Construction Of Highly Sensitive Electrochemical/Electrochemiluminescence Sensors Based On Silica Isoporous Membranes And Their Applications

The development of rapid,accurate and sensitive analytical methods has always been a hot issue in the field of analytical chemistry.Electrochemical method has attracted a lot of attention due to its advantages of simple equipment,easy operation,rapid response,easy integration and miniaturization of the instrument.As an important branch of electrochemical technology,electrochemiluminescence（ECL）combines the merits of the electrochemical and chemical luminescence（CL）method,not only exhibits a wide linear range and high sensitivity of the classic CL method,but also has an excellent reaction controllability and spatiotemporal controllability derived from electrochemical technology.Nowadays,a diversity of sensors based on electrochemistry and electrochemiluminescence have been developed,and play a significant role in the field of disease diagnosis,food analysis and environmental monitoring.However,due to the uncertainty and complexity of the tested samples,those electrochemical sensors are easily contaminated by proteins,ions or large particles in the matrix during practical application,resulting in the distortion of signals.The direct interaction between target analytes and luminophores which in turn causes the enhancement or quenching of ECL intensity is the basis for establishing quantitative relationship of traditional ECL sensors.However,these analytes tend to be a large group of substances,which leads to the problem of the poor selectivity,limiting their practical application scenarios.In addition,in order to improve the sensitivity of sensors,researchers tend to modify the electrodes with complex and expensive nanomaterials to introduce specific effects and functions（such as catalysis,surface coordination,enrichment and separation,molecular recognition,etc.）.Vertically-ordered mesoporous silica films（VMSF）,a kind of nanochannel arrays with simple synthesis and low cost,possess excellent permeability,good mechanical stability,and large specific surface area.It is an ideal modification material for the construction of sensors.Abundant silanol groups on the surface of VMSF provide numerous covalent reaction sites to introduce functional groups and specific recognition elements through silane coupling reaction,resulting in the improvement of specificity and selectivity of the sensors.What is more,silanols with a p K_a of 2～3 tend to ionize in the conventional solution environment,resulting in negatively charged channels,which endows VMSF with significant charge-based permselectivity and shows great superiority and potential in analytical systems involving charged species.Based on the current research status,three multiple sensitization strategies were proposed to enhance the sensitivity based on VMSF modified electrodes.Combined with the excellent anti-matrix effect of VMSF,highly sensitive electrochemical or electrochemiluminescence sensor were constructed in this thesis to realize the determination of metal ions,metabolic small molecules and biomarkers in complex samples.The specific research contents are as follows:（1）A simple and efficient chelator-based sensitivity enhancement strategy was developed to realize ultrasensitive electrochemical determination of trace iron（II）in environmental water samples and tablets using VMSF modified electrodes.An economical and optically transparent indium tin oxide（ITO）electrode was selected as the substrate.The rapid equipment of VMSF on ITO was realized by electrochemical assisted self-assembly（EASA）method to obtain the target working electrode VMSF/ITO.The morphology and permeability of the VMSF/ITO were investigated by electron microscopy and electrochemical methods,respectively.Phenanthroline（Phen）,a chelator,was proposed for the first time to improve the detection performance of the target metal ion Fe²⁺.Compared with free Fe²⁺,the electrochemical behavior of complex Fe（Phen）₃²⁺formed after Fe²⁺’s chelation with Phen changed significantly,and had a much larger enrichment factor in the negatively charged nanochannels,thus realizing the improvement of electrochemical response.Compared with the traditional spectrophotometric method based on phenanthroline,thanks to the excellent electrostatic enrichment ability of VMSF to positively charged Fe（Phen）₃²⁺,the proposed method succeeded to lower the detection limit by 3 orders of magnitude,and is immune to the color of the tested samples,thus having a wider range of application scenarios.Combined with the excellent anti-fouling ability of VMSF,the sensitive detection of trace Fe²⁺in environmental water samples and tablets was finally realized.（2）A sensitive method for glucose detection was established by covalently immobilizing glucose dehydrogenase（GDH）on the outer surface of VMSF and catalyzing the production in situ to generate reduced nicotinamide adenine dinucleotide（NADH）as a coreaction reagent of Ru（bpy）₃²⁺.VMSF/ITO was obtained by assembling VMSF on economical and optically transparent ITO electrodes through EASA method.The morphology and permeability were investigated by electron microscopy and electrochemical methods,respectively.GDH was immobilized successfully on the outer surface of VMSF with the assist of bifunctional reagent as crosslinker,which was verified by cyclic voltammetry（CV）and electrochemical impedance spectroscopy（EIS）.GDH can catalyze the dehydrogenation of glucose and generate NADH concomitantly in the presence of coenzyme I,NAD⁺.The ECL enhancement essentially dependents on the amount of zymolyte,thereby achieving the quantification of glucose.The robust VMSF not only acted as an effective enrichment material for Ru（bpy）₃²⁺,but also the substrate for covalently immobilized monolayer of natural enzymes.Compared with the traditional enzyme immobilization methods such as entrapment and encapsulation using polymers,the proposed sensor is less susceptible to cracking during storage or swelling during testing under solution phase,exhibiting better stability and lower economic cost.Combined with the excellent antifouling ability of VMSF,the sensitive detection of glucose in healthy human serum was finally achieved.By changing the immobilized dehydrogenase,it is expected to achieve universal detection of a diversity of zymolytes.（3）A dual-antibody sandwich enzyme-linked immunization assisted signal sensitization strategy was constructed to detect alpha-fetoprotein（AFP）by ECL sensor combined with VMSF.VMSF/ITO prepared by St?ber-solution growth approach was selected as the substrate electrode,followed by modification with a monolayer of capture antibody（c Ab）with the aids of crosslinker.In the presence of the target antigen and detection antibody（d Ab）conjugated with streptavidin,immune sandwich structure with large size was formed on the electrode interface.Then,biotinylated alkaline phosphatase（ALP）was immobilized with the help of the streptavidin-biotin system.CV and EIS techniques were applied to monitor the properties of electrode/electrolyte interface during the multilayer assembly process,and finally confirmed the successful construction of the immunosensor.On the one hand,the large immune sandwich structure formed on the electrode interface exerted a significant steric hinderance effect on the diffusion of ECL probe Ru（bpy）₃²⁺,leading to the decrease of the ECL signal.On the other hand,ALP can catalyze the hydrolysis of the substrate disodium phenyl phosphate（DPP）to produced phenol under near-neutral conditions,which acted as an effective quencher to quench the ECL of Ru（bpy）₃²⁺.The above two quenching effects significantly amplified the change of the signal.Compared with the traditional single quenching analysis strategy that only relies on the steric hindrance caused by antibody-antigen immune complexes,this work combined with sandwich immunization to introduce biological enzymes for efficient catalysis to achieve double quenching analysis with higher sensitivity.Alpha fetoprotein（AFP）,the biomarker for primary liver cancer,was selected as a proof-of-concept analyte.Sensitive determination of AFP in the plasma of healthy people and patients was achieved.By changing the specific recognition antibodies,it is expected to extend to the universal detection of different biomarkers.