RU2386120C2 - Optical biosensor of irreversible cholinesterase inhibitors in air - Google Patents

Abstract

FIELD: physics.

SUBSTANCE: optical biosensor of irreversible cholinesterase inhibitors in air has an optical detector and an active component consisting of a complex immobilised on a neutral substrate, where the complex contains cholinesterase and a molecular rotor (MR) type fluorogenic indicator which fluoresces intensely with that enzyme in the acetylcholinesterase-molecular rotor complex. The fluorogenic indicator used is thioflavine T, whose fluorescence intensity in the presence of acetylcholinesterase increases by more than 1000 times. Irreversible inhibition of acetylcholinesterase also speeds up.

EFFECT: disclosed biosensor is more sensitive to irreversible inhibitors.

3 cl, 1 dwg, 2 tbl, 1 ex

Description

The present invention relates to the field of environmental pollution control, in particular, organophosphorus insecticides and carbamates.

The high toxicity and cumulative effect of many organophosphorus inhibitors (POIs) of cholinesterase (ChE) determine the urgency of creating sensitive technical tools designed to monitor the ambient air for the presence of ChE inhibitors.

In the field, it is advisable to use the most simple in design and easy-to-use analytical technical equipment for determining ChE inhibitors, such as a biosensor - a device consisting of an active component and a converter of the analytical effect into a recorded signal (light, sound, etc.). The active component of the biosensor (BS) mainly plays the role of a “recognizing” agent that specifically interacts with the analyte, for example, an enzyme — a substrate, an enzyme — an inhibitor, etc. This role is well matched by ChE, which is highly sensitive and specific in relation to it inhibitors. To convert the analytical effect into an information signal, optical schemes are usually used.

Of the optical BSs, BSs are most sensitive to FSI, based on recording as an analytical fluorescence response. Optical BS definitions of ChE inhibitors are known, based on the use of a fluorescent label, which is a reversible ChE inhibitor [1, 2]. For example, to analyze water samples, the complex of acetylcholinesterase (AChE) and fluorescein isocyanate (FITC) immobilized on silica fibers is used as the active component of an optical BS [1]. AChE activity is monitored by the pH dependence of the fluorescent signal of the FITC-AChE complex immobilized on the surface of the fiber. Enzymatic hydrolysis of the substrate (acetylcholine) with acetylcholinesterase results in the formation of protons that inhibit AChE labeled with FITC, and this causes quenching of the fluorescence of the reaction solution. Reversible inhibitors of edrofonium and neostigmine carbamate at a concentration of 0.1 mM inhibit FITC-labeled AChE, which leads to a decrease in the degree of fluorescence quenching. The degree of decrease in fluorescence intensity is inversely proportional to the concentration of these inhibitors. Such a BS determines the concentration of POI (echothiophate and paraoxon) in the range of nM-μM.

The disadvantage of this BS is its low specificity, high sensitivity to acid reagents that may be present in the analyzed air and, thus, introduce a certain error in the analysis results. The listed disadvantages of such a BS do not make it possible to use it for determination of ChE inhibitors in the air and monitoring.

Closest to the proposed analytical device is an optical BS for determining ChE inhibitors in air, the active component of which is made from a complex of this enzyme with a reversible lumogen inhibitor I _f immobilized on a neutral substrate [2]. The fluorescence intensity of I _f , for example N-methylacridine, decreases when exposed to ChE, which is due to the formation of a non-fluorescent enzyme-inhibitor complex [ChE-I _f ]. In turn, when exposed to a non-fluorescent irreversible inhibitor I _n , for example O, O-diisopropyl fluorophosphate, an increase in the fluorescence intensity is observed. The observed effect is due to high-speed phosphorylation of ChE, which leads to a shift in equilibrium towards dissociation of the [ChE-I _f ] complex, an increase in the concentration of free reversible fluorophore inhibitor I _f, and, as a consequence, to an increase in the fluorescence intensity of the reaction solution. The advantage of this BS is the insignificant effect of the increased acidity of atmospheric air due to the use of a buffer with a high buffer capacity in the active component.

However, this BS does not meet modern requirements for sensitivity. This BS is adopted as a prototype.

The proposed solution is aimed at increasing the sensitivity of BS to ChE inhibitors.

The technical result is achieved by the fact that in the proposed BS, the active component is an intensely fluorescent complex immobilized on a substrate formed by AChE and fluorogen from the group of indicators of the "molecular rotor" type (MR). A number of such fluorogens MR are known, for example, propidium, gallamine, thioflavin T (TF) and others [3], the fluorescence intensity of which, for example TF, increases in the presence of AChE by two to three orders of magnitude [3]. When the POI acts on the active component, the fluorescence intensity of the [AChE-TF] complex decreases in proportion to the concentration of the irreversible inhibitor. At the same time, an increase in the rate of AChE inhibition by organophosphorus inhibitors in the presence of certain MRs, such as propidium or TF, was found. These MR properties allowed us to propose using the [AChE-MR] complex as an active component of the BS, which is characterized by a higher sensitivity and shorter response time of the analytical effect to irreversible ChE inhibitors compared to the active component of the prototype BS.

In addition, it is proposed to introduce a laser light source to excite the active component, a modulator and a synchronous amplifier into the optical part of the BS, which will increase the sensitivity of the BS as a whole.

The design of the optical part of the BS is shown in the drawing in the form of a block diagram.

Example. The manufacture of the active component

The following Sigma reagents were used: eel acetylcholinesterase, thioflavin T, Na ₂ HPO ₄ , KH ₂ PO ₄ , paraoxone, N-isopropyl acrylamide, N, N'-methylene-bisacrylamide.

The AChE complex with thioflavin was immobilized in 0.03 M Tris-HCI buffer (pH 7) at 4 ° С.

In 1 ml of 0.03 M Tris-HCI buffer (pH 7) containing 200 mg of N-isopropylacrylamide, AChE (20 U / ml) and 1 mM thioflavin were added at 4 ° С with stirring 0.02 ml of a solution of N, N'-methylene- bisacrylamide (18 mg / ml) was applied to the surface of a neutral polymer film and polymerized for 20 minutes under UV irradiation.

The analysis of the results of the determination of an irreversible paraoxon inhibitor by the biosensor prototype and the proposed biosensor are given in table 1.

Table 1 The results of the determination of paraoxon by the active component of the biosensor prototype and the proposed biosensor Reference solution BS prototype proposed BS The amount of paraoxone, nm The amount of paraoxone, nm The amount of paraoxone, nm 1.00 Not found 1.14 ± 0.10 10.00 Not found 9.20 ± 1.05 100.00 95.05 ± 10.25 102.0 ± 9.05

From the data presented in table 1, it follows that the sensitivity to the paraoxon of the proposed active component is significantly higher than the prototype.

Thus, the active component of a biosensor based on an intensely fluorescent complex immobilized on a substrate formed by AChE and a fluorogen from the group of molecular rotor (MR) type indicators can increase the fluorescence intensity by two to three orders of magnitude and has the ability to accelerate irreversible inhibition of AChE, which allowed to design a BS more sensitive to irreversible inhibitors, including organophosphorus inhibitors, compared with a prototype BS.

Table 2 compares the essential features of the BS prototype and the proposed BS.

Table 2. The essential features of the biosensor of the prototype and the proposed biosensor No. p / p Salient features Biosensor prototype Suggested biosensor one Active component Cholinesterase complex with inhibitor lumogen immobilized on a neutral substrate A complex of cholinesterase with an indicator of the "molecular rotor" type, immobilized on a neutral substrate, causes an increase in fluorescence intensity by two to three orders of magnitude 2 Response time The composition of the active component does not allow to further reduce the response time of the biosensor The composition of the active component allows to reduce the response time of the biosensor by increasing the rate of irreversible inhibition 3 Optical sensor Classical optical fluorescence detection scheme It is proposed to use a laser light source to excite the active component, a modulator and a synchronous amplifier to increase the stability and sensitivity of fluorescence detection

Used literature

1. Rogers K.R., Sao C.J., Valdes J.J., Eldefrawi A.T., Eldefrawi M. E. Fundam Appl Toxicol 1991. v.16, p. 810-20.

2. RF patent No. 2198394, 12/18/2003. IPC ⁷ C12Q 1/46, G01N 21/76.

3. De Ferrari G.V., Mallender W. D., Inestrosa N.C., Rosenberry T.L. J. Biol. Chem. 2001. v.276, p.23282-23287.

Claims (3)

1. The optical biosensor of irreversible cholinesterase inhibitors in the air, including an optical detector and an active component, consisting of a complex immobilized on a neutral substrate, characterized in that the complex is an intensely fluorescent complex, including cholinesterase and a fluorogenic indicator of the molecular rotor type. 2. The optical biosensor according to claim 1, characterized in that the fluorogenic indicator of the "molecular rotor" type is thioflavin T. 3. The optical biosensor according to any one of claims 1 and 2, characterized in that it includes a laser light source to excite the active component, a modulator and a synchronous amplifier.