplification Techniques And DNAzyme Nanodevice

As a powerful tool for analysis and diagnosis,the electrochemical biosensor can convert the specific interaction between the recognition element and analyte into an easy-to-handle electrical signal to realize quantitative detection of the analyte.Because of some advantages such as simple operation,fast response,low cost and miniaturization,the electrochemical biosensor is widely used in many fields such as disease diagnosis,drug discovery,food analysis and environmental monitoring.In recent years,with the development of DNA nanotechnology,it is of great significance to integrate some innovations?DNA nanomachines and various signal amplification strategies?into the electrochemical biosensors for further improving their sensitivity and specificity.In this paper,aiming at constructing more sensitive and accurate detection platform,some DNA functional structures were designed and combined with signal amplification strategy to propose several new methods of electrochemical biosensors.The specific works are as follows:1.A novel electrochemical biosensor based on highly efficient target recycling amplification and netlike Y-DNAThe target recycling amplification strategy can significantly improve the sensitivity of biosensors,but such a strategy often has serious waste problem of recycling products.To overcome this problem,we proposed a highly efficient target recycling amplification strategy in the construction of biosensor.Here,all the recycling products were fully used for the assembly of netlike Y-DNA.At the same time,netlike Y-DNA could regulate the electrocatalysis of Fe₃O₄@CeO₂-Pt nanoparticles?Fe₃O₄@CeO₂-PtNPs?toward methylene blue?MB?for signal amplification,so as to achieve sensitive detection of the target DNA related to oral tumors.Specifically,with the assistance of nicking endonuclease,one target DNA input was circularly converted to corresponding plenty of DNA strands S1-Fe₃O₄@CeO₂-Pt and S2-MB output,which could be employed to interact with HP2 immobilized on the electrode surface to form stable netlike Y-DNA without any waste.In addition,the formation of netlike Y-DNA could regulate electrocatalytic efficiency of Fe₃O₄@CeO₂-PtNPs,inducing the proximity of Fe₃O₄@CeO₂-PtNPs to MB and significantly enhancing electrochemical signal.Therefore,by virtue of this ingenious design,the proposed electrochemical DNA biosensor achieved sensitive detection of target DNA ranging 10 fmol/L to 50 nmol/L with a detection limit of 3.5 fmol/L,and further promoted the development of the target amplification strategy in biosensing platform..2.Programming a target-initiated bifunctional DNAzyme nanodevice for sensitive ratiometric electrochemical biosensingThe construction of multifunctional DNA nanodevices is usually a difficult task,requiring not only multiple DNA strands,but also intricate operation,severely hindering their further development in biosensing system.Here,a bifunctional DNAzyme nanodevice?BFDN?was intelligently designed and applied to construct a ratiometric electrochemical biosensor for highly reliable and sensitive mercury ion(Hg²⁺)detection.In the presence of the target Hg²⁺,the T-Hg²⁺-T pair could actuate the preassembled DNA four-branched nanostructure?DNA-4B?without cleavage capability transform to the BFDN with strong cleavage capability for triggering two synchronous Hg²⁺detection paths,including a“signal-off”path?1?that consisted of a cascade DNAzyme cleavage reaction to dramatically decrease the ferrocene?Fc?response and a“signal-on”path?2?that accomplished the capture of significant amounts of methylene blue?MB?on the electrode surface under the assistant of DNAzyme2 in BFDN.This strategy not only effectively avoided the false positive signal compared with traditional single paths,but also proposed a new ratiometric method to successfully circumvent the deficiency that existed in previous ratiometric electrochemical biosensors.As a result,the reliable and sensitive Hg²⁺detection was achieved in the range from 0.1 pmol/L to 200 nmol/L with a detection limit of 23 fmol/L.Above all,here,the assembly of the BFDN is ingeniously coupled with amplification strategy,paving a promising avenue to promote the performances of simple multifunctional DNA nanomachines and facilitate the corresponding development of multifunctional DNA nanomachines in biosensor platform..3.A novel electrochemical biosensor based on 3D DNAzyme Motor.On biosensor platform,DNAzyme-amplified detection strategy is particularly attractive due to the characteristics of DNAzyme including low synthesis cost,stability and excellent cleavage specificity.But unfortunately,defects such as long reaction time and low DNAzyme cleavage efficiency still exist in the DNAzyme-amplified computing strategy.Herein,driven by the need of highly-efficient DNAzyme-amplified detection strategy,a DNAzyme motor is successfully assembled by combining DNAzyme nanowires and nanoparticles localization DNA design to realize sensitive target DNA detection in biosensor platform.In short,the DNAzyme motor consists of target-activated DNAzyme nanowires and corresponding substrates that are co-immobilized on the surface of the nanoparticle.On the one hand,nanoparticles localization DNA design increases the effective concentrations of DNAzyme and corresponding substrates?H1-Fc?,which solved the problem of low DNAzyme cleavage efficiency due to low reactant concentration that is common in traditional DNAzyme amplification detection strategies.On the other hand,like a 3D walking machine,the target-activated DNAzyme nanowires of DNAzyme motor can autonomously walk on the 3D track to complete the task of recycling cleavage.But compared with the traditional 3D walking machine,the target-activated DNAzyme nanowires have greater flexibility and more powerful cleavage capability without troublesome sequence optimization,which overcome the space limitation and can simultaneously interact with adjacent and distant substrates to output a large amount of cleavage products for high signal response.By virtue of such ingenious design,the reported DNAzyme motor solves many defects existing in traditional DNAzyme-amplified DNA computing strategies,achieving effective DNAzyme signal amplification.As a result,wide linear range from 5 fmol/L to 50 nmol/L and low detection limit of 1.7 fmol/L is obtained for target DNA detection.Moreover,the 3D DNAzyme motor proposed here also has potential for payload release due to its powerful cleavage ability,which provides a reference for the assembly of various functions of DNA machines in the future.