CN109900653A - A kind of molecular imprinting photonic crystal film and its preparation method and application of quick detection lysozyme - Google Patents
A kind of molecular imprinting photonic crystal film and its preparation method and application of quick detection lysozyme Download PDFInfo
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Abstract
The present invention relates to a kind of lysozyme molecular imprinting crystal film with photon, belong to materials chemistry and analysis detection field.Silica photon crystal micro-ball in conjunction with molecular imprinting technology, is prepared for the molecular imprinting photonic crystal film for identifying and combining to three antalzyme protein matter molecular specificities with counter opal structure by the present invention.As the antalzyme protein matter molecular amounts of combination increase, the macroporous structure on film changes, and leads to the Bragg diffraction red shift of wavelength of the molecular imprinting photonic crystal film of preparation.Changed according to spectral response of the molecular imprinting photonic crystal gel mould of preparation to lysozyme, the quick detection to lysozyme concentration in sample may be implemented in the present invention.This method has the advantages that highly sensitive and selectivity, low detection limits, can be used as a kind of quick detection of biosensor for antalzyme protein matter molecule in biological sample.
Description
Technical field
The invention belongs to materials chemistries and technical field of analysis and detection, are related to a kind of molecular engram of quickly detection lysozyme
Crystal film with photon and its preparation method and application can be used for bio-sensing and optical device.
Background technique
Lysozyme (Lysozyme) is that a kind of molecular weight is 14.3KDa, contains only the single-stranded small molecule egg of 129 amino acid
White matter.Lysozyme has serum in human body and body fluid Lysozyme Levels exception and leukaemia, nephrosis, conjunctivitis, meningitis etc.
A variety of diseases are related.Lysozyme has important physiological significance in the intracorporal content of human body, it has also become one in clinical diagnosis
Important clinical index.Currently, the research of bacteriolyze enzyme assay method has been achieved for many achievements, but there are still sample pretreatments to answer
The problems such as miscellaneous, selectivity low, time-consuming, cost.Therefore, biosensor of the preparation based on molecular imprinting photonic crystal film is quickly examined
Surveying lysozyme is a kind of outstanding solution.
Molecular imprinting technology (molecularly imprinted technique, MIT), refers to a certain target molecule
As template, under the action of being incorporated in crosslinking agent using monomer and template molecule, by going removing template to be prepared to target point
Son has the technology of specific selectivity polymer.Photonic crystal refers to by two or more with differing dielectric constant (folding
Penetrate rate) material be formed by the material with ordered structure according to certain cycle sequences arrangement in space structure.Photon
Crystal has distinctive Bragg diffraction effect, and the wavelength at its Bragg diffraction peak is joined by the refractive index and lattice of dielectric material
Several influences, can be used as sensing material.
Summary of the invention
In order to solve the problems in the existing technology, the present invention provides a kind of molecular engrams of quickly detection lysozyme
Crystal film with photon and its preparation method and application.
Molecular imprinting technology in conjunction with photonic crystal, is removed photonic crystal template by hf etching, obtained by the present invention
The photonic crystal with inverse opal structure film of lysozyme molecular imprinting is arrived.The photon of lysozyme molecular imprinting prepared by the present invention is brilliant
Body film combines the advantages of two kinds of technologies, when acting on specific bond template lysozyme by molecular engram, can cause hole on film
The specific bond process of molecule can be converted into readable optical signalling, to reach detection target analysis by cave structure change
The purpose of object.In addition, the diffraction maximum position of film is related with its internal cavity in the case where incidence angle is certain, work as opening structure
Variation will lead to the displacement of diffraction maximum.The bacteriolyze enzyme molecule that hole combines on film is more, and displacement is more obvious, according to this phenomenon
It can establish the equation that lysozyme is displaced diffraction maximum, the lysozyme content in quick test sample.
The present invention provides a kind of preparation method of the molecular imprinting photonic crystal film of quickly detection lysozyme, including following step
It is rapid:
(1) silicon wafer is cleaned and is cleaned, obtain hydrophiling matrix；
(2) monodisperse silica microspheres are distributed in dehydrated alcohol, form colloidal dispersion system, step (1) is made
Standby hydrophiling matrix is inserted at a certain angle in silicon dioxide microsphere solution, and using the method for vertical self assembly, constant temperature is stood
It volatilizees completely to ethyl alcohol, obtains photonic crystal template；
(3) it is covered on after cleaning polymethyl methacrylate film in the photonic crystal template that step (2) obtains；
(4) successively template molecule, function monomer and crosslinking agent are dissolved in phosphate buffer solution, are obtained after being sufficiently mixed
Pre-polymer solution；
(5) initiator and accelerator are added in the pre-polymer solution of step (4), precursor solution is obtained after dispersion；
In the gap for drawing a certain amount of precursor solution injection PMMA film and silicon wafer, until solution is in the work of capillary and atmospheric pressure
With the lower gap full of between PMMA and silicon wafer, water-bath polymerization reaction obtains lysozyme molecular imprinting crystal film with photon；
(6) the lysozyme molecular imprinting crystal film with photon that step (5) is prepared is soaked in hydrofluoric acid solution, is removed
Silicon dioxide microsphere obtains the molecular imprinting photonic crystal hydrogel blotting membrane of counter opal structure；
(7) the molecular imprinting photonic crystal hydrogel blotting membrane for the counter opal structure that step (6) obtains is eluted,
Until completely removing lysozyme template protein, the molecular imprinting photonic crystal film being prepared.
Preferably, the partial size of the monodisperse silica microspheres is 200~400nm in step (2)；The colloid
In dispersion, the mass fraction of monodisperse silica microspheres is 1.0~5.0%；It the angle ranging from 30-90 °, the constant temperature
For the constant temperature at 25-40 DEG C.
Preferably, in step (4), the template molecule is lysozyme, and function monomer is acrylamide, crosslinking agent N,
N '-methylene bisacrylamide amine；The weight ratio of the template molecule, function monomer and crosslinking agent are as follows: (10~200mg): (50~
500mg): (50~500mg)；The pH of the phosphate buffer solution is 6.0~8.0.
Preferably, in step (5), the condition of the water-bath polymerization reaction be 30 DEG C water-bath 2.0~10.0 hours；
The initiator is ammonium persulfate, and accelerator is tetramethylethylenediamine, and the amount ratio of initiator and accelerator is (5~50mg):
(5~50 μ L).
Preferably, being impregnated 1~6 hour in the hydrofluoric acid solution of mass concentration 0.5~5% in step (6).
Preferably, in step (7), when the elution, eluent is the mixed solution of lauryl sodium sulfate and acetic acid；
In mixed solution, water is solvent, and the mass concentration of lauryl sodium sulfate is 1~10%, the volumetric concentration of acetic acid is 1~
15%.
The present invention also provides the molecular imprinting photonic crystal films being prepared using the above method.
The present invention also provides application of the above-mentioned molecular imprinting photonic crystal film in quickly detection lysozyme.
The present invention also provides a kind of methods of quickly detection lysozyme content, comprising the following steps:
(1) the molecular imprinting photonic crystal film that any one of claim 1-6 is prepared is respectively put into various concentration
In lysozyme standard solution, make its adsorption saturation, take out molecular imprinting photonic crystal film, measure its ultra-violet absorption spectrum respectively,
Absorption peak displacement-lysozyme concentration change curve is calculated；
(2) the molecular imprinting photonic crystal film that any one of claim 1-6 is prepared is added in sample to be tested, is led to
The diffraction peak wavelength variation for crossing measurement lysozyme molecular imprinting crystal film with photon, substitutes into the standard variation curve that the first step obtains,
The content of Lysozyme in Solution to be measured can be obtained.
The present invention provide in a kind of fast qualitative detection solution to be measured whether the method containing lysozyme, it is characterised in that:
It is that the molecular imprinting photonic crystal film that any one of claim 1-6 is prepared is added in sample to be tested, keeps its absorption full
With if apparent red shift occurs under natural light, contain lysozyme in solution to be measured, and content is greater than 0.5 μm of ol/L.
Beneficial effects of the present invention are as follows:
1. the present invention to form photonic crystal by assembling monodisperse silica microspheres, molecularly imprinted polymer is introduced,
By being prepared for the molecular imprinting photonic crystal film with the hole configurations for forming three-dimensional order after etching.
2. molecular imprinting photonic crystal film prepared by the present invention, can be quickly in conjunction with template lysozyme after removing removing template
Molecule, and there is the displacement of apparent diffraction maximum after combining, the quick detection to micro lysozyme is realized according to this phenomenon.
In addition, when bacteriolyze enzyme molecule binding capacity reaches 0.5 μm of ol/L or more on film, due to the molecular engram photon of preparation
The displacement of crystal film diffraction maximum, under natural light irradiation, apparent red shift can occur for the visible molecular engram film of naked eyes (because being not
With the microballoon of size, variation is green to red from indigo plant to green, and Huang is all red shift to red), it according to this can quick judgement sample
In whether have the presence of lysozyme, further reduced testing cost and operation require.
3. molecular imprinting photonic crystal membrane stability of the invention is high, reusing is good, easy to operate, low in cost,
A kind of ideal biosensor be can be used as lysozyme detection in biological sample.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is in embodiment 1 using the purple for the molecular imprinting photonic crystal film that partial size is the preparation of 240nm silicon dioxide microsphere
Outer absorption peak.
Fig. 2 is the stereoscan photograph of the molecular imprinting photonic crystal film of 1 step of embodiment (7) preparation.
Fig. 3 is the bacteriolyze that molecular imprinting photonic crystal film is placed in 0,0.2,0.4,0.6,0.8,1.0,1.2,1.5,2.0 μM
Ultraviolet absorption peak displacement-lysozyme concentration the change curve obtained after enzyme standard solution.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
Specific preparation process is as follows for the molecular imprinting photonic crystal film of quick detection lysozyme of the invention:
1) after the silicon wafer of 1 × 10 × 75mm being cleaned by ultrasonic with deionized water, piranha solution (98% concentrated sulfuric acid with
The mixed solution of 30% hydrogen peroxide, volume ratio 7:3) the middle immersion removing surface impurity of processing in 12 hours is clear with deionized water later
Wash clean, is dried with nitrogen spare, obtains hydrophiling matrix；
2) monodisperse silica (SiO for being 200~400nm by partial size2) microballoon ultrasonic disperse is into dehydrated alcohol, shape
At colloidal dispersion system, wherein SiO2Mass fraction 1.0~5.0%, at a certain angle by the hydrophiling matrix of step 1) preparation
In (30~90 °) insertion silicon dioxide microsphere solution, by the way of vertical self assembly, under the conditions of 25~40 DEG C constant-temperature enclosed
Standing is volatilized completely to ethyl alcohol, prepares photonic crystal template；
3) polymethyl methacrylate (PMMA) film is cut into 0.75cm wide × 1.0cm size, the PMMA film cut out
It cleans up, is dried with nitrogen, PMMA film is gently covered in photonic crystal template later, clamp is spare；
4) successively that template molecule (10~200mg), function monomer (50~500mg) and crosslinking agent (50~500mg) is molten
In 1~10mL phosphate buffer solution in (pH 6.0~8.0), pre-polymer solution is obtained after being sufficiently mixed；
Wherein, template molecule is lysozyme, and function monomer is acrylamide, crosslinking agent N, N '-methylene bisacrylamide amine；
5) initiator (5~50mg) and accelerator (5~50 μ L) are added in pre-polymer solution, after ultrasonic disperse,
Obtain precursor solution, with micro syringe draw certain volume precursor solution (10~50 μ L), syringe needle from PMMA film with
It is slowly injected into precursor solution in the gap of silicon wafer, until solution is full of PMMA and silicon wafer under the action of capillary and atmospheric pressure
Between gap, 30 DEG C water-bath 2.0~10.0 hours, lysozyme molecular imprinting crystal film with photon is prepared；
Wherein, initiator is ammonium persulfate, and accelerator is tetramethylethylenediamine；
6) the lysozyme crystal film with photon that above-mentioned polymerization obtains is soaked in the hydrofluoric acid solution of mass concentration 0.5~5%
Bubble 1~6 hour removes silicon dioxide microsphere, and the molecular imprinting photonic crystal hydrogel blotting membrane of counter opal structure is made
(MIPH)；
7) using lauryl sodium sulfate and acetic acid mixed solution as eluant, eluent (in mixed solution, water is solvent,
The mass concentration of lauryl sodium sulfate is 1~10%, and the volumetric concentration of acetic acid is 1~15%.), MIPH merging elution is molten
Shaking table is incubated for 6~12 hours in agent, and repeatedly, until completely removing lysozyme template protein, what is be prepared divides for elution repeatedly
Sub- trace crystal film with photon saves backup in pure water.
Embodiment 1
Specific preparation process is as follows for the molecular imprinting photonic crystal film of quick detection lysozyme of the invention:
1) after the silicon wafer of 1 × 10 × 75mm being cleaned by ultrasonic with deionized water, piranha solution (98% concentrated sulfuric acid with
The mixed solution of 30% hydrogen peroxide, volume ratio 7:3) the middle immersion removing surface impurity of processing in 12 hours is clear with deionized water later
Wash clean is dried with nitrogen spare；
2) monodisperse silica (SiO for being 240nm by partial size2) microballoon ultrasonic disperse is into dehydrated alcohol, formation glue
Body dispersion, wherein SiO2The hydrophiling matrix of step 1) preparation is inserted into titanium dioxide with 45° angle degree by mass fraction 2.0%
It in silicon microspheres solution, by the way of vertical self assembly, stands under the conditions of 35 DEG C constant-temperature enclosed and volatilizees completely to ethyl alcohol, prepare
Obtain photonic crystal template；
3) polymethyl methacrylate (PMMA) film is cut into 0.75cm wide × 1.0cm size, the PMMA film cut out
It cleans up, is dried with nitrogen, PMMA film is gently covered in photonic crystal template later, clamp is spare；
4) successively antalzyme protein 50mg, acrylamide 300mg and N, N '-methylene bisacrylamide amine 150mg are dissolved in
In 2.5mL phosphate buffer solution in (pH 6.4), pre-polymer solution is obtained after being sufficiently mixed；
5) ammonium persulfate 10mg and 8 μ L of tetramethylethylenediamine are added in pre-polymer solution, after ultrasonic disperse, are obtained
Precursor solution.20 μ L precursor solutions are drawn with micro syringe, syringe needle is slowly infused from the gap of PMMA film and silicon wafer
Enter precursor solution, until solution is full of the gap between PMMA and silicon wafer, 30 DEG C of water under the action of capillary and atmospheric pressure
Bath reaction 6.0 hours, is prepared lysozyme molecular imprinting crystal film with photon；
6) crystal film with photon for obtaining above-mentioned polymerization impregnates 2.0 hours in the hydrofluoric acid solution of 1.5% mass concentration,
Silicon dioxide microsphere is removed, the molecular imprinting photonic crystal hydrogel blotting membrane (MIPH) of counter opal structure is made；
7) using lauryl sodium sulfate and acetic acid mixed solution as eluant, eluent (in mixed solution, water is solvent,
The mass concentration of lauryl sodium sulfate is 2.5%, and the volumetric concentration of acetic acid is 8%), MIPH to be placed in shaking table in eluting solvent
It is incubated for 4 hours, elutes 3 times, until completely removing antalzyme protein matter, the molecular imprinting photonic crystal film being prepared is in pure water
In save backup.
Embodiment 2
Specific preparation process is as follows for the molecular imprinting photonic crystal film of quick detection lysozyme of the invention:
1) after the silicon wafer of 1 × 10 × 75mm being cleaned by ultrasonic with deionized water, piranha solution (98% concentrated sulfuric acid with
The mixed solution of 30% hydrogen peroxide, volume ratio 7:3) the middle immersion removing surface impurity of processing in 12 hours is clear with deionized water later
Wash clean, is dried with nitrogen spare, obtains hydrophiling matrix；
2) monodisperse silica (SiO for being 220nm by partial size2) microballoon ultrasonic disperse is into dehydrated alcohol, formation glue
Body dispersion, wherein SiO2The hydrophiling matrix of step 1) preparation is inserted into titanium dioxide with 90 ° of angles by mass fraction 3.0%
It in silicon microspheres solution, by the way of vertical self assembly, stands under the conditions of 35 DEG C constant-temperature enclosed and volatilizees completely to ethyl alcohol, prepare
Obtain photonic crystal template；
3) polymethyl methacrylate (PMMA) film is cut into 0.75cm wide × 1.0cm size, the PMMA film cut out
It cleans up, is dried with nitrogen, PMMA film is gently covered in photonic crystal template later, clamp is spare；
4) successively antalzyme protein 50mg, acrylamide 300mg and N, N '-methylene bisacrylamide amine 150mg are dissolved in
In 2.5mL phosphate buffer solution in (pH 6.4), pre-polymer solution is obtained after being sufficiently mixed；
5) ammonium persulfate 10mg and 8 μ L of tetramethylethylenediamine are added in pre-polymer solution, after ultrasonic disperse, are obtained
Precursor solution draws 20 μ L precursor solutions with micro syringe, and syringe needle is slowly infused from the gap of PMMA film and silicon wafer
Enter precursor solution, until solution is full of the gap between PMMA and silicon wafer, 30 DEG C of water under the action of capillary and atmospheric pressure
Bath reaction 6.0 hours, is prepared lysozyme molecular imprinting crystal film with photon；
6) crystal film with photon for obtaining above-mentioned polymerization middle immersion 2.0 in the hydrofluoric acid solution of 1.5% mass concentration is small
When, silicon dioxide microsphere is removed, the molecular imprinting photonic crystal hydrogel blotting membrane (MIPH) of counter opal structure is made；
7) using lauryl sodium sulfate and acetic acid mixed solution as eluant, eluent (in mixed solution, water is solvent,
The mass concentration of lauryl sodium sulfate is 2.5%, and the volumetric concentration of acetic acid is 8%.), MIPH is placed in eluting solvent and is shaken
Bed is incubated for 4 hours, is eluted 3 times, until completely removing antalzyme protein matter, the molecular imprinting photonic crystal film being prepared is pure
It is saved backup in water.
Embodiment 3
Specific preparation process is as follows for the molecular imprinting photonic crystal film of quick detection lysozyme of the invention:
1) after the silicon wafer of 1 × 10 × 75mm being cleaned by ultrasonic with deionized water, piranha solution (98% concentrated sulfuric acid with
The mixed solution of 30% hydrogen peroxide, volume ratio 7:3) the middle immersion removing surface impurity of processing in 12 hours is clear with deionized water later
Wash clean is dried with nitrogen spare；
2) monodisperse silica (SiO for being 220nm by partial size2) microballoon ultrasonic disperse is into dehydrated alcohol, formation glue
Body dispersion, wherein SiO2The hydrophiling matrix of step 1) preparation is inserted into titanium dioxide with 45° angle degree by mass fraction 2.0%
It in silicon microspheres solution, by the way of vertical self assembly, stands under the conditions of 35 DEG C constant-temperature enclosed and volatilizees completely to ethyl alcohol, prepare
Obtain photonic crystal template；
3) polymethyl methacrylate (PMMA) film is cut into 0.75cm wide × 1.0cm size, the PMMA film cut out
It cleans up, is dried with nitrogen, PMMA film is gently covered in photonic crystal template later, clamp is spare；
4) successively antalzyme protein 100mg, acrylamide 300mg and N, N '-methylene bisacrylamide amine 300mg are dissolved in
In 2.5mL phosphate buffer in (pH 6.4), pre-polymer solution is obtained after being sufficiently mixed；
5) ammonium persulfate 15mg and 12 μ L of tetramethylethylenediamine are added in pre-polymer solution, after ultrasonic disperse, are obtained
To precursor solution, 20 μ L precursor solutions are drawn with micro syringe, syringe needle is slow from the gap of PMMA film and silicon wafer
Inject precursor solution, until solution under the action of capillary and atmospheric pressure full of gap between PMMA and silicon wafer, 30 DEG C
Water-bath 10.0 hours, lysozyme molecular imprinting crystal film with photon is prepared；
6) crystal film with photon for obtaining above-mentioned polymerization middle immersion 3.0 in the hydrofluoric acid solution of 2.5% mass concentration is small
When, silicon dioxide microsphere is removed, the molecular imprinting photonic crystal hydrogel blotting membrane (MIPH) of counter opal structure is made；
7) using lauryl sodium sulfate and acetic acid mixed solution as eluant, eluent (in mixed solution, water is solvent,
The mass concentration of lauryl sodium sulfate is 2.5%, and the volumetric concentration of acetic acid is 8%.), MIPH is placed in eluting solvent and is shaken
Bed is incubated for 4 hours, is eluted 3 times, until completely removing antalzyme protein matter, the molecular imprinting photonic crystal film being prepared is pure
It is saved backup in water.
Embodiment 4
Specific preparation process is as follows for the molecular imprinting photonic crystal film of quick detection lysozyme of the invention:
1) after the silicon wafer of 1 × 10 × 75mm being cleaned by ultrasonic with deionized water, piranha solution (98% concentrated sulfuric acid with
The mixed solution of 30% hydrogen peroxide, volume ratio 7:3) the middle immersion removing surface impurity of processing in 12 hours is clear with deionized water later
Wash clean, is dried with nitrogen spare, obtains hydrophiling matrix；
2) monodisperse silica (SiO for being 220nm by partial size2) microballoon ultrasonic disperse is into dehydrated alcohol, formation glue
Body dispersion, wherein SiO2The hydrophiling matrix of step 1) preparation is inserted into titanium dioxide with 45° angle degree by mass fraction 3.0%
It in silicon microspheres solution, by the way of vertical self assembly, stands under the conditions of 35 DEG C constant-temperature enclosed and volatilizees completely to ethyl alcohol, prepare
Obtain photonic crystal template；
3) polymethyl methacrylate (PMMA) film is cut into 0.75cm wide × 1.0cm size, the PMMA film cut out
It cleans up, is dried with nitrogen, PMMA film is gently covered in photonic crystal template later, clamp is spare；
4) successively antalzyme protein 50mg, acrylamide 300mg and N, N '-methylene bisacrylamide amine 150mg are dissolved in
In 2.5mL phosphate buffer solution in (pH 6.4), pre-polymer solution is obtained after being sufficiently mixed；
5) ammonium persulfate 10mg and 8 μ L of tetramethylethylenediamine are added in pre-polymer solution, after ultrasonic disperse, are obtained
Precursor solution draws 20 μ L precursor solutions with micro syringe, and syringe needle is slowly infused from the gap of PMMA film and silicon wafer
Enter precursor solution, until solution is full of the gap between PMMA and silicon wafer, 30 DEG C of water under the action of capillary and atmospheric pressure
Bath reaction 6.0 hours, is prepared lysozyme molecular imprinting crystal film with photon；
6) crystal film with photon for obtaining above-mentioned polymerization impregnates 1.0 hours in the hydrofluoric acid solution of 3.0% mass concentration,
Silicon dioxide microsphere is removed, the molecular imprinting photonic crystal hydrogel blotting membrane (MIPH) of counter opal structure is made；
7) using lauryl sodium sulfate and acetic acid mixed solution as eluant, eluent (in mixed solution, water is solvent,
The mass concentration of lauryl sodium sulfate is 10%, and the volumetric concentration of acetic acid is 10%.), MIPH is placed in eluting solvent and is shaken
Bed is incubated for 3 hours, is eluted 4 times, until completely removing antalzyme protein matter, the molecular imprinting photonic crystal film being prepared is pure
It is saved backup in water.
Embodiment 5
1) after the silicon wafer of 1 × 10 × 75mm being cleaned by ultrasonic with deionized water, piranha solution (98% concentrated sulfuric acid with
The mixed solution of 30% hydrogen peroxide, volume ratio 7:3) the middle immersion removing surface impurity of processing in 12 hours is clear with deionized water later
Wash clean is dried with nitrogen spare；
2) monodisperse silica (SiO for being 200nm by partial size2) microballoon ultrasonic disperse is into dehydrated alcohol, formation glue
Body dispersion, wherein SiO2The hydrophiling matrix of step 1) preparation is inserted into titanium dioxide with 30 ° of angles by mass fraction 2.0%
It in silicon microspheres solution, by the way of vertical self assembly, stands under the conditions of 35 DEG C constant-temperature enclosed and volatilizees completely to ethyl alcohol, prepare
Obtain photonic crystal template；
3) polymethyl methacrylate (PMMA) film is cut into 0.75cm wide × 1.0cm size, the PMMA film cut out
It cleans up, is dried with nitrogen, PMMA film is gently covered in photonic crystal template later, clamp is spare；
4) successively antalzyme protein 100mg, acrylamide 300mg and N, N '-methylene bisacrylamide amine 300mg are dissolved in
In 2.5mL phosphate buffer solution in (pH 6.4), pre-polymer solution is obtained after being sufficiently mixed；
5) ammonium persulfate 15mg and 10 μ L of tetramethylethylenediamine are added in pre-polymer solution, after ultrasonic disperse, are obtained
To precursor solution, 20 μ L precursor solutions are drawn with micro syringe, syringe needle is slow from the gap of PMMA film and silicon wafer
Inject precursor solution, until solution under the action of capillary and atmospheric pressure full of gap between PMMA and silicon wafer, 30 DEG C
Water-bath 6.0 hours, lysozyme molecular imprinting crystal film with photon is prepared；
6) crystal film with photon for obtaining above-mentioned polymerization middle immersion 2.0 in the hydrofluoric acid solution of 5.0% mass concentration is small
When, silicon dioxide microsphere is removed, the molecular imprinting photonic crystal hydrogel blotting membrane (MIPH) of counter opal structure is made；
7) using lauryl sodium sulfate and acetic acid mixed solution as eluant, eluent (in mixed solution, water is solvent,
The mass concentration of lauryl sodium sulfate is 10%, and the volumetric concentration of acetic acid is 10%.), MIPH is placed in eluting solvent and is shaken
Bed is incubated for 3 hours, is eluted 2 times, until completely removing antalzyme protein matter, the molecular imprinting photonic crystal film being prepared is pure
It is saved backup in water.
Embodiment 6
Using the method for molecular imprinting photonic crystal film detection Lysozyme in Solution content of the invention are as follows:
By molecular imprinting photonic crystal film be respectively put into concentration be respectively 0,0.2,0.4,0.6,0.8,1.0,1.2,1.5,
In 2.0 μM of lysozyme standard solution, after saturation to be adsorbed, molecular imprinting photonic crystal film is taken out, measures its ultraviolet suction respectively
Spectrum is received, absorption peak displacement-lysozyme concentration change curve is calculated.It is brilliant with the molecular engram photon that embodiment 1 obtains
It is as shown in Figure 3 that body film carries out the absorption peak obtained after above-mentioned experiment displacement-lysozyme concentration change curve.
Molecular imprinting photonic crystal film is added in sample to be tested, by measuring lysozyme molecular imprinting crystal film with photon
Diffraction peak wavelength variation, substitute into the obtained standard variation curve of the first step, the content of Lysozyme in Solution to be measured can be obtained.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (10)
1. a kind of preparation method of the molecular imprinting photonic crystal film of quickly detection lysozyme, it is characterised in that: including following step
It is rapid:
(1) silicon wafer is cleaned and is cleaned, obtain hydrophiling matrix；
(2) monodisperse silica microspheres are distributed in dehydrated alcohol, form colloidal dispersion system, by step (1) preparation
Hydrophiling matrix is inserted at a certain angle in silicon dioxide microsphere solution, and using the method for vertical self assembly, constant temperature is stood to second
Alcohol volatilizees completely, obtains photonic crystal template；
(3) it is covered on after cleaning polymethyl methacrylate film in the photonic crystal template that step (2) obtains；
(4) successively template molecule, function monomer and crosslinking agent are dissolved in phosphate buffer solution, obtain pre-polymerization after being sufficiently mixed
Close solution；
(5) initiator and accelerator are added in the pre-polymer solution of step (4), precursor solution is obtained after dispersion；It draws
In the gap of a certain amount of precursor solution injection PMMA film and silicon wafer, until solution is under the action of capillary and atmospheric pressure
Full of the gap between PMMA and silicon wafer, water-bath polymerization reaction obtains lysozyme molecular imprinting crystal film with photon；
(6) the lysozyme molecular imprinting crystal film with photon that step (5) is prepared is soaked in hydrofluoric acid solution, removes dioxy
SiClx microballoon obtains the molecular imprinting photonic crystal hydrogel blotting membrane of counter opal structure；
(7) the molecular imprinting photonic crystal hydrogel blotting membrane for the counter opal structure that step (6) obtains is eluted, until
Lysozyme template protein is completely removed, the molecular imprinting photonic crystal film being prepared.
2. according to the method described in claim 1, it is characterized by: in step (2), the grain of the monodisperse silica microspheres
Diameter is 200~400nm；In the colloidal dispersion system, the mass fraction of monodisperse silica microspheres is 1.0~5.0%；Institute
Stating angle is 30-90 °, and the constant temperature is the constant temperature at 25-40 DEG C.
3. according to the method described in claim 1, it is characterized by: the template molecule is lysozyme, function in step (4)
Monomer is acrylamide, crosslinking agent N, N '-methylene bisacrylamide amine；The weight of the template molecule, function monomer and crosslinking agent
Measure ratio are as follows: (10~200mg): (50~500mg): (50~500mg)；The pH of the phosphate buffer solution is 6.0~8.0.
4. according to the method described in claim 1, it is characterized by: the condition of the water-bath polymerization reaction is 30 in step (5)
DEG C water-bath 2.0~10.0 hours；The initiator is ammonium persulfate, and accelerator is tetramethylethylenediamine, initiator with plus
The amount ratio of fast agent is (5~50mg): (5~50 μ L).
5. according to the method described in claim 1, it is characterized by: in step (6), in the hydrofluoric acid of mass concentration 0.5~5%
It is impregnated 1~6 hour in solution.
6. according to the method described in claim 1, it is characterized by: in step (7), when the elution, eluent is dodecane
The mixed solution of base sodium sulphate and acetic acid；In mixed solution, water is solvent, the mass concentration of lauryl sodium sulfate is 1~
10%, the volumetric concentration of acetic acid is 1~15%.
7. the molecular imprinting photonic crystal film that the method for any one of application claim 1-6 is prepared.
8. application of the molecular imprinting photonic crystal film as claimed in claim 7 in quickly detection lysozyme.
9. a kind of method of quickly detection lysozyme content, it is characterised in that: the following steps are included:
(1) the molecular imprinting photonic crystal film that any one of claim 1-6 is prepared is respectively put into the bacteriolyze of various concentration
In enzyme standard solution, make its adsorption saturation, take out molecular imprinting photonic crystal film, measure its ultra-violet absorption spectrum respectively, calculates
Be absorbed peak shift amount-lysozyme concentration change curve；
(2) the molecular imprinting photonic crystal film that any one of claim 1-6 is prepared is added in sample to be tested, passes through survey
The diffraction peak wavelength variation for determining lysozyme molecular imprinting crystal film with photon, substitutes into the standard variation curve that the first step obtains
Obtain the content of Lysozyme in Solution to be measured.
10. in a kind of fast qualitative detection solution to be measured whether the method containing lysozyme, it is characterised in that: be by claim
The molecular imprinting photonic crystal film that any one of 1-6 is prepared is added in sample to be tested, makes its adsorption saturation, if in natural light
It is lower that apparent red shift occurs, then contain lysozyme in solution to be measured, and content is greater than 0.5 μm of ol/L.
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