Enzyme biosensors inhibition

Z. Guang-Ming, T. Lin, S. Guo-Li, H. Guo-He and N. Cheng-Gang, Determination of trace chromium (VI) by an inhibition-based enzyme biosensor incorporating an electro polymerized aniline membrane and... 

Our research group is working on the development of electrochemical biosensors for the detection of microcystin and anatoxin-a(s), based on the inhibition of protein phosphatase and acetylcholinesterase, respectively. These enzyme biosensors represent useful bioanalytical tools, suitable to be used as screening techniques for the preliminary yes/no detection of the toxicity of a sample. Additionally, due to the versatility of the electrochemical approach, the strategy can be applied to the detection of other cyanobacterial toxins. 

The main drawback of acetylcholinesterase-based biosensors is the lack of selectivity because, as we mentioned, this enzyme is inhibited not only by anatoxin-a(s) but also by insecticides such as organ-ophosphorates and carbamates. This problem can be overcome by the choice of specific mutant enzymes. The combined use of mutants highly sensitive to anatoxin-a(s) and resistant to most insecticides and vice versa allows us to unambiguously discriminate between the cyanobacterial toxin and insecticides. 

Although the main analytes that are determined by enzyme biosensor are substrates of enzymes, numerous attempts were reported in the literature about using them for indirect determination of inhibitors.112113 Such determination can be also carried out in organic solvents.114 Many of such inhibition-based methods can be questioned because of numerous fundamental and practical disadvantages.115 These are, for instance, difficult or more often impossible regeneration of inhibited enzymes, and the fact that different inhibition degree is caused by different inhibitors. One can find example that in some cases, better results are obtained with the use of raw tissues when compared with purified enzyme.116 It was also pointed out that the most rational use of inhibitory effects is after a separation step for proper removal of species, which can act on the activity of enzyme. 

For pesticide analysis, the potential of enzyme biosensors has been tested. In this field, biosensors based on the inhibition of acetylcholinesterases, acylcholinesterases, or butylrylchol-inesterases by organophosphorus compounds are widely used. Their specific activity can be monitored by electrochemical methods such as the ion-selective electrode and the ion-selective field effect transistor (ISFET). 

Enzymes-based biosensors are well reported in the literature for chemical toxicity screening. The sensor devices produced using enzymes are usually simple and easy to fabricate, inexpensive, and sensitive to low levels of toxicants. Immobilization of enzymes on the electrode surface can include adsorption, covalent attachment, or film deposition using a range of procedures [68-70]. The sensor system relies primarily on two enzyme mechanisms catalytic transformation of a pollutant and detection of pollutants that inhibit or mediate the enzyme s activity. In catalytic enzyme biosensor, the enzyme specific for the substrate of interest (toxin in this case)... 

Tang L., Zeng G. M., Huang G. H., Shen G. L., Xie G. X., Hong Y. X., and Christine W. C., Toxicity testing in environmental analysis—application of inhibition based enzyme biosensors. Transactions of Nonferrous Metals Society of China, 14(1), 14-17, 2004. 

Enzyme One single biosensor One-enzyme biosensor in the array Several enzyme biosensors in the array Inhibition BioET... 

The application of FIA and biosensors in environmental analysis is attractive because enzyme biosensors operate on the principle of inhibition. The process is economical because only small quantities of substrate are required and because the incubation, reaction and reactivation times can be controlled. The process can therefore yield high reproducibility. This method has been used to measure insecticide levels in sea water [269], Finally, FIA is particularly suitable for the construction of automated systems. 

Toxin (Enzyme Inhibition) Biosensors Enzyme affectors (inhibitors and activators) that influence the rate of biocatalytic reactions can also be measured. Sensing probes for organophosphate and carbamate pesticides, for the respiratory... 

Biosensors may provide the basis for in-field analyses and real-time process analysis. However, biosensors are generally limited to the determination of a limited range of analytes in defined matrices. Enzyme-based biosensors, principally acetylcholinesterase (AChE) inhibition, have been successfully used in environmental analysis for residues of dichlorvos and paraoxon, " carbaryl " and carbofuran. " Immunochemically based biosensors may be the basis for the determination of pesticide residues in liquid samples, principally water and environmental samples, but also fruit juices. The sensors can be linked to transducers, for example based on a piezo-... 

The design and implementation of a portable fiber-optic cholinesterase biosensor for the detection and determination of pesticides carbaryl and dichlorvos was presented by Andreou81. The sensing bioactive material was a three-layer sandwich. The enzyme cholinesterase was immobilized on the outer layer, consisting of hydrophilic modified polyvinylidenefluoride membrane. The membrane was in contact with an intermediate sol-gel layer that incorporated bromocresol purple, deposited on an inner disk. The sensor operated in a static mode at room temperature and the rate of the inhibited reaction served as an analytical signal. This method was successfully applied to the direct analysis of natural water samples (detection and determination of these pesticides), without sample pretreatment, and since the biosensor setup is fully portable (in a small case), it is suitable for in-field use. 

Most of the pesticide biosensors are designed based on the inhibitory property of enzymes. AChE and butyrylcholinesterase (BChE) are widely used in the development of pesticide biosensors [17, 18], Inhibition leads to a decrease in activity, which... 

Mainly, two principles are used in electrochemical pesticide biosensor design, either enzyme inhibition or hydrolysis of pesticide. Among these two approaches inhibition-based biosensors have been widely employed in analysis due to the simplicity and wide availability of the enzymes. The direct enzymatic hydrolysis of pesticide is also extremely attractive for biosensing, because the catalytic reaction is superior and faster than the inhibition [27],... 

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... 

Although the inhibition-based biosensors are sensitive, they are poor in selectivity and are rather slow and tedious since the analysis involves multiple steps of reaction such as measuring initial enzyme activity, incubation with inhibitor, measurement of residual activity, and regeneration and washing. Biosensors based on direct pesticide hydrolysis are more straightforward. The OPH hydrolyzes ester in a number of organophospho-rus pesticides (OPPs) and insecticides (e.g. paraoxon, parathion, coumaphos, diazinon) and chemical warfare agents (e.g. sarin) [53], For example, OP parathion hydrolyzes by the OPH to form p-nitrophenol, which can be measured by anodic oxidation. Rainina... 

T. Noguer, B. Leca, G. Jeanty, and J.L. Marty, Biosensors based on enzyme inhibition detection of organophosphorus and carbamate insecticides and dithiocarbamate fungicides. Field Anal. Chem. Tech. 3, 171-178 (1999). 

Simple or automated offline or online biodegradability tests can be performed by measuring CO2 or CH4 gas production or O2 consumption [13]. Biosensors may utilize either whole bacterial cells or enzymes to detect specific molecules of hazardous substances. Toxicity can be monitored specifically by whole-cell sensors whose bioluminescence may be inhibited by the... 

Cortina, M., del Valle, M., and Marty, J.-L. (2008). Electronic tongue using an enzyme inhibition biosensor array for the resolution of pesticide mixtures. Electroanalysis 20(1), 54-60. 

W.E. Lee, H.G. Thompson, J.G. Hall and D.E. Bader, Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor, Biosens. Bioelectron., 14(10-11) (2000) 795-804. 

Electrochemical biosensors for heavy metals based on enzyme inhibition... 

---