| Electrochemiluminescence (ECL) immunosensors is the combination of electrochemiluminescence technology and immunoassay with the merits of easy operation, good selectivity, high sensitivity, rapid detedtion and wide dynamic concentration response range, are very useful and available tools for monitoring the antibody or antigen. The pivotal steps for an ECL immunosensor are the biological recognition system and the singal trancduction between the biorecognition event and detected ECL signal. With the rapid development of nano technology, various nano-materials have been widely applied in the biosensor field and realized sensitive detection due to their unique properties, such as excellect conductivity, large surface areas and good biocompatibility and so on. This thesis is devoted to the construction of sensing interface for the immobilization of antibody and the design nano-probes for the analyte recognization and signal amplification using nanomaterials or nanocomposites. Based on this point, this thesis developed some sensitive ECL immunosensor, and the detailed researchs are described as follows:1. A novel solid-state electrochemiluminescence immunosensor based on MWCNTs-Nafion and Ru(bpy)32+-nano-Pt nanocomposites for detection of a-fetoproteinA new approach was successfully employed for constructing a solid-state electrochemiluminescence (ECL) immunosensor by layer-by-layer self-assembly of multi-wall carbon nano tubes (MWCNTs)-Nafion composite film, Ru(bpy)32+/nano-Pt aggregates (Ru-PtNPs) and Pt nanoparticles (PtNPs). Firstly, Ru-PtNPs were synthesized via electrostatic interaction between the positively-charged Ru(bpy)32+and the negatively-charged Pt nanoparticles. Then, large numbers of Ru-PtNPs were immobilized onto the MWCNTs-Nafion uniform composite film by cation-exchange function and electrostatic interaction. Subsequently, the negatively-charged Pt nanoparticles were adsorbed as crystal seeds onto the positively-charged Ru-PtNPs via electrostatic interaction. Finally, a great quantity of Pt nanoparticles were electrodeposited on crystal seeds, which could be used as an effective antibody-immobilization matrix with excellent stability, large surface area and good biocompatibility. Meanwhile, the influence of Pt nanoparticles on the ECL intensity was quantitatively evaluated by calculating the electro-active surface area of different electrodes with or without Pt nanoparticles to immobilize Ru(bpy)32+. Under the optimal experimental conditions, the results indicated that the immunosensor had good ECL response for AFP. The linear response range was0.01-10ng/mL with the detection limit of3.3pg/mL (S/N=3). The immunosensor exhibited advantages of simple preparation and operation, high sensitivity and good selectivity.2. Ultrasensitive luminol electrochemiluminescence immunosensor for protein detection based on in-situ generated hydrogen peroxide as coreactant with glucose oxidase anchored AuNPs@MWCNTs labelingIn this study, an ultrasensitive luminol electrochemiluminescence (ECL) immunosensor was constructed based on in-situ generated H2O2as coreactant, using carboxyl group functionalized multi-walled carbon nanotubes (MWCNTs) as platform and glucose oxidase (GOD) supported on Au nanoparticles (AuNPs) decorated MWCNTs (AuNPs@MWCNTs-GOD) as labels. Firstly, using poly(ethylenimine)(PEI) as linkage reagents, AuNPs@MWCNTs were prepared and introduced for binding of the secondary antibody (Abi) and glucose oxidase (GOD) with high loading amount and good biological activity due to the improved surface area of AuNPs@MWCNTs and excellent biocompatibility of AuNPs. Then the GOD and Ab2labeled AuNPs@MWCNTs were linked to the electrode surface via sandwich immunoreactions. These localized GOD and AuNPs amplified luminol ECL signals dramatically, which were achieved by efficient catalysis of the GOD towards the oxidation of glucose to in situ generate improved amount of H2O2as coreactant and the enhancement of AuNPs to the ECL reaction of luminol-H2O2. The experimental results demonstrated that the proposed immunosensor exhibited sensitive and stable response for the detection of a-fetoprotein (AFP), ranging from0.0001to80ng/mL with a limit of detection down to0.03pg/mL (S/N=3).3. Amplified cathodic electrochemiluminescence of luminol based on graphene nanocomposites for ultrasensitive detection of proteinAn ultrasensitive electrochemiluminescence (ECL) immunosensor was constructed for ultrasensitive detection of carcinoembryonic antigen (CEA) based on an amplified cathodic ECL of luminol. Firstly, single walled carbon nanotube-graphene composites (CNTs-Gra) was coated onto glass carbon electrode (GCE) to motivated cathodic ECL of luminol. Then, Au nanoparticles (AuNPs) were electrodeposited to the surface of CNTs-Gra composites, with enhanced surface area and good biocompatibility to capture primary antibody (Ab1) and then bind the antigen analytes. Pd and Pt nanoparticles (Pd&PtNPs) decorated reduced graphene oxide (Pd&PtNPs@rGO) and glucose oxidase (GOD) labeled secondary antibody (Pd&PtNPs@rGO-GOD-Ab2) could be captured onto the electrode surface by a sandwich immunoassay protocol to generate an amplified cathodic ECL signals of luminol in presence of glucose. The Pd&PtNPs@rGO composites and loaded GOD promoted luminol cathodic ECL response by efficiently catalyzing glucose to in-situ produce amount of hydrogen peroxide (H2O2). Then in turn Pd&PtNPs catalyzed H2O2to generate various reactive oxygen species (ROSs), which accelerated the cathodic ECL reaction of luminol, enhanced the cathodic ECL intensity of luminol and improved the sensitivity of the immunosensor. The as-proposed ECL immunosensor exhibited sensitive response on the detection of CEA ranging from0.0001to160ng/mL with a detection limit of0.03pg/mL (S/N=3). Moreover, the stability, specificity, and reproducibility tests demonstrated the feasibility of the developed immunoassay, which can be further extended to the detection of other disease biomarkers. |
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