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Cholinesterase Methods forNerve Agents Detection Using Optical Evaluation: Comparison
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Please note this is a comparison between Version 2 by Catherine Yang and Version 1 by Aneta Brizova.

The extreme toxicity of nerve agents and the broad spectrum of their physical and chemical properties, enabling the use of these agents in a variety of tactical situations, is a continuing challenge in maintaining the knowledge and capability to detect them, as well as in finding new effective methods. With the military standardization of V-series agents (VX), the sensitivity of traditional chemical methods was no longer satisfactory; therefore, increased attention was paid to cholinesterase methods. The reduction of enzyme activity after contact with NAs allows for the detection of these cholinesterase inhibitors with high sensitivity since, in this case, one and the same enzyme acts as both a sensitive element and an amplifier of the analytical signal.

  • nerve agents
  • chemosensors
  • biosensors
  • colour reactions
  • fluorescence
  • cholinesterase reaction

1. Enzyme, Substrate, and Acid–Base Indicator

Already during World War II, Richard Kuhn’s team developed a biochemical method to determine AChE activity based on the colour indication of acetic acid released by the hydrolysis of acetylcholine with bromothymol blue (pH 6.0–7.6, yellow–blue)—in the plasma or serum of experimental animals, as a way to evaluate the efficiency of a new generation of CWAs [42][1]. Based on this method, a rapid test for cholinesterase activity in human blood [43][2], as well as field methods for the detection of organophosphorus inhibitors, were later developed. In 1964, a simple field detector containing, in addition to the enzyme, the substrate acetylcholine and the acid–base indicators Bromothymol Blue or Phenol Red were described [44][3]. During further studies, Phenol Red (pH 6.8–8.8, yellow–red) proved to be more suitable. For example, in about 1965, the Soviet GSP-11 automatic tape analyser was introduced with a textile tape sampling of contaminated air, a BChE solution dispenser, and a butylcholine solution dispenser with Phenol Red. Phenol Red is still used in the detection tubes.
The advantage of a system with acid–base indicators is a distinct and unambiguous colour transition. The disadvantage is the problematic use of quantitative analysis and the interference of acidic and basic agents.

2. Enzyme, Thio-Substrate, and Redox Indicator

A highly significant modification of the cholinesterase method was the replacement of the specific substrate acetylcholine with the synthetic substrate acetylthiocholine (later also its analogue butyrylthiocholine), which breaks down to create thiocholine, which reduces 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) to a yellow-coloured product [45][4]. This so-called Ellman’s reagent is perhaps the basis of the most widely used optical methods employed not only in clinical biochemistry and many other fields but also in the military for the detection of organophosphorus NAs [46,47,48][5][6][7]; the basic reaction scheme and examples of use are shown in Figure 31. Some analogues of Ellman’s reagent have also been validated for the detection of NAs, but none of them have yielded improved analytical performance. Other reagents, such as 5-(2-aminoethyl)dithio-2-nitrobenzoate [49][8], will certainly be the subjects of further study.
Figure 31. Standard Czech devices work on the principle of cholinesterase reaction using Ellman’s reagent (from above): (a) detection tube DT-11; (b) personal detector/biosensor DETEHIT positive result; (c) personal detector/biosensor DETEHIT blank; (right) (d) the newly developed DAPH detection device of nerve agents.
Another group of redox indicators suitable for the cholinesterase method is 2,6-dichlorophenolindophenol and its analogues, which are decoloured by reduction. For example, 2,6-dichlorophenolindophenol (blue in the oxidized form) has been proposed for the automated determination of cholinesterase inhibitors for routine clinical laboratories [50][9] or an area biosensor for the detection of NAs [51][10]. The analogous N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)-2-chloro-5-sulfo-4-iminobenzoquinone is also relatively resistant to acidic and basic gases and vapours, but the redox reaction is slower (about 5 min); it has been proposed for a biosensor for NAs detection with a red–white transition [52][11].
Of particular importance in military applications of the cholinesterase method are triphenylmethane dyes, which are decoloured by reduction. The most widely used is probably Guinea Green B, designed for the biosensor for NAs detection [53][12] and earlier for the Soviet GSP-12 (Figure 42) automatic tape analyser, which replaced the obsolete GSP-11. A mixture of Guinea Green B with another triphenylmethane dye, Fuchsin Basic, which changes the blue colour to red–violet by reduction, was proposed for the preparation of the detection tube and the surface biosensor for NAs detection [54][13]. In all these means with triphenylmethane dyes, BChE was preferentially used.
Figure 42. (a) Structure of Guinea Green B; (b) Guinea Green biosensor for nerve-agents detection: 1—blank, 2—presence of nerve agents. Reprinted from ref. [53][12]; (c) the older Soviet GSA-12 automatic alarm, which works on a similar reaction principle.
The advantage of a system with redox indicators is its possible use for quantitative analysis and a higher resistance to common interferences.

3. Enzyme and Chromogenic Substrate

An interesting and very successful variant of the cholinesterase method is based on the use of so-called chromogenic substrates, which hydrolyse directly to generate a coloured product; the chromogenic substrate is therefore both a substrate and a chromogenic reagent.
Among the oldest and still used chromogenic substrates are indoxylacetate (3-indolylacetate) and its analogues [55,56,57,58][14][15][16][17]. Indoxylacetate is colourless; in the absence of a cholinesterase inhibitor, it hydrolyses to a blue–green fluorescent product which is further oxidized to indigo blue. Proposals have been made to accelerate the formation of indigo by the addition of a mixture of potassium ferrocyanide and potassium ferricyanide [59][18]. A structurally different resorufinbutyrate has been proposed as a fluorogenic substrate along with indoxylacetate [56][15].
In the 1950s, indophenolacetate with a red-to-blue colour transition was proposed for the measurement of AChE activity [60][19]. It has recently been described in a colourimetric detector, validated on the pesticide dichlorpyrifos, with an evaluation by smartphone [61][20]. Even more well known is 2,6-dichlorophenolindophenolacetate, a red-coloured compound which, by reduction (i.e., in the absence of inhibitors), provides the analogous blue 2,6-dichlorophenolindophenol (see redox indicator above). It has appeared, for example, in the design of a detection-tube preparation [62][21] or in the Canadian Nerve Agent Vapor Detector (NAVD) kit [30][22] and similar devices. It is still very popular and used in NA detectors, with the drawback of its lesser availability and high cost.
Classical chromogenic substrates include p-nitrophenyl acetate or 2-azobenzene-1-naphthylacetate, yellow-coloured compounds that hydrolyse to red products [16][23]. However, some compounds are known that can be considered as chromogenic substrates only conditionally (they belong rather to the group of nonspecific substrates). A simple field detector of cholinesterase inhibitors, which contains the substrate 6-bromo-2-naphthylacetate and as an indicator a stabilized diazonium salt (Fast Blue B salt), reacting with the resulting 6-bromo-2-naphthol to a distinct azo-coloured dye, is also described in the previously mentioned work [44][3]. Later, 1-naphthylacetate was proposed as a fluorogenic substrate for pesticide detection [63][24].
The advantage of using chromogenic substrates is the significant simplification of the analytical system (it contains only two key components); the disadvantage is the lower selectivity.

References

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