The Establishment Of Enzyme Inhibition And Fluorescence Quenching Systems And Their Applications In The Determination Of Organophosphorus Compounds

Organophosphorus compounds (OPs) have been widely used as pesticides and chemical warfare agents. They are toxic to mammals, especially when ingested, inhaled, or absorbed through the skin. As a matter of fact, OPs are irreversible inhibitors of well-known esterase phosphorylation in the central nerve system. The primary enzyme inhibited by OPs is the acetylcholinesterase (AChE). AChE can catalyze the hydrolysis of acetylcholine (ACh), when the AChE activity is inhibited, the accumulation of ACh results in transmitting nerve impulses frustration, leading to serious health problems or even ultimately death. Paraoxon is a typical OPs, which can inhibit the AChE activity.Numerous analytical methods have been used for the detection of OP compounds, including gas chromatography-mass spectrum (GC-MS), high performance liquid chromatography (HPLC), electrochemicalmethod and immunoassay. GC-MS, HPLC and electrical techniques are all sensitive and reliable methods, however, they are time-consuming and expensive. Immunoassay is an effective method with high sensitivity, selectivity and reproducibility, but it needs corresponding antibody. It is believed that enzymatic bioassay has the ability to overcome these problems.In this thesis, different fluorescence quenching systems have been systematic studied. In addition, we have successfully applied these systems to OPs detection including the quantification of paraoxon.The first chapter is an introduction part to summarize the enzyme inhibition, fluorescence quenching and the researches of fluorescent probes. The aspects of OPs detection using enzyme inhibition and fluorescence quenching have been described in detail, including qualitative and quantitative detection, dynamic analysis, theories of reaction and so on.In the second chapter, the system realized by combination enzyme inhibition with fluorescence quenching has been detailed studied. By binding to OP compounds, AChE is irreversibly inhibited and forms the phosphorylated and unreactive enzyme. And the fluorescent compound DACM-TCh decreases. In the process of enzyme inhibition, p-nitrophenol was produced which could lead to the fluorescence quenching of DACM-TCh. Therefore, OPs could be detected without separating from the real samples and we quantitatively detected paraoxon. Meanwhile, we proved both of the enzyme inhibition and fluorescence quenching occured in the system. To study the interaction, different methods have been utilized and then the experimental phenomena have been reasonably interpreted.In the third chapter, the system combined enzyme inhibition with gold nanoparticle enlargement has been studied in detail. In this system, AChE can catalyze the hydrolysis of acetylthiocholine (ATCh), and produce thiocholine (TCh). TCh could combine TCh-TCh, and electrons were produced which were the reducing agents of the AuCl4-, the reducing products were produced on the gold nanoparticles. Therefore, the gold nanoparticles get enlarge and Rhodamine B would absorb to the surface of gold nanoparticles, leading to fluorescence quenching. When OPs were added, the reducing agents decreased and the fluorescence intensity of Rhodamine B enhanced, quantitative analysis of paraoxon has been achieved. In addition, different methods have been utilized and then the experimental phenomena have been reasonably interpreted.The main characteristics of this thesis are as follows:1. The effects of enzyme inhibition and fluorescence quenching systems have been studied and two novel quantification approaches of OPs have been established by utilizing their combined interaction.2. The enzyme inhition system has been detailed studied and effective OPs detection methods have been established without separating the target from the real samples, which has a high signal to nosie ratio and high selectivity.3. The above researches provide a certain theoretical and experimental basis to explore novel OPs detection systems and select novel fluorescence probes, which could have potential value of applications to the OPs analysis.