CN110016469A - Can decontamination never poison enzyme-polymer complex and its preparation method and application - Google Patents

Abstract

The application provide can decontamination never poison enzyme-polymer complex, wherein the enzyme be selected from DFPase class phosphotriester hydrolase；The polymer is water-soluble polymer.Present invention also provides the preparation method of the compound and its purposes in decontamination toxic agent.The compound can be realized the decontamination to never poison extremely fast, and not stimulate skin mildly.

Description

Enzyme-polymer complex for decontaminating nerve agent and preparation method and use thereof

technical field

The invention relates to the field of chemical prevention, in particular to an enzyme-polymer complex, which can be used as a decontamination material for nerve agents. The complex can achieve extremely fast decontamination of nerve agents, has mild performance, and is non-irritating to skin, equipment, environment, and the like.

Background technique

1. Introduction to nerve agents

Nerve agent is one of chemical warfare agents CWA (chemical warfare agents), which refers to toxic chemicals that destroy the normal conduction function of the nervous system. The injury routes of nerve agents include skin, eyes, respiratory tract, digestive tract, etc. Among them, the skin has the largest surface area and is most likely to be contaminated with the agent, causing poisoning or even death.

Nerve agents are mainly divided into G-type agents and V-type agents. They have in common: stability, that is, they are not easily decomposed at room temperature; quick-killing, that is, if they are not rescued in time, they can quickly cause casualties to die. Class G poisons mainly include tabun GA (tabun), sarin GB (sarin), and soman GD (soman). GD has almost no specific detoxification enzymes in the body, and it is very difficult to treat the body after exposure. The V-type poison is mainly Viex VX, which is characterized by long-term effect, very slow degradation in the environment, and the contamination of the poisoning area after its use lasts for a long time; ten times. To sum up, in order to quickly remove nerve agents from the human body and the environment and ensure the safety of civilians or combat personnel, research on nerve agent decontamination materials is particularly important.

2. Decontamination of nerve agents

Existing methods for decontamination of nerve agents mainly include: natural decontamination, physical decontamination, chemical decontamination and biological decontamination. Natural decontamination mainly decontaminates nerve agents on poisoned objects through ventilation, sun exposure, rain washing, self-evaporation and decomposition, etc. The decontamination speed is extremely slow and uncontrollable; physical decontamination mainly uses activated carbon and other adsorption materials to remove Adsorption, solvent absorption, high-temperature decomposition, washing, etc., can be quickly decontaminated, but the decontamination is not thorough, which is likely to cause leakage or secondary pollution; chemical decontamination mainly includes the use of compounds containing effective chlorine (such as hypochlorites) or chloramines, etc.), alkali-alcohol-amine disinfection systems (such as sodium hydroxide, sodium bicarbonate or ethanolamine, etc.), oxidants and other materials (such as hydrogen peroxide), which can completely eliminate the poison through chemical reaction, but have certain irritation and corrosion Some materials are difficult to apply to equipment and human body; biological decontamination, mainly through the catalyzed hydrolysis of nerve agents by biological decontamination enzymes, is known as "the most ideal Detergent".

In 1992, the International Union of Biochemistry and Molecular Biology Committee uniformly classified the above-mentioned biological decontamination enzymes as phosphotriester hydrolase PTH (Phosphoric triester hydrolase), including organophosphorus hydrolase OPH (Organophosphorus hydrolase), methyl parathion hydrolysis Phosphotriesterase PTE (Phosphotriesterase) represented by Methyl parathion hydrolase and organophosphorus hydrolase C2 (OPHC2), with diisopropyl fluorophosphatase DFPase (Diisopropyl-fluorophosphatase), human serum paraoxonase 1 (Human serum paraoxonase1, PON1) and organophosphoric acid anhydrase OPAA (Organophosphorous acid anhydrolase) represented by diisopropyl fluorophosphatase DFPase class. Among them, the hydrolase represented by OPH mainly breaks the P-O bond of the poison molecule, and the hydrolase represented by DFPase mainly breaks the P-O and P-CN bond of the poison molecule. Compared with OPH enzyme, DFPase has a small molecular weight, is easy to separate, prepare and process in the later stage, and is resistant to high temperature (70 degrees) and is not easy to be inactivated. Bundeswehr equipment. In general, the advantages of biodetergents are that they can efficiently and specifically catalyze the decomposition of toxins under mild conditions, and are less irritating to the skin and body. Although biological enzyme-based detergents are mild, their decontamination efficiency is relatively slow, and nerve agents are quick-killing agents. But people have been poisoned and died.

The high lethality and quick-killing properties of nerve agents place higher requirements on decontamination technology and decontamination materials: (1) The decontamination agent must completely decontaminate the poisonous agent, and a slight residue will cause a serious poisoning reaction; (2) The decontamination agent must complete the decontamination of the poison molecules in a very fast time before the poison molecules diffuse and take effect as much as possible, and prevent them from contacting the skin; (3) The performance is as mild as possible, not irritating and Damages the skin, does not corrode and destroy equipment, and reduces environmental pollution. Therefore, how to design a detergent with high efficiency, rapidity and low irritation has always been an unsolved problem in the art.

SUMMARY OF THE INVENTION

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps involved in this article are all routine steps widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term DFPase, is diisopropyl fluorophosphatase.

As used herein, the term NAS, is N-acryloyloxysuccinimide.

As used herein, the term DMAEMA, is 2(N,N-dimethyl)ethyl methacrylate.

As used herein, the term MBA, is N,N'-methylenebisacrylamide.

As used herein, the term KPS, is potassium persulfate.

As used herein, the term TMEDA, is N,N,N',N'-tetramethyldiethylamine.

As used herein, the term NHS, is N-hydroxysuccinimide.

As used herein, the term DFPase-DMAEMA, is a diisopropyl fluorophosphatase-hydrogel complex.

As used herein, the term OPH-DMAEMA, is an organophosphorus hydrolase-hydrogel complex.

As used herein, the term F127, is a polyoxyethylene/polyoxypropylene/polyoxyethylene amphiphilic block copolymer (Pluronic F127, abbreviated as F127)

As used herein, the term ATCh, is thioacetylcholine iodide.

As used herein, the term DTNB is 5,5-dithiobisnitrobenzoic acid.

As used herein, the term h, means hours.

As used herein, the term min, means minutes.

As used herein, the term n, is the number of moles.

As used herein, the term M, is molar mass, ie g/mol.

As used herein, the term g, means grams.

As used herein, the term V, is volume.

In order to meet the high-efficiency, large-scale, rapid, and broad-spectrum decontamination requirements for nerve agents, the inventor obtained the enzyme-polymer composite decontamination material of the present application through in-depth research and creative work, which can be used for nerve agents. Extremely fast decontamination of poisons, and mildness without irritating the skin, thereby providing the following invention:

In one aspect, the present application provides an enzyme-polymer complex for decontamination of nerve agents, wherein the enzyme is selected from the group consisting of diisopropyl fluorophosphatase (DFPase) phosphotriester hydrolase ( DFPase enzyme for short); the polymer is a water-soluble polymer.

In some preferred embodiments, the DFPase-like phosphotriester hydrolase is selected from the group consisting of DFPase, human serum paraoxonase 1 (PON1) and organophosphoric anhydrase (OPAA).

In some preferred embodiments, the polymer is selected from poly-2-(N,N-dimethylamino)ethyl methacrylate (pDMAEMA), polyacrylic acid, F127, Tween, Triton X100, PLGA and cross-linked polymers formed with cross-linking agents. In some preferred embodiments, the polymer is selected from the group consisting of poly-2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA), F127, and cross-linked polymers formed with cross-linking agents. In some preferred embodiments, the crosslinking agent is N,N'-methylenebisacrylamide (MBA).

In some preferred embodiments, the nerve agent is an organophosphorus agent, including sarin, tabun, soman, and VX, with soman being preferred.

In some preferred embodiments, the DFPase-like enzymes introduce monomeric groups through surface amino groups. In some preferred embodiments, the monomer group is selected from -CO-( _CH2 ) _m -CH= _CH2 , and m is selected from an integer of 0-6. In some preferred embodiments, the complex is polymerized by DFPase to which the monomer group is attached, 2-(N,N-dimethylamino)ethyl methacrylate, and an optional cross-linking agent. to make.

In some preferred embodiments, the complex is formed from the F127 linked to DFPase via a linker -CO-( _CH2 ) _n- CO-, wherein n is selected from an integer of 0-6.

In some preferred embodiments, the particle size of the composite is 50-400 nanometers (eg 50-350 nanometers, 50-300 nanometers, 50-250 nanometers, 50-200 nanometers, 50-150 nanometers, 100-350 nanometers) nm, 100-300 nm, 100-250 nm, 100-200 nm, 100-150 nm, 150-350 nm, 150-300 nm, 150-250 nm or 150-200 nm).

In some preferred embodiments, the complex is a core-shell structure.

In some preferred embodiments, the composite shell is 50-200 nm thick (eg, 50-150 nm, 50-100 nm, or 100-150 nm).

In another aspect, the application provides the preparation method of the above-mentioned complex, it comprises the following steps:

method 1:

(1) introducing a monomer group into the enzyme, and then performing a polymerization reaction with the monomer of the polymer and an optional cross-linking agent to obtain the crude composite product;

(2) the complex crude product obtained in step (1) is dialyzed to obtain the complex; or,

Method 2:

(1) introducing a linker into the polymer, and then adding the polymer introduced into the linker into an aqueous solution of an enzyme to react to obtain the crude complex product;

(2) Dialyzing the crude complex obtained in step (1) to obtain the complex.

In method 1:

In some preferred embodiments, the monomer group is selected from -CO-(CH ₂ ) _m -CH=CH ₂ , and m is selected from an integer of 0-6;

In some preferred embodiments, the molar ratio of the crosslinking agent (if present) to the monomers of the following polymers is (0.2-1):1 (eg 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1): pDMAEMA, polyacrylic acid;

In some preferred embodiments, an initiator is also added to the polymerization reaction. In some preferred embodiments, the initiator is potassium persulfate. In some preferred embodiments, the molar ratio of the initiator to the monomer of the polymer is (0.01-0.5):1 (eg 0.01:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1 or 0.5:1);

In some preferred embodiments, a pH modifier is also added to the polymerization reaction. In some preferred embodiments, the pH adjusting agent is N,N,N',N'-tetramethyldiethylamine. In some preferred embodiments, the amount of the pH adjuster is such that the pH of the reaction system is 7.5-9.5.

In method 2:

In some preferred embodiments, the linker is selected from -CO-(CH ₂ ) _n -CO-, and n is selected from an integer of 0-6;

In some preferred embodiments, the polymer is selected from F127, Tween, Triton X100 and PLGA;

In some preferred embodiments, the aqueous concentration of the enzyme is 0.1-1 mg/ml, such as 0.1-0.5 mg/ml, 0.2 mg/ml;

In some preferred embodiments, one end of the linker is connected to the surface amino group of the enzyme through an amide bond, and the other end is connected to the polymer through an ester bond;

In some preferred embodiments, the crude complex product is dialyzed to remove small-molecule impurities with a molecular weight below 5000.

The composite obtained by the method 1 or 2 has a particle size of 50-400 nanometers (for example, 50-350 nanometers, 50-300 nanometers, 50-250 nanometers, 50-200 nanometers, 50-150 nanometers, 100-350 nanometers, 100- 300 nm, 100-250 nm, 100-200 nm, 100-150 nm, 150-350 nm, 150-300 nm, 150-250 nm or 150-200 nm) core-shell structure; its shell thickness is 50- 200 nanometers (eg 50-150 nanometers, 50-100 nanometers or 100-150 nanometers).

In another aspect, the present application provides a cleaning composition or cleaning solution comprising the aforementioned complex.

In another aspect, the present application provides a protective gear comprising the aforementioned complex or detergent composition or detergent.

In another aspect, the present application provides the use of the aforementioned complexes, decontamination compositions or decontamination solutions or protective equipment in a decontamination agent. In some preferred embodiments, the agent is a nerve agent or an erosive agent. In some preferred embodiments, the nerve agent is an organophosphorus agent such as selected from sarin, tabun, soman and VX, preferably soman. In some preferred embodiments, the erosive agent is mustard gas, Lewis reagent, or nitrogen mustard.

Beneficial Effects of Invention

The enzyme-polymer complexes of the present application have one or more of the following beneficial effects:

The present application provides a nerve agent decontamination material, which is an enzyme-polymer complex, which can achieve extremely fast decontamination of the nerve agent, and is mild and does not irritate the skin. In certain preferred embodiments, the complexes can approach 100% decontamination of the soman agent. In certain preferred embodiments, the complexes can achieve decontamination efficiencies approaching 100% in a very short period of time (eg, within 2 s).

The enzyme-polymer complex of the present application has high safety, is mild and does not irritate the skin. The nanocarrier used in the complex of the present application is a natural enzyme material, which has no immunogenicity and high biocompatibility, and the degradation products are various amino acids, which have no toxic and side effects.

Compared with the traditional synthesis scheme of enzyme polymer solidification, the inventors unexpectedly found that the decontamination rate of the product obtained by changing the feeding sequence of the polymer and the enzyme (as described in step (1) of the compound preparation method 2 of the present application) is improved. 100% within 2 seconds, far exceeding existing reports.

Description of drawings

Figure 1 shows the scanning electron microscope photos of DFPase complexes; among them, Figure A is DFPase-pDMAEMA, with a size of about 200-300nm; Figure B is DFPase-F127, with a circular shape and a size of about 200-300nm.

Figure 2 shows the transmission electron microscope photos of the DFPase complex; among them, Figure A is DFPase-pDMAEMA; Figure B is DFPase-F127; it is a core-shell structure with a dark protein in the center and a light-colored polymer layer in the outer layer.

Figure 3 shows the X-ray diffraction results of the DFPase complex.

Figure 4 shows DFPase-pDMAEMA and DFPase-F127 washout rates at high and low doses.

Figure 5 shows a comparison of Soman decontamination rates for commonly used decontamination materials.

Figure 6 shows the pathology and photos of skin irritation and damage by high concentration NaOH (Panel A), DFPase-pDMAEMA (Panel B), DFPase-F127 (Panel C) and normal group (Panel D).

Figure 7 shows the inhibition rates of DFPase-pDMAEMA and DFPase-F127 on blood enzymes in vivo after decontamination of rat skin.

Figure 8 shows the washout rate of OPH-pDMAEMA.

Detailed ways

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1 Synthesis of DFPase-pDMAEMA

1. Isolation and purification of DFPase

(1) Weigh 650g of fresh pork liver into a flask, cut it into small pieces, add distilled water and mix.

(2) 100 ml of cell lysing solution was added to it to fully rupture the porcine liver tissue cells.

(3) Homogenize in batches with a homogenizer, and collect a total of 2000 ml of homogenate.

(4) Centrifuge the homogenate (10000 r/min, 20 min, 4° C.), and collect the supernatant to obtain a total of 1500 ml of supernatant A (suspension).

(5) The supernatant liquid A is heat-treated. The collected supernatant was placed in a water bath at 60°C for 10 min, and then quickly cooled in ice cubes. Then, it was centrifuged (10000 r/min, 10 min, 4° C.) to remove denatured protein impurities, and 1175 ml of supernatant B solution (red, clear) was collected.

(6) Filter the supernatant B to remove impurities. Aspirate solution B with a 50ml syringe, filter it in batches with a 0.22 μm filter, and obtain a total of 1160ml of solution C.

(7) In order to remove the small molecular salts and part of the water in the C liquid, centrifugal ultrafiltration (10000r/min, 1h, 4°C) with a 15ml ultrafiltration tube (molecular weight cut-off is 3000) was used to collect the concentrated D liquid 98ml.

(8) with α-naphthol diazonium salt color reaction reagent, the purpose is to determine the position of the test tube containing DFPase in the next chromatographic process. The reaction principle is: DFPase can hydrolyze α-naphthol ethyl ester under certain conditions to generate the product α-naphthol, which can be coupled with the stable diazonium salt Fast Violet B Salt to generate A stable purple-red compound, the substance has a good absorption value at the wavelength of 580nm.

Reagent preparation:

a. Substrate solution: 1 part of 0.03mol/L α-naphthol ethyl ester (tert-butanol:n-butanol=9:1 (V:V) as solvent)+50 parts of 0.2mol/L, pH7.4 phosphoric acid Buffer + 49 parts water (V:V:V).

b. Color reagent: Quick violet B salt was dissolved in 0.2 mol/L, pH 7.4 phosphate buffer at a concentration of 0.1%.

c. Determination method: Add the solution to be tested into the substrate solution, and add the color reagent immediately after incubating at 37°C for 5 minutes. The solution turns from colorless to red, which proves that it contains DFPase.

(9) Determine by the color reaction of α-naphthol diazonium salt in step (7) that D solution (color development) is a solution containing DFPase, and D filtrate (no color development) does not contain DFPase.

(10) DE52 gel column chromatography separation of D filtrate. Divide 10 times, add about 10ml of sample each time, carry out DE52 chromatography, and use a collector to collect samples in each 5ml tube. Determine the sample receiving test tube where DFPase is located by the α-naphthol diazonium salt color development method, and collect it. A total of about 141 ml of E solution is collected.

(11) G50 gel column chromatography separation of DFPase. The E solution was divided into 14 batches, and each time about 10 ml was added for G50 chromatography. Determine the sample receiving test tube where DFPase is located by the α-naphthol diazonium salt color development method, and collect it. A total of about 127ml of F solution is collected.

(12) Determine the concentration of DFPase enzyme in F solution by BCA protein quantification method, the standard curve y=0.8978x+0.085, R ² =0.997, the final measured DFPase concentration is 0.2 mg/ml.

2. Synthesis of DFPase-pDMAEMA

(1) Thaw 30 ml of DFPase (0.2 mg/ml) into a pear-shaped bottle, and stir (600 r/min) in the dark.

(2) Subsequently, 25 mg of the vinyl acyl-introducing agent NAS was weighed and dissolved in 500 μl of DMSO solvent, and 180 μl was then added to the above reaction system.

(3) Then slowly drop the initiator KPS solution (30 μl, 10% w/v), the monomeric DMAEMA solution in DMSO (40 μl, 10% w/v), and the cross-linking agent MBA in DMSO solution (30 μl, 10% w/v) w/v), acid-base regulator TMEDA solution in DMSO (6 μl, 10% w/v), and stirred at room temperature in the dark (600 r/min, 6 h).

(4) Pretreatment of dialysis bag. First, 500 ml of 2% w/t NaHCO ₃ aqueous solution and 500 ml of 1 mM disodium EDTA aqueous solution were prepared. Subsequently, a dialysis bag with a molecular weight cut-off of 5000 was cut to a length of 30 cm. The dialysis bag was successively placed in the above-mentioned two groups of solutions of NaHCO ₃ and disodium EDTA and boiled (100 ° C, 10 min) in turn, rinsed with distilled water after each boiling, and weighed 30 g of HEPES and 1.2 g of NaOH and added to 2.5 g of NaOH. L distilled water, fully stir, disperse and dissolve to prepare dialysate for use.

(5) Product dialysis. Add the solution of (3) reaction to the dialysis bag (do not exceed 1/2-1/3 of the total volume of the dialysis bag), close both ends of the dialysis bag with clips, then put it into the dialysate, and stir at low speed at room temperature (150r/min). During the period, the dialysate was replaced every 12h, and the dialysate was replaced three times in total to completely remove the excess small molecule monomer impurities in the solution. The solution in the dialysis bag was collected after dialysis for 48 hours to obtain the product DFPase-pDMAEMA, which was colorless and transparent, with a total volume of 30 ml. The overall solution volume did not change significantly before and after the reaction, and it was placed in a refrigerator for frozen storage (-20°C).

Example 2. Synthesis of DFPase-F127

(1) Weigh 5g of F127 and put it into a two-necked pear-shaped bottle with a volume of 50ml. The system is sealed, evacuated and then filled with nitrogen. The subsequent reactions in this pear-shaped bottle are all carried out in a nitrogen-filled system. Mix 15ml of 1,4-dioxane and 110μl of triethylamine, then add 97mg of DMAP to dissolve completely, inject the solution into the above pear-shaped bottle with a syringe, and keep stirring (1000r/min, 90min).

(2) The succinic anhydride solution (99 mg of succinic anhydride dissolved in 5 ml of 1,4-dioxane) was slowly dropped into the pear-shaped bottle through a constant pressure dropping funnel, and the stirring was continued at room temperature (1000 r/min). , 41h).

(3) After stopping stirring, transfer the solution to a 100 ml pear-shaped bottle (the solution is nearly oily and colorless). Remove excess 1,4-dioxane with a rotary evaporator (100°C).

(4) Mix the sample with ether, centrifuge twice (10000r/min, 10min) to remove unreacted excess impurities, and retain the precipitated F127-COOH product, about 5g in total.

(5) F127-COOH (about 5 g in total) was dissolved in 65 ml of dichloromethane, and stirred at room temperature (1500 r/min, 10 min). 800 mg of EDC was dissolved in 5 ml of dichloromethane and then added dropwise to the reaction and stirring continued for 30 min.

(6) Dissolve NHS (500mg) in 10ml of dichloromethane, drop it into the reaction system, stir at room temperature for 24h, the total volume of the solution is about 80ml, and it is a milky white suspension.

(7) The solution mixture was precipitated with cold ether for 3 times, and then dried with a rotary evaporator (40° C.) to collect milky white precipitate F127-NHS, which was dried to obtain F127-NHS powder.

(8) Add 800 mg of F127-NHS powder (ie, 40 mg/ml, 20 ml) to the DFPase solution (0.2 mg/ml, 20 ml), and stir (1000 r/min, 16° C.) for 18 h.

(9) Dialysis, the dialysis method is the same as steps 4-5 in Example 1 to obtain the product DFPase-F127.

Example 3 Physical evaluation of DFPase-F127 and DFPase-pDMAEMA

(1) Morphological characterization. A small amount (about 150-200 μl) of DFPase-F127 and DFPase-pDMAEMA aqueous solution (the concentration of DFPase is 0.2 mg/ml) was respectively taken and dropped on the silicon wafer, and air-dried at room temperature. After drying, a nano-gold layer was sprayed to enhance the conductivity of the sample, and the sample was observed under a scanning electron microscope (SEM), as shown in Figure 1. It can be seen that the synthesized DFPase-F127 and DFPase-pDMAEMA are Nanoparticles with a size of about 200-300nm.

(2) Characterization of internal structure. Take 50μl of DFPase-F127 and DFPase-pDMAEMA aqueous solution (the concentration of DFPase is 0.2mg/ml) and drop them on the copper wire to air dry naturally. It is a near-circular nanoparticle with a diameter of about 200-300nm; the inner core of the nanoparticle has a high degree of symmetry, and the outer layer has a low degree of obviousness, which proves that the nanomaterial has a core-shell structure.

(4) XRD measurement. Take DFPase-F127 and DFPase-pDMAEMA aqueous solution (concentration of 0.2mg/ml), DMAEMA, F127 aqueous solution and drop them on a 2*2cm glass slide, and repeat the dripping several times. After drying, the samples were observed under XRD, and the results are shown in Figure 3. F127 is a disordered structure without obvious diffraction peaks, while the synthesized DFPase-F127 and DFPase-pDMAEMA have obvious sharp peaks (blue) at 2θ=6.47874°. For the lattice diffraction of the synthesized complex, it was also proved that through the activation modification of the F127 end group, it was successfully combined with the amino group on the protein to form a nanocomposite structure; by introducing a monomer on the protein, the protein was subsequently involved in free radicals. The polymer was successfully modified in the outer layer of the protein by the polymerization reaction. The introduction of macromolecules forms a new diffraction peak around 2θ=6.47874°, which preliminarily proves that the formation of this peak is not related to the type of macromolecules, but to the formed nanostructure.

Example 4 Decontamination evaluation of nerve agents by each complex

Specific experimental steps:

1. Preparation of solution reagents for decontamination reaction

(1) Saturated benzidine solution: Add excess (1 g) benzidine to 200 ml of distilled water, and the solubility increases in a 70°C water bath for 1 hour. Immediately after that, the solution was drawn with a 50 ml syringe and filtered with a 0.22 μm filter. 200 ml of saturated benzidine solution was obtained.

(2) 0.25% sodium perborate solution: 0.5 g of sodium perborate was added to 200 ml of distilled water and fully dissolved.

(3) 0.01M PBS solution: Dilute to 1/20 with the existing 0.2M PBS solution.

(4) Acetone-0.01M PBS solution: add 90ml of 0.01M PBS solution to 200ml of acetone in a ratio. A total of 290ml.

(5) The concentrations of various decontamination materials were respectively prepared at 0.2 mg/ml.

2. Prepare the standard linear curve (standard curve) of Soman's benzidine reaction.

(1) Take 10 μl of Soman with 90% purity, dissolve it in 9.99 ml of distilled water, that is, dilute it 1000 times, demarcate it as mother liquor, and store it in ice cubes.

(2) Dilute soman according to different concentrations

Numbering Dilution ratio Concentration (V/V) Matching method 1 1/10 1*10(-4) 0.5ml mother liquor + 4.5ml distilled water 2 1/20 1/2*10(-4) 1ml①+1ml distilled water 3 1/40 1/4*10(-4) 1ml①+3ml distilled water 4 1/120 1/12*10(-4) 1ml③+2ml distilled water 5 1/840 1/84*10(-4) 1ml④+6ml distilled water 6 1/5880 1/588*10(-4) 1ml⑤+6ml distilled water 7 0 distilled water

(3) Benzidine color reaction: mix 150 μl of distilled water and 50 μl of soman (different concentrations), centrifuge (3000 r, 1 min), and water bath at 37° C. for 20 min. Subsequently, 600 μl of acetone+0.01M PBS mixed solution, 300 μl of saturated benzidine hydrochloride solution, and 300 μl of 0.25% sodium perborate solution were added in sequence, and mixed well. Put in a 37°C oven for 5 min, and add to a 96-well plate. The absorbance value at 414 nm was measured. Excess soman was destroyed with concentrated NaOH.

(4) By calculation, the equation of the standard curve of the reaction of Solmantaniline is y=1.4184x+0.0525, R ² =0.9992. Soman concentration was in the range of 0-0.1 μg/μl, and its absorbance value was in this linear range. By this method the content of residual soman can be determined.

3. Soman decontamination rate by DFPase, DFPase-pDMAEMA and DFPase-F127

Dilute 90% pure soman to 5*10 ^-5 (V/V) according to the method in item 2 above, and dilute DFPase, DFPase-pDMAEMA and DFPase-F127 (in all materials, DFPase concentration is 0.2mg/ml) ) 3.125ml (high concentration group) and 0.625ml (low concentration group) were added to two 10ml EP tubes respectively, and then 1.25ml of soman was added to each tube respectively, and then distilled water was added to adjust the total volume to 7.5ml, and mixed well. Put into a water bath (37°C) in a three water tank.

Take 200 μl from the EP tube at certain time points, then add 600 μl of acetone-0.01M PBS solution, 300 μl of saturated benzidine solution, and 300 μl of 0.25% sodium perborate solution in sequence, mix well, and then put it into a 37°C water-proof constant temperature Incubator (stand for 5 min), add the solution in the EP tube to a 96-well plate, 200 μl per well, put it into a microplate reader, and measure the absorbance value at a wavelength of 414 nm. Pick a certain point in time. The results are shown in Figure 4. At the instant (within 2s) of the decontamination agent, the decontamination rate of DFPase-F127 in the high-dose group reached nearly 100%, and the decontamination rate of DFPase-pDMAEMA reached 90%. In the low-dose group, DFPase-F127 and DFPase-pDMAEMA only reaches about 20%, while pure DFPase (with the same concentration as the high-dose group), its decontamination rate is only 5% within 2s, but with the prolongation of time, due to the enzyme's biocatalytic cracking of the toxic agent, the decontamination rate is reduced. The elimination rate gradually increased, reaching about 70% at 160min, indicating that DFPase has been gradually decomposing the toxin, which partly proves that its decontamination effect is derived from the catalytic decomposition ability of the enzyme itself, but the efficiency is still lower than that of DFPase-F127 and DFPase-F127 at the same concentration. DFPase-pDMAEMA.

Example 5 Evaluation of nerve agent decontamination by common decontamination materials

(1) Evaluation of NaHCO ₃ on the decontamination rate of the nerve agent soman

The test sample is NaHCO ₃ (0.2 mg/ml), and the rest of the test methods, the dosage of each solvent and the sampling point time are the same as those in Example 5.

(2) Evaluation of the decontamination rate of the nerve agent Soman with NaOH

The test sample is NaOH (low concentration: 0.2 mg/ml; high concentration: 2 mg/ml), and the rest of the test methods, the dosage of each solvent and the time for taking samples are the same as those in Example 5.

(3) Evaluation of activated carbon on the decontamination rate of the nerve agent soman

The test sample was a 0.2 mg/ml activated carbon suspension. Except for taking out the water-proof constant temperature incubator (standing for 5 min) and centrifuging (1000 r/min) for 5 min in the last experimental step, taking the supernatant and adding 200 μl to the 96-well plate to measure the results, the rest of the test methods, the dosage of each solvent and the samples were taken. The point time is the same as that of Example 5.

(4) Evaluation of H ₂ O ₂ on the decontamination rate of the nerve agent soman

The test sample is a 30% H ₂ O ₂ solution, and the rest of the test method, the dosage of each solvent and the time for taking the sample are the same as those in Example 5.

(5) Decontamination results

In this experiment, various common decontamination materials were tested. In order to ensure the comparability of the experimental results, the concentration of each material was 0.2 mg/ml. The results are shown in Figure 5. At the same concentration, the decontamination rate of NaHCO ₃ did not change significantly with time, and the decontamination rate reached 5.63%; the decontamination rate of activated carbon gradually increased with time, reaching 22.76% in half an hour; and another common decontamination material, hydrogen peroxide , it can only reach a decontamination rate of 6.01% at the highest concentration of 30%, and has no decontamination effect at the same concentration as the detergent (30% hydrogen peroxide diluted about 1500 times). Both low-concentration and high-concentration NaOH can reach nearly 100% decontamination efficiency in a relatively short period of time, especially for high-concentration NaOH; while the DFPase-F127 and DFPase-pDMAEMA synthesized in this experiment were decontaminated immediately (2 seconds) immediately. Reaching nearly 100% decontamination, the decontamination efficiency is far superior to the above-mentioned groups of materials.

Example 6 Safety evaluation on skin

After the rats were anesthetized, they were laid down and fixed on the experimental table. After depilatory cream was applied to the abdomen, the abdomen was cleaned and dried for use. Subsequently, 30 μl of the detergent to be tested was dropped on the exposed abdominal skin, the skin changes were observed, and photographs were taken. After 10 minutes, the skin in the area was taken for pathological examination. The detergents added dropwise in turn were DFPase-F127, DFPase-pDMAEMA and high-concentration sodium hydroxide. From the results (Fig. 6), although sodium hydroxide can achieve a decontamination rate that is approximately the same as that of DFPase-F127 and DFPase-pDMAEMA (see Example 5), it will cause severe alkali burns to the skin, directly destroying the surface DFPase-F127 and DFPase-pDMAEMA are non-irritating to the skin and have high safety.

Example 7 Evaluation of decontamination effect on mouse skin after soman exposure

1. After the rats were anesthetized, they were laid down and fixed on the experimental table. After depilatory cream was applied to the abdomen, the abdomen was cleaned and dried for use. First, 10 μl of blood was taken as a blank group for each test animal to be tested. 50 μl of soman poison (1*10 ⁻³ v/v) was added dropwise to the abdomen, followed by 400 μl of the corresponding detergent (DFPase-F127, DFPase-pDMAEMA, NaHCO ₃ ). Put a layer of 5*10cm plastic wrap on the abdomen to ensure that all the liquid soaks on the bare skin without overflowing. Blood samples were collected from each test animal at the 10 min time point.

2. Enzyme activity assay.

a. Dilute whole blood with distilled water to (1:100v/v) to be tested;

b. Pipette 20 μl of the diluted whole blood and add it to the ELISA plate, add 30 μl of PBS to the blank control space, and add 30 μl of ATCh (3mM) to the test space. Duplicate wells are set for both the blank and the test, and finally each well is added to 100 μl with PBS;

c. 37℃ incubator reaction for 30min;

d. Add 20 μl DTNB (0.03%) 200 μl to each well;

e. Measure the absorbance value X (OD) at a wavelength of 415 nm;

f. Calculate the inhibitory rate of the poison in each group according to the measured absorbance value. The calculation formula is as follows:

q=1-X exposure group/X normal group

3. Results

From the results in Figure 7, using the traditional decontamination agent NaHCO ₃ , the inhibition rate of the toxic agent still reaches nearly 40% of the enzyme inhibition rate, which is at the level of moderate to severe poisoning, while the decontamination agents using DFPase-F127 and DFPase-pDMAEMA The inhibitory rate of the post-toxic agent on blood enzymes in vivo is about 13%, which is a mild poisoning level and does not affect the survival of the tested animals.

Example 8 Synthesis and Decontamination Efficiency Evaluation of OPH-pDMAEMA

(1) Put 30ml of OPH (0.2mg/ml) into a pear-shaped bottle, and stir (600r/min) in the dark.

(2) Subsequently, 25 mg of the vinyl acyl-introducing agent NAS was weighed and dissolved in 500 μl of DMSO solvent, and 180 μl was then added to the above reaction system.

(3) Then slowly drop the initiator KPS solution (30 μl, 10% w/v), the monomeric DMAM solution in DMSO (40 μl, 10% w/v), and the cross-linking agent MBA in DMSO solution (30 μl, 10% w/v) w/v), acid-base regulator TMEDA solution in DMSO (6 μl, 10% w/v), and stirred at room temperature in the dark (600 r/min, 6 h).

(4) Pretreatment of dialysis bag. First, 500 ml of 2% w/t NaHCO ₃ solution and 500 ml of 1 mM disodium EDTA aqueous solution were prepared. Subsequently, a dialysis bag with a molecular weight cut-off of 5000 was cut to a length of 30 cm. The dialysis bag was successively placed in the above-mentioned two groups of solutions of NaHCO ₃ and disodium EDTA and boiled (100 ° C, 10 min) in turn, rinsed with distilled water after each boiling, and weighed 30 g of HEPES and 1.2 g of NaOH and added to 2.5 g of NaOH. L distilled water, fully stir, disperse and dissolve to prepare dialysate for use.

(5) Product dialysis. Add the solution of (3) reaction to the dialysis bag (do not exceed 1/2-1/3 of the total volume of the dialysis bag), close both ends of the dialysis bag with clips, then put it into the dialysate, and stir at low speed at room temperature (150r/min). During the period, the dialysate was replaced every 12h, and the dialysate was replaced three times in total to completely remove the excess small molecule monomer impurities in the solution. After 48 hours of dialysis, the solution in the dialysis bag was collected to obtain the product OPH-pDMAEMA, which was colorless and transparent, with a total volume of 30 ml. The overall solution volume did not change significantly before and after the reaction, and it was stored in a refrigerator (-20°C).

(6) Dilute 90% pure Soman to 5*10-5 (v/v), add 3.125ml of OPH-pDMAEMA (concentration is 0.2mg/ml) into 10ml EP tube, and then add shuttle to each tube respectively Man 1.25ml, then add distilled water to adjust the total volume to 7.5ml, mix well, and put it into a water bath (37°C) in a three-water tank.

Take 200 μl from the EP tube at certain time points, then add 600 μl of acetone-0.01M PBS solution, 300 μl of saturated benzidine solution, and 300 μl of 0.25% sodium perborate solution in sequence, mix well, and then put it into a 37°C water-proof constant temperature Incubator (stand for 5 min), add the solution in the EP tube to a 96-well plate, 200 μl per well, put it into a microplate reader, and measure the absorbance value at a wavelength of 414 nm. Pick a certain point in time. The results are shown in Fig. 8. At the instant (within 2s) of the detergent input, OPH-pDMAEMA reaches about 80%.

Although the specific embodiments of the present invention have been described in detail, according to all the disclosed teachings, those skilled in the art can make various modifications and substitutions to the details of the technical solutions of the present invention, and these changes are all within the protection scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (9)

1. An enzyme-polymer complex for use in nerve agent decontamination, wherein the enzyme is selected from the group consisting of a phosphotriester hydrolase of the diisopropyl fluorophosphatase (DFPase) class; the polymer is a water-soluble polymer;

preferably, the DFPase-type phosphoric acid triester hydrolase is selected from the group consisting of DFPase, human serum paraoxonase 1(PON1) and organophosphorous acid anhydrase (OPAA); the polymer is selected from poly (2- (N, N-dimethylamino) ethyl methacrylate) (pDMAEMA), polyacrylic acid, F127, tween, triton X100, PLGA and a cross-linked polymer formed by the Poly (PLGA) and a cross-linking agent, and is further preferably poly (2- (N, N-dimethylamino) ethyl methacrylate) (DMAEMA), F127 and a cross-linked polymer formed by the Poly (PLGA) and the cross-linking agent.

2. The complex of claim 1, wherein the nerve agent is an organophosphorous poison comprising sarin, tabun, soman and VX, preferably soman.

3. The complex of claim 1 or 2, wherein the DFPase-like enzyme introduces a monomeric group through a surface amino group; preferably, the monomer group is selected from-CO- (CH)₂)_m-CH＝CH₂M is an integer of 0 to 6; preferably, the complex is polymerized via DFPase, 2- (N, N-dimethylamino) ethyl methacrylate, to which the monomer groups are attached, and optionally a crosslinking agent.

4. The complex of claim 1 or 2, which is formed by said F127 via a linker-CO- (CH)₂)_n-CO-is linked to DFPase, wherein n is selected from an integer from 0 to 6.

5. The enzyme-polymer complex of any one of claims 1-4, which has a particle size of 50-400 nm (e.g., 50-350 nm, 50-300 nm, 50-250 nm, 50-200nm, 50-150nm, 100-350 nm, 100-300 nm, 100-250 nm, 100-200 nm, 100-150nm, 150-350 nm, 150-300 nm, 150-250 nm or 150-200 nm);

preferably, it is a core-shell structure;

preferably, the shell thickness is 50-200nm (e.g., 50-150nm, 50-100nm, or 100-150 nm).

6. A method for preparing a complex as claimed in any one of claims 1 to 5, comprising the steps of:

the method comprises the following steps:

(1) by introducing monomeric groups into the enzyme, followed by reaction with the polymerCarrying out polymerization reaction on the monomer and an optional cross-linking agent to obtain a crude product of the compound; preferably, the monomer group is selected from-CO- (CH)₂)_m-CH＝CH₂M is an integer of 0 to 6;

(2) dialyzing the crude compound obtained in the step (1) to obtain the compound; or,

the method 2 comprises the following steps:

(1) introducing a connector on the polymer, and then adding the polymer introduced with the connector into an enzyme aqueous solution (for example, 0.1-1mg/ml) for reaction to obtain a crude compound; preferably, the linker is selected from the group consisting of-CO- (CH)₂)_n-CO-, n is selected from an integer from 0 to 6;

(2) dialyzing the crude compound obtained in the step (1) to obtain the compound;

preferably, in method 1, the molar ratio of the crosslinking agent (if present) to the monomers of the polymer below is (0.2-1): 1 (e.g., 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, or 1: 1): pDMAEMA, polyacrylic acid;

preferably, an initiator is also added in the polymerization reaction; preferably, the initiator is potassium persulfate; preferably, the molar ratio of the initiator to the monomers of the polymer is (0.01-0.5):1 (e.g., 0.01:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, or 0.5: 1);

preferably, in the method 1, a pH regulator is also added in the polymerization reaction; preferably, the pH adjuster is N, N' -tetramethyldiethylamine; preferably, the amount of the pH regulator is such that the reaction system has a pH of 7.5 to 9.5;

preferably, in the method 1, the crude compound is dialyzed to remove small molecular impurities with the molecular weight of below 5000;

preferably, in method 2, the polymer is selected from F127, tween, triton X100 and PLGA;

preferably, in the method 2, one end of the linker is connected with the amino group on the surface of the enzyme through an amide bond, and the other end of the linker is connected with the polymer through an ester bond;

preferably, in the method 2, the crude compound is dialyzed to remove small molecular impurities with the molecular weight of below 5000;

preferably, the composite is a core-shell structure with a particle size of 50-400 nm (for example, 50-350 nm, 50-300 nm, 50-250 nm, 50-200nm, 50-150nm, 100-350 nm, 100-300 nm, 100-250 nm, 100-200 nm, 100-150nm, 150-350 nm, 150-300 nm, 150-250 nm or 150-200 nm); the thickness of the shell layer is 50-200nm (for example, 50-150nm, 50-100nm or 100-150 nm).

7. A decontamination composition or decontamination solution comprising a complex as claimed in any one of claims 1 to 5.

8. Protective equipment comprising a complex according to any one of claims 1 to 5 or a decontamination composition or a decontamination solution according to claim 7.

9. Use of a complex according to any one of claims 1 to 5, a decontamination composition or a decontamination solution according to claim 7 or a protective device according to claim 8 in a sanitizer; preferably, the agent is a nerve agent or blister agent; preferably, the nerve agent is an organophosphorous agent, for example selected from sarin, tabun, soman and VX; preferably, the blister agents are mustard gas, lewis agents, or nitrogen mustard gas.