| The acetylcholinesterase (AChE) is one kind of serine esterases in the animal’scentral nervous system, and it can not only participate in the development and matureof cells, but also promote the development of nerve and the regeneration of neurons.Acetylcholine is the substrate of the AChE, one of the important neurotransmitters inthe acetylcholine systerm. Its main function is to maintain the conscious awareness,and it plays an important role in learning and memory. There is a lot of acetylcholine inthe brain tissue of man, but the level of acetylcholine will decrease with age. AChE canform ChE by combining with organophosphorus, and ChE becames very stable andloses the catalyzing activity for the acetylcholine hydrolyzation. At the same time,AChE gradually becames aging, making acetylcholine accumulates, and causingcholinergic nerve to be first excited and then inhibited. Alzheimer’s disease (AD), is aneurological disease recession, and seriously influences the people’s cognitive,memory, emotion, and other functions. The AD’s pathology recognized by the world isthe lack of neurotransmitte-acetylcholine in the brain. At present, the number of sickpeople has already reached one thousand seven hundreds to twenty-five million,causing serious burden to the family and society. So, it is very important for earlydiagnosis and treatment of the AD pathogenesis and many other nerve diseases toachieve rapid and simple test for the cholinesterase activity and related substance.Therefore, the development of easy operation, low cost, high precision and effiencycholinesterase sensing technology is of great significance.The enzyme sensor technology has been widely used for its good selectivity, simplestructure and high sensitivity advantage. Nanosized gold particle becomes one of nanomaterials with most extensive application for its unique optics, electricity and catalyticproperties. In this paper, we combine the enzyme sensor and nano gold technologyorganically to construct the electrochemical and liquid crystal enzyme sensors. Werealize the sensitive detection of cholinesterase inhibitors, substrate and antibodies byusing the nano gold amplification technology. The detailed content is described asfollows:(1) The design of the AChE electrochemical biosensor. The sensor is based on theuse of reductibility of thiocholine which is generated by AChE mediating hydrolysis ofacetylthiocholine (ATCl) to reduce AuCl4-to form gold nanopartcles (Au NPs). Nano gold particles are used as the seeds and catalyst of generating copper nanoparticles. Inthe absence of nano gold seeds, the copper irons can’t be reduced to form metal copper.The deposited copper was quantified by linear sweep voltammetry (LSV). Scanningelectron microscopy (SEM) image proved that gold nanoparticles generated bybiometallization and the copper deposited on gold nanoparticles successfully. Theeletrochemical stripping signal depends on the AChE inhibitor concentration, and thedecrease in the stripping peak currents of copper is proportional to the concentration ofAChE inhibitor. The detection limit reached as low as0.02ng/mL (S/N=3).(Chapter2)(2) The design of the signal of amplified liquid crystal biosensor for AChE. Thesensor is based on the signal amplification of nano gold deposits to achieve lowconcentration measurement of the AChE substrate (acetylcholine) and the inhibitororganophosphorus pesticide. The detection of acetylcholine is achieved by using thecompetition assay method between acetylthiocholine and acetylcholine. In this method,the generation of nano gold particles is controled by the amount of hydrolysis ofthiocholine, and the nano particles can directe the arrangment of liquid crystalmolecules, causing a signal-amplified polarization effect of the LC. It can realize thesimple, sensitive and effective detection of acetylcholin, and the detection limitreaches15nM/mL. The catalytic activity of AChE can be specifically inhibited byorganophosphorus pesticide, and thus the organophosphorus can also be detected usingthe liquid crystal biosensor. Organophosphorus pesticide inhibits the catalytic activityof AChE and abates hydrolysis of the AChE substrate. Correspondingly, the content ofthe reducing agent thiocholine will be reduced, resulting in a decrease in the number ofnano gold particles. This also weakens the disturbing effect on the LC’s orders, anddecreases the polarization effect. The liquid crystal detection limit of rganophosphoruspesticide reaches0.1ng/mL. This is a simple, rapid and relatively sensitive assaytechnology for testing organophosphorus pesticide, and has strong commonality.(Chapter3)(3) The development of liquid crystal for acetylcholinesterase antibody. Thismethod is based on the AChE antibody which has the similar catalytic hydrolysisactivity with AChE. The AChE antibody also can hydrolyze the substrateacetylthiocholine to form reducing agent thiocholine which can reduce AuCl4-to formgold nanoparticles (Au NPs). We construct a liquid crystal biosensor based on the nanogold amplification technology. By obtaining polarizing images, this method simplyrealized the detection of the AChE antibody’s catalytic activity, and the detection limit reaches0.01pg/mL. This liquid crystal sening method is not only more effective thanthe traditional methods to test the sensitive AChE antibodies, but also it is a novel ideawhich provides a new platform for testing other proteins in future. The results showthat this method is a low cost, high selectivity, high sensitivity detection technologyfor target protein analysis.(Chapter4)... |
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