Acetylthiocholine enzymic hydrolysis

Theoretical Modeling of Enzymic Hydrolysis of Acetylcholine Compared to Acetylthiocholine... 

As proof of principle, Lehn and coworkers individually synthesized all acyl hydrazone combinations from the 13 DCL building blocks and measured their inhibition of acetylthiocholine hydrolysis by ACE in a standard assay. They then established a dynamic deconvolution approach whereby the pre-equilibrated DCL containing all members is prepared, frozen, and assayed. Thirteen sublibraries were then prepared containing all components minus one hydrazide or aldehyde component, and assayed. Active components in the DCL were quickly identified by an increase in ACE activity, observed in sublibraries missing either hydrazide 7 or dialdehyde i, pointing to the bis-acyl hydrazone 7-i-7 as the most likely active constituent. This was in line with the individual assay data recorded earlier resynthesis of this compound characterized it as a low nanomolar inhibitor of the enzyme. 

In this section, we discuss about the screen printed electrode (SPE) based AChE sensors for the selective determination of OP and CA pesticides. In the past decades, several attempts were made by the researchers to develop SPE based pesticide sensors, where the enzyme AChE was immobilized either directly onto the electrode or above other matrices incorporated SPE surfaces. Both approaches resulted in the good, rapid detection of OP and CA pesticides. Earlier, Hart et al. employed AChE/SPE to detect OP and CA pesticides [21], They measured the enzyme activity from the rate of hydrolysis of acetylthiocholine iodide. Three polymers such as hydroxyethyl cellulose, dimethylaminoethyl methacrylate, and polyethyleneimine were used as enzyme immobilization matrices. Initially, electrodes were exposed to drops of water or pesticide solution, dried and their activity was screened after 24 h. They found that, when the enzyme matrix was hydroxyethyl cellulose, electrode activity inhibited both by water as well as by pesticides. While with co-polymer matrix, a significant response towards pesticides alone was observed. Further, the long-term storage stability of electrodes was highest when the enzyme matrix consisted of the co-polymer. The electrodes retained their activity for nearly one year. In contrast, the electrodes made of hydroxyethyl cellulose or polyethyleneimine possess less stability. 

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. 

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