Vladimír Pitschmann

Enzymatic Determination of Anticholinesterases Using a Composite Carrier

ABSTRACT A modified enzyme method is reported for the determination of cholinesterase inhibitors based on the use of a granulated composite carrier with immobilized butyrylcholinesterase. The detection system includes butyrylthiocholine as the substrate and N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazoline-4-yl)-2-chloro-5-sulpho-4-iminobenzoquinone as a redox indicator. This method was characterized using carbamate physostigmine and sarin (O-isopropyl methylphosphonofluoridate). The method was also used in a simple tube detector. Evaluation was based on the observation with the naked eye of the decolorization of the redox indicator in comparison to a control time and by tristimulus colorimetry. The limits of detection were 0.5 µg/mL for physostigmine and 7 × 10−4 mg/m3 for sarin visually, or 0.06 µg/mL for physostigmine and 1.1 × 10−4 mg/m3 for sarin by colorimetry.

New Carrier Made from Glass Nanofibres for the Colorimetric Biosensor of Cholinesterase Inhibitors

Cholinesterase inhibitors are widely used as pesticides in agriculture, but also form a group of organophosphates known as nerve chemical warfare agents. This calls for close attention regarding their detection, including the use of various biosensors. One such biosensor made in the Czech Republic is the Detehit, which is based on a cholinesterase reaction that is assessed using a colour indicator—the Ellman’s reagent—which is anchored on cellulose filter paper together with the substrate. With the use of this biosensor, detection is simple, quick, and sensitive. However, its disadvantage is that a less pronounced yellow discoloration occurs, especially under difficult light conditions. As a possible solution, a new indicator/substrate carrier has been designed. It is made of glass nanofibres, so the physical characteristics of the carrier positively influence reaction conditions, and as a result improve the colour response of the biosensor. The authors present and discuss some of the results of the study of this carrier under various experimental conditions. These findings have been used for the development of a modified Detehit biosensor.

Military importance of natural toxins and their analogs

Toxin weapon research, development, production and the ban on its uses is an integral part of international law, with particular attention paid to the protection against these weapons. In spite of this, hazards associated with toxins cannot be completely excluded. Some of these hazards are also pointed out in the present review. The article deals with the characteristics and properties of natural toxins and synthetic analogs potentially constituting the basis of toxin weapons. It briefly describes the history of military research and the use of toxins from distant history up to the present age. With respect to effective disarmament conventions, it mentions certain contemporary concepts of possible toxin applications for military purposes and the protection of public order (suppression of riots); it also briefly refers to the question of terrorism. In addition, it deals with certain traditional as well as modern technologies of the research, synthesis, and use of toxins, which can affect the continuing development of toxin weapons. These are, for example, cases of new toxins from natural sources, their chemical synthesis, production of synthetic analogs, the possibility of using methods of genetic engineering and modern biotechnologies or the possible applications of nanotechnology and certain pharmaceutical methods for the effective transfer of toxins into the organism. The authors evaluate the military importance of toxins based on their comparison with traditional chemical warfare agents. They appeal to the ethics of the scientific work as a principal condition for the prevention of toxin abuse in wars, military conflicts, as well as in non-military attacks.

A SIMPLE COLORIMETRIC DETECTOR OF SYMMETRIC 2-(DIALKYLAMINO)ETHANETHIOLS AS PRECURSORS OF V-TYPE NERVE AGENTS

A colorimetric detector tube of 2-(dialkylamino)ethanethiols as precursors of V-type nerve agents and their hydrolytic products was designed. The detector is based on a reaction of 2-(dialkylamino)ethanethiols with a chromogenic reagent 4-chloro-7-nitrobenzofurazan (NBD-Cl) in alkaline medium leading to characteristic coloured products. The detection limit in water is of 5 μg/ml, the optimum measurement range being up to 250 μg/ml. The detector also presented a response to some other warfare chemical agents, the colour shade being, however, different. Effects of disturbing substances and organic solvents were studied.

Pellet patented technology for fast and distinct visual detection of cholinesterase inhibitors in liquids

Graphical abstract Figure. No Caption available. HighlightsDouble‐coated pellets for detection of cholinesterase inhibitors were prepared.Lower amount of enzyme for a formulation of the detection system was needed.A more distinctive color transition during the detection in liquids was achieved. ABSTRACT The main objective of the presented research was to prepare an innovative carrier as a filler for detection tubes in the form of double‐coated pellets with a very significant color transition during the detection of cholinesterase inhibitors such as nerve agents, organophosphorus or carbamate insecticides in liquids that is observable visually and also spectrophotometrically at 412 nm. The pellet cores were prepared by the extrusion/spheronization method. Consecutively, two different coats were applied on the pellet cores in the coating device using the Wurster column method. To increase the color change intensity, the second semipermeable coat based on Eudragit® RL was applied on top of the first coat, which was formed by butyrylcholinesterase immobilized in hydroxypropyl methylcellulose. Prepared samples differing in thickness of the second coat were evaluated for their quality parameters, enzymatic activity and inhibition. The detection mechanism was based on the standard Ellman’s colorimetric reaction. It was observed that the semipermeable coat prevented leaching of the enzyme into the solution and led to an increased intensity of color transition from white – yellow to white – deep yellow/orange, thus enabling a more accurate visual detection. This system allows easy, rapid and safe identification of cholinesterase inhibitors in liquids, especially chemical warfare agents.

Modified Biosensor for Cholinesterase Inhibitors with Guinea Green B as the Color Indicator

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%).