Studies On Electrochemical Biosensor Based On Enzyme-catalyzed Amplification

Electrochemical biosensor is a novel biosensor that converts various physical and chemical signals generated before and after the target and the molecular recognition element into electrochemical signals.Due to its inherent advantages of low sample usage,low cost,simple operation,and fast analysis speed,and has attracted much attention in the field of analytical chemistry.In order to improve the performance of electrochemical biosensors,we have introduced multiple enzyme-catalyzed signal amplification strategies to enable ultrasensitive detection of MMPs and DNA.The specific works are as follows:1.DNA Enzyme-Decorated DNA Nanoladders as Enhancer for Peptide CleavageBased Electrochemical BiosensorIn general,one initiator can only obtain one corresponding DNA nanowire through self-assembly processes,which acting as nanocarrier limits the loading of biomolecules,resulting in a low sensor performance.Therefore,we developed a peptide cleavagebased electrochemical biosensor for sensitive detection of matrix metalloproteinase-7(MMP-7)on basis of DNA nanoladders as enhancer.Here,the DNA nanoladders was a type of DNA nanostructures with multiple DNA nanowires that one initiator could synchronously trigger two hybridization chain reactions(HCRs)through self-assembly processes.It could act as ideal nanocarriers for loading numerous manganese porphyrin(Mn PP),improving the sensitivity of the electrochemical biosensor.Then,Mn PP with excellent peroxidase-like activity catalyzed oxidization of the 4-chloro-1-naphthol(4-CN)into insoluble precipitation on electrode,amplifying the electrochemical signal.In addition,the strategy not only avoided using proteases with the disadvantages of easy inactivation and high cost,but also eliminated the fussy labeling procedure.As a result,the developed electrochemical biosensor exhibited a wide linear ranging from 0.2 pg/m L to 20 ng/m L,and the detection limit was 0.05 pg/m L.2.PtNPs as Scaffolds to Regulate Interenzyme Distance for Construction of Efficient Enzyme Cascade Amplification for Ultrasensitive Electrochemical Detection of MMP?2The regulation of interenzyme distance is an important approach for efficient enzyme cascade amplification and it is also a research hotspot in recent years.Conventional scaffolds regulating interenzyme distance such as metal-organic frameworks(MOFs),DNA nanostructures,showed limited catalytic efficiency owing to the disadvantages of sophisticated preparation and instable construction.In this work,the rigid Pt NPs with different sizes were employed as scaffolds to regulate interenzyme distance,and selected the optimal size of Pt NPs for efficient enzyme cascade amplification to construct electrochemical biosensor for highly sensitive detection of matrix metalloproteinases-2(MMP-2).On the one hand,Pt NPs served as scaffolds for immobilization of horseradish peroxidase(HRP)and glucose oxidase(GOx)via hostguest interaction,resulting in the formation of a stable three-dimensional netlike structure containing amounts of enzymes with enhanced catalytic efficiency of the enzyme cascade reaction.On the other hand,the Pt NPs could act as catalyst to accelerate enzyme cascade reaction with further enhanced catalytic efficiency.This strategy effectively overcame the shortcomings of traditional enzyme cascade reaction with low catalytic efficiency due to diffusion and diffraction effects.With such ingenious design,a wide linear ranged from 0.1 pg·m L-1 to 20 ng·m L-1 was acquired with a low detection limit of 0.03 pg·m L-1 for MMP-2 detection.3.Dynamical Regulation of Enzyme Cascade Amplification by a Regenerated DNA Nanotweezer for Ultrasensitive Electrochemical DNA DetectionIn the previous work,the rigid Pt NPs with different sizes were employed as scaffolds to regulate interenzyme distance for efficient enzyme cascade amplification for electrochemical detection of MMP-2.However,it was difficult to maintain the size and morphology of Pt NPs due to its easy aggregation property.Moreover,the sizeinherent feature as well as strong bonding capability with enzymes made Pt NPs hard to regulate interenzyme distance dynamically and reversibly.To address these issues,a regenerated DNA tweezer which can be reversibly switched between “opened” and “closed” state with nucleic acid strand as external triggers was designed and served as scaffold to regulate the interenzyme distance for efficient enzyme cascade amplification.GOx and HRP as model enzymes were firstly modified with the arms of opened DNA tweezer,respectively.With the aid of target induced Mg2+-dependent DNAzyme cleavage recycling amplication,the output methylene blue(MB)labeled DNA(S5)was used as fuel to trigger switching of the DNA tweezer from opened to closed state,leading to cascaded enzymes close enough for enhancing the catalytic efficiency of enzyme cascade reaction,which also provided detectable electrochemical signal for sensitive analysis of target DNA.Amazingly,the DNA tweezer could switch back to opened state by adding anti-fuel strands via one-step strand displacement,which thus served as new DNA tweezer for subsequently recycling target detection.With the dynamical regulation of interenzyme distance in an “open-close-open” way,the catalytic efficiency of enzyme cascade reaction became dynamically controllable and the DNA tweezer realized simply reutilization over five times.As a result,the developed biosensor exhibited high sensitivity with a detection limit down to 30 fmol·L-1.