Electrochemiluminescence Immunoassay Methods Based On Biological Barcode Mode And Its Applications

In the thesis, electrochemiluminescence immunoassay (ECLIA), biological barcode assay(BBA) and their applications are summarized systematically. The nanomaterials includinggraphene and gold nanoparticles and so on, are widely used in the construction of immunosensordue to their good conductivity, good biocompatibility and large surface area. BBA has highsensitivity, good specificity and wide detection range, so it is widely used in the detection ofprotein, nucleic acid and so on. In this research work, a series of ECL immunosensors forultrasensitive determination of tumor markers of ultra-low concentrations in the human body weredeveloped by combining BBA and ECLIA with nanomaterials. The major contents are describedasfollows:1. A sandwich-type label-free electrochemiluminescence immunosensor for neurotensinbasedon sombreromodel withgraphene-hyaluronate-luminolcompositeAn ultrasensitive sandwich-type label-free electrochemiluminescence (ECL) immunosensorbased on sombrero model for the detection of neurotensin (NT) in human serum and urine wasdeveloped. Graphene-hyaluronate-luminol (G-HY-luminol) composite was coated on the surfaceof the glassy carbon electrode to build the ECL immunosensor, which gave in-situ ECL signals byluminol. A secondary NT unlabelled antibody was used to form the final sandwichimmunocomplex as a sombrero model, facilitating reduction of the ECL intensity to improve thesensitivity of detection significantly. Results show that the inhibition of ECL intensity increasedwith thelogarithm of NT concentration withina widelinearrange from0.001pg/mL to100pg/mL.In addition, specificity, stability, reproducibility, regeneration and application were satisfactory.Therefore, this developed ECL immunosensor has a potential for practical detection of smallmolecularweight NT andNT-likepeptideswith in theclinical diagnostics. 2.Electrochemiluminescenceimmunosensorfortumormarkersbasedonbiologicalbarcodemodewithconductive nanospheresA novel sandwich-type electrochemiluminescence (ECL) immunosensor was developed forhighly sensitive and selective determination of tumor markers based on biological barcode mode.N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and the second antibody (Ab2) were simultaneouslyimmobilized on conductive nanospheres to construct ABEI/Ab2-CNSs probes, which could formsandwich immunocomplex by Ab2and emit ECL signals by ABEI. The gold layer coated on thesurface of the conductive nanospheres could extend the outer Helmholtz plane of the ECLimmunosensor effectively. Benefited from it, all ABEI molecules immobilized on conductivenanospheres would act as biological barcode to give in-situ ECL signals without interfering withthe activity of the second antibody. In such a case, the sensitivity of the ECL immunosensor wouldbe greatly improved because an antigen molecule would correspond to ECL signals of thousandsof ABEI molecules. Using prostate specific antigen (PSA) as a model tumor marker, the ECLintensity was found to increase with the logarithm of PSA concentration with a wide linear rangefrom0.04to10fg/mL. In addition, specificity, stability, reproducibility, regeneration andapplication were satisfactory. Therefore, this developed ECL immunosensor has a potential forpracticaldetectionof disease-relatedproteins besides tumormarkersin theclinical diagnostics.3. Electrochemiluminescenceimmunosensorforprostatespecificantigen(PSA) basedonimmunomagneticbeadsandmagneticelectrodeA sanwhich-type ECL immunosensor was developed for highly sensitive determination ofPSA based on immunomagnetic beads and magnetic electrode. ABEI and Ab2weresimultaneously immobilized on conductive nanospheres to construct ABEI/Ab2-CNSs probes,which could emit ECL signals by ABEI. Sandwich-type immunocomplex were constructed bybiotin modified Ab1, PSA and ABEI/Ab2-CNSs probe through immunoreaction, which werebinded to streptavidin functionalized magnetic beads by biotin-streptavidin linkage. Finally, ECLimmunosensor was assembled successfully through the magnetic effect. The ECL intensity wasfound to increase with the logarithm of PSAconcentration with a wide linear range from0.04to10 fg/mL, with a linear correlation coefficient0.9952. In addition, stability, specificity, repeatabilityand reproducibility were acceptable. Therefore, the ECL immunosensor has the potential value inclinicaldiagnosis.