Study On The Activity Of Acetylcholinesterase And Its Inhibition Based On The Two Kinds Of Fluorescence Probe

Acetylcholinesterase(ACh E) shows an important role in terminating neurotransmission at cholinergic synapses. Because of specially physical and chemical properties, the fluorescence probes have attracted more and more attentions. With the specific chemodosimeter for detection, the fluorescence probe would be selective and sensitive. Depending on the high sensitivity, selectivity, simple operation, fluorescence spectroscopy became one of the hottest research among various detection methods. In this paper, the two fluorescence probes based on 1,8-naphthalimide derviatives have been synthesized and characterized by IR spectra, UV spectra, fluorescence spectra and 1HNMR spectra. On the basis of the use of the fluorescence probes and enzymes, we achieve efficient and sensitive analysis of acetylcholine and pesticide. The main contents are as follows:1) A highly sensitive and selective fluorescence method for detection of acetylcholine(ACh) based on enzyme generated H2O2 and a new boronate fluorescence probe, 4-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl)-N-butyl-1, 8-naphthalimide(BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase(ACh E) to produce choline, and then further oxidized by choline oxidase(Ch Ox) to obtain betaine and H2O2. The enzyme-generated H2O2 reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product(4-hydroxyl-N-butyl-1, 8-naphthalimide, ON). The fluorescence intensity is proportional to the concentration of ACh in the range of 0.01- 80 μM(R2 = 0.9961). The detection limit was 2.7 n M(signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, the boronate probe is based on the hydrolytic deprotection to detect ACh and has no interference from other biologically relevant reactive oxygen species(ROS). This study will provide a new method for detection of acetylcholine with high selectivity and sensitivity.2) We report an ultrasensitive detection strategy for pesticides based on a novel boronate fluorescence probe(4-boronate-N-butyl-l, 8-naphthalimide, BN). This analytical strategy involves the reaction of acetylcholine(ACh) with acetylcholinesterase(ACh E) to produce choline. Then, the choline is in turn catalytically oxidized by choline oxidase(Ch Ox) to generate H2O2. The enzyme-generated H2O2 can react with BN and result in a fluorescence increase. Pesticides which inhibit the activity of ACh E, would reduce the production of enzyme-generated H2O2 and hence lead to relatively small fluorescence increase of BN. The inhibition efficiency of pesticide to ACh E activity could be evaluated based on the changed fluorescence of BN in the absence and presence of pesticide. Experimental results showed that the enzyme inhibition efficiency of parathion-methyl, phorate and acephate were proportional to the logarithm concentration of the pesticides in range from 9.54 × 10-7 to 9.54 × 10-4, 9.24 × 10-8 to 9.24 × 10-3 and 3.16 × 10-6 to 8.45 × 10-3 g/L with detection limits 2.31 × 10-7, 2.20 × 10-8 and 9.92 × 10-7 g/L, respectively. This study will provide a new method for detection of pesticides with high sensitivity.3) This report presents a highly sensitive, optical probe for the detection of organophosphate pesticides. The analytical strategy is based on the fact that the organophosphate pesticides can inhibit the activity of acetylcholinesterase(ACh E), hence prevent the generation of thiocholine, which can specifically react with fluorescent nitro probe. The nitro probe is a specific probe contained thiol group, which can result in fluorescence increasing and offering a low fluorometric background after reacting with the product of the enzyme reaction. By use of this system, the further detection of organophosphate pesticides can be ralized and the lowest detectable concentrations for several kinds of pesticides including parathion-methyl, phorate and acephate are measured to be 3.12 × 10-10, 3.20 × 10-9 and 3.04 × 10-9 g/L, respectively. The low detection limit makes it possible to detect pesticides in real samples such as agricultural products. Experimental results showed that pesticide residues collected from food samples determined via this method agree well with those by high-performance liquid chromatography(HPLC). We believe that the assay has potential value to exend more effective strategy for the detection of organophosphate pesticides.