To simulate the inhibition of the enzyme
To simulate the inhibition of the enzyme by organophosphorus, carbamate insecticides or nerve agents, a reversible cholinesterase inhibitor physostigmine was used for most experiments for safety reasons. However, the results of the measurements were verified using the soman organophosphate, which is one of the strongest known inhibitors and therefore has an extraordinary military significance. All the used solutions and methodology are summarized in Sections 2.5 and 2.6. The sample was considered as inhibited if it remained its white color for at least 2 min after 10 min of incubation in a physostigmine test solution. This was followed by the addition of a test solution, i.e. Ellman’s reagent and butyrylthiocholine iodide. It was found that the lowest concentration of physostigmine for all samples, which met the above-mentioned requirements, was 8.0 μg mL−1. This decrease in sensitivity compared to previous studies [23,25] could have been caused by the reduced inhibition of the enzyme which was immobilized in the first layer under the second coat. Physostigmine is a large molecule and is difficult to penetrate through the coating. Therefore there is a less disposition for a contact with the enzyme. The course of the inhibition test for sample E2.5 was depicted in Fig. 3. The stated RGB values in comparison with the RGB values from tests of activity showed no significant change during the first two minutes indicating sufficient inhibition of the immobilized enzyme.
In terms of sensitivity, a different situation occurs when dealing with nerve paralytic substances for which these detection systems are especially intended. It is expected that the detection limit for nerve agents will be from 10−2 to 10−4 mg L−1 in water . At a soman dose of 10−2 mg L−1, the sample layer of EP1.5 remained white for at least 2 min. This dose can be considered as a limit of detection at the given enzyme activity and workflow. It seems that this pellet-coated system is more selective for smaller cholinesterase inhibitor aspirin solubility such as soman.
However, a potential disadvantage for enzymatic detection via the Ellman’s method is the occurrence of false positive or false negative results caused by the presence of other compounds in the tested samples of contaminated liquids. Among these compounds belong strong bases, acids, oxidants, reductants and some organic solvents (e.g. benzene, ethanol or acetone). False positive results (yellow color occurs even in the presence of cholinesterase inhibitor) could be caused by reductants such as cyanides, thiols, sulfites and sulfides. False negative results meant no yellow color occurring or being indistinctive in the absence of cholinesterase inhibitor, therefore allowing this to be caused by oxidants such as nitrites or chlorine .
Tristimulus colorimetry was performed to determine to what extent the semipermeable coat decreases the level of coloring of a solution in which the samples are immersed during the detection. In case of this measurement lower color intensity in the solution was preferred since more colored pellets were then achieved. The results are summarized in Fig. 4. A significant decrease in the color of the above-mentioned solutions was observed in comparison with the sample of uncoated carriers from the previous study . A thicker coat also led to a reduction in color intensity (E2.5 compared to E5.0). The results of the test solution served as initial color.
Conclusion In this study an innovative carrier for the detection of cholinesterase inhibitors was prepared. The enzyme butyrylcholinesterase was immobilized in the HPMC layer on the surface of the pellet cores and consequently coated by a semipermeable layer comprised of Eudragit® RL, Kollidon® 25 and PEG 400. The second coating layer showed a unique influence on the properties of the carriers, especially the most important one being a very distinctive color transition compared to the previous results in other studies. Sample E2.5 showed the best results while achieving a deep yellow/orange coloring compared to a more common indistinctive light yellow and thereby enabled an easier and more accurate detection of cholinesterase inhibitors in water. It is therefore particularly suitable for a rapid detection of nerve agents in case of terrorist attacks.