| The acetylcholinesterase (AChE) is a kind of serine esterases in human andanimal’s central nervous system, which is able to hydrolyze the neurotransmitteracetylcholine (ACh) to choline at cholinergic synapses. Medical studies have show nthat low level of acetylcholine has close relationship with Alzheimer’s disease (AD).At present, the number of the patient s of AD increases year by year all over the world,which brings serious burden to human and the society. Organophosphate pesticides(OPs) has the ability to inhibit the activity of AChE. The inhibition of AChE activityleads to the accumulation of ACh throughout the body, affecting the physiology of thenervous system with serious or fatal consequence. Therefore, the development of asimple, rapid and reliable assay method for AChE activity and its inhibitor screeningis important to improve the health condition of human.The enzym atic sensor has advantages of good selectiv ity and high sensitivity. Inthis paper, we developed several novel fluorescent biosensor s for AChE activity,AChE inhibitors and ACh based on enzyme reaction. The detailed method is describedas follows:(1) A new sensitive fluorometric assay method for AChE and its inhibitor wasdeveloped using a fluorescent dye, nile red (NR). Due to the fluorescence resonanceenergy transfer between the NR and the gold nanoparticle (AuNPs), the fluorescencewas quenched. AChE can break down acetylthiocholine to produce a thiol-bearingcompound, thiocholine. In the presence of thiocholine, the nile red is replaced fromthe AuNPs surfaces and is simultaneously transformed to be a derivative of nile red.The fluorescence intensity of the derivative is much stronger than that of the nativenile red with the same concentration and its maximum emission wavelength has a blueshift so that the sensor achieves a good signal-to-background ratio. In addition, whenOPs exists, the activity of AChE can be inhibited, the generation of thiocholine willbe prevented and no fluorescence enhancement occurs. The results show that themethod is sensitive to AChE and paraoxon with the detection limits of0.2mU/mL and0.05ng/mL, respectivel y.(2) A novel sensitive fluorometric “turn-on” assay method was constructed forthe detection of ACh and OPs based on the fluorescence quenching of squaraine dye.In this method, thiocholine can react with squaraine dye, which result ed in fluorescence quenching. In the presence of ACh, ACh and ATCl competitivelycombined with AChE, the stimulate hydrolysis of acetythiocholine to form thiocholinedecreas ing, and the squaraine dye remains intact and give s signal-on fluorescence.The fluorescence intensity of squaraine dye is positively correlated with the ACh insolution. The detection limit is found to be as low as0.05μM. In the presence ofAChE inhibitor, paraoxon, the hydrolysis catalytic activity of AChE is inhibited andthen the squaraine derivative also remains intact. The detection limit of paraoxon isfound to be as low as5pg/mL. The proposed sensor is simple, rapid and high lysensitiv e to the test of acetylcholine and organophosphorus pesticides, and has strongcommonality.(3) A novel fluorometric assay method was developed to distinguish differentorganophosphate pesticide s, which is based on the different efficacy of reactivators tothe inhibitors of different structure. The activity of AChE can be inhibited by OPs insolution, which would prevent the generation of thiocholine and hence improved thesignal-on fluorescence of squaraine. If the inhibited AChE is reactivated by areactivator, the AChE activity can be recovered to a certain degree, which benefit s thegeneration of thiocholine and then decreases the fluorescence intensity of the wholesystem. According to the changes of the fluorescence intensity, it can be achieved todistinguish three kinds of organophosphates: methamidophos, trichlorfon andparaoxon by reactivator s. This method provides a new platform for the identificationof OPs. |
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