Research On Novel Acetylcholinesterase Biosensors And Application For The Detection Of Organophosphorus

Acetylcholinesterase (AChE) is a serine hydrolase which mainly exists in the human and animal central nervous system. Its basic function is to catalyze the hydrolysis of the neurotransmitter acetylcholine and results in the termination of nerve impulse transmission to maintain the normal physiological function of cholinergic nerve. Neural system diseases, such as Alzheimer’s disease, Parkinson’s disease and so on, are associated with human acetylcholinesterase function.AChE combined with organophosphorus pesticides will form the phosphorylation of ChE. The structure of phosphorylation of ChE is very stable and will be gradually aged, which lose the ability to catalyze the hydrolysis of substrate and then cause the accumulation of acetylcholine, resulting in cholinergic nerve from excitement to suppression. The high concentration of organophosphorus pesticides can cause acute poisoning such as convulsions, breathing difficulties, arrhythmia, hypoxia and other symptoms, but the low concentration of organophosphorus pesticides also damage organ by the long-term chronic toxicity to heart, liver, kidney and other organ.In recent years, acetylcholinesterase biosensor is one of the most active research fields due to its high sensitivity, speed, low cost, on-line continuous detection in complex system. However, the establishment of a new and effective acetylcholinesterase biosensor for health, pharmacy, food, agriculture and environmental monitoring is still a hot and meaningful research topic. A series of research work on the development of AChE biosensing methods of easy operation, low cost and high sensitivity signal transformation have been carried out in the present dissertation and described as follows:In chapter2, a novel fluorescent method was developed for AChE and inhibitor detection. To construct the AChE sensing system, a fluorescent substrate, Nile Red non-covalently adsorbed on the surface of gold nanoparticles (Au NPs) was used as the fluorescent probe, which can offer a low fluorescent background due to Au NPs can efficiently quench the fluorescence of Nile red. In the presence of AChE detection system, AChE can catalyze the hydrolysis of acetylthiocholine (ATC1) to generate thiocholine, which competitively adsorbs onto the Nile Red adsorbed gold nanoparticles thought the form of strong Au-S bond, resulting in Nile Red desorption from the surface of gold nanoparticle to generate a new fluorescent product. The experiment results showed that the new product had a stronger fluorescence than the nature Nile red. In the presence of inhibitor, the activity of AChE was inhibited, as a result, the catalysis hydrolysis of ATC1to generate thiocholine decreased. Accordingly, the Nile red desorption from the surface of AuNPs to produce the new fluorescent substances also decreased. The unique fluorescent properties of the Nile Red-AuNPs probe contribute to the distinguishing features of easy operation and high sensitivity quenching the fluorescence to the proposed AChE method for AChE and its inhibitor.In chapter3, a novel fluorescent method was developed for AChE inhibitor detection using a fluorescent squaraine derivative as a specific chemodosimeter for thiol-containing compounds. In this system, the AChE molecules catalyze the hydrolysis of acetylthiocholine (ATC1) to form thiocholine, which in turn can specifically react with fluorescent squaraine derivative, a specific chemodosimeter for thiol-containing compounds, resulting in fluorescence quenching and offering a low fluorometic background for the further detection of AChE inhibitor. In the presence of AChE inhibitor, the catalytic hydrolysis of ATC1is blocked, and then the squaraine derivative remains intact and shows signal-on fluorescence. The amount of the remaining fluorescent squaraine derivative is positively correlated with that of the AChE inhibitor in solution. This new designed sensing system shows an obviously improved sensitivity towards the AChE inhibitor with a detection limit of5pg mL-1(0.018nM). The method is the first successful fluorescence detection of AChE inhibitors based on such a signal-on principle and using a specific reaction.In chapter4, a novel electrochemical biosensor was developed for the detection of AChE inhibitor based on the continuous amplifications of biometallization and copper-enhancement. Oganophosphate pesticide (OP), an important AChE inhibitor was used as a model to confirm our strategy. In this method, the immobilized AChE catalyzed the hydrolysis of ATC1to yield a reducing agent thiocholine, and the latter further reduced AuCLf to form gold nanopartcles. And then the copper enhancer solution was added to proceed to the catalytic deposition of copper onto the Au NPs for continuous signal amplification. The deposited copper was quantified by linear sweep voltammetry (LSV). On the basis of the dependence of the amount of the deposited copper on the AChE inhibitor concentration, the decrease of stripping peak currents of copper was proportional to the concentration of AChE inhibitor from0.1ng mL-1to500ng mL-1. The detection limit was found to be as low as0.02ng ml-1(S/N=3). The continuous signal amplification of biometallization and copper-enhancement dramatically enhanced the detection sensitivity of AChE inhibitor.In chapter5, a novel AChE liquid crystal (LC) biosensor based on enzymatic growth of Au NPs has been developed for the amplified detection of acetylcholine (ACh) and AChE inhibitor. In this method, AChE catalyzes the hydrolysis of ATC1to form thiocholine, and the latter further reduces AuCl4-to Au NPs without Au nano-seeds. This process leads to a great enhancement in the optical signal of LC biosensor due to the large size of Au NPs, which can greatly disrupt the orientational arrangement of LCs. On the other hand, the hydrolysis of ATC1is inhibited in the presence of ACh or AChE inhibitor, which will decrease the catalytic growth of Au NPs, and as a result, reduce the orientational response of LCs. Based on such an inhibition mechanism, the AChE LC biosensor can be used as an effective way to realize the detection of ACh and AChE inhibitors. The results showed that the AChE LC biosensor was highly sensitive to ACh with a detection limit of15μM and OPs with a detection limit of0.3nM. Owing to the sensitivity of the LC molecules orientation to the property of a bounding interface and the enhanced disturbing behavior of the enzymatic growth of Au NPs to the LC molecules orientation, the method achieves an amplified detection of AChE inhibitors with a simple and visual procedure.In chapter6, an improved AChE liquid crystal biosensor has been developed for the selective detection of OPs using a reactivator. In chapter5, a sensitive detection method for AChE inhibitors was achieved due to the enhanced disturbing behavior of the enzymatic growth of Au NPs to the LC molecules orientation. In this method, when the AChEs inhibited by three kinds of organophosphorus are reactived by a reactivator, the catalytic activity of AChEs is recovered with different activation efficiency because different phosphorylation structures are formed in the inhibited AChEs. Accordingly, the reactived AChEs can catalyze the hydrolysis of acetylthiocholine to generate thiocholine product in varying degrees, which will result in the different catalytic growth of Au NPs and the distinct orientational response of LCs. Based on such a reactivation mechanism, the AChE LC biosensor achieves an obvious identification of OPs with a simple and visual procedure.