CN107064013A - Phosphorated agent detection gel photonic crystal, preparation method and application - Google Patents
Phosphorated agent detection gel photonic crystal, preparation method and application Download PDFInfo
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- CN107064013A CN107064013A CN201710052472.5A CN201710052472A CN107064013A CN 107064013 A CN107064013 A CN 107064013A CN 201710052472 A CN201710052472 A CN 201710052472A CN 107064013 A CN107064013 A CN 107064013A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract
The invention provides enzyme is solidified on a kind of new phosphorated agent detection gel photonic crystal, a kind of new optical sensing detection means is provided for the high sensitivity of phosphorated agent, trace detection.Polystyrene microsphere and polyacrylamide gel are combined and prepare a kind of gel photonic crystal by the present invention, acted on after chemical modification by condensing agent and solidify cholinesterase on gel photonic crystal, in the detection to extremely low concentration phosphorated agent sarin, low test limit, highly sensitive effect are shown.
Description
Technical field
The present invention relates to phosphorated agent detection field, in particular to phosphorated agent detection gel photonic crystal, system
Preparation Method and application.
Background technology
Gel photonic crystal is a kind of characteristic for combining photonic crystal and intelligent aqueous gel and the new sensitivity developed
Material.Environmental change produces response to hydrogel in gel photonic crystal to external world, drives the lattice of photonic crystal in gel normal
Number changes, so as to produce corresponding difraction spectrum change.Based on this characteristic, gel photonic crystal can be applied to all kinds of biochemistry
In senser element, the characteristics of with simple to operate, Miniaturizable is detected, the common detection for having glucose, temperature, a metal ion
Deng.
Phosphorated agent be a class severe toxicity organic phospho acid esters compound, including sarin (Sarin, GB), soman (Soman,
GD), tabun (Tabun, GA), VX (VX) etc..Phosphorated agent toxicity is extremely strong, is class activity very strong cholinesterase
(AChE) inhibitor, can cause cholinergic nerve system dysfunction, organism is lost function even dead.
If enzyme solidified in gel photonic crystal, it becomes possible to which the single-minded evident characteristics of enzyme are mutually tied with gel photonic crystal
Close, so that develop using enzyme as discriminating element and using gel photonic crystal as the biochemical sensor of display element, and this
New possibility is provided for the trace of phosphorated agent, quick detection.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of preparation method of phosphorated agent detection gel photonic crystal, this hair
In bright method, by preparing the gel photonic crystal of functionalization first, then by gel photonic crystal chemical modification and solidification,
And solidify cholinesterase in gel photonic crystal, so that the gel photonic crystal after solidification can be to phosphorated agent
Response with high sensitivity and high selectivity, and can be used in the detection of phosphorated agent.The inventive method has preparation method
Operating procedure is simple and convenient, the advantages of obtained gel photonic crystal detection sensitivity is high.
The second object of the present invention is to provide a kind of phosphorated agent detection gel photonic crystal, the gel photon crystalline substance
Body is prepared by the inventive method, with to phosphorated agent response sensitivity height, good selective.
Third object of the present invention is the application for providing the phosphorated agent detection gel photonic crystal, the present invention
The sensitivity that gel photonic crystal is responded for phosphorated agent is high and selectivity is good, it is thus possible to trace, quickly detect phosphorous
Toxic agent.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme:
Gel photonic crystal preparation method is used in phosphorated agent detection, and methods described comprises the following steps:
Styrene emulsion polymerization prepares monodisperse polystyrene microsphere, obtains the emulsion containing polystyrene microsphere;With from
Sub-exchange resin is handled emulsion, is then added acrylamide pre-polymerization liquid and is mixed, then in-situ polymerization, obtains polyphenyl second
Alkene-polyacrylamide gel photonic crystal;
By gel photonic crystal with base extraction after, immersion cholinesterase solution in and preculture;Then to after preculture
Gel photonic crystal in, add condensing agent and the solidified reagents dissolved with cholinesterase, and condensation reaction;Then, it is condensation is anti-
Gel photonic crystal after answering, which is put into cushioning liquid, to be cultivated, that is, obtains phosphorated agent detection gel photonic crystal.
Optionally, in the present invention, the monodisperse polystyrene microsphere is the single dispersing polyphenyl that particle diameter is 100~300nm
Ethene microballoon.
Optionally, in the present invention, the in-situ polymerization is specially:Emulsion and acrylamide after ion-exchange treatment is pre-
Then diaphragm is placed under ultraviolet light by gained mixed solution diffusion profile in substrate membrane surface after poly- liquid mixing, and low temperature is former
1~5h of position polymerization.
Optionally, in the present invention, the time of the preculture is 24~72h.
Optionally, in the present invention, the condensing agent is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
And/or 3- (diethoxy phosphoryl oxy) -1,2,3- phentriazine -4- ketone.
Optionally, in the present invention, the solidified reagents dissolved with cholinesterase are the Tris- dissolved with duck serum cholinesterase
HCl cushioning liquid.
Optionally, in the present invention, the temperature of the condensation reaction is 20~25 DEG C.
Optionally, in the present invention, the cushioning liquid is Tris-HCl cushioning liquid.
Meanwhile, present invention also offers as the phosphorated agent detection gel photonic crystal obtained by the inventive method.
Further, present invention provides as the phosphorated agent detection gel photonic crystal obtained by the inventive method
Application in detection phosphorated agent.
Compared with prior art, beneficial effects of the present invention are:
In the inventive method, by the way that photonic crystal technology is combined with intelligent aqueous gel, gel photon has been prepared
Crystal new material.In this new material, alkali esterase is have cured on gel photonic crystal, so that it possesses gel photon
The advantage of crystal and two kinds of materials of biology enzyme.
Further, the gel photonic crystal of present invention solidification cholinesterase is to having the combination of selectivity containing Phosphorus toxic agent
Performance, serine sites and the phosphorated agent of cholinesterase combine to form a kind of anion tetrahedron material of stabilization, phosphonylation
The anionic electrodeposition of enzyme sexually revises the Donnan equilibrium of gel photonic crystal, makes the pressure increase of gel internal penetration, and gel swelling absorbs water, directly
Connect and detected with spectrometer, gel photonic crystal diffraction peak wavelength changes.And the gel that this also to have cured cholinesterase
Photonic crystal can realize trace, the purpose of quick, convenient detection phosphorated agent.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described.
Fig. 1 is the control spectrum that different condensing agents influence on gel photonic crystal diffraction peak wavelength after solidification cholinesterase
Figure;
Fig. 2 is the hydrolysis rate figure of various concentrations substrate;
Fig. 3 is gel photonic crystal to sarin aqueous solution response light spectrogram.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
In the preferred scheme of the present invention, the polystyrene microsphere is that particle diameter is 100~300nm, and with height
The polystyrene microsphere of electrical and monodispersity.Further, in the present invention, by using ion exchange resin to including polyphenyl
The emulsion of ethene microballoon is handled, and polystyrene microsphere can be obtained with three-dimensional periodic by self assembly
Crystal colloidal array.
In the preferred scheme of the present invention, the emulsion and acrylamide pre-polymerization liquid by after ion-exchange treatment is mixed
Gained mixed solution diffusion profile is specially in substrate membrane surface after conjunction:By the siphon diffusion of solution, it will mix molten
Liquid is covered with quartz glass diaphragm surface.
In the preferred scheme of the present invention, the time of the preculture can be, but be not limited to 36,48,60 or
72h etc..
In the preferred scheme of the present invention, the alkali is sodium hydroxide.Further, in the present invention, by with alkali
Polystyrene-polypropylene acrylamide gel photonic crystal is handled, so as to by the amide groups on polyacrylamide backbone
Group is converted into carboxyl by reaction, and these carboxyls can further in the condensation reaction, with choline in the presence of curing agent
Amino reaction on esterase, so that cholinesterase be solidified in gel photonic crystal.
In the preferred scheme of the present invention, the time of the condensation reaction is 1~5h, for example, the condensation reaction
Time can be but to be not limited to 2,3 or 4h etc..
In the preferred scheme of the present invention, the culture of the gel photonic crystal by after condensation reaction is specially:
Gel photonic crystal after condensation reaction is put into Tris-HCl cushioning liquid and 24~72h is cultivated, such as described culture
Time can be, but be not limited to 36,48 or 60h etc..Further, in the present invention, by by the gel light after condensation reaction
Sub- crystal is cultivated in cushioning liquid, so as to be exchanged cholinesterase uncured after reaction by cultivating.
In the preferred scheme of the present invention, the present invention is still further comprised obtained phosphorated agent detection gel
Photonic crystal is placed in step stand-by in cushioning liquid.
The present invention a preferred scheme in, phosphorated agent of the present invention be sarin, soman, tabun, VX,
One kind in parathion, thimet etc..
In the preferred scheme of the present invention, the phosphorated agent detection method is as follows:Phosphorated agent detection is used solidifying
Glue photonic crystal is placed in phosphorated agent solution and detected, and gathers the spectrogram of gel photonic crystal after balance.
It is further preferred that in the present invention, the phosphorated agent is sarin;Further, the detection method of the sarin
It is as follows:Gel photonic crystal is placed in 10-9Detected in mg/mL sarin solution, and gather gel photonic crystal after balance
Spectrogram.
Further, phosphorated agent detection of the present invention is specific such as with the overall preparation flow of gel photonic crystal preparation method
Under:
Styrene emulsion polymerization is made particle diameter and is 100~300nm monodisperse polystyrene microsphere, and obtains containing polyphenyl
The emulsion of ethene microballoon;Emulsion is handled with ion exchange resin, acrylamide pre-polymerization liquid is then added and mixes, and institute
Mixed solution diffusion profile is obtained in substrate membrane surface, then diaphragm is placed under ultraviolet light, and low-temperature in-site polymerize 1~5h,
Obtain polystyrene-polypropylene acrylamide gel photonic crystal;
By gel photonic crystal with naoh treatment after, immersion cholinesterase solution in and 24~72h of preculture;Then
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and/or 3- (two are added after to preculture in gel photonic crystal
Ethyoxyl phosphinylidyne epoxide) -1,2,3- phentriazine -4- ketone, and dissolved with the Tris-HCl cushioning liquid of duck serum cholinesterase,
And 1~5h of condensation reaction;Then, by the gel photonic crystal after condensation reaction be put into Tris-HCl cushioning liquid culture 24~
72h, that is, obtain phosphorated agent detection gel photonic crystal, and obtained phosphorated agent detection is put with gel photonic crystal
Enter stand-by in Tris-HCl cushioning liquid.
Embodiment 1
Appropriate styrene monomer is taken, and uses the method for emulsion polymerization to prepare particle diameter for the high electrical, single point of 200nm
Polystyrene microsphere is dissipated, and obtains including the emulsion of above-mentioned microballoon;
Emulsion is handled by ion exchange resin, so that the polystyrene microsphere included passes through self assembly
Obtain the crystal colloidal array of three dimensional periodic structure;
By acrylamide pre-polymer solution and after the mixing of the emulsion comprising polystyrene microsphere, gained mixed solution is passed through
Siphon diffusion, diffusion is covered with quartz glass diaphragm;Then quartz glass diaphragm is placed under 5 DEG C~10 DEG C ultraviolet lights,
And under cryogenic, by the mixed solution poly- 2h of light in situ, polystyrene-polypropylene acrylamide gel photonic crystal is made;
Obtained polystyrene-polypropylene acrylamide gel photonic crystal is chemically modified with sodium hydroxide, Ji Jiangju
The amide group of acrylamide gel skeleton is changed into carboxyl (providing access site for solidification enzyme) by chemical modification method;
Then, the gel photonic crystal after chemical modification is immersed in cholinesterase solution and preculture 48h;Then, will
Gel photonic crystal after preculture adds 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) and molten
Have in the Tris-HCl cushioning liquid of duck serum cholinesterase, and condensation reaction 2h;
Then, the gel photonic crystal after condensation reaction is put and cultivates 48h in Tris-HCl buffer solutions to be swapped out not admittedly
The cholinesterase of change, that is, obtain the phosphorated agent detection gel photonic crystal of embodiment 1, and it is further stored in
It is stand-by in Tris-HCl buffer solutions.
Embodiment 2
Appropriate styrene monomer is taken, and uses the method for emulsion polymerization to prepare particle diameter for the high electrical, single point of 200nm
Polystyrene microsphere is dissipated, and obtains including the emulsion of above-mentioned microballoon;
Emulsion is handled by ion exchange resin, so that the polystyrene microsphere included passes through self assembly
Obtain the crystal colloidal array of three dimensional periodic structure;
By acrylamide pre-polymer solution and after the mixing of the emulsion comprising polystyrene microsphere, gained mixed solution is passed through
Siphon diffusion, diffusion is covered with quartz glass diaphragm;Then quartz glass diaphragm is placed under 5 DEG C~10 DEG C ultraviolet lights,
And under cryogenic, by the mixed solution poly- 2h of light in situ, polystyrene-polypropylene acrylamide gel photonic crystal is made;
Obtained polystyrene-polypropylene acrylamide gel photonic crystal is chemically modified with sodium hydroxide, Ji Jiangju
The amide group of acrylamide gel skeleton is changed into carboxyl by chemical modification method;
Then, the gel photonic crystal after chemical modification is immersed in cholinesterase solution and preculture 48h;Then, will
Gel photonic crystal after preculture add 3- (diethoxy phosphoryl oxy) -1,2,3- phentriazine -4- ketone (DEPBT) and
Dissolved with the Tris-HCl cushioning liquid of duck serum cholinesterase, and condensation reaction 2h;
Then, the gel photonic crystal after condensation reaction is put and cultivates 48h in Tris-HCl buffer solutions to be swapped out not admittedly
The cholinesterase of change, that is, obtain the phosphorated agent detection gel photonic crystal of embodiment 2, and it is further stored in
It is stand-by in Tris-HCl buffer solutions.
Experimental example 1
Gel photonic crystal before solidification cholinesterase is tested using fiber spectrometer (to be placed in the Tris-HCl that pH is 7.4 to delay
In fliud flushing) and using different condensing agents solidification cholinesterase, i.e., using containing obtained by embodiment 1 respectively and the method for embodiment 2
The diffraction peak wavelength of phostoxin agent detection gel photonic crystal, is as a result shown in Fig. 1.
From the result of Fig. 1 control tests, after using condensing agent solidification cholinesterase, gel photonic crystal diffraction maximum
Generation red shift;Meanwhile, the phosphorated agent detection obtained by the method for embodiment 2 is more than with the diffraction maximum red shift amount of gel photonic crystal
Phosphorated agent detection gel photonic crystal obtained by the method for embodiment 1.
Experimental example 2
The starting velocity of cholinesterase catalysis various concentrations substrate hydrolysis is tested using the Ellman methods of improvement, Michaelis is set up
The access rate of equation detection solidification enzyme, specific method of testing is as follows:
4 25mL are taken to dry test tube, respectively thereto in 2 control tubes (control tube A and control tube B) and 1 testing tube
15mLBuCHI-DTNB solution is added, 15mLDTNB solution is added into remaining 1 blank tube, 4 test tubes are put into 37 DEG C
Preheated in thermostat water bath;
Wherein, control tube A is added without gel photonic crystal, and the gel photon that control tube B adds modified uncured enzyme is brilliant
Body, and the gel photonic crystal that testing tube is then added after solidification enzyme.
Using the secondary DTNB solution of blank tube as reference, 2 control tubes and 1 are monitored at wavelength 412nm using 1cm cuvettes
The absorbance of branch testing tube.Its concentration is mapped with substrate hydrolysis speed, Fig. 2 is as a result seen.
From Fig. 2 control test results:The hydrolysis rate of substrate is its natural hydrolysis rate in control tube A, is compared pair
The substrate hydrolysis speed in A and control tube B is looked after it can be found that the hydrolysis rate in two control tubes is essentially identical, this explanation
Hydrolysis of the modified gel photonic crystal to substrate does not have an impact;
The gel photonic crystal after solidification enzyme, substrate hydrolysis speed increase, when concentration of substrate reaches are added in testing tube
During 6mmol/L, because substrate makes the rate reduction that it is catalyzed substrate hydrolysis to the excessive suppression of cholinesterase.
Wherein, the Michaelis-Menten equation is speed side of the initial rate with concentration of substrate relation for representing an enzymatic reaction
Journey, its formula is as follows:
The testing tube substrate hydrolysis speed calculated is mapped with corresponding concentration of substrate, wherein Michaelis constant Km=
1.2mmol/L, maximum reaction rate vmax=3.7 × 10-3mmol/min.The unit of activity (U) of one enzyme represents to turn in 1 minute
Change the enzyme amount needed for 1 micromole substrate, thus solidify the unit of activity of enzyme for 3.1U, cholinesterase connects on gel photonic crystal
It is 5.7 × 10 to enter amount-13Solidify the access amount of acetylcholinesterase on mol, with conventional electrodes in same order.
Experimental example 3
Gel photonic crystal after two kinds of condensing agent solidification cholinesterases of test is 10 to concentration-9The mg/mL sarin aqueous solution
Response condition, specific detection method is as follows:
Under the conditions of 25 DEG C, the embodiment 1 and embodiment that will will be soaked in respectively in the Tris-HCl buffer solutions that pH is 7.4
Phosphorated agent detection obtained by 2 methods is transferred to after the immersion of 10mL ultra-pure waters with gel photonic crystal, gathers its spectrogram.
Then, the phosphorated agent detection obtained by embodiment 1 and the method for embodiment 2 will be soaked with gel photonic crystal respectively
It is 10 to steep in 10mL concentration-9In the mg/mL sarin aqueous solution, the gel photonic crystal of uncured enzyme is gathered using fiber spectrometer
And the gel photonic crystal spectrogram after two kinds of condensing agent solidification enzymes, as a result see Fig. 3.
The control test result shown in Fig. 3:Slightly blue shift occurs for the gel photonic crystal diffraction maximum of uncured enzyme,
Blue shift amount is 3nm;Red shift occurs for the diffraction maximum of solidification enzyme gel photonic crystal, and contains phostoxin obtained by the method for embodiment 2
The diffraction maximum red shift amount of agent detection gel photonic crystal is more than the phosphorated agent detection gel light obtained by the method for embodiment 1
Sub- crystal.
As can be seen here, the phosphorated agent detection obtained by the method for embodiment 2 is high with the corresponding sensitivity of gel photonic crystal
In the phosphorated agent detection gel photonic crystal obtained by the method for embodiment 1.
In the present invention, by the way that photonic crystal technology is combined with intelligent aqueous gel, gel light is had concurrently so as to be made
Sub- crystal and the gel photonic crystal new material of the advantage of two kinds of materials of biology enzyme, obtained have cured the solidifying of cholinesterase
Glue photonic crystal being capable of trace, quick, convenient detection phosphorated agent.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that without departing substantially from the present invention's
Many other changes and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. gel photonic crystal preparation method is used in phosphorated agent detection, it is characterised in that methods described comprises the following steps:
Styrene is prepared into monodisperse polystyrene microsphere by emulsion polymerization, the emulsion containing polystyrene microsphere is obtained;With
Ion exchange resin is handled emulsion, is then added acrylamide pre-polymerization liquid and is mixed, polyphenyl second is obtained after in-situ polymerization
Alkene-polyacrylamide gel photonic crystal;
By gel photonic crystal with alkali process after, immersion cholinesterase solution in preculture;Then to the gel light after preculture
In sub- crystal, condensing agent, and condensation reaction are added;Then, the gel photonic crystal after condensation reaction is put into cushioning liquid
Cultivated, phosphorated agent detection gel photonic crystal is obtained after culture.
2. according to the method described in claim 1, it is characterised in that the monodisperse polystyrene microsphere be particle diameter be 100~
300nm monodisperse polystyrene microsphere.
3. according to the method described in claim 1, it is characterised in that the in-situ polymerization is specially:After ion-exchange treatment
Emulsion and acrylamide pre-polymerization liquid mixing after, by gained mixed solution diffusion profile in substrate membrane surface, then by diaphragm
It is placed under ultraviolet light, and low-temperature in-site polymerize 1~5h.
4. according to the method described in claim 1, it is characterised in that the time of the preculture is 24~72h.
5. according to the method described in claim 1, it is characterised in that the condensing agent is 1- (3- dimethylamino-propyls) -3- second
Base carbodiimide hydrochloride and/or 3- (diethoxy phosphoryl oxy) -1,2,3- phentriazine -4- ketone.
6. according to the method described in claim 1, it is characterised in that the solidified reagents dissolved with cholinesterase are dissolved with duck blood
The Tris-HCl cushioning liquid of clear cholinesterase.
7. according to the method described in claim 1, it is characterised in that the temperature of the condensation reaction is 20~25 DEG C.
8. according to the method described in claim 1, it is characterised in that the cushioning liquid is Tris-HCl cushioning liquid.
9. phosphorated agent detection gel photonic crystal made from the method according to any one of claim 1-8.
10. application of the phosphorated agent detection gel photonic crystal described in claim 9 in detection phosphorated agent.
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CN112909178B (en) * | 2021-01-18 | 2022-07-08 | 香港科技大学深圳研究院 | Light trap substrate, preparation method and application thereof, and semitransparent solar cell |
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