Study Of New Electrogenerated Chemiluminescence Method

This thesis included a review and a research section. In the review section, the basic principles, characteristics, main reaction systems and the mechanism of electrogenerated che mi luminescence (ECL) were introduced. Moreover, a general introduction was proposed about the ECL coupled with some other techniques as well as their applications in pharmaceutical analysis. In addition, the physiological significance of protein kinase was introduced, and a general introduction was proposed about the developed of the methods for the detection of protein kinase activities.The research section contained the following subunits:1. For the first time, a simple and ’green’ approach based on one-step strategy was designed and developed for the modification of a fused-silica capillary with polynorepinephrine (PNE) to separate amino acid enantiomers using capillary electrochromatography coupled with electrogenerated chemiluminescence detection (CE-ECL). The formation of the PNE coating was characterized by scanning electron microscopy, UV-vis spectra and contact angle measurements. Compared with the native capillary, the modified capillary had much better wettability, less nonspecific adsorption toward amino acids, and the enantiomers of histidine, phenylalanine, and valine samples received baseline separation with the resolution factors of1.6,1.8and1.6, respectively.2. Herein, we present a novel electrogenerated chemiluminescence (ECL) biosensor for monitoring the activity and inhibition of protein kinases based on signal amplification of the enzyme-functionalized Au NPs nanoprobe and mediated signal transition of biotin-avidin system. In this design, nanoprobes were prepared by conjugating Au NPs with Biotin-DNA and glucose oxide (GOx). Meanwhile, biotinylated antibody would bind the phosphoserine site due to the specific antibodyantigen interaction. With the biotin-avidin system, the nanoprobes could conveniently be bound to the phosphoserine site to the electrode surface, and then, the glucose oxidase (GOx) assembled on the nanoprobe can catalyze glucose to generate H2O2when the ECL reaction occurred in the luminol ECL biosensor. Therefore the activity of protein kinases could be monitored by ECL with high sensitivity due to the high amount of GOx. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the proof-of-concept strategy. The as-proposed biosensor presented high sensitivity, low detection limit of0.013U/mL, wide linear range (from0.02to40U/mL), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. 3. For the first time, we developed a novel and versatile ECL-RET protocol in the ECL biosensor through the energy transfer interaction between graphene oxide (GO) and graphene quantum dots (GQDs), and demonstrated its feasibility in sensitive detection of protein kinases activity and inhibition. In our design, monoclonal anti-phosphoserine-conjugated GO as an acceptor and GQDs as donors were chosen to fabricate an ECL-RET bio sensor for detecting protein kinases activity and inhibition. The antibody-conjugated GO would bind to the phosphoserine site on the electrode due to the specific antibody-antigen interaction, which bring GO and GQDs into ECL-RET proximity to facilitate the luminescence quenching of GQDs based on ECL-RET. Therefore the activity of protein kinases could be monitored by ECL-RET. Casein kinase2(CK2) was chosen for the study because its activity promotes and regulates the cellular activity of other proteins such as Wnt, nuclear factor-κB, and phosphatidylinositol3-kinase. The as-proposed ECL-RET biosensor presented high sensitivity, low detection limit of0.023U/mL, wide linear range (from0.01to5U/mL), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC50of ellagic acid, a CK2inhibitor, was estimated. It is expected that this novel strategy will open new opportunities for development of the ECL-RET technique and promote the application of carbon-based nanomaterials in bioassays.