Immunosensor Based On Polyaniline And Its Nanocomposites For Escherichia Coli O157:H7

Electrochemical immunosensors based on screen-printed electrodes(SPCE)have greatly challenged the research and application of traditional solid electrodes in the field of electrochemical immunosensors due to their low cost,mass production,elimination of complex pretreatment,good consistency and easy modification.However,in practical applications,SPCE still faces some problems that need to be solved:Because of its high absolute sensitivity and poor relative sensitivity,the parallelism of the test results is bad,and even the experimental reproducibility is affected.Due to its small electrode area,the antibody loading is low,which affects the sensitivity of the experiment.Due to the maturity of mass production technology,few people pay attention to the reuse and regeneration of electrodes.Polyaniline(PANI)is one of the typical representatives of conjugated polymers.Because of its low cost,convenient synthesis,good thermal stability,rapid doping and dedoping,it has attracted much attention in the field of electrochemical immunosensors.In this research paper,based on the conjugated polymer PANI,combined with a variety of composite nanomaterials,selected Escherichia coli 0157:H7(E.coli 0157:H7)as the detection model,started with the stability of the electrode surface modification,improved the detection performance of electrochemical immunosensors and realized the regeneration of SPCE.In order to promote the development of SPCE in the field of food safety,promote the commercialization and practical application of electrochemical immunosensors.The main contents of this research are as follows:1.Electropolymerization of stable leucoemeraldine base polyaniline film and application for quantitative detection of Escherichia coli 0157:H7In this chapter,in order to solve the problem of low firmness and poor stability of the modifier on SPCE,further improve the surface stability of the working electrode and construct a new E.coli 0157:H7 electrochemical immunosensor,we used the potentiostatic method to electrodeposit polyaniline(PANI)on SPCE,combined with gold nanoparticles(AuNPs),started from the simple "direct method"immunosensor,achieved the target of quantitative detection of E.coli 0157:H7.First,the electropolymerization of aniline to form a PANI film firmly adheres to the surface of the SPCE by potentiostatic method,which not only enhances the electron transport rate on the electrode surface,but also increases the specific surface area and provides more active sites(-NH3).Compared with the PANI deposited by cyclic voltammetry(CV),the PANI prepared by the potentiostatic method is more stable and can withstand strong acid(HCl),strong alkali(NaOH)and sonication treatment.Ultra-stable PANI film is expected to achieve sensor reuse.Second,AuNPs can be cross-linked with-NH3 to form Au-N bonds,so using them as a linker for PANI and capture antibody(Ab)can not only further improve the conductivity of the electrode surface,but also avoid the inactivation or self-crosslinking of Ab caused by activation of-COOH with reagents(such as EDC/NHS)when PANI and Ab are link directly.Field emission scanning electron microscopy(SEM)was used to characterize the morphological changes during the stepwise modification of the electrode and was further verified by CV and Electrochemical impedance spectroscopy(EIS).The immunosensor(SPCE-PANI-AuNPs-Ab-BSA/E.coli 0157:H7)quantitatively detects E.coli 0157:H7 by differential pulse voltammetry(DPV).Under the optimal experimental conditions,the current value decreases with the increase of E.coli 0157:H7 concentration and the current value is linear with the logarithm of E.coli 0157:H7 concentration.The linear range is from 4.0 × 104 to 4.0 × 109 CFU/mL,the minimum detection limit(LOD)was 7.98 × 103 CFU/mL.At the same time,the immunosensor has good specificity,reproducibility and stability.The actual sample analysis of the milk sample was carried out by the sensor,and the recovery rate was between 81.00%and 95.50%.2.A sensitive and reusable electrochemical immunosensor for quantitative detection of Escherichia coli 0157:H7 based on stable polyaniline coated screen-printed carbon electrode and rGO-NR-Au@PtThe "direct" type of method in electrochemical detection is simple and has good reproducibility,but the sensitivity is not very high,which will affect the accuracy.In order to further improve the sensitivity and detection performance of electrochemical immunosensors,this chapter builds a novel sandwich immunosensor based on the previous chapter(SPCE-PANI-AuNPs-Ab1-BSA/E.coli 0157:H7/rGO-NR-Au@Pt-Ab2-BSA).In this chapter,gold-platinum composite metal nanoparticles(Au@Pt)were synthesized,and neutral red(NR)functionalized reduced graphene oxide(rGO-NR)was prepared.The signal amplifier rGO-NR-Au@Pt was prepared by adsorption of negatively charged Au@Pt on rGO-NR by electrostatic adsorption.The nanocomposite probe has high specific surface area,good biocompatibility and catalyzed performance of hydrogen peroxide(H2O2).The non-enzymatic immunosensor indirectly quantifies E.coli 0157:H7 by detecting the redox signal exhibited by the electron transfer compound thionine(TH)in the test solution.Under the optimal experimental conditions,the current value increases with the increase of E.coli O157:H7 concentration and the current value is linear with the logarithm of E.coli O157:H7 concentration.The linear range is from 8.9 × 103 to 8.9 × 109 CFU/mL,the minimum detection limit(LOD)was 2.84 × 103 CFU/mL.At the same time,the immunosensor has good specificity,reproducibility and stability.The actual sample analysis of the milk and pork samples were carried out by the immunosensor,and the recovery rate were between 80.90%-91.01%in the milk samples and 88.76%-106.74%in the pork samples.Compared to the previous chapter,in addition to the improvement of the detection limit,we have used the characteristics of ultra-stable PANI to regenerate the electrode.The formed immune complex(Ab1-BSA/E.coli O157:H7/rGO-NR-Au@Pt-Ab2-BSA)was successfully detached from the electrode surface by ultrasound.The biological activity of SPCE-PANI-AuNPs was retained and it can be used for the next detection.In this chapter,the immunosensor can be reused at least 5 times.3.Reusable polyaniline film/gold nanoparticles modified screen print carbon electrode and application for quantitative detection of Escherichia coli 0157:H7 with AuNPs@Polyaniline/Au@Pt nanocomposites as electrical signal labelIn sandwich type electrochemical immunosensors,capture probes are one of the key factors affecting detection performance.In this chapter,we have developed a new type of probe that is more economical,safer,more efficient and environmentally friendly(AuNPs@PANI-Au@Pt-Ab2-BSA).Firstly,using chloroauric acid solution(HAuC14)as oxidant,oxidizing precursor aniline(ANI),AuNPs@Polyaniline microspheres(AuNPs@PANI)were prepared under hydrochloric acid(HCl)environment.Secondly,the synthesized Au@Pt was adsorbed on the AuNPs@PANI surface by the Au-N bond.The resulting sandwich-type non-enzymatic immunosensor is SPCE-PANI-AuNPs-Ab1-BSA/E.coli 0157:H7/AuNPs@PANI-Au@Pt-Ab2-BSA.Under the optimal experimental conditions,the current value increases with the increase of E.coli 0157:H7 concentration and the current value is linear with the logarithm of E.coli 0157:H7 concentration.The linear range is from 8.9× 104 to 8.9 × 109 CFU/mL,the minimum detection limit(LOD)was 3.0× 103 CFU/mL.At the same time,the immunosensor has good specificity,reproducibility and stability.The actual sample analysis of the milk samples were carried out by the immunosensor and the recovery rate were between 75.28%-105.62%.Compared with the second chapter,the immunosensor constructed in this chapter has two advantages:(1)the preparation of probe was more cost-effective,safer and environmentally friendly.In the process of preparing rGO-NR-Au@Pt,the process of synthesizing graphene oxide(GO)by Hummer method takes a long time and has certain safety hazards.The preparation of AuNPs@PANI-Au@Pt can reduce the time consumption by about 90%,and the reaction conditions are mild and simple;(2)the electrode regeneration was further explored,and the number of reuses was increased.