Construction Of Electrochemiluminescence Immunosensor Based On Nanocomposites And Signal Amplified Strategy

Electrochemiluminescence?ECL?immunosensor as an ingenious combination of ECL technology and immunoassay,has achieved sensitive detection of protein markers and given tremendous utilization potentiality to the early diagnosis of disease.With ECL biosensing field making concerted effort,designing strategy of signal amplification is the key point for highly sensitive detection of target with trace amount.Recently,following the spring up of nanotechnology in various areas,nanomaterials of multiple and intrinsic superiority,such as biocompatibility,high specific surface area,electrocatalytic activity and so on,have bocome effective means for researchers to realize the high output signals.Accordingly,in this thesis,improvements have been made to endow nanocomposites with multifunction for the purpose of abtaining amplified ECL signal.Besides,simplifying operation process and improving efficiency are also of necessity.Based upon above,various functionalized nanocomposites including functionalized Pt@Cu hierarchical trigonal bipyramid nanoframes,luminescent functionalized Au@Ag nanochains with self-enhanced property and conjugated polymer dots dual-amplified luminescence emitter are synthesized and implemented.Moreover,the detailed reacted mechanisms of coreactant and catalyzer for ECL system are demonstrated.In asisitance of various amplified strategies of functionalized nanocomposites,several ECL immunosensing platforms are developed for the quantitative detection of different protein markers with sufficiently good performance.The main results are divided into the following sections:1.The research on electrochemiluminescence immunosensor based on ABEI and FCA functionalized Platinum@Copper hierarchical trigonal bipyramid nanoframes.Compared to monometallic and entitative nanomaterials,the bimetallic counterparts with hierarchical structure have the superior performance in stability and electrocatalytic activity.In this work,platinum@copper hierarchical trigonal bipyramid nanoframes?Pt@Cu HNFs?are successfully synthesized through a facile chemical reduction method to serve as the loading platform of luminophore N-?aminobutyl?-N-?ethylisoluminol??ABEI?and its catalyzer ferrocenecarboxylic acid?FCA?.Thereinto,FCA is a novel coreaction accelerator to promote the decomposition of coreactant?hydrogen peroxide,H₂O₂?,and then efficiently catalyze the luminous reaction of ABEI/H₂O₂ system,which shows more stable catalytic ability than conventional enzymes.The unique hierarchical structure of Pt@Cu HNFs consists of multitudinous interlinked branches nano-units,and their nobel metal component?Pt?possess special catalytic ability for H₂O₂,which make themselves possess high electrocatalytic activity and stability to accelerate the decomposition of H₂O₂.Thus,Pt@Cu HNFs and FCA both as enhancer could form a synergistical catalysis for the ABEI/H₂O₂ syangystem,obtaining dramaticly amplified response signals in ECL reaction.Moreover,the immobilization of abundant luminophore and catalyzer means a shortened spatial distance between reagent and electrode,which is in favour of reducing the adverse effect of steric hindrance on ECL reaction efficiency.Based on functionalized Pt@Cu HNFs and immunoreaction,a sandwiched immunosensor is proposed to sensitively detect mucin-1?MUC1?with desirable results of wide linear range from 10 fg mL^-1 to 10 ng mL^-1,and realizes the detection in human serum samples,making this approach hold great potential for other protein analysis.2.The research on electrochemiluminescence immunosensor based on nanochains in situ prepared by self-enhanced luminescent reagent.Conventionally,the enhancement effect of coreactant to luminescent agents is mainly based on their intermolecular interactions,which is inevitably accompanied with energy loss caused by spatial distance and thus limits the reaction efficiency.To avoid the energy loss of intermolecular interactions,a novel ECL reagent?ABEI-PEI?with self-enhanced property is prepared through simply crosslinking luminophore?ABEI?and coreactant?polyethylenimine,PEI?inside one molecule,leading to significantly shortened electron-transfer path and efficient intramolecular promotion from PEI to ABEI.Besides,via amine-rich ABEI-PEI as reductant and stabilizer to react with HAuCl₄ and AgNO₃,Au@Ag nanochains?Au@AgNCs?are in situ prepared along the lineament of ABEI-PEI to obtain the luminescent functionalized nanostructure,which not only achieves the immobilization of massive ECL reagent,but also gives play to the catalysis of precious metal constituent.More notably,nanochains prepared by ABEI-PEI are fully wrapped with cations that could adsorb catalyzer CO²⁺with the coordination of anionic entities?polyacrylic acid,PAA?,obtaining a mulriple amplified ECL signal.Using the functionalized nanochains with high large specific surface area and biocompatibility to immobilize the secondary antibody,a highly sensitive ECL immunosensor is contructed for the measurement of?2-microglobulin??2-MG?in human urine samples to explore the potential of analysis in clinical assays.This new-type luminescent functionalized nanocomposites possessing high luminous efficiency,self-enhancement,catalytic ability could provide new thought for the application of ECL technology in bioanalysis aspect.3.The research on sensitive electrochemiluminescence immunosensor based on ABEI/H2O2 system with the dual-amplifed effect of PFO dots.In this work,we explores an appealing phenomenon that PFO dots have an obvious enhancement effect for the ECL intensity of ABEI.Meanwhile,PFO dots could react like mimic peroxidases to catalyze H₂O₂ for further amplifying the ECL signal of ABEI.This dual amplified effect incorporated by PFO dots achieves preferable luminous efficiency of ABEI/H₂O₂ system,which is serviceable to improve the detection sensitivity of biosensors in aspect of trace analysis.Owing to the electronegative property of PFO dots,electropositive ABEI-PEI as ECL reagent could be adsorbed on their surface and thus form a novel luminescence emitter?ABEI-PEI-PFO dots?with high luminous efficiency.Meanwhile,the amine-rich property of the ECL reagent eliminates the influence of high hydrophobicity of PFO dots and gives favourable hydrophilicity to the obtained emitter,which is beneficial for the stable and efficient ECL reaction in aqueous solution.Furthermore,by virtue of excellent electroconductibility of graphene and nobel metal nanostructure,RGOs/PtNPs via a onepot synthetic strategy are chosen as an ideal immobilizing platforms for the ECL emitter.Herein,the obtained dual-amplifed nano-complex is served as an ideal nanocarrier to capture detection antibody?Ab₂?.According to sandwiched immunoreaction,a highly sensitive ECL immunosensor is constructed for the detection of kidney injury molecule-1?KIM-1?,with a linearity from 50 fg mL^-1 to 1 ng mL^-1 and a detection limit of 16.7 fg mL^-1.The developed ECL emitter containing dual amplified property has the merits of effectiveness,simplicity,and water stability.Meanwhile,RGOs/PtNPs could admirably accelerate the electron transfer to attain a further enhanced luminous efficiency.The proposed nanocomposites sharply enhance the ECL signal and broaden the application of ABEI/H₂O₂ system in ECL assays.