Construction And Application Of A New Biosensing Method Based On Nano-interface Regulation

Nanomaterials have drawn great attention in the field of biosensing.The unique physical and chemical properties make nanomaterials ideal for designing and building high performance electrochemical biosensors with multiple functions.In addition,surface functions can be introduced directly during the modification or modification process.Today,the controlled synthesis of nanomaterial shapes and sizes has led to a significant increase in its use in electrochemical biosensors.Electrochemical sensors are by far the largest sensor group and are a particularly attractive means of converting biometrics directly into electrical signals.The research work of this thesis mainly includes the following five parts:1.CoA-dependent coordination polymer as a novel electrochemical sensing platform for sensitive detection of hydrogen peroxide in biological environmentsA growing body of evidences suggest that hydrogen peroxide?H2O2?plays an effective role in the regulation of multifarious physiological processes.Development of sensitive probes for H₂O₂ is an urgent hotspot.In this work,we recommended a coenzyme A?CoA?-mediated gold coordination polymer?CoA-Au?I?CP?for sensitive electrochemical detection of H2O2.The biosensing mechanism is based on the following highlights:the formation of an unordinary structure with-thiol-Au?I?-repeated units derived from thiol in CoA;an extraordinary interaction between graphene?GO?and CoA-Au?I?CP due to massive adenines.A potential application of the resultant CoA-Au?I?CP as a novel sensing platform to monitor H2O2 is investigated.Such CoA-Au?I?CP-based sensor exhibited an excellent electro-catalytic property for H2O2 reduction.The steady-state current response increases linearly with H₂O₂ concentration from 0.1 to 300?M with a low detection limit of 0.02?M.Importantly,the proposed electrochemical biosensor has shown a new perspective for detection of H2O2 levels in human real samples and that released from human cervical cancer cells with satisfactory results.This novel CoA-Au?I?CP nanomaterial may provide a cost-efficient,robust and high-sensitive platform for detecting various species involving H₂O₂-generation reactions for biomedical applications.2.In situ grown DNA nanotail-templated silver nanoclusters enabling label-free electrochemical sensing of terminal deoxynucleotidyl transferase activityA novel label-free electrochemical strategy was established based on the unique electro-catalytic activity of graphene oxide?GO?-supported terminal deoxynucleotidyl transferase?TdT?-generated C-rich DNA nanotail-templated silver nanoclusters?DNA-AgNCs?.TdT can catalyze the deoxycytidine triphosphate?dCTP?to the 3'-OH terminus of single-stranded DNA?ssDNA?with no template;then,in the presence of Ag?I?,TdT-generated C-rich DNA sequence was employed for the synthetic template of AgNCs because of the formed complexes of nitrogen atoms of cytosine based with silver atoms.We proved that in situ grown DNA nanotail-templated AgNCs can be adsorbed on GO-modified electrode and possess high electro-catalytic activity to H₂O₂ reduction,presenting a good electrochemical indicator for signal readout.Under optimal conditions,the proposed biosensor could be employed for quantitatively monitoring TdT activity and within a dynamic range from 0.4 to 90 U/mL and a low limit of detection is 0.08 U/mL.With high sensitivity and excellent selectivity,this strategy offers a facile,convenient and specific electrochemical method for TdT activity detection and its relevant inhibitors screening.It holds a promising potential in the practical application of TdT-based biochemical research,disease diagnosis and drug discovery.3.Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowires-templated copper nanoclustersElectrochemical methods allow fast and inexpensive analysis of enzymatic activity.Here,a simple and yet efficient“signal-on”electrochemical assay for sensitive,label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase?TdT?-mediated extension strategy.TdT can catalyze the deoxythymidine triphosphate?dTTP?to the 3'-OH terminus of single-stranded DNA?ssDNA?without template;then,the TdT-yielded T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters?CuNCs?.Grown DNA nanowires-templated CuNCs?noted as DNA-CuNCs?were attached onto graphene oxide?GO?surface and exhibit unique electro-catalytic activity to H2O2 reduction.Under optimal conditions,the proposed biosensor was utilized for quantitatively monitoring TdT activity,with the observed LOD of0.1 U/mL.It also displayed high selectivity to TdT with excellent stability,and offered a facile,convenient electrochemical method for TdT-relevant inhibitors screening.Moreover,the proposed sensor was successfully used for BamHI activity detection,in which a new3'-OH terminal was exposed by the digestion of a phosphate group.Ultimately,it has a good prospect in DNA-related enzyme-based biochemical studies,disease diagnosis and drug discovery.4.Protein-mimicking nanowire-inspired electro-catalytic biosensor for probing acetylcholinesterase activity and its inhibitorsA highly sensitive electrochemical biosensor based on the synthetized L-Cys-Ag?I?coordination polymer?L-Cys-Ag?I?CP?,which looks like a protein-mimicking nanowire,was constructed to detect acetylcholinesterase?AChE?activity and screen its inhibitors.This sensing strategy involves the reaction of acetylcholine chloride?ACh?with acetylcholinesterase?AChE?to form choline that is in turn catalytically oxidized by choline oxidase?ChOx?to produce hydrogen peroxide?H₂O₂?,thus L-Cys-Ag?I?CP possesses the electro-catalytic property to H2O2 reduction.Herein,the system was capable of analyzing successive of H₂O₂ effectively by amperometric i-t?current-time?response,and the protein-mimicking nanowire-based sensor was further applied for the turn-on electrochemical detection of AChE activity.The proposed sensor is highly sensitive?limit of detection is 0.0006U/L?and is feasible for screening inhibitors of AChE.The model for AChE inhibition was further established and two traditional AChE inhibitors?donepezil and tacrine?were employed to verify the feasibility of the system.The IC₅₀ of donepezil and tacrine were estimated to be 1.4nM and 3.5 nM,respectively.The developed protocol provides a new and promising platform for probing AChE activity and screening its inhibitors with low cost,high sensitivity and selectivity.5.Tetrahedron DNA-mediated graphene nanolayer for advanced signal amplification:ultrasensitive electrochemiluminescence immunosensor toward p300 analysisProtein p300 is a versatile transcriptional co-activator and participates in many physiological processes,including cell cycle control,differentiation and apoptosis,in which it serves as a protein bridge linking specific transcription factors to the fundamental transcription machinery,a scaffold to complete multiple transcription cofactors and a catalytic enzyme acetylating histone and non-histone proteins.So,the whole p300 analysis in transcriptional regulation is significant greatly.Based on it,an original ultrasensitive electrochemiluminescence?ECL?immunosensor is constructed on account of tetrahedron DNA?TDN?-mediated graphene nanolayer which consists of graphene oxide?GO?and Au nanocrystals?AuNCs?.Strong and stable ECL signal response was achieved with the antigen-antibody interaction and caused by the abundant signal molecules Ru?phen?₃²⁺and hollow structure of TDN.It is particularly noteworthy that the development of our ultrasensitive p300 immunosensor relies on:the graphene nanolayer allows for greater carrying capacity and wider outer Helmholtz plane?OHP?.This immunosensor exhibited desirable and excellent performance for p300 ranging from 0.005 to 80 nM and a preferable detection limit?0.002 nM?.Additionally,the proposed immunosensor can also be exploited for investigating the p300 detection in Hela cell lysate and serum.These suggested that the strategy paved the way for p300-associated clinical diagnosis and drug discovery.