Chemiluminescence Functionalized Nanomaterials And Their Applications In Chemiluminescence Immunoassays

In this dissertation, the state of arts in the field of chemiluminescence (CL), chemiluminescence immunoassays (CLIA), chemiluminescence functionalized nanomaterials and their applications in chemiluminescence immunoassays were reviewed. The development of chemiluminescence functionalized nanoparticles (CF-NPs) is a new trend with innovative concepts in nanoscience, which makes hundreds of CL signal-generating molecules be coated on or encapsulated in a nanoparticle host. The emergence of nanotechnology is opening new horizons for highly sensitive CLIA. However, limited CF-NPs have been reported. Most of the reported CF-NPs focuses on the use of Ruthenium complex, QD and enzyme as signal-generating molecules. CF-NPs based on the luminol type CL reagents have been rarely reported. Therefore, it is an important research subject to expand the signal-generating molecules, to find appropriate nanomaterials as host, to explore novel method for the synthesis of CF-NPs with good CL activities, and to further develop ultrasensitive CLIA based on the novel materials. The aim of this dissertation is to explore the synthesis of new CF-NPs and their applications in immunoassays. Two kinds of CL functionalized graphene-based nano-composites were synthesized. The CL and electrochemiluminescence (ECL) activities of these nanomaterials were investigated. Novel immunosensor protocols were designed by using CF-NPs to build nano-probe or CL functionalized nano-interface. A sandwich-type ECL immunosensor and a label-free ECL immunosensor were developed. The main results are as follows:1. A novel nanoparticle-based ECL immunosensor was designed for highly sensitive and selective detection of human Cardiac troponin I (cTnI), an important Acute Myocardial Infarction (AMI)-related biomarker, by using N-(aminobutyl)-N-(ethylisoluminol)-functionalized gold nanoparticles (ABEI-AuNPs) as labels. ABEI-AuNPs were successfully synthesized via a simple seed growth method. A great number of luminescence molecules ABEI as stabilizers were coated on the surface of the AuNPs, which exhibited better ECL activities than previously reported luminol functionalized gold nanoparticles. ABEI-AuNPs were used as new ECL labels to build bio-probes by conjugation with secondary antibodies, which showed good ECL activity, immunological activity, and stability. Another kind of AuNPs functionalized with streptavidin was modified on the electrode surface for biotinylated antibodies capture through the specific interaction of biotin/streptavidin and enhancing the electrical connectivity. By combining with the novel ECL labels and amplification of AuNPs and biotin-streptavidin system, a high sensitive sandwich-type electrochemiluminescence immunoassay was developed for detecting human cTnI with a low detection limit of2.0pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of cTnI in real samples, which is of great potential application in clinical analysis. Importantly, the sensitive detection would have far more diagnostic value than would absolute measurements during the early stage of AMI.2. A bottom-up approach for preparing multifunctional grapheme-based materials noncovalently functionalized with CL reagents with aromatic rings such as ABEI, luminol and isoluminol is reported. X-ray photoelectron spectroscopy, FT-infrared, Raman, and X-ray diffraction, UV-visible spectroscopy, and atomic force microscopy were used to characterize the morphology, surface component and surface state of the as-prepared nano-composites. The results indicated that CL reagents with aromatic rings serve a dual purpose during the process:initially as a reductant to convert the exfoliated GO to graphene (CCG), and further as a stabilizer to be adsorbed on the basal planes of the as-prepared CCG nanosheets via a noncovalent π-π stacking mechanism. These nano-composites exhibit good CL activity, high solubility and stability in water. This work has discovered a bottom-up approach for preparing multifunctional graphene-based materials with CL activity. It is possible to extend the present approach for the synthesis of various chemiluminescent functionalized graphene composites by using other CL reagents with aromatic rings. Furthermore, based on the novel CL property of grapheme-based materials functionalized with ABEI (ABEI-CCG), we have developed a CL sensor for the detection of H2O2to demonstrate its direct usage. The unique properties of the as-prepared nano-composites offer excellent prospects in bioassays.3. An advanced label-free ECL immunosensor is proposed for the sensitive detection of human IgG (hIgG) by using ABEI-CCG to build chemiluminescent functionalized nano-interface. The immunosensor was prepared via self-assembly technique as follows:ABEI-CCG is first assembled onto an indium tin oxide (ITO)-coated glass slide to afford chemiluminescent functionalized nano-interface. Positively charged chitosan is then electrostaticly adsorbed to the chemiluminescent functionalized nano-interface, which also acts as the binding linker for negatively charged antibody coated gold nanoparticles. After blocking with bovine serum albumin (BSA), the electrode could be used as an ECL immunosensor for the detection of hIgG. The modification procedure is monitored by electrochemical impedance spectroscopy and ECL behavior, respectively. It demonstrates that the chemiluminescent functionalized nano-interface shows high ECL intensity and good electronic conductivity, and the gold nanoparticles could further enhance the ECL intensity, all of which holds great promise for the fabrication of ECL immunosensors with improved sensitivity. The as-prepared label-free ECL immunosensor has an extremely sensitive response to hIgG in a linear range of1.0×10-13～1.0×10-8g/mL with a detection limit of5.0×10-14g/mL. It also shows good precision, acceptable stability and reproducibility. This research provides a promising technique for protein detection, which may open up a new avenue for the ultrasensitive label-free Immunoassays.4. An one-pot method for preparing ABEI functionalized gold nanoparticles/graphene oxide (ABEI-AuNPs/GO) nano-composites is proposed by in situ reduction of AuCl4-ions with chemiluminescent reagent ABEI in the presence of GO as templates. The obtained nano-composites were characterized by transmission electron microscopy, UV-visible spectroscopy, X-ray photoelectron spectroscopy, Raman, and X-ray diffraction. The results demonstrate that the AuNPs with an average diameter of about10nm are uniformly dispersed on the surfaces of GO nanosheets. During the process, ABEI molecules are served as both reductants and stabilizers, and thousands of ABEI molecules are immobilized on the as-prepared nano-composites, which endows the nano-composites with good chemiluminescence and electrochemiluminescence activities. Due to the good electronic conductivity and biocompatibility, large surface area, unique catalytic activity, and combining with the novel CL and ECL activities, the nano-composites may find future applications in CL/ECL biosensors.