Colorimetric biosensors of cholinesterase inhibitors are ideal for fast, reliable, and very simple detection of agents in air, in water, and on surfaces. This paper describes an innovation of the Czech Detehit biosensor, which is based on a biochemical enzymatic reaction visualized by using Ellman’s reagent as a chromogenic indicator. The modification basically consists of a much more distinct color response of the biosensor, attained through optimization of the reaction system by using Guinea Green B as the indicator. The performance of the modified biosensor was verified on the chemical warfare agents (sarin, soman, cyclosarin, and VX) in water. The detection limits ascertained visually (with the naked eye) were about 0.001 µg/mL in water (exposure time 60 s, inhibition efficiency 25%).
Cholinesterase inhibitors adversely affect the transmission of nerve impulses by preventing hydrolytic decomposition of acetylcholine. The accumulation of this neurotransmitter in the area of a synapse is the primary cause of intoxication of the organism. Cholinesterase inhibitors include common organophosphorus and carbamate pesticides, both synthetic and natural substances used in medicine but also as nerve warfare agents. Considerable attention is paid to the detection and determination of cholinesterase inhibitors. In addition to advanced instrumental methods, simple chemical methods are also used in practice, including methods based on color reactions. It is color changes that simple colorimetric detectors are based on, such as indicator papers and strips, detection tubes or various detection kits, and pocket laboratories. Some cholinesterase inhibitors, especially nerve chemical warfare agents, are extremely toxic: for instance, the agents GB (sarin), GD (soman) and GF (cyclosarin) have LCt 50 by inhalation about 35 mg·min/m 3, the agent VX even a mere 15 mg·min/m 3. So, biosensors based on very sensitive enzymatic (cholinesterase) reactions are required for simple detection of the substances in dangerous concentrations.
The enzymatic (cholinesterase) reaction utilized in simple biosensors is based on color indication of the product of hydrolysis of a suitable substrate. The inhibitor concentration is then proportional to the degree of inhibition of the enzyme and the color change rate. The first biosensors with acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) contained the substrates acetylcholine and butyrylcholine, which split into choline and the respective acid (acetic or butyric), detectable with a pH-indicator. Recently, however, acetylcholine and butyrylcholine have largely been replaced with acetylthiocholine or butyrylthiocholine, respectively, in which case thiocholine is produced instead of choline. Thiocholine changes the color of redox indicators, such as Ellman’s reagent, 2,6-dichlorophenolindophenol or its analogues, or triphenylmethane dyes such as Guinea Green B and Malachite Green. Chromogenic substrates, e.g., 2,6-dichlorophenolindophenyl acetate and indoxyl acetate, which decompose directly on colored products, are frequently used alternatives. A lower reaction rate and poorer availability are the drawbacks.
An example of a biosensor of cholinesterase inhibitors used widely in practice is the Czech Detehit biosensor, which is based on AChE (obtained directly from a pig brain), the substrate acetylthiocholine and Ellman’s reagent as a redox indicator. In spite of its undisputed advantages, this biosensor provides an unclear cut white-yellow color transition, which may cause difficulties especially during visual evaluation in poor light conditions. Earlier, the authors had designed a modification of the Detehit biosensor by using filter paper made from glass nanofibres (as the carrier of the substrate and of Ellman’s reagent), which deepens the intensity of the developing yellow color. They also designed a modified biosensor with the indicator 2,6-dichlorophenolindophenol exhibiting a distinct blue-white color transition. During the further development of this biosensor, the comments, and requirements of potential users (armed forces, rescue corps) were accepted to resist the ambient temperature at around 60 °C for a short time (at least several days). As it turned out, the biosensor with 2,6-dichlorophenolindophenol did not meet this requirement, therefore another, more appropriate indicator was sought. This paper describes an innovated biosensor with Guinea Green B as an indicator providing a distinct green-white color transition, and has increased heat resistance, while maintaining the basic analytical parameters (simple and rapid detection and detection limits).
Butyrylcholinesterase (BuChE) from horse plasma, butyrylthiocholine iodide (BuTChI), N-ethyl-N-[4-[[4-[ethyl[(3-sulphophenyl)methyl]amino]phenyl]-phenylmethylene]-2,5-cyclohexadiene-1-ylidene]-3-sulpho-benzenemethane ammonium sodium hydroxide—internal salt (Guinea Green B, C.I. 42085), dextran (all by Sigma-Aldrich, St. Louis, MO, USA), the non-ionic surfactant C12-14 alcohol 7EO, trade name Spolapon 247 (Enaspol, Teplice, Czech Republic), absolute ethanol (Penta, Prague, Czech Republic) and redistilled water were used. A buffer solution at pH 7.3 was prepared using Na 2 HPO 4·H 2 O and KH 2 PO 4 (both by Sigma-Aldrich). Physostigmine (Sigma-Aldrich, St. Louis, MO, USA), sarin, soman, cyclosarin and VX (all obtained from the Military Research Institute, Brno, Czech Republic) were used to test the biosensor performance.
The biosensor was prepared by using a white cellulose fabric whose surface density was 173 g/m 2, cellulose filter paper whose surface density was 85 g/m 2, and a white plastic pad 0.5 mm thick.
An LMG 173 portable tristimulus colorimeter (Dr. Lange, Dusseldorf, Germany) was used for objective color change measurements. An AB150 instrument (Fisher Scientific, Pardubice, Czech Republic) was employed to measure the pH.
The biosensor was prepared from a plastic pad 10 × 80 mm in size, onto which a carrier with the immobilized enzyme (indicator zone), an etalon, and a carrier impregnated with the substrate and the indicator were glued. The indicator zone consisted of a white cellulose fabric immersed for 25 min in a solution containing BuChE at a specific activity of 15 nkat/mL, 3.5% (m/m) of dextran and 1.5% (m/m) of a non-ion surfactant in a phosphate buffer solution with pH 7.3. The standard was made from a white cellulose fabric impregnated with a solution containing 9.2% (m/m) of dextran and 5.5% (m/m) of a non-ionic surfactant in a buffer solution pH 7.4. The two impregnated pieces of fabric were dried at 25 °C for 12 h. The working part with the substrate and with the indicator was prepared by immersing the filter paper for 5 min in a solution containing 1.2% of the substrate and 0.2% of Guinea Green B in 50% ethanol. The impregnated paper was dried at 20 °C to 25 °C for 6 h.
The performance of the biosensor was studied in water and in aqueous solutions of selected cholinesterase inhibitors. The working procedure was similar to that used with the Detehit biosensor, with some minor differences matching the design modifications. In step 1, the biosensor (fabric with the enzyme and the etalon) was immersed for 60 s in the sample, analyzed, then removed and rinsed with distilled water. In step 2, the plastic pad was folded and the opposite carriers were pressed onto one another so that the paper with the substrate and indicator overlapped both the fabric with the enzyme and the standard. After 120 s, the carriers were separated and the color of the fabric with the enzyme (indicator zones) was observed. The etalon, on which the stable green color remained after the separation of the carriers, served to facilitate the evaluation.
The inhibitor concentration, which is a function of their inhibitions efficiency (I), was determined visually (with the naked eye) based on the relation: I (%) = (1 − T 0/T) × 100 where T 0 is the control time (in seconds) of discoloration of the indicator with the blank (T 0 = 120 s applies to the proposed system), and T is the time (in seconds) of its discoloration in the presence of the inhibitor. The detection limit corresponded to a concentration that had a 25% inhibitory effect (the indicator was completely decolored in 160 s, 40 s later than the blank). This 25% inhibitory efficacy was selected on the basis of long-term field practice experience in emergency situations that not only required sensitivity but also high detection reliability. The detection limit determined visually (with the naked eye) is not exact, but it is quite common in the field of chemical test methods of analysis.
The proposed method of determining the inhibitors concentration or determination of the detection limit did not require the use of any instrumentation technique (only the time of discoloration was measured). The instrumentation technique (tristimulus colorimeter) was used only to study selected biosensor parameters where the evaluation with the naked eye is difficult or impossible. Tristimulus colorimetry, a.k.a. reflectance colorimetry (spectrophotometry), based on the CIE-L*a*b* color system was employed for the objective measurement of the color changes. In this system, L* represents the neutral brightness axis, a*, the chromatic green-red axis (+a* red, −a* green), and b*, the chromatic blue-yellow axis (+b* yellow, −b* blue). In practice, the color difference Δ E was calculated according to the equation Δ E=Δ L∗2+Δ a∗2+Δ b∗2 is also used, where Δ L*, Δ a* and Δ b* are the differences between the individual L*, a* and b* values of the standard and the color measured. Differences Δ E up to 0.2 are imperceptible with the naked eye, 0.2–0.5 negligible, 0.5–1.5 very small, 1.5–3.0 distinct, 3.3–6.0 very distinct, 6.0–12.0 profound and above 12.0 very profound.
The principle of the biosensor consists in BuTChI hydrolysis catalyzed by the BuChE enzyme. The hydrolysis produces thiocholine, which reduces Guinea Green B to its colorless leuco-form. The reaction schema is shown. The practical use of the biosensor was then based on monitoring the kinetics of this enzymatic reaction; i.e., on the rate of discoloration of the indicator zone. Such discoloration proceeds significantly more slowly in the presence of a cholinesterase inhibitor. Figure 3a shows the response of the biosensor (i.e., blank, the indicator zone is completely colorless) and on a sample contaminated by the inhibitor, where the indicator zone remains green after the carriers have been separated. It is evident from a comparison with a standard Detehit biosensor (Figure 3b) that the color change is much more marked with the modified biosensor.
