Investigation On Acridinium Ester Chemiluminescence System And Capillary Electrophoresis Coupled With Chemiluminescence

Chemiluminescence (CL) is a powerful detection technique due to its extremely high sensitivity, simple instrument and easy automation. It has been widely applied to various fields, such as environmental monitoring, pharmaceutical assays, clinical diagnosis and biotechnology. However, to take full advantage of the benefits offered by CL assay, it is necessary to explore the effective approaches to improve its selectivity, and look for new CL reactions to increase the applied value of CL method. In recent years, the study of CL has been extended to nanoparticle system, which is of significance to enhance CL efficiency and establish new CL methods. The combination of CL detection with capillary electrophoresis (CE) as separation technique has improved the CL selectivity effectively. The efforts to develop and improve CE-CL interface will promote the advancement of CE-CL and its applications.This investigation mainly focuses on two aspects. One is to introduce gold nanoparticles (AuNPs) into acridinium ester (AE) CL system, and study the superquenching effect and its analytical applications. The other is to design and fabricate CE-CL interface for maintaining the high separation efficiency and high sensitivity simultaneously.1) The AuNPs involved AE CL system were investigated with the flow injection. It was found that the chemiluminescence of AE was quenched effectively by AuNPs with Stern-Volmer constants approaching 1010M-1. The superquenching was ascribed to the strong binding affinities of AuNPs to AE molecules and efficient nonradiative energy transfer from N-methylacridone* to AuNPs. The DNA-modified AuNPs are also highly efficient chemiluminescent nanoquenchers of the AE system, providing a potential alternative to the luminol system to detect AuNPs labels.2) The AuNPs quenching of AE CL was exploited to realize sequence-specific DNA detection, which was based on the differential adsorption of single-stranded DNA and double-stranded DNA on the AuNPs. In the absence of target DNA or in the presence of noncomplementary DNA, the AE labeled ssDNA probe was adsorbed on the AuNPs, and the proximity of AE with AuNPs resulted in the CL quenching. However, in the presence of target DNA, the hybrids between the probe and target could not be adsorbed on AuNPs. When the CL reaction was initiated, the CL of the hybrids remained. This DNA detection method is simple, rapid, and is also straightforward to distinguish single-base mismatch. 3) An on-column fracture/end-column reaction interface for CE-CL was developed to optimize the operating conditions for CE separation and CL detection independently. The interface consists of an on-column fracture being installed in a reservoir near the capillary end to create an electrical connection and also serve as reagent addition entrance. The capillary terminal was inserted into an end-column reservoir for CL reaction and detection. The additives flow with the post-column running buffer propelled by gravity and mix with the CL substrates in the detection cell to produce CL signal. The assembly is capable of adding two or more reagents through the fracture and the reservoir. The application potential this system was demonstrated by luminol CL system. Peroxide hydrogen was introduced stably in HNO3 solution through the fracture. The comparable sensitivity and separation efficiency were achieved without the need to add H2O2 or catalyst into CE buffer solution.4) The applicability of this interface was further evaluated by using AE CL system. The on-column fracture was dipped in buffer reservoir to create electrical connection while the capillary terminal was placed into an extra end-column cell for CL reaction and detection. The backflow migration of anions (-OH, HO2-) towards anode, a phenomenon accounting for the suppression of AE CL has been effective eliminated. The pH adjustment was achieved by the addition of HNO3 through fracture prior to detection while the separation finished. Thus, the pH range of CE buffer can be independent to the optimal pH value of AE CL reaction, which is usually below 3. As the model anlytes, the AE derivatives of lysine and sarcosine could be separated at pH 8.7 with the same detection limits of 0.45 nM (S/N=3), and theoretical plate numbers of 40 000-56 000,70 000-100 000, respectively.