Metal Nanoparticle Enhanced Fluorescence Of Organic Phosphorus Biosensors

Organophosphate has high insecticidal abilities, which has been a far-ranging pesticide nowadays. The food safe problems resulting from damages of organophosphate are always taking people's attentions. Consequently, there is a growing interest in fast, reliable, sensitive, simple and practical detection methods, which has important practical significance on environmental protection and food safety.Biosensors have attracted much attention in recent times because of the potential applications of these devices in the clinical diagnostics, environmental monitoring, pharmaceuticals, and food processing industries due to their fast response and ease of operation. And the use of acetylcholinesterase (AChE) as organophosphate biological recognition element in biosensor technology has gained more and more attention, in particular with respect to pesticides detection. Many kinds of biosensors using different transducer for insecticide detection involving optics, electrics, electrochemistry, calorifics, magnetics,etc, has been developed. Among those, fluorescent biosensor is widely used as a class of biological sensors because of its good physical property such as high sensitivity, good penetrability, space resolution and low cost. Herein, a kind of biological sensors has been developed using fluorescence as detection signal.Bioconjugation involves the linking of two or more molecules to form a novel complex having the combined properties of its individual components. The objective of the present work is to develop a novel nanobiosensor for insecticide detection, combining noble metal nanoparticles(e.g. AuNPs or AgNPs) and with AChE by covalent bonding to form bioconjugation. Thus, the influence of metal nanoparticles was applied to improve the sensitivity of the fluorescent probe for better detetion of organophosphate.This thesis contains three chapters. Chapter one is introduction. It firstly reviews the progress of fluorescent biosensors and the basic characteristics and their applications of nobel metal nanoparticles, then summarize the functions and applications of AChE, the properties and chemical modification method of nobel metal nanoparticles, particularly in analytical chemistry application, finally describes the method, aim and significance of this research.Chapter two and chapter three are experimental sections as follows: Chapter two Organophosphate biosensor based on fluorescence enhancement with gold nanoparticles7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) is a red fluorescent dye with weak fluorescence. By means of metal nanoparticles affecting the fluorescence properties of DDAO, a novel method based on AChE about the detection of organophosphate has been established. The sensing mechanism has been also explored. Firstly, based on the thiol 11-mercapto-undecane acid (MUA) and Au forming a covalent bond, MUA was covalently immobilized onto the gold nanoparticles, then the carboxyl was activated through the EDC/NHS, reacted with the free amino groups of AChE via covalent binding, and form AuNPs/AChE conjugate. Finally the synthesized gold nanoparticles and AuNPs/AChE were characterized by absorption spectroscopy and transmission electron microscopy methods, and the influence of temperature and pH on enzyme activity between AChE and AuNPs/AChE was studied, then the biosensor performance of AuNPs/AChE biological conjugate on detection of paraoxon was studied. It was also calculated the bimolecular rate constants and inhibition constants of paraoxon combination with AuNPs-AChE-DDAO, and binding constants of paraoxon and AuNPs-AChE-DDAO biological conjugates. The inhibition type between AChE and DDAO was also tested. Using autodock4.2 software, the interactions between DDAO and AChE was studied by molecular docking. The absorption spectra and transmission electron microscopy images showed that the AgNPs/AChE conjugate had formed. Compared with the free enzyme, the immobilized AChE had better stability in the same temperature and acidity. In the presence of AuNPs/AChE bio-conjugates, the fluorescence intensity of DDAO had greatly enhanced,then the quenching phenomenon of DDAO fluorescence occured when the paraoxon was gradually added in this system. Consequently, a simple and sensitive method for determination of paraoxon was established according to the change of fluorescence intensity.The detection limit was 4×10-7 M. The sensor had good stability, fast response, high sensitivity. The interaction studies between AChE and DDAO showed that DDAO strongly inhibited the activity of AChE, the inhibition constant Ki was 1.1×10-7 M and resulted in a mixed inhibition type. Binding constant was 3.2×106 L-mol-1, which showed that there was a strong interaction between paraoxon and DDAO/AChE/AuNPs. The results of molecular docking showed that it was different from paraoxon binding to Ser200 in the active center of AChE. DDAO interacts with peripheral anion sites of AChE. It reasoned that the high sensing performance between paraoxon and DDAO-AChE-AuNPs was due to disturbance of the conformation of AChE conjugates. It ultimately lead to the changes of the micro-environment of DDAO and resulted in the changes of fluorescence emission performance.Chapter three Organophosphate biosensor based on fluorescence enhancement with silver nanoparticles In the second chapter, it was found that the gold nanoparticles could improve fluorescence of fluorescent probe DDAO. Many literatures reported that gold nanoparticles had fluorescence quenching features, few literatures reported that gold nanoparticles had a fluorescent enhancement effect.On the contrary, more studied showed that silver nanoparticles resulted in the fluorescent enhancement effect. To validate the experimental results, AChE the labeled silver nanoparticles was synthesized and characterized by absorption spectroscopy and transmission electron microscopy. The influence of temperature and pH value on enzyme activity of free AChE and AuNPs/AChE bioconjugate was studied. Then, the biosensing performance of AgNPs/AChE bioconjugate on detection of paraoxon was tested. The binding constants of AgNPs-AChE-DDAO bioconjugates and paraoxon was also calculated. The absorption spectra and images of transmission electron micros-copy showed that the AgNPs/AChE conjugate had been fabricated. At the same temperature and acidity, the immobilized AChE was more stabe than free AChE. The calculatesd binding constant was 1.7×106 L·mol-1, which also showed that there was a strong interaction between paraoxon and DDAO/AChE/AgNPs. In the presence of AgNPs/AChE bio-conjugates, the fluorescence intensity of DDAO had greatly enhanced, then the quenching phenomenon of DDAO fluorescence occured when the paraoxon was gradually added. It indicated that DDAO/AChE/AgNPs bioconjugate was also sensitive for biosensing paraoxon.