Chemiluminescent Immunoassay For Disease Biomarker And Pathogenic Bacterium Detection

Chemiluminescent immunoassay (CLIA) is a promising analytical technique combining chemiluminescence (CL) with conventional immunoassay, which opens up new pathway for pharmaceutical analysis, bioanalysis and clinical chemistry, due to its high sensitivity, ideal selectivity, simple manipulation, rapid detection and possibility of assaying complex samples without time-consuming pre-treatments.Extensive research interests and great efforts have already been focused on disease biomarkers and pathogenic bacteria detection. Herein, a novel chemiluminescence resonance energy transfer (CRET)-based immunoassay and double-site recognition-based CL sandwich pseudo-immunoassay were developed for disease biomarker and pathogenic bacterium detection, respectively.Part1Resonance energy transfer-based chemiluminescent immunoassay of transferrinAmorphous carbon nanoparticles (ACNPs) showing highly efficient quenching of CL were prepared from candle soot with a very simple protocol. The prepared ACNP was employed as the novel energy acceptor for a CRET-based immunoassay. In this work, ACNP was linked with transferrin (TRF), and horseradish peroxidase (HRP) was conjugated to TRF antibody (HRP-anti-TRF). The immunoreaction rendered the distance between the ACNP acceptor and the HRP-catalyzed CL emitter to be short enough for CRET occurring. In the presence of TRF, this antigen competed with ACNP-TRF for HRP-anti-TRF, thus led to the decreased occurrence of CRET. A linear range of20-400ng mL-1and a limit of detection of20ng mL-1were obtained in this immunoassay. The proposed method was successfully applied for detection of TRF levels in human sera, and the results were in good agreement with ELISA method. Moreover, the ACNPs show higher energy transfer efficiency than other conventional nano-scaled energy acceptors such as graphene oxide in CRET assay. It is anticipated that this approach can be developed for determination of other analytes with low cost, simple manipulation and high specificity.Part2Double-site recognition-based chemiluminescent sandwich pseudo-immunoassay of Staphylococcus aureusA novel CL sandwich pseudo-immunoassay based on double-site recognition by antibiotic and IgG was developed for the directly determination of pathogenic bacterium. In this protocol, HRP-labeled vancomycin was utilized as the molecure recognition agent to anchor the cell of Staphylococcus aureus. Also, immobilized IgG was adopted to bind the protein A to Staphylococcus aureus to develop a sandwich pseudo-immunoassay for Staphylococcus aureus. A linear range of1.0x103-2.0×106CFU mL-1and a limit of detection of330CFU mL-1(S/N=3) were obtained. This proposed method shows a series of advantages such as high sensitivity, ideal selectivity, simple manipulation, rapid detection and low cost. Thus, it opens up new pathway for clinical disease screening and early diagnostic application.