Construction Of Electrochemical Biosensor Based On Multiple Signal Amplification Strategy And Its Application In Analytical Detection

Electrochemical biosensor is a self-contained integrated device that can provide specific quantitative or semi-quantitative analytical information.Due to the advantages of fast response,high sensitivity,simple instrument and easy miniaturization,it has been extensively used in food analysis,environmental monitoring,and biomedicine field.As an ideal analytical device,electrochemical biosensor fruitfully endeavors to detect disease-related biomolecules.However,the low concentration of disease-related biomolecules in the early stage of disease,the strong interference in complex biological matrices,as well as the limited types and unsatisfactory activity of signal tags have brought great challenges to the detection,making the sensitivity and accuracy of existing methods are not high enough.The amplification efficiency of a single signal amplification strategy is limited,which is difficult to meet the detection demands of low-abundance targets in complex matrices.In view of this,this thesis is based on molecular recognition.Combining high-performance functional nanomaterials,a series of multiple signal amplification strategies have been designed.The effective integration and synergy between each strategy overcomes the shortcomings of low amplification efficiency of a single signal amplification strategy,and greatly improves the detection sensitivity by accelerating the electron transfer,improving the target utilization,and providing amplified electrochemical signals.On this basis,magnetic separation technology and dual-modality output are further introduced,which are used for target separation and signal output to improve the detection accuracy.Electrochemical biosensors with high sensitivity,high specificity and high accuracy are constructed for ultrasensitive and reliable detection of biomarkers and virus molecules.The detailed work of this thesis is summarized as follows:（1）In situ generation of silver nanoclusters by hybridization chain reaction for highly sensitive detection of DNA methyltransferase.The common signal tags are limited,and their activities are easily affected during the labeling and modification process.To solve this problem,an efficient and enzyme-free hybridization chain reaction（HCR）amplification strategy was introduced to generate abundant DNA superstructures,which were used as templates to in situ generate a large number of silver nanoclusters（Ag NCs）as signal tags,providing amplified electrochemical signal.Ulteriorly,gold nanoparticles/reduced graphene oxide（Au NPs/ERGO）nanocomposites were prepared for electrode modification.By increasing the electrode specific surface area and accelerating the electron transfer,the current intensity was improved by about109.4μA.Combining Ag NCs,HCR and Au NPs/ERGO triple amplification strategies to improve detection sensitivity,this biosensor was capable of achieving highly sensitive and highly specific quantitative detection of DNA methyltransferase（DNA MTase）with a detection limit as low as 7.3 m U/m L,showing the potential for inhibitor screening.As a signal tag,Ag NCs can provide amplified signal output,but they have poor stability and are easily oxidized.（2）One-pot synthesis of Au NCs-Mn O₂ nanosheets and Cu²⁺-DNAzyme for pyrophosphatase sensitive detection.Based on the excellent catalytic amplification effect and environmental stability of nanocatalysts,the nanocatalyst of gold nanoclusters-manganese dioxide nanosheets（Au NCs-Mn O₂ NSs）was prepared by protein-guided biomineralization processes,which was used as signal tags to catalyze the H₂O₂/TMB system,providing amplified electrochemical signal.Combining the Cu²⁺-DNAzyme cycle initiated by target-induced Cu²⁺release with exonuclease III（Exo III）-assisted amplification process,the conversion of low-concentration protein targets was achieved.Through Au NCs-Mn O₂ NSs catalytic amplification,Cu²⁺-DNAzyme and Exo III-assisted target conversion to improve sensitivity,an electrochemical biosensor was developed for the highly sensitive analysis of pyrophosphatase（PPase）with a detection limit of 0.0058 m U/m L.This biosensor also presented potential application in complex biological system detection and inhibitor screening,providing new ideas for the design of other PPase detection methods.（3）Magnetic DNAzyme walker and Pt@COF catalytic nanospheres for highly sensitive micro RNA-21 detection.In detection applications,numerous interferences in complex matrices often contaminate electrode surface or cause nonspecific adsorption,resulting in false negative or false positive results.Therefore,the accuracy of the analysis is an important factor to consider in addition to sensitivity.In this chapter,the magnetic DNAzyme walker was introduced,on the one hand,the low-abundant target micro RNA-21（mi RNA-21）could be converted by the cleavage process to improve the sensitivity;one the other hand,separation and enrichment of targets in complex matrices were realized by magnetic separation to reduce interference from coexisting components,ensuring detection accuracy.Covalent organic framework（COF）nanospheres were prepared at room temperature and used as the supports for loading platinum nanoparticles（Pt NPs）by the in situ reduction of metal precursors in their nano-structure.Pt NPs were highly dispersed in COF nanospheres with fewer aggregation and higher catalytic activity.Using Pt@COF nanospheres as signal tags,nearly 4-fold amplification was achieved for the catalytic reduction of p-nitrophenol（p-NP）.And the redox cycling of catalysate p-aminophenol（p-AP）further enhanced signal by 2-fold.Through magnetic DNAzyme walker for target separation and recycling,and Pt@COF nanospheres for catalytic amplification,the proposed biosensor achieved highly sensitive and accurate detection of mi RNA-21 with a detection limit of 47.5 a M,and exhibited excellent detectability and anti-interference ability in serum and cell samples.（4）Magnetic nanobeads and de novo growth of electroactive polymers for ultrasensitive micro RNA detection at the cellular level.Considering that electrochemically mediated atom transfer radical polymerization（e ATRP）as a signal amplification strategy can generate a large number of electroactive polymer chains as signal tags under mild reaction conditions,it has better stability and simpler synthesis process than Ag NCs and nanocatalysts as signal tags.In this chapter,a multiple amplified electrochemical platform with high sensitivity and accuracy was proposed for micro RNA-21（mi RNA-21）detection at the cellular level by integrating the efficient magnetic separation of magnetic nanobeads（MBs）with the multisignal amplification of catalytic hairpin self-assembly（CHA）and e ATRP.Under potentiostat conditions,the continuous polymerization of monomers through the e ATRP process produced a large number of stable ferrocene electroactive polymer chains,which provided amplified electrochemical signals.CHA achieved target amplification by displacing mi RNA-21 into solution.Compared to simple hybridization,SDA and e ATRP can enhance the signals by～35-fold,achieving high signal-to-noise ratio for low-abundant target detection.MBs as capture carriers can directly separate and enrich targets from complex samples by magnetic separation,endowing the method with excellent specificity and anti-interference ability.Combining gold nanoflowers（Au NFs）-modified electrodes as working electrodes,SDA and e ATRP as signal amplification strategies to improve sensitivity,and MBs as separation tools to improve accuracy,the developed biosensor achieved ultrasensitive detection of mi RNA-21 at single cellular level with a detection limit down to a M,indicating the great prospect of this method in the early diagnosis of cancers,life science research,and single-entity electrochemical detection.（5）Fluorescent-magnetic-catalytic nanospheres for dual-modality detection of H9N2avian influenza virus.The previous chapters mainly relied on single-modality signal output,which may be affected by operators,instruments,and non-standard analytical procedures.In order to further improve the accuracy of the method from the perspective of signal output,fluorescent-magnetic-catalytic multifunctional nanospheres（FMCNs）were prepared by layer-by-layer（LBL）assembly method,which could be simultaneously used as capture carriers and signal tags for H9N2 avian influenza virus（H9N2 AIV）dual-modality detection.The excellent fluorescence properties of FMCNs provided amplified fluorescence signal output.Combined with Au-Mn O₂/r GO modified electrodes and enzyme-induced metallization signal amplification strategy,FMCNs provided an amplified electrochemical signal output via alkaline phosphatase（ALP）-catalyzed silver deposition.By integrating electrochemistry and fluorescence into one analytical system,two modes corroborated each other and reduced the experiment operation and uncontrollability,thus improving the accuracy of the experimental results.The excellent magnetic properties of FMCNs could achieve rapid separation and capture of H9N2 AIV in complex samples,reducing the interference of coexisting components.This proposed dual-modality immunosensor can simultaneously realize electrochemical and fluorescence quantitative analysis of H9N2 AIV with detection limits of 10 pg/m L and 69.8ng/m L,respectively,providing the diversity to meet the detection requirements at different situations.Combining FMCNs,enzyme-induced metallization,and Au-Mn O₂/r GO modified electrodes to improve detection sensitivity,and magnetic separation and dual-modality output to improve detection accuracy,this method has excellent sensitivity,anti-interference ability,accuracy and diversity,showing unlimited potential for early diagnosis of suspect infections.