Assembly Of Multifunctionalized Gold Nanoparticles With Chemiluminescent,Catalytic And Immune Activity For Label-Free Immunoassays

In this dissertation,the concept and principle of chemiluminescence(CL),the development of chemiluminescent functionalized nanomaterials(CF-NMs)and their applications in immunoassays are reviewed.In recent years,nanomaterials,especially gold nanoparticles(GNPs),have been widely used in immunoassays due to their unique physical and chemical properties.GNPs have been used as labels,catalysts and carrier of functionalized molecules for the fabrication of analytical probes and interfaces in chemiluminescent immunoassays(CLIA).On the other hand,the present CLIA are mainly based on labeling technology,which is complicated,time-consuming and high cost.Therefore,the development of simple,sensitive,rapid and low cost label-free CL immunoassays is a new trend in this field.CF-NMs were successfully used as analytical probes and interfaces for immunoassays and DNA assays.In order to achieve high sensitivity,CF-NMs with higher CL efficiency are highly desired.Accordingly,in our research group,CL reagent and catalyst bifunctionalized nanomaterials(BF-NMs)with high CL efficiency were developed.However,such BF-NMs are difficult to connect with recognition elements such as antibodies,DNA and aptamers owing to the high surface coverage fraction of BF-NMs,limiting their applications in bioassays.The aim of this dissertation is to explore the assembly strategy of multi functionalized gold nanoparticles(MF-GNPs)with excellent CL,catalytic and immune activity and their applications in label-free CLIA.It was found that MF-GNPs exhibited excellent CL activity.Based on the obtained MF-GNPs as analytical interfaces,a label-free CL immunosensor was established for the determination of human immunoglobulin G(hlgG)and heart-type fatty acid-binding protein(H-FABP),a biomarker of early Acute Myocardial infarction(AMI).The main research contents are as follows:The MF-GNPs were fabricated by successive assembly of ABEI-GNPs with antibody,BSA and Co2+.The morphology of MF-GNPs was characterized by transmission electron microscopy(TEM).The coating of antibody,BSA and Co2+ onto ABEI-GNPs was confirmed by(circular dichroism)CD and inductively coupled plasma atomic emission spectrometry(ICP-AES).The assembly process was investigated by Zeta potential and agarose gel electrophoresis on migration properties.Antibody was attached to ABEI-GNPs to obtain anti-hlgG/ABEI-GNPs via electrostatic,hydrophobic and weak covalent interactions.Then BSA was used to block the non-specific adsorption of proteins on the surfaces of GNPs to form BSA/antibody/ABEI-GNPs.Finally,the catalyst Co2+ was further coated onto BSA/antibody/ABEI-GNPs through the coordination of the amino and carboxyl groups of BSA to form Co2+-BSA/antibody/ABEI-GNPs nanocomposites.The synthesis conditions of the Co2+-BSA/antibody/ABEI-GNPs nanocomposites were optimized.The CL behavior of nanocomposites at different assembly stages was studied and the mechanism of the strong CL emission of Co2+-BSA/antibody/ABEI-GNPs was clarified.Co2+-BSA/antibody/ABEI-GNPs nanocomposites showed unique CL emission,which was 300 times the CL intensity of ABEI-GNPs.It was suggested that Co2+ could catalyze the decomposition of H2O2,facilitating the formation of OH·-and O2·-,accelerating the CL reaction.-COO-of BSA could react with O2·-,producing-CO4·2-,which further accelerated the CL reaction.Besides,GNPs as nanosized platform could also promote radical generation and electron transfer in CL reactions.Accordingly,the excellent CL efficiency of Co2+-BSA/anti-hIgG/ABEI-AuNPs nanocomposites resulted from the synergetic effect of BSA,Co2+,and GNPs on the ABEI-H2O2 reaction.The MF-GNPs could respond to antigens of different levels via antigen-antibody specific bindings.In the absence of hIgG,strong CL emission was observed.However,CL intensity decreased in the presence of hIgG.The mechanism of quenching effects on CL signal was discussed.The immunoreaction led to the aggregation of MF-GNPs,which decreased the distance of interparticles,enhancing the intra-and interparticlc quenching effects of GNPs on the CL emission of the oxidation products of ABEI molecules on their surfaces.Based on the decreased CL intensity,a label-free CL immunosensor for the detection of antigens was established.Taking hIgG as a model of antigen,corresponding antibody anti-hlgG was used as the recognition element.The experimental conditions were optimized and the analytical performance for detecting hIgG was investigated.The linear range is from 1.0 fM to 1.0 nM and the detection limit is 0.13 fM.The immunosensor with a wide linear range and low detection limit showed good stability and selectivity and could be applied to real human serum samples.This label-free CL immunosensor has been demonstrated to be a general strategy by replacing hIgG with H-FABP.The detection limit is 7.8 fM and selectivity of this proposed immunoassay is also good.The immunosensor presents a promising application potential in the field of clinical diagnosis.