The Study Of Immunosenor Based On Fe₃O₄@SiO₂ And Functional Graphene For Detection Of Pathogenic Bacteria

Listeria monocytogenes?L.monocytogenes?and Cronobacter sakazakii?C.sakazakii?are common food-borne pathogens with high mortality and serious harm.Therefore,rapid,sensitive and specific detection of L.monocytogenes and C.sakazakii to achieve its prevention and control purposes has important significance for human health.Because of its good selectivity,high sensitivity,low cost and rapid detection,the electrochemical immunosensor has attracted extensive attention in food safety control,environmental monitoring and medicine.At present,electrochemical immunosensors still have some problems in practical application:the purity of the sample should be high,so the pre-treatment of samples including separation and purification is generally needed,especially when bacteria as objects to be detected.The operation is complex and time-consuming.And the environment and conditions of detection process require a strict controls,meanwhile the stability and accuracy of this technique is also a problem to be solved.Therefore,it is necessary to further improve the performance of electrochemical immunosensor.In this study,L.monocytogenes and C.sakazakii were used as targets,the novel eectrochemical immunosensors were established by using multiple nanomaterial as composite probes,which also provide a research model for the detection of other pathogens and promote the development and application of electrochemical biosensors in the field of rapid detection of pathogenic bacteria.This study was divided into the following four parts:1.Construction of electrochemical immunosensor based on Fe₃O₄@SiO₂ using rGO/AuNPs as non-enzymatic label for rapid detection of L.monocytogenesIn order to improve the shortcomings of electrochemical immunosensor that the time-consuming process of separating and pure culturing for detection of bacteria,and the deficiency of the false-positive and non-specific binding of enzymatic labels.We have prepared a novel capture probe and a non-enzymatic label as indicating probe.This chapter studied the preparation process of reduced graphene oxide?rGO?and gold nanoparticles?AuNPs?composite materials?rGO/AuNPs?,using sodium citrate reduction of HAuC14 and graphene oxide?GO?.And the antibody of L.monocytogenes?Ab?was modified into the surface of rGO/AuNPs,to obtain immune activity of the redox probe for specially recognizing L.monocytogenes.The magnetic nanoparticles?MNPs?were prepared by coprecipitation method.On the basis of this,the silica-coated core-shell magnetic nanomaterials?Fe₃O₄@SiO₂?were prepared.After modification by amination-aldehyde,the surfaces of Fe₃O₄@SiO₂ obtained active microparticles whose active site binds to Anti-L.monocytogenes by 3-aminopropyltriethoxysilane?APTES?and glutaraldehyde?GLU?.We can obtain Fe₃O₄@SiO₂/Ab for enriching L.monocytogenes as a capture probe from the samples.The sandwich method was used to form the sandwich structure of Fe₃O₄@SiO₂/Ab which was enriched with L.monocytogenes by the rGO/AuNPs/Ab as the nonenzymatic label.The sandwich structure complex was adsorbed on the surface of the screen printed carbon electrode?SPCE?by a magnet.The AuNPs were oxidized and then reduced by differential pulse voltammetry?DPV?in the HCl reaction solution.Under the optimized conditions,the DPV response signal had a good linearity in the range of 2.9×103 to 2.9×109 CFU·mL-1 for L.monocytogenes.The low detection limit was 1.8×104 CFU·mL-1?S/N=3?.The immunosensor reproducibility was good because the RSD was 5.8%.The immunesensor was stored at 4? after 30 days and the stability was 92.9%;the specificity is good;the accuracy of the immunosensor is 93.3%.Thus,the novel immunosensor can effectively shorten and omit the pure culture process,improve the accuracy and consistency,and provide a basic research model for the detection of other pathogens.2.The Electrochemical immunosensor based on AuNPs-Thi@rGO as a non-enzymatic label for rapid detection of L.monocytogenesThe results of the electrochemical immunosensor analysis showed that the detection of L.monocytogenes had good accuracy and stability.But the sensitivity is still not ideal,to be improved.In order to solve this problem,rGO was utilized as substrate to immobilize thionine?Thi?by ?-? stacking to form a novel redox active hybrid material?Thi@rGO?and gold nanoparticles?AuNPs?were anchored to the surface of Thi@rGO by the chemisorption of the amine groups of the Thi and the opposite charged adsorption.And the Anti-L.monocytogenes was modified into the surface of AuNPs-Thi@rGO,to obtain immune activity of the redox probe for specially recognizing L.monocytogenes.The AuNPs-Thi@rGO with immunoactivity?Ab-AuNPs-Thi@rGO?was used as a non-enzymatic labeled indicator to form a sandwich structure of Fe₃O₄@SiO₂-Ab/L.monocytogenes/Ab-AuNPs-Thi@rGO.The sandwich structure of the reaction solution was 0.1M PBS?pH 6.5?.DPV detection method was used to produce the current of reduction peak and the reduction peak potential was-0.23V.Under the optimized conditions,the peak current of DPV showed a good linearity in the range of 2.9X102-2.9×108 CFU·mL-1 with the low detection limit of 4.7×10CFU·mL-1?S/N=3?.The reproducibility was good,and the RSD was 3.4%.The immunesensor was stored at 4? after 70 days and the stability was 95%;the specificity was good;the accuracy rate of the immunosensor with the national standard was 97%.The sensitivity of the novel immunosensor composed of Fe₃O₄@SiO₂-Ab/L.monocytogenes/Ab-AuNPs-Thi@rGO sandwich structure was significantly improved,and its linearity range was wide,the accuracy and stability were good.The study provided a good basic research model for the diversity of non-enzyme labeled bacterial immunosensor.It also provided a model for the diversity of non-enzymatic labels in electrochemical immunosensors.3.Construction of a double-probe immunosensor based on AuNPs-NR@rGO as non-enzymatic label for rapid detection of C.sakazakiiOn the basis of the above research,in order to validate the extended application of sensor and follow-up to achieve multiple detection,a new immunosensor was constructed based on a non-enzymatic label of AuNPs-neutral red?NR?@rGO.According to the results reported in the literature and comparative experiments,neutral red?NR?,an excellent non-enzymatic-labeled redox active substance,was screened on the basis of the difference of reduction peak potential and the way of combination with rGO.The indicator probe of Anti-C.sakazakii/AuNPs-NR@ rGO was obtained for the first time after labeling the antibody of C.sakazakii?Anti-C.sakazakii?with AuNP s-NR@rGO.The capture probe of Fe₃O₄@SiO₂/Anti-C.sakazakii was constructed on the basis of the preliminary research model to prepare the sandwich structure of Fe₃O₄@SiO₂/Anti-C.sakazakii/C.sakazakii/Anti-C.sakazakii/AuNPs-NR@rGO.The sandwich structure had been adsorbed on a four channel screen printed electrode?4-SPCE?under the action of an applied magnetic force.The sandwich structure of the reaction solution was 0.1M PBS?pH7.4?.DPV detection method was used to produce the current of reduction peak and the reduction peak potential was-0.23V.The results showed that under the optimal conditions,the peak current response of DPV had a good linearity when the concentration of C.sakazakii was in the range of 3.9×102-3.9×107 CFU·mL-1 and the low detection limit was 2.2×10 CFU·mL-1?S/N=3?.The RSD of the measurements was 6.7%,indicating good reproducibility.After 50 days,the reduction peak current is 94%of the initial value;the specificity was good;the accuracy rate of the immunosensor with the national standard was 97.4%.It can be seen that the novel immunosensor has good stability,reproducibility and high sensitivity.4.Construction of an immunosensor for simultaneous detection of C.sakazakii and L.monocytogenes based on nano-double probeBased on the above results,an immunosensor capable of simultaneous detection of C.sakazakii and L.monocytogenes was constructed.Under the optimized conditions,the reduction potential of Thi and NR were-0.23V and-0.62V,respectively,which were suitable for the construction of immunosensor for simultaneous detection of two target bacteria.The immunosensor was constructed by dual-sandwich sandwich structure of Fe₃O₄@SiO₂/Anti-C.sakazakii/C.sakazakii/Anti-C.sakazakii/AuNPs-NR@rGO and Fe₃O₄@SiO₂/Anti-L.monocytogenes/L.monocytogenes/Anti-L.monocytogenes/AuNPs-NR@rGO.The results showed that under the optimal conditions,the peak current response of DPV had a good linearity when the concentration of C.sakazakii was in the range of 3.9×102-3.9×108 CFU·mL-1 and the concentration of L.monocytogenes was in the range of 2.9×102-2.9×106 CFU·mL-1.The low detection limit was 1.1×102 CFU·mL-1 and 1.5×102 CFU·mL-1?S/N=3?,respectively.The RSD of the measurements was 6.2%and 8.3%,respectively,indicating good reproducibility.After 30 days,the stability of the immunosensor was good.The specificity was good,and no obvious cross-correlation was observed.the accuracy rate of the immunosensor with the national standard was 96.9%.