Preparation Of Electrochemical Biosensors Based On Vertically Ordered Mesoporous Silica Thin Films And Their Analytical Applications In The Biomedical Field

With the increasing improvement of people’s living standards,health issues have attracted widespread attention from society.Therefore,preventing the occurrence of diseases or achieving early diagnosis of diseases is crucial.Developing fast and sensitive detection methods to achieve accurate detection of low levels of pathogenic factors in the human body is what current researchers expect.Electrochemical methods have been widely applied in various fields of society due to their advantages such as fast response speed,high detection sensitivity,and integrability.However,due to the complex composition of biological samples,there is a non-specific adsorption between proteins,small biomolecules,etc.contained in biological samples and the sensor,which will occupy the active site on the electrode surface,resulting in passivation of the electrode surface while reducing the detection sensitivity of the sensor to the target analyte,and interfering with the reliability of the experimental results.Finding a material that can protect electrodes and reduce substrate environmental interference has become one of the current research hotspots.Vertical ordered mesoporous silica films（VMSF）with uniform and ultrasmall pore size,high porosity,ultrathin,and rigid structure displays unique permselectivity,size exclusion,and antifouling capacity.In addition,VMSF is rich in silanol（Si-OH）groups,which can be alkylated with silane coupling agents to introduce functional groups with different functions on VMSF,meeting the needs of different analysis and detection.This article uses VMSF as the substrate material for further functionalization including changing the internal charge of VMSF nanochannels and introducing biological recognition elements on their surfaces through silane coupling agents.Three kinds of electrochemical sensors consisting of direct electrochemical sensor,immunosensor,and cytosensor are constructed to achieve rapid and sensitive detection of electroactive small biological molecules,tumor markers,and tumor cells,respectively.The specific research content is as follows:（1）A portable electrochemical sensor based on functional group modified VMSF integrated with a disposable screen-printed electrode（SPCE）was constructed for the rapid and sensitive detection of uric acid（UA）in human whole blood samples.Using SPCE as the working electrode and graphene oxide（GO）as the nano adhesive layer,the amino functionalized VMSF（NH₂-VMSF）was further modified on SPCE through electrochemical assisted self-assembly（EASA）method.During the growth of VMSF,GO is reduced to electrochemically reduced graphene oxide（Er GO）with high electrocatalytic activity.Successful reduction of GO are characterized by X-ray photoelectron spectroscopy（XPS）.The uniform pore size,long-range ordered pore structure,and film integrity of NH₂-VMSF were investigated by transmission electron microscopy（TEM）and electrochemical methods.Based on the electrocatalytic effect of Er GO on UA and the electrostatic enrichment effect of NH₂-VMSF on UA,dual amplification of UA electrochemical detection signal was achieved.Furthermore,the anti-interference,electrode regeneration,stain resistance and selectivity of the sensor were evaluated.Finally,the sensor was used to detect UA in human whole blood samples.Compared with traditional methods that require expensive uricase in the detection of uric acid,this work integrates the advantages of SPCE（disposable,integrated,low production cost,etc.）and VMSF（anti-fouling,electrostatic pre-enrichment,etc.）,greatly sparing detection costs and providing a new strategy for the construction of portable electrochemical sensors with anti-fouling and high detection performance.（2）An electrochemical immunosensor based on VMSF architecture for highly sensitive detection of tumor biomarkers in serum samples was constructed by combining biological functionalization with gated analysis strategy.VMSF was modified on indium tin oxide electrode（ITO）by St?ber solution growth method.The surface morphology and thickness of VMSF were characterized by scanning electron microscopy（SEM）and TEM.The film integrity and selective permeability of VMSF were proved by cyclic voltammetry（CV）and electrochemical impedance spectroscopy（EIS）.Using（3-Glycidoxypropyl）methyldiethoxysilane（GPTMS）as a silane coupling agent and a covalent binding aptamer as a crosslinking agent,an amino modified aptamer is covalently immobilized on the outer surface of VMSF to construct a biological recognition interface.Based on the specific recognition capture effect of the selected aptamer on liver cancer marker alpha fetoprotein（AFP）,selective detection is achieved.The construction process of the biosensor was characterized by two electrochemical characterizations,CV and EIS.The incubation time of aptamer and AFP was optimized during the experiment.Differential pulse voltammetry（DPV）was used as a quantitative analysis method to achieve sensitive detection of AFP.Finally,the stability and repeatability of the aptasensor were evaluated,and the reliability of determining AFP content in serum was verified through recovery experiments.Compared with antibodies that are susceptible to environmental factors,aptamers as recognition molecules chosen in this study for tumor markers have the advantages of small size,good stability,and low generation cost,which provides a new detection strategy for constructing universal aptasensors.（3）An electrochemical cytosensor was constructed based on the biological functionalization and gating analysis strategy of VMSF and successfully used for sensitive detection of Hela cells.VMSF was prepared on ITO using the EASA method,and its morphology（pore size,orderliness,thickness）and film integrity were characterized by TEM and electrochemical methods.Folic acid（FA）functionalized with 3-aminopropyltriethoxysilane（APTES）were first prepared and further covalent modification on the outer surface of VMSF through silanization alkylation reaction,leading to the biological recognition interface with high specificity for Hela cells.Benefiting from the overexpression of FR on the membrane of Hela cells,Hela cell could be specifically recognized by the proposed FA-APTES/VMSF/ITO sensor and attached to the electrode surface,producing steric hindrance effect and eventually inhibiting the access of electrochemical redox probe to the underling ITO electrode.Therefore,the reduced electrochemical signals have relation to the concentration of Hela cells,allowing the quantitative detection of Hela cells.Subsequently,the selectivity and reproducibility of the constructed cytosensor were evaluated.This work innovatively proposes the construction of a receptor based cancer cell recognition interface on the surface of VMSF,and utilizes electrochemical methods to achieve rapid analysis and detection of Hela cells.Compared with traditional detection methods for cancer cells based on the aptamer or antibody,the immobilized folate strategy proposed in this paper has lower costs and further expands the application fields of various sensors based on VMSF.