Fabrication Of Homogeneous Electrochemical And Electrochemiluminescence Sensors Based On Signal Amplification By Vertically-ordered Mesoporous Silica Film And Their Applications In Food Analysis

Harmful bacteria,chemicals,toxins or heavy metals in food can seriously affect human health.Therefore,developing sensitive and simple analysis methods to monitor the analytes related to food safety is of great significance.Homogeneous electrochemical（EC）and electrochemiluminescence（ECL）sensors do not need to fix the recognitive and signal probe on the electrode surface,through the changes of EC/ECL signals caused by the diffusion of probe and catalytic reaction in solution phase to achieve the detection.It has the advantages of simplicity,rapid response and high sensitivity.However,When EC or ECL sensors are applied to analyze actual samples,electrodes are often easily contaminated by particles or macromolecules（e.g.proteins and starches）in complex matrices.In addition,the sensitivity of unmodified electrodes is limited and it is difficult to detect analytes with low concentration.Introducing signal sensitization performance through nanomaterials and improving the anti-interference ability of the electrode is the key to building a high-sensitivity method for food safety analysis.Vertically-ordered mesoporous silica films（VMSF）have ultra-small and uniform nanochannels（2～3 nm in pore size）with high density.On the one hand,VMSF modified on the surface of the working electrode does not affect the diffusion of small molecule to the electrode surface.On the other hand,ultrasmall VMSF nanochannel can block large-size interfering molecules and improve the anti-fouling and anti-interference performance of the electrode.In addition,VMSF contains a large number of silicon hydroxyl groups（p K_a～2）,which are negatively charged in common solutions,leading to enhanced sensitivity through enrichment of positively charged EC or ECL probes.In this thesis,three homogeneous sensing strategies were designed by using the anti-interference,anti-fouling and signal amplification capabilities of VMSF.The EC or ECL detections of alkaline phosphatase（ALP）for evaluating pasteurization levels in beverages,mycotoxin zearalenone（ZEN）food preservative and food preservative-antimicrobial peptide Nisin were achieved.The detailed researches are given as follows:（1）Using VMSF modified indium tin oxide（ITO）as working electrode,a homogeneous ECL quenching sensor was constructed for the detection of ALP in milk and serum samples.VMSF/ITO electrode was prepared by electrochemically assisted self-assembly method（EASA）.VMSF was characterized by Transmission electron microscopy（TEM）,Scanning electron microscopy（SEM）,and EC methods.The results showed that VMSF was formed by uniform ordered and intact nanochannel structure with a pore size of 2 to 3 nm and had charge-selective permeability to positive charges.VMSF can enrich the ECL probe,tris2,2’-bipyridyl ruthenium(Ru（bpy）₃²⁺).ALP catalyzed the hydrolysis reaction of the substrate disodium Phenyl phosphate hydrate（DPP）to generate Phenol（Phe）,which can quench the ECL signals of Ru（bpy）₃²⁺and co-reactant tripropanamine（n-dipropyl-1-propanamine,TPA）.Based on the correlation between ECL signal quenching degree and ALP content,ALP was detected.VMSF/ITO electrode can significantly amplify the ECL signal change rate of the ruthenium probe system so the detection sensitivity can be effectively improved.The linear range for the ECL detection of ALP was from 0.01 U/L to 30 U/L,with a detection limit of 0.008 U/L.Combined with the anti-fouling and anti-interference ability of VMSF,ALP in milk samples was detected.As ALP is a biomarker,the fabricated sensor has also been applied for the sensitive detection of ALP in serum.（2）Based on the release of the EC probe Ru（NH₃）₆³⁺caused by the recognition between aptamer and ZEN as well as the enrichment of Ru（NH₃）₆³⁺by VMSF/ITO electrode,a homogeneous aptamer sensor was constructed for the EC detection of ZEN.Ru（NH₃）₆³⁺-ZEN aptamer-graphene oxide（GO）nanocomposite was prepared based onπ-πstacking between GO and aptamer and electrostatic interaction between GO,aptamer and Ru（NH₃）₆³⁺.The successful preparation of the Ru（NH₃）₆³⁺-ZEN aptamer-GO nanocomposite probe was confirmed by zeta potential and electrochemical investigations.Target ZEN could bind with ZEN aptamer and pull out Ru（NH₃）₆³⁺from Ru（NH₃）₆³⁺-ZEN aptamer-GO nanocomposite probe,leading to the enhanced redox currents at the VMSF/ITO electrode.Thus,quantitative determination of ZEN was realized with a linear range from 1 pg/ml to 1μg/mL.The detection limit was 1.2fg/mL.Combined with the anti-fouling and anti-interference ability of VMSF,the sensor can be used for sensitive detection of ZEN in the extract of corn and chestnut.（3）Based on the mechanism that antimicrobial peptides-nisin can destroy the phospholipid bilayer membrane,an EC/ECL dual-mode homogeneous biosensing system was constructed using Ru（bpy）₃²⁺@liposome as EC or ECL probe,VMSF/ITO that can significantly enrich Ru（bpy）₃²⁺probe as working electrode for sensitive detection of the food preservative Nisin.Ru（bpy）₃²⁺@liposome was prepared by ultrasound method.The size of liposomes and the content of inner Ru（bpy）₃²⁺were characterized by TEM and UV-Vis spectroscopy,the results showed that the diameter of the liposome was about 97 nm,and the content of Ru（bpy）₃²⁺was about 0.036 mg.Nisin can cause the rupture of liposome to release Ru（bpy）₃²⁺from Ru（bpy）₃²⁺@liposome.By recording the EC and ECL signals of the released Ru（bpy）₃²⁺,an EC/ECL dual-mode detection of Nisin can be achieved.The linear range for ECL detection of Nisin was from 10 ng/mL to 50μg/mL,and the detection limit was 9.3ng/mL.The linear range for EC of Nisin was from 800 ng/mL to 100μg/mL with a detection limit of 70 ng/mL.Combined with the anti-fouling and anti-interference ability of VMSF,Nisin in milk and egg white was detected.