Biosensors acetylthiocholine

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

OPH-based biosensors have been fully discussed in previous reviews [2,165]. AChE-based biosensors are based on the principle that OP pesticides have an inhibitory effect on the activity of AChE that may be permanent or partially reversible. The extent of the inhibition is directly related to the concentration of the pesticide and therefore enzyme activity may be used as a measure of the inhibition [166]. The amperometric measurement of AChE activity can be based on the measurement of any of the following three mechanisms [167] (1) production of hydrogen peroxide from choline, (2) oxygen consumption during the enzyme reaction or (3) production of electroactive compounds directly from the oxidation of acetylthiocholine chloride such as thiocholine. The measurement of hydrogen peroxide and oxygen consumption has been described in more details in other reviews [167]. 

Another class of enzymes that has found wide application in the biosensor field in the last decades is that of the cholinesterases which have been mainly used for the detection of pesticides. For the amperometric detection of cholinesterase activity, both the substrates acetylcholine and acetylthiocholine have been extensively used [6-9], the latter being preferred because this avoids the use of another enzyme, choline oxidase, which is usually coupled with acetylcholinesterase. However, the amperometric measurement of thiocholine, produced by... 

As mentioned in the previous section, the response, the stability and the enzyme activity found greatly enhanced at the MWCNT platform. Other than CNTs, AuNPs also possess some unique properties and recent years it has been widely employed in the biosensors to immobilize biomolecules. Thus in this section we discuss about the application of AuNP matrix for the immobilization of AChE for pesticide sensor development. With the use of AuNPs, the efficiency and the stability of the pesticide sensor gets greatly amplified. Moreover, the nanoparticles matrix offers much friendly environment for the immobilized enzyme and thus the catalytic activity of the enzyme got greatly amplified. Interestingly, Shulga et al. applied AChE immobilized colloidal AuNPs sensor for the nM determination of carbofuran, a CA pesticide [16], The enzyme-modified electrode sensor was also utilized for the sensitive electrochemical detection of thiocholine from the enzyme catalyzed hydrolysis of acetylthiocholine chloride (ATCl). The fabrication and the enzyme catalyzed reaction at the AuNPs coated electrode surface is shown in Fig. 6. 

Randey R. C., Upadhyay S., Rathak H. C., Randey C. M. D., and Tiwari I., Acetylthiocholine/acetylcholine and thiocholine/choline electrochemical biosensors/ sensors based on an organically modified sol-gel glass enzyme reactor and graphite paste electrode. Sens. Actuators B, 62(2), 109-116, 2000. 

Anatoxin-a(S) can therefore be measured by its inhibition of acetylcholinesterase whose enzyme activity can be measured by several ways. One example is its degradation of the acetylcholine analog, acetylthiocholine, and subsequent measurement of the released thiocholine by the sulfur reacting chemical Ellman s reagent. Acetylcholinesterase has been cloned and its mutation can increase the enzyme s sensitivity for anatoxin-a(S). Combining different acetylcholinesterase mutants with divergent specificity for anatoxin-a(S) and the above-mentioned organophosphate insecticides has enabled better analyte discrimination. This multiple enzyme method has been implemented in a biosensor that carries several of the acetylcholinesterase mutants. 

Table 62.2 collects some properties of AChE-modified biosensors based on acetylthiocholine. As shown in reaction (7), thiocholine can be directly oxidized to dithio-choline. However, the oxidation potential of thiocholine is relatively high (approximately + 0.7V), and thus a... 

A number of papers describe techniques for the determination of choline esterase activity based on amperometric measurement of the product formed as a result of enzymatic hydrolysis. The oxidation potential of choline is too high to be electro-chemically oxidized directly. In this case, artificial (nonnative) choline esterase substrates are used. Thus, butyiyl or acetyl thiocholine forms thiocholine as a result of choline esterase hydrolysis. When the analyte is not present in the solution, the substrate acetylthiocholine is converted into thiocholine and acetic acid. Thiocholine is oxidized by the appUed voltage. In the presence of an inhibitor, conversion of acetylthiocholine is decreased or even null. Furthermore, the anodic oxidation current is inversely proportional to the concentration of pesticides in samples and the exposed time as well. The procedure of the preparation of an AChE biosensor and pesticide detection is shown in Fig. 13.4. 

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