Proximity Inducement Strategy For Chemiluminescence And Imaging Immunoassay Methods

Combing the specificity of immunoassay and the sensitivity of chemiluminescence (CL), Chemiluminescence immunoassay (CLIA) possesses the advantages of high sensitivity, high selectivity, low cost, pollution free as well as the feasibility to couple with automation and sensing technology, and has been widely used in clinical diagnosis, biological analysis, environmental monitoring, food security and other fields. The conventional immunoassay methods generally include many steps, such as washing and drying, making it difficult to realize higher throughput. With the capacity of transferring the detection of proteins to nucleic acids, single-analyte immunoassay methods, the proximity inducement strategy could take advantage of varieties of amplification strategies based on DNA to achieve enhanced sensitivity. This thesis introduces proximity inducement strategy to CLIA, focusing on one-step homogeneous assay as well as multiplex CL imaging.1. Proximity hybridization-regulated chemiluminescence resonance energy transfer for homogeneous immunoassayChemiluminescence resonance energy transfer (CRET) and the proximity ligation assay have been widely used in developing sensors for the analysis of biomolecules respectively. Here, a wash-free and homogeneous strategy was proposed to analyze the concentration of carcino-embryonic antigen (CEA) based on proximity hybridization-regulated CRET. As an excellent chemiluminescence (CL) quencher, graphene oxide (GO) was used to produce the "signal off’ mode that little CL emission was observed through CRET between GO and the Cy5-labelled DNA3. Once CEA was introduced, the target-induced proximity hybridization occurred and formed a proximate complex, which inhibited the CRET by taking probe DNA3 away from GO. Meanwhile, taking advantage of nicking endonuclease Nt.BbvCI for in situ recycling, the signal could be further amplified for highly sensitive CL detection. Our results showed that this strategy enabled a specific response to CEA with a detection range of 5 orders of magnitude, along with a detection limit at 3.2 pg mL-1. Apart from its easy operation, high sensitivity and acceptable accuracy, the proposed method needed only 0.3μL sample, indicating its great opportunity for commercial application.2. Construction of chemiluminescence immunoassay microarray based on proximity inducement strategyMultianalyte-immunoassay and sensor-array detection possess some remarkable advantages, such as high sample throughput, improved assay efficiency, low sample consumption and low cost, which have promising practical application in screening and quantitative analysis of tumor markers. However, due to its poor chemiluminescent intensity, the application of chemiluminescent microarray has been limited. We combed the HCR with the proximity inducement strategy to detect protein based on nucleic acids. With enhanced signal, our work focuses on constructing the chemiluminescence immunoassay microarray to realize the screening and analysis of tumor markers in large scale.