Biosensors acetylcholinesterase inhibition

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

Faschi S., Ogoriczyk D., Palchetti I., Mascini M., (2007) Evaluation of pesticide-induced acetylcholinesterase inhibition by means of disposable carbon-modified electrochemical biosensors. Enzyme Microb. Technol., 40, 485-489... 

Pundir, C.S., Chauhan, N., 2012. Acetylcholinesterase inhibition-based biosensors for pesticide determination a review. Anal. Biochem. 429, 19—31. 

Enzyme inhibition sensors are of interest in the environmental context, the most used being those involving acetylcholinesterase inhibited by pesticides e.g. these can involve rather complex architectures, and characterisation of such systems by EIS is becoming more widespread. Complex sensor architectures have been used for endocrine disrupters, with similar characterisation by electrochemical impedance. Recently, EIS was used for the first time to characterise the response of glucose biosensors in the presence of heavy metal ion inhibition. ... 

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-... 

Acetylcholinesterase (AChE) isolated from various organisms has been used in the majority of pesticide biosensors. In the early 1950s potentiometric detection was adopted for pesticide detection. In the middle of the 1980s it was used for the construction of the first integrated biosensors for detection of pesticides based on inhibition of AChE. Later rapid changes in science and technology introduced novel genetically... 

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. 

The developed biosensor was applied to the analysis of cyanobacterial bloom samples from freshwater lakes of Spain, Greece, France, Scotland and Denmark. Two samples from Scotland and one from Denmark irreversibly inhibit the acetylcholinesterase. The estimated concentrations were between 1.5 and 30nmol/g of dry weight, values extremely high when compared to the intraperitoneal 50% lethal dose of anatoxin-a(s) in mice (121 nmol/kg). 

A. Crew, J.P. Hart, R. Wedge and J.-L. Marty, A screen-printed, am-perometric, biosensor array for the detection of organophosphate pesticides based on inhibition of wild type, and mutant acetylcholinesterases, from Drosophila melanogaster, Anal. Lett., 37 (2004) 1601-1610. 

Antibodies to OP-tyrosine will be made. These antibodies will be used to diagnose OP exposure in a biosensor assay with saliva, sweat, or urine. New biomarkers of OP exposure will be identified using mass spectrometry and the new OP-tyrosine antibodies. The identification of new biomarkers for low-dose OP exposme is expected to lead to an understanding of how neurotoxicity is caused by OP doses that are too low to inhibit acetylcholinesterase. For example, it is possible that disruption of microtubule polymerization by OP-adduct formation may explain cognitive impairment from OP exposure. 

The effect of irreversible inhibition of acetylcholinesterase has been used in dendrimer-based electrochemical biosensors for environmental applications. Acetylcholinesterase is a very efficient protein catalyst for the hydrolysis of its physiological substrate acetylcholine. Organophosphorus and carbamic pesticides, heavy metals and detergents exert strong specific... 

The aim of this chapter is to review some basic concept concerning the electrochemical biosensors and to illustrate a protocol for the detection of environmental organic pollutants on the basis of electrochemical biosensors. In particular, a method based on the inhibition of the enzyme acetylcholinesterase (AChE) for the detection of organophosphorus and carbamate pesticides will be described in detail. 

Caetano J, Machado SAS. Determination of carbaryl in tomato "in natura" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity. Sensor Actuat B-Chem 2008 B129 40-6. 

Abstract. The biosensors described in this work, for the monitoring of pesticides, are based on acetylcholinesterase immobilized on the surface of screen-printed electrodes. The principle of the biosensor is that the degree of inhibition of an enzyme sensor by a pesticide is dependent on the concentration of that pesticide. The DPV technique was used as a detection method and methyl-paraoxon as a reference pesticide for sensor calibration. 

In living beings, these pesticides bind irreversibly to the active site of acetylcholinesterase (AChE) - enzyme involved in the transmission of nerve impulse. Electrochemical biosensors for measurement of these pesticides are based all on the inhibition of AChE and the inhibition degree is proportional to the pesticide concentration. 

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). 

Acetylcholinesterase is by far the most widely used enzyme in the preparation of biosensors for determining pesticides, both because organophosphorus insecticides and carbamates represent over half of the entire insecticide market and because the acetylcholinesterase commercially available has a high degree of purity and specificity of action and may be paired with many transducers (potentiometric, amperometric) in both flow and nonflow systems [62]. The specific tendency of organophosphorus pesticides and carbamates to inhibit acetylcholinesterase has been exploited for the purpose of determining these compounds, which are first separated by means of HPLC, then detected through a post-column reaction with immobilized acetylcholinesterase [63]. 

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. 

In the world market, more than 90% of commercial biosensors are enzymatic, in particular, those that measure glucose, used by diabetics [79]. This kind of biosensors is very useful because if immobilized enzymes are sensitive to certain pollutants, these analytes can be easily measured. For example, biosensors based on the inhibition of acetylcholinesterase detect phosphorus insecticides and other inhibitors [80]. A comprehensive analysis showed that the developed enzymatic biosensors demonstrated reproducible, stable, and fast responses to the substrates to be measured. Unfortunately, the application of these biosensors can be restricted because of the dramatic decrease in the sensor response at increasing buffer capacity and ionic strength, pH-dependence of the enzyme kinetics, and cosubstrate limitation of the measured enzymatic reaction rate (the glucose sensor) [79]. Recently, Soldatkin et al. reported a complete review of some biopolymers used in enzymatic... 

In the following section an example of the use of disposable graphite sensor based for food analysis will be described. In particular, the use of these sensors to develop enzymatic biosensors for pesticide detection based on AChE (acetylcholinesterase) enzyme inhibition will be described. 

Pesticides are another important group of pollutants that can be detected by fiber-optic chemical sensors. Since pesticides are designed to interact with biological molecules, fiber-optic biosensors are mostly used for their detection. One example is the detection of organophosphate and carbamate pesticides by monitoring their inhibition effect on the enzymatic reaction of acetylcholinesterase (AChE) with its substrate, acetylcholine. The enzyme is coimmobilized at the distal end of the fiber together with... 

Cremisini, C., Sario, S., Mela, J., et al, 1995. Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an ampero-metric choline oxidase based biosensor. Analyt. Chim. Acta 311, 273-280. Darvesh, S., Walsh, R., Kumar, R., et al, 2003. Inhibition of human cholinesterases by drugs used to treat Alzheimer s disease. Alzheimer Dis. Assoc. Disord. 17,117-126. 

A very specialized application of enzyme-based biosensor arrays has been reported for the resolution of pesticide mixtures containing dichlorvos and methylparaoxon, with a three-element array and a flow injection system [41]. The screen-printed, amperometric electrode array was modified with three acetylcholinesterase enzyme variants, one from electric eel and two from Drosophila (fruit fly) mutants, and were used to measure signal inhibition in conjunction with an artificial neural network. Good results down to the low uM range of pesticide concentrations were reported. 

The concept was further extended by the French researchers to three-pesticide mixtures, chlorpyrifos-oxon (CPO), chlorfenvinphos (CFV) and azinphos methyl-oxon (AZMO) [72], The array of biosensors was designed using only two acetylcholinesterases from Drosophila melanogaster (wild type and genetically modified) and used the dynamic comprment of the inhibition profiles and an ANN to solve mixtures of CPO, CFV and AZMO insecticides at the 1-10 nM level. 

Monitoring the amount of pesticides in water and soil is an effective way to detect the abuse of pesticides in agriculture. Because pesticides can inhibit the activity of many enzymes, such as acetylcholinesterase (AChE), butyrylcholi-nesterase (BCliE), organophosphate hydrolase (OPH), and tyrosinase (Tyr), various inhibition biosensor systems emerged in recent years as promising alternatives for in situ detection of pesticides. Modem methods for the detection of pesticides usually involve liquid or gas chromatography coupled to mass spectrometric detection (HPLC-MS, GC-MS), requiring an appropriate sample preparation (as seen in Table 8.1). However, optical and electrochemical detection methods were also developed for this purpose. 

---