Porphyrin Based Highly Efficient Chemiluminescent Immunoassay

As a promising approach for selective and sensitive analysis, immunoassay especially chemiluminescent immunoassay has been further developed. Enzyme-tag chemiluminescent immunoassay has been challenged for their easily-lost activity and weak stability. It is necessary to develop a series of mimicking enzymes with better stability. Porphyrins, especially metalloporphyrins, as catalytic centers of lots of enzymes, are potential candidates for mimicking enzymes. In chemiluminescent systems, solvent metalloporphyrins, such as manganese porphyrin and iron porphyrin, possess excellent catalytic activity of peroxidase. This thesis focused on the preparation of porphyrin-related probes and the development of highly efficient immunoassay s for ultrasensitive and high throughput detection of cancer markers.1. Manganese porphyrin-dsDNA complex:a mimicking enzyme for highly efficient bioanalysisBased on the catalysis of porphyrins and the interaction between porphyrin and dsDNA, we combined DNA amplification techniques with immune-reactions for preparing porphyrin-dsDNA complex. Manganese porphyrin (MnTMPyP)-dsDNA complex was reported as an excellent mimicking enzyme of peroxidase. It possessed high catalytic activity and much quicker catalytic kinetics and better stability with exposure to light irradiation and high temperature than both horseradish peroxidase and hemin/G-quadruplex DNAzyme. The groove binding of MnTMPyP to the dsDNA scaffold efficiently maintained the catalytic activity of the MnTMPyP center and improved its stability. By combining with an isothermal hybridization chain reaction (HCR) and in situ formation of MnTMPyP-dsDNA, a highly efficient chemiluminescent (CL) immunosensing method was proposed. After a sandwich immunoreaction, a biotinylated DNA strand, which was bound to biotinylated signal antibody by streptavidin, triggered the HCR and growth of MnTMPyP-dsDNA on the immunocomplex. The in situ, HCR-assisted enzyme formation brought numerous enzymatic catalytic centers, MnTMPyP, on the immunocomplex, resulting in significant CL signal amplification and highly sensitive CL detection. Using carcinoembryonic antigen as the model target, the proposed CL immunoassay method showed a wide linear range from10pg/mL to100ng/mL with a detection limit of6.8pg/mL. The new MnTMPyP-dsDNA complex could be conveniently synthesized, functionalized, and combined with DNA amplification strategies, showing a promising potential in bioanalysis and other relative fields.2. Manganese porphyrin based chemiluminescent generating nanoreactor for imaging immunoassay of multiple biomarker using microarray.Multi-analyte immunoassay possesses some remarkable advantages, such as high sample throughput, improved assay efficiency, low sample consumption and reduced overall cost per assay. They have potential application in simultaneous quantitative analysis of tumor markers. However, the development of chemiluminescent microarray has been challenged due to the poor chemiluminescent intensity. This work focus on the synthesis of manganese porphyrin based chemiluminescent generating nanoreactor and construction of a highly effictient protein microarray for CL imaging immunoassay of multiple biomarker and cancer screening. The proposed micro-array based CL assay was simple, high sample throughput and sensitive, showing good potential application in the point of care detection of biomarkers and large scale screening of cancers in early age.