Research Of Cluster Enzyme Lables And Their Application In Chemiluminescence Immunoassay

This dissertation focuses on the development of a new strategy for preparing cluster enzyme labels using nanoparticles as carriers and their application in chemiluminescence immunoassay (CLIA). CLIA was used more widely among immunoassays, because it simultaneously has chemiluminescence method of high sensitivity and immunoassay of high selectivity. And the immunoassay based on nanoparticles as cluster enzyme labels is a new technique that bears a lot of advantages. In our research, we combined these two techniques and developed a novel technique. The thesis consists of two chapters:In chapter one, we reviewed the development of chemiluminescence immunoassay, emphatically summarized the application of the cluster markers in the immunoassay.In chapter two:Research reports which are composed of four components as follows:1. The synthesis characterization of the mesoporous silica nanoparticles.A new kind of mesoporous silica nanoparticles was synthesized and its morphology and size was characterized by TEM. The TEM image of the mesoporous silica nanoparticles showed that the nanoparticles were 70±10nm in diameter and uniform without aggregation. The results demonstrated that our nanopaticles were suited to use as enzyme, antibody and other biological samples labeling carriers.2. An ultra-sensitive chemiluminescence immunosensor of carcinoembryonic antigen using HRP-functionalized mesoporous silica nanoparticles as cluster enzyme labelsA novel chemiluminescence immunosensor using horseradish peroxidase HRP-functionalized mesoporous silica nanoparticles (MSN) as cluster enzyme labels was developed, which increases the sensitivity of the chemiluminescence immunoassay (CLIA). The enzyme-functionalized MSN were fabricated by simultaneous co-immobilization of HRP and the carcinoembryonic antigen antibody (anti-CEA) onto the surface of MSN using 3-aminopropyltriethoxysilane (APTES) as the linkage. Because the large surface area of MSN carriers increased the amount of HRP bound per sandwiched immunoreaction and the proportion between the enzyme and antibody molecules labeling on the surface of MSN were optimized, the conjugates provided a much higher signal and increased sensitivity. The results showed that this approach was demonstrated as a simple, cost-effective, specific, and potent method to detect CEA in practical samples. The sensitivity of the immunosensor using MSN-HRP-Ab2 as labels was about 10-fold higher than that of traditional labels.3. Determination of staphylococcal enterotoxin B by chemiluminescence immunoassay using mesoporous silica nanoparticles as cluster enzyme labelsA novel chemiluminescence immunoassay using horseradish peroxidase (HRP)-functionalized mesoporous silica nanoparticles (MSN) as cluster enzyme labels was developed, which increases the sensitivity of the chemiluminescence immunoassay (CLIA). The enzyme-functionalized MSN were fabricated by simultaneous co-immobilization of HRP and the anti-Staphylococcal Enterotoxin B (Ab2) onto the surface of MSN using 3-aminopropyltriethoxysilane (APTES) as the linkage. The conjugates provided a much higher signal and sensitivity through optimizing the proportion between the enzyme and antibody molecules labeling on the surface of mesoporous silica nanoparticles. This approach was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect SEB in practical samples. The method applies to all biological sample analysis and detection.4. Determination of Clenbuterol Residual in Pork by Flow Injection Chemiluminescence ImmunoassayA sensitive and rapid immunoassay based on Flow injection chemiluminescence detection has been developed for the determination of Clenbuterol residual in pork. The chemiluminescence reaction of luminal and hydrogen peroxide-Urea adduct catalyzed by horseradish peroxidase (HRP) can be enhanced by 4-Biphenylboronic acid. After competitive immunoreaction, the supernate enzyme labeled compound was detected by flow injection chemiluminescence. The proposed method has advantages on sensitivity, simplicity, reliablity as well as cheap on instrument, and has been successfully applied for determination of clenbuterol residual in pork with satisfactory results, providing a useful monitoring method for food safety.In conclusion, this paper put forward a kind of mesoporous silica nanoparticles based on new cluster enzyme labels and its preparation methods. Compared with conventional method that enzyme molecules were directly coupled to antibody or antigen molecules, the preparation of cluster enzyme labels by using silica nanoparticles as carriers had several advantages. For example, easy to handle and the labeling process was controllable, high repeatability and easy to purify, could enhanced the signal by several times. Therefore, this labeling method has great application value.