Studies On Biosensing Techniques And Kinetics Of Chemical Reaction Based On Scanning Electrochemical Microscopy

Scanning Electrochemical Microscopy(SECM)has been applied into many advanced fields related to electrochemistry,like electrochemical biosensor,electrochemical reaction kinetics and surface/interface study.In the field of electrochemical biosensor,the tip of SECM acts as signal receiver while the substrate acts as signal carrier.This separation of signal receiver and signal carrier overcomes the limitation of electrochemical biosensors based on traditional electrodes,which the signal carrier covered on the electrode surface may lead to a low efficiency of received signal.And also because of this advantage of“separation”,the substrate of SECM can be designed into a selective and high throughput platform that responses to multiple targets.So that this kind of SECM-based biosensors combines the high selectivity,high sensitivity and high throughput together,having good prospects in the biosensing study of severe diseases or complex systems.SECM can exhibit a high-resolution electrochemical image towards the sample on substrate.Its resolution can reach into nanoscale with the tip size decreasing.But there is a limitation in traditional SECM,which is that only a single molecule can be responded during one single imaging.This limitation results in too single information acquisition.And multiple scans spend a long time,leading to an unsimultaneous information on some samples requiring simultaneity(like cells).Therefore,it is necessary to develop a new SECM method which can response to multiple molecules in one single imaging.Due to the nanoscale tip and piezoelectric ceramics,SECM exhibits a very high resolution in time and space.This high resolution provides a method to study the kinetics of electrochemical reaction and capture the unstable intermediates.For analytical methods or biological activities related to electrochemical reactions,exploring the mechanism and kinetic process of electrochemical reaction is helpful to recognize their nature.Therefore,using SECM to study the kinetics of these electrochemical process makes more sense.In this thesis,a biosensor of tumor marker was firstly designed based on the advantage of“separation”.And a dual catalytic signal amplification DNA biosensing platform was established,using a polymer-modified tip with the function of precise localization and signal amplification.Besides,a programmable SECM for simultaneous multi-target response was developed based on traditional SECM and potential waveform design.This programmable SECM strategy was used in the identification of different cell status,providing multiple information simultaneously hence improve the accuracy of identification.At last,based on SECM's high resolution in time and space,the kinetics of TPr A oxidation in Ru(NH₃)₆³⁺-TPr A ECL system was investigated.And the mechanism of this ECL process was verified based on the kinetic result.The research work of this thesis is as follows.(1)A simultaneous detection of four tumor markers in lung cancer(alpha-fetoprotein(AFP),carcinoembryonic antigen(CEA),neuron-specific enolase(NSE),cytokeratin-19-fragment(Cyfra21-1))was achieved for the first time using immune sandwich structures coupled with generation collection(GC)mode of scanning electrochemical microscopy(SECM).The proposed method exhibited excellent performance in quantitative detection of the four target proteins.A good linear correlation between the signal current issued from reduction of p-benzoquinone(BQ)oxidized from hydroquinone(H₂Q)and the amount of target tumor markers at logarithmic protein concentrations ranging from 5 ng/m L to 1?g/m L was achieved.The detection limit was also low,meeting the needs of clinical use.The specificity was satisfactory and signal current of the target protein was unaffected by other simultaneously detected target proteins and common interfering species.This proposed method looks promising for high-throughput protein determination based on SECM,which could potentially be applied in clinical lung cancer diagnosis.(2)A PEDOT-modified Pt microelectrode was used in SECM for DNA biosensing and combined with long self-assembled DNA concatemers to realize dual signal amplification.The approach curve could also be plotted for tip location using this modified microelectrode with no impact on the catalytic effect so that both signal amplification and tip locating were achieved in one single tip.The detection limit of this method reached 0.076 f M,providing an ultra-sensitive DNA biosensing platform.Furthermore,this strategy could be applied in scanning a DNA chip,showing great potential in high-throughput bioanalysis with high sensitivity.(3)A multi-target-response electrochemical imaging technique was presented using scanning electrochemical microscopy(SECM)combining with self-designed waveform.This technique broke the limitation of traditional SECM that only one molecule was measured in one imaging.Using this method,PC12 cells status was identified through three different molecules accurately.(4)An important electrochemiluminescence(ECL)“co-reactant”reaction,tri-n-propylamine(TPr A)oxidation was studied by scanning electrochemical microscopy using tip generation/substrate collection(TG/SC)mode.The reaction mechanism was demonstrated by capturing the two key reaction intermediates,TPr A^+?and TPr A^?,within the gap between the SECM tip and substrate.The half-life of TPr A^+?was obtained by comparing the experimental data and the simulated one.The results indicate that TPr A^+?was reasonably long-lived to serve as an oxidant to participate the ECL processes for TPr A-Ru(bpy)₃²⁺system,which are consistent with the reaction pathways that proposed earlier.This present SECM methodology offers a new means to study electron associated bond breakage and bond formation.It can also be further utilized to understand the coreactant ECL reaction mechanism.