To detect nerve agents in practice, the analytical methods such as gas, liquid and thin-layer chromatography, mass spectrometry or capillary electrophoresis are usually used. Apart from these analytical methods, we developed an analytical device (tape photocolorimetric biosensor) based on the modified Ellman`s cholinesterase biochemical reaction for multidetection of cholinergic organophosphorus compounds. Enzyme butyrylcholinesterase was used as a biorecognizing component and its activity was evaluated by RGB(Full) sensor. This method eliminates errors in the evaluation and provides automatic data collection with their subsequent evaluation. The unique method of dosing allows appropriate dispensing of reagents in microlitres volumes and the whole system is simple to operate. Suitability of the constructed biosensors was evaluated using the six organophosphates (Tabun, sarin, Soman, cyclosin, VX and R33 compound). Biosensor showed the ability to measure substances at concentrations ranging between ~ 1×10^-8 mg/l - 1×10^-6 mg/l in the air, according to their inhibition effect.

Keywords:    Nerve agent,  Ellman´s reaction,  tape biosensor,  colorimetric detection 

Nerve agents are the most toxic synthetic compounds that were selected for combat use¹. Many non-toxic organophosphorus compounds with the same basic structure are used in industry as softeners, hydraulic fluids, plasticizers, etc² The wide-reaching application of these substances is in agriculture and forestry as pesticides³. The main toxic effect of nerve agents is interference with nervous system. Nerve agents affect the transmission of nerve impulses by inhibiting acetylcholinesterase (AChE), which ensures neurotransmitters acetylcholine (ACh) degradation. Acetylcholine is responsible for the transmission of nerve impulses. Acetylcholine is decomposed in normal cholinergic transmission immediately after transmission of nervous impulses by the catalytic action of the enzyme acetylcholinesterase^4,5. Under optimal conditions, a molecule of the enzyme hydrolyzes about 2,000 molecules of ACh per second⁵. Nerve agents inhibit the active hydrolytic centre of AChE and irreversible enzyme-inhibitor complex is formed. This process is known as ageing. Enzyme inactivation is irreversible and recovery mainly depends on synthesis of new enzyme. The half-time ageing (time for half of involved cholinesterase to age) varies from 2 min for Soman, to >40 hours for VX and Tabun⁶. To detect nerve agents in practice, the analytical methods such as gas, liquid and thin-layer chromatography, mass spectrometry or capillary electrophoresis are usually used^1,7. .

The detectors operating on the ion mobility spectrometry principle (RAID - 1, Raid M, detector Dräger X-AM, ChemPro detector) are widely used to detect of nerve agents in the air. The detectors using flame spectrophotometer (AP2C, AP4C) and the detectors using surface acoustic wave technology (JACD ChemSentry detector) are used, too. These miniaturized detectors compare measured data with the database stored in the memory. Difficult determination of less known toxic derivatives (i.e. the false negative detection) and higher acquisition prices are their main disadvantages. It´s detection limit is 10^-5 mg/l in the air.

The above mentioned shortcomings can be eliminated by using biosensors. Biosensors are portable, fast and highly sensitive bioanalytical devices. The biosensors based on cholinesterase inhibition achieve extremely high sensitivity⁸. Generally, these biosensors use two types of cholinesterase: acetylcholinesterase and butyrylcholinesterase (BuChE)⁹. Many AChE-based biosenso^12,13rs have been proposed on electrochemiluminescence^10,11, fluorescence, voltametric¹⁴ and amperometric methods^15-18.

This study describes the results achieved during the testing of the laboratory sample of the tape photocolorimetric portable biosensor using utilizing cholinesterase reaction (CHR) for detection of nerve agents.

CHR is a colorimetric method based on hydrolysis of the substrate (usually an ester of choline, or thiocholine) by cholinesterase (acetyl-or butyrylcholinesterase). This catalyzed hydrolysis produces acid and choline (thiocholine). Thiocholine produced by the CHR can be determined by Ellman´s method. The principle of this method is based on the reaction of thiocholine with chromogenic thiol reagent ^5,5 '-dithiobis-(2-nitro) benzoic acid (DTNB). 5-sulphanyl-2-nitrobenzoic acid (yellow colour) is formed during the CHR, and measured at 412 nm (as shown in Fig. 1). In the presence of nerve agent, the reaction is blocked (no colour change)^19,20.

2.1 Chemicals and Materials

O-ethyl N,N-dimethylphosphoroamidocyanidate (Tabun; GA – purity 97.24 %), O-isopropyl methylphosphonofluoridate (Sarin; GB - purity 93.61 %), O-pinakolyl methylphosphonofluoridate (Soman; GD – purity 99.00 %) O-cyklohexyl methylfosfonofluoridate (Cyclosarin; GF - purity 98.01 %) O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX - purity 90.76 %) and O-isobutyl S-[2-(diethylamino)ethyl] methylphosphonothioate (R33 - purity 30.30 %) were manufactured in the Slovak Republic (Zemianske Kostol‘any). 5,5´-dithiobis-2-nitrobenzoic acid (DTNB - purity: > 97.5 %), butyrylthiocholin iodide (BTCHJ - purity: ≥ 98.0 %), sodium bicarbonate (NaHCO₃ - purity 99.0-100.0 %), sodium hydrogen phosphate (Na₂HPO₄.2H₂O - purity min. 99.5 %) and potassium dihydrogen phosphate (KH₂PO₄ - purity >= 99.0 %) were supplied by Sigma Aldrich. Sodium hydroxide (NaOH - purity min. 98.0 %) and boric acid (H₃BO₃ - purity min. 99.5 %) were supplied by Lach-Ner. Methanol (purity 99.8 %) was supplied by Chromservis. Butyrylcholinesterase (BChE, EC 3.1.1.8., specific activity 150 $\mu$ kat/l) was supplied by Bioveta.

2.2 Solutions

• Buffers: phosphate (pH 7; 0.1 M), borate (pH 7; 0.05 M )
• Substrate: butyrylthiocholin iodide, 0.075 M (23.77 mg were dissolved in 1 ml distilled water).
• Reagent: 5,5'-dithiobis-2-nitrobenzoic acid, 0.01 M (39.6 mg were dissolved in 10 ml phosphate buffer and 15 mg of sodium bicarbonate was added)
• Mixture: 0.1 ml substrate and 0.5 ml of reagent were dispersed into 13.3 ml of borate buffer
• Enzyme: 50 mg of butyrylcholinesterase was dissolved in 2 ml of borate buffer.

2.3 Principle of Detection with Photocolorimetric Detector

The proposed biosensor described in this article uses Ellman´s modification of CHR. This reaction goes on the white cotton tape 15 mm wide and 0.35 mm thickness. This is an ordinary tape biosensor with improving elements. In particular, this is a new principle of evaluating the results of this reaction using as photocolorimetric sensor. Photocolorimetric sensor can evaluate the colour change of a reaction. The method of dosing, which allows the appropriate dispensing reagents in microlitres volumes, is also an advantage. The whole system is consists of three basic parts as shown in Fig. 2. The first part is pneumatic and provides sampling across the tape by sampling pump, flow stabilization and the measurement of its value, using a flow meter. The second part is the dosage system that ensures each reagent dosing pump and dispenser containment vessels with the pressure relief system. The last part serves as the evaluation part and is described below.

Note: 1- digital RGB sensor, 2 – cotton tape, 3 – spraying of enzyme, 4 – spraying of reagents, 5 – sampling samples, and 6 – tape shifting mechanism.

2.4 Description of the Measurement Process

The algorithm of the measuring procedure is as follows:
(a) Insertion of the cotton tape for detection and its mechanical shift
(b) Spraying a defined volume of enzyme solution BuChE (the enzyme is applied at ambient temperature and at pressure not higher than 0.15 bar)
(c) Sampling samples across the tape
(d) Spraying of a defined volume of the mixture of substrate (butyrylthiocholin iodide - BTChI) and Ellman´s reagent
(e) Scanning an optical signal
(f) Data evaluation
The whole measurement process takes 5 min (2 min for sampling a sample, 1 min for incubation, and 2 min for scanning an optical signal). In case of VX compound, a part of the sampling device was heated to 40 °C during the whole measurement. Limit of detection (LOD - is the lowest analyte concentration likely to be reliably distinguished from the blank and at which detection is feasible)21 and limit of quantification (LOQ - is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met)21 were obtained for each substance from statistical processing of measured data (depending on the rate constant of the absolute amount of nerve agent):
LOD = k + 3 × s_y,
LOQ = k + 10 × s_y,
where s_y is the residual standard deviation of measurement, and k (the rate constant) is the value on the y axis of the regression line (mg/l).

3.1 Design of Evaluation

The course of a reaction was evaluated using a digital colour sensor which is sensitive to red, blue and green spectrum. Each colour is given by the intensity of these three primary colours. Ellman´s modification of CHR is based on the colour transition yellow-white. For this transition, there is critical representation of blue colour, i.e. the value of the signal B. The changes of the values of the signal B depending on time were measured for each concentration of nerve agents as shown in Fig. 3. Methanol was used for the preparation of the calibration solutions (its presence does not affect the activity and stability enzyme during detection). Its presence also improved sample evaporation during the spraying. The rate constant k was obtained from the linear regression of the equation (a polynomial approximation of the measured data): y = (k×x) + q.

The time for reading of the rate constants was optimized on the values of determination coefficients R² (it´s main purpose is the prediction of future outcomes on the basis of other related information) for Soman as shown in Table 1. Table 1 shows the values of the determination coefficients of the regression equations for different time intervals during the measurement of Soman (time counted from spraying a mixture of substrate and DTNB).

As the optimal time, 90-100 seconds after spraying the mixture of substrate and reagents were chosen. From the calculated rate constants, it can be determined whether or not the sample contains nerve agents.

3.2 Calibration Curves of Nerve Agents in the Air

Dependencies of rate constants on the concentration of nerve agents for Tabun, Sarin, Soman, Cyclosin, VX compound, and R 33 are shown in Figs. 4, 5, 6, 7, 8, and 9 respectively, were measured by the proposed tape photocolorimetric detector. The point of each concentration level was measured 10 times and each curve was constructed from their average values. The Figs. 4-9 shows that the highest inhibitory effect (according to the lowest nerve agent amount giving the positive signal of detector) was reached for Cyclosin and VX substance, while Tabun and R33 showed the lowest inhibitory activity. This fact correlates with the inhibition effect and thus the toxicity of individual organophosphorus inhibitors.

Limit of detection (LOD) and limit of quantification (LOQ) for proposed photocolorimetric biosensor were obtained by statistical processing of linear parts of the measured dependencies of rate constants on the nerve agents concentration as shown in the Table 2.

In summary, the simple and sensitive portable means for detection of nerve agents has been demonstrated. It is characterized by high robustness and reliability. The evaluation of the results is not dependent on subjective evaluation and allows an operator to work under difficult visual conditions. The whole analysis needs only 5 min and is simple to operate without sophisticated instrument. The method of dosing allows to dose the appropriate dispensing reagents in microlitres volumes. On the basis of the measurement it is clear, that this photocolorimetric biosensor can be used in future to detect all type of organophosphorus cholinesterase inhibitors in environment.

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	Mrs. Kamila Vymazalová received her Ing title (Environmental Analytical Chemistry) from the University of Technology, Faculty of Chemistry, Brno, Czech Republic, in 2008. She is pursuing her PhD from University of Defense, Brno. Presently she is working as research worker in the Military Technical Institute of Protection, Brno.
	Prof. Emil Halámek completed his graduate and postgraduate studies at the Military Academy in Brno, Czech Republic. He has been working at the University of Defense, the Institute of NBC Defence, Vyškov, Czech Republic. For a long time, he has been cooperating with the Military Technical Institute of Protection, Brno. He applies one's mind to the problems of chemical warfare agents, with an emphasis on research and development of technical means of chemical research and chemical control.
	Mr. Jiří Kadlcak received his PhD from MAGF Vyskov at 1991. He is working as head of Detection and Analytical Department of Military Technical Institute of Protection, Brno. His areas of interest includes: Stand-off detection CWA based on CO2 DIAL/DISC, point detection and identification real CWA and development the Analytical systems for CWA analysis.