Construction And Application Of Homogeneous Amplification Reaction-Based Electrochemical Biosensors

With the continuous development of science and technology and the advancement of society,life science,medical diagnosis,food safety and environmental monitoring have put forward higher and higher requirements on high-sensitivity and anti-interference for biological detection.In the field of biosensing,electrochemical detection has the advantages of simplicity,portability,and rapid response,and is widely used for analysis and detection.Due to the controllable synthesis and easy modification of DNA,DNA electrochemical biosensors have been greatly developed in recent years.Traditional DNA electrochemical sensors are generally based on heterogeneous recognition and amplification.In heterogeneous assays,identification of analytes occurs at the interface between the electrode and the solution.Due to the steric hindrance of the electrode surface,the structural freedom of the fixed recognition unit is often limited,resulting in a relatively low binding efficiency of the recognition unit to the target molecule and a decrease in detection sensitivity.In order to solve this problem,electrochemical biosensors based on homogeneous identification have emerged.All events in the homogeneous electrochemical detection process including target recognition,signal amplification and signal readout occur in the solution phase.Homogeneous reaction is more effective in terms of molecular recognition and in maintaining enzyme reactivity compared with heterogeneous reactions.In view of the advantages of homogeneous reaction,we are committed to further combine homogeneous reaction with electrochemical detection method to construct new electrochemical biosensors.The main content of this paper research includes two parts:(1)Exonuclease-assisted positive feedback amplification strategy for ultrasensitive electrochemical detection of nucleic acids.Through the ingenious design of Hairpin DNA and Hybrid DNA and the use of Exo ?-assisted target recycling amplification,we integrate the advantages of traditional heterogeneous analysis and homogeneous amplification.In this strategy,a small amount of target DNA can trigger the release of assist DNA at the electrode interface which in turn triggers the homogeneous amplification reaction,and the homogeneous amplification reaction further promotes the amplification at the electrode interface,thereby realizing the positive reaction.We verified the strategy by gel electrophoresis and electrochemical methods,and explored the analytical performance of the constructed sensor.In the range of 5 aM-5 fM,the response signal is linear with the logarithm of the target DNA concentration.The method showed higher sensitivity than the control experiment with only one cycle.The detection limit of the developed method is 1.2 aM,which is lower than most of the detection strategies involving nuclease signal amplification.(2)Construction and application of nanoporous gold sensing platform based on homogeneous recognition.To effectively amplify the detection signal,we constructed a nanoporous gold electrochemical sensing platform to effectively enrich the electroactive molecules through the unique two-channel structure of nanoporous gold and functional molecular(such as G4-DNA,MPA,and MBA)modification.We used SEM,EIS,etc.to characterize the construction of the sensing interface,and analyzed the effect of different modified porous gold electrodes on molecular diffusion by electrochemical method.The diffusion of the electroactive molecule MB in the nanoporous gold electrode changes linearly with time in a certain period of time,and G4-DNA,MPA,MBA all have an enrichment effect on MB,the enrichment effect of G4-DNA on MB is the best.At the same time,the nanoporous gold sensing platform also shows a blocking effect on the diffusion of DNA molecules.The analysis and detection applications of the sensing platform have also been explored.Homogeneous recognition amplification strategys are designed based on the specific recognition reaction of biomolecules and the nuclease-assisted cyclic amplification reaction.The nanoporous gold sensing platform responds well to the target such as DNA and exonuclease,confirming the enormous potential of the constructed sensor platform for bioanalysis.