CN116794289A - Fluorescent gain NC film and preparation method and application thereof - Google Patents
Fluorescent gain NC film and preparation method and application thereof Download PDFInfo
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- CN116794289A CN116794289A CN202210529261.7A CN202210529261A CN116794289A CN 116794289 A CN116794289 A CN 116794289A CN 202210529261 A CN202210529261 A CN 202210529261A CN 116794289 A CN116794289 A CN 116794289A
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Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention belongs to the field of materialogy, and particularly relates to a fluorescent gain NC film and a preparation method and application thereof. The photonic crystal type NC film comprises an NC film and a photonic crystal structure positioned in a photonic crystal region of the NC film. When detecting on NC film, when detecting antibody-target detecting object-biological material forms sandwich structure on photon crystal, the distance between the receptor substance connected with detecting antibody and photon crystal is shortened, the gain effect of photon crystal can make the fluorescence gain reported by detecting antibody 10-100 times based on the original fluorescence of receptor substance, the detecting limit is reduced by one to two orders of magnitude, realizing trace detection of low concentration detecting object.
Description
Technical Field
The invention belongs to the field of materialogy, and particularly relates to a fluorescent gain NC film and a preparation method and application thereof.
Background
Nitrocellulose (nitrocellulose filter membrane, NC film for short) is a carrier used as a C/T line in colloidal gold test paper, and is also an immune reaction occurring place, NC film is one of the most important consumables in biological tests, and at present NC films mostly recognize by setting antibodies corresponding to targets on the C/T line through color change, and have low sensitivity.
Because of the unique optical characteristics, the photonic crystal has great research significance in the field of high-sensitivity detection of biological materials such as low-concentration ions, DNA, proteins, biological probes and the like. By means of the inherent fluorescence enhancement and amplification fluorescent signal property of the photonic crystal, the detection sensitivity of the photonic crystal biosensor can be greatly improved, and the biological detection limit is reduced, however, the existing photonic crystal mainly carries out specific recognition with an object to be detected to form a corresponding luminous system, for example, a resonance energy transfer recognition system for detection, the detection system is complex, the preparation difficulty is high, and the cost is high.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a photonic crystal type NC film, which comprises an NC film and a photonic crystal structure positioned in a photonic crystal region of the NC film, wherein the photonic crystal region comprises a region where a C line and a T line are positioned.
According to an embodiment of the invention, the photonic crystal structure is provided with a C-line and a T-line.
According to an embodiment of the invention, the C-line, T-line comprises a biomaterial having groups thereon capable of reacting with a photonic crystal. For example, the C line and the T line are formed by connecting biological materials on the photonic crystal.
According to an embodiment of the invention, the biomaterial is attached to the photonic crystal by chemical bonds.
According to an embodiment of the present invention, the biological material on the T-line is a biological material that does not bind to the substance to be detected.
According to an embodiment of the invention, the biomaterial on the C-line is a material capable of specifically reacting with the target analyte.
According to an embodiment of the invention, the specifically reactive material is selected from at least one of enzymes, DNA/RNA, antigens, antibodies, aptamers, biotin-streptavidin, protein receptors, etc.
The invention also provides a preparation method of the photonic crystal NC film, which comprises the following steps:
and transferring the photonic crystal structure on the photonic crystal film to a photonic crystal region of the NC film to obtain the photonic crystal NC film.
According to an embodiment of the present invention, the transfer is performed by a conventional transfer method.
According to an embodiment of the invention, the preparation method further comprises the steps of: a biological material is immobilized on the photonic crystal structure.
According to an embodiment of the present invention, before the biological material is immobilized on the photonic crystal structure, the method further comprises the steps of: and activating the photonic crystal structure to expose a reactive group on the photonic crystal, wherein the reactive group is a group capable of reacting with biological materials.
According to an embodiment of the invention, the activating comprises coating an activator on the photonic crystal structure.
The invention also provides an application of the photonic crystal NC film or the photonic crystal NC film prepared by the method in biological detection.
The invention also provides a method for detecting the biomarker by using the photonic crystal NC film, which comprises the following steps: and enabling the sample to be detected to be in contact with the photonic crystal NC film through capillary chromatography.
According to an embodiment of the invention, the method comprises the steps of:
a) Measuring fluorescence values by contacting standard biomarker samples with different concentrations with the photonic crystal NC film through capillary chromatography;
b) And contacting the sample to be detected with the photonic crystal NC film through capillary chromatography, and detecting a fluorescence signal of a C/T line.
According to an embodiment of the present invention, before the sample to be measured is contacted with the photonic crystal NC film by capillary chromatography in step b), the method further comprises the steps of: mixing a sample to be tested with a detection antibody, wherein the detection antibody is connected with a receptor substance.
According to an embodiment of the invention, the acceptor substance comprises at least one of nanocrystals and fluorescent dye-labeled molecules.
According to an embodiment of the present invention, the nanocrystals are selected from at least one of silica nanocrystals, titania nanocrystals, zirconia nanocrystals, zinc oxide nanocrystals, cadmium sulfide nanocrystals, cadmium telluride nanocrystals, gold nanoparticles, silver nanoparticles.
According to an embodiment of the present invention, the fluorescent dye labeling molecule is selected from at least one of Cy3, cy5, FITC, rhB, etc.
The invention also provides a biological detection platform which contains the photonic crystal NC film or the photonic crystal NC film prepared by the method.
Advantageous effects
1. The viscosity and the surface tension of the photonic crystal (such as latex ball) ink are regulated and controlled by the humectant and the wetting agent, the average surface tension of the photonic crystal ink is less than 35.5mN/m, and the viscosity at 20 ℃ is less than 4 m.Pa.s -1 A viscosity at 25 ℃ of less than 3.5 mPa.s -1 The method comprises the steps of carrying out a first treatment on the surface of the Thereby effectively regulating and controlling the spreading and infiltration behaviors of the photonic crystal ink on the surface of the substrate; so that the photonic crystal (such as latex balls) can be assembled on the substrate in a rapid and orderly manner through a roll printing machine and a coating machine to form a uniform photonic crystal film.
2. The photonic crystal prepared by the method has a fluorescence gain effect, the photonic crystal film is transferred onto the NC film in a hot stamping mode, the photonic crystal NC film is prepared, and due to the existence of the photonic crystal, when the detection antibody-target detection object-biological material forms a sandwich structure on the photonic crystal, the distance between a receptor substance connected with the detection antibody and the photonic crystal is shortened, the fluorescence gain reported by the detection antibody can be 10-100 times based on the original fluorescence of the receptor substance by the gain effect of the photonic crystal, the detection limit is reduced by one to two orders of magnitude, and trace detection of a low-concentration object to be detected is realized.
3. The biological platform constructed by the invention has universality, and biological materials (antibodies, aptamers, polypeptides and the like) are fixed through simple coupling reaction, so that visual detection is realized.
4. The photonic crystal NC film has the advantages of simple preparation method, environment friendliness, large-scale preparation and easy commercial use.
5. The photonic crystal of the invention not only has fluorescence enhancement effect on fluorescent markers, but also can be used as a carrier for coupling biological materials (antibodies, aptamer, polypeptide and the like), and is not only a biological detection probe, but also a biological detection platform.
Drawings
FIG. 1 is a photonic crystal film assembled from 260nm latex spheres in example 1.
Fig. 2 is a SEM image of the photonic crystal thin film in example 1.
FIG. 3 is a photonic crystal film assembled from 230nm latex spheres in example 2.
Fig. 4 is a physical diagram of a photonic crystal NC film in example 3.
FIG. 5 is a schematic diagram of a biofunctional photonic crystal type NC membrane (photonic crystal coupled Cy 3-labeled IgG antibody) in example 4.
FIG. 6 is a fluorescence microscope image of the NC film of the photonic crystal type of example 4, wherein the left image represents a fluorescence imaging image of the photonic crystal unconjugated Cy 3-labeled IgG antibody in a direct drop-wise manner; the right panel represents a fluorescence imaging of a photonic crystal coupled Cy3 labeled IgG antibody.
FIG. 7 shows the result of the preparation of example 7, wherein the photonic crystal type NC membrane recognizes the CK-MB antigen of the target specimen in different concentrations (the initial mass concentration of the CK-MB antigen of the target specimen is 100. Mu.g/mL, which means from left to right that the CK-MB antigen of the target specimen is diluted to 10 and 10 of the initial concentration, respectively 2 、10 3 、10 4 、10 5 、10 6 Fold) of the standard curve.
Detailed Description
[ Photonic Crystal film ]
As described above, the present invention provides a photonic crystal film including a substrate and a photonic crystal structure located on at least one side surface of the substrate.
According to an embodiment of the present invention, the photonic crystal structure is located on one side surface or both side surfaces of the substrate.
According to an embodiment of the present invention, the photonic crystal structure includes a lattice structure or a thin film structure, respectively denoted as a photonic crystal lattice and a photonic crystal thin film.
According to an embodiment of the present invention, the lattice structure is, for example, a dot pattern, a line pattern or a plane pattern, respectively denoted as photonic crystal dot, photonic crystal line and photonic crystal plane.
According to an embodiment of the present invention, the photonic crystal structure is formed on the surface of the substrate by photonic crystal ink. Specifically, the photonic crystal ink may be formed on the surface of the substrate in a conventional spraying, printing or printing manner to form the photonic crystal structure. The photonic crystal structure is formed by printing photonic crystal ink onto a substrate, for example, by a roll printer or coater.
According to an embodiment of the invention, the photonic crystal structure has hydrophilicity.
According to an embodiment of the present invention, the photonic crystal structure is formed by self-assembly of a photonic crystal on a substrate; preferably, the photonic crystal structure is a structure in which photonic crystals are self-assembled on a substrate to form a periodic regular arrangement.
According to an embodiment of the present invention, the photonic crystal structure has a scanning electron microscope image substantially as described in fig. 2.
According to an embodiment of the invention, the substrate comprises a flexible material, preferably a flexible transparent material, selected from the group comprising transparent plastics, such as PP plastics (Polypropylene), PS plastics (general purpose polystyrene, polystyrene based plastics), PVC plastics (Polyvinyl chloride ) or PET plastics (Polyethylene terephthalate, polyethylene terephthalate).
According to an embodiment of the present invention, the photonic crystal ink includes a photonic crystal, a humectant, and a wetting agent.
According to an embodiment of the present invention, the photonic crystal in the photonic crystal ink is a photonic crystal with or without a linking group.
Specifically, the linking group includes at least one of a carboxyl group, a hydroxyl group, a mercapto group, and an amino group, for example, the linking group is a carboxyl group.
According to an embodiment of the present invention, the photonic crystal is at least one of a protein Dan Guangzi crystal, an inverse opal photonic crystal, and a two-dimensional photonic crystal.
According to an embodiment of the invention, in the ink, the photonic crystal is present in the form of an emulsion.
According to an embodiment of the present invention, in the ink, the photonic crystal exists in the form of a latex sphere.
Preferably, the latex spheres are monodisperse latex spheres.
According to an embodiment of the present invention, the particle size of the monodisperse latex spheres is 150 to 320nm, preferably 180 to 300nm, further 180 to 280nm, and exemplified by 180nm, 190nm, 200nm, 205nm, 215nm, 220nm, 230nm, 250nm, 260nm, 280nm.
According to an embodiment of the present invention, the photonic crystal latex sphere may be at least one selected from the group consisting of photonic crystal-poly (methyl methacrylate-acrylic acid-styrene) latex spheres, photonic crystal-silica microspheres, photonic crystal-polystyrene microspheres, and the like, for example, photonic crystal-poly (methyl methacrylate-acrylic acid-styrene) latex spheres.
According to an embodiment of the present invention, the surface of the monodisperse latex spheres has a carboxyl functional group.
Preferably, the silica microsphere is a carboxyl modified silica microsphere, and the polystyrene microsphere is a carboxyl modified polystyrene microsphere.
According to an embodiment of the present invention, in the photonic crystal ink, the mass concentration of the photonic crystal is 10 to 25%, preferably the mass concentration of the photonic crystal is 10 to 20%, and further, the mass concentration of the photonic crystal is 14 to 18%, for example, 10%, 12%, 14%, 16%, 18%, 20%.
According to an embodiment of the present invention, the humectant may be at least one selected from ethylene glycol, propylene glycol, glycerol, sorbitol, and the like, for example, ethylene glycol.
According to an embodiment of the present invention, in the photonic crystal ink, the mass concentration of the humectant is 8 to 15%, preferably 10 to 12%, and exemplified by 10%, 11%, 12%.
According to an embodiment of the present invention, the wetting agent may be at least one selected from BYK-3400 (polyether modified polydimethylsiloxane), tween T-20, triton X-100, etc., for example BYK-3400.
According to an embodiment of the present invention, the mass concentration of the wetting agent in the photonic crystal ink is 0.1 to 0.5%, preferably 0.2 to 0.4%, for example 0.5%.
According to an embodiment of the present invention, the photonic crystal ink further includes a solvent for dissolving the photonic crystal, the humectant and the humectant, the solvent being selected from water, for example, deionized water or ultrapure water.
As one example, the photonic crystal ink includes monodisperse poly (methyl methacrylate-acrylic acid-styrene) latex spheres, ethylene glycol, and BYK-3400.
According to an embodiment of the invention, the photonic crystal ink has an average surface tension of less than 35.5mN/m, preferably the photonic crystal ink has an average surface tension of less than 25.5mN/m, for example, the photonic crystal ink has an average surface tension of 35.158mN/m, 23.658mN/m, 20.892mN/m, 26.086mN/m, 21.492mN/m, 21.250mN/m.
The average surface tension refers to the average value obtained after 5 times of testing under the same condition.
According to an embodiment of the invention, the photonic crystal ink has a viscosity of less than 4 m.Pa.s at 20 DEG C -1 Preferably, the photonic crystal ink has a viscosity of less than 3 mPa.s at 20deg.C -1 For example, the photonic crystal ink has a viscosity of 1.7431 mPa.s at 20 DEG C -1 、1.7280m·Pa·s -1 、3.7642m·Pa·s -1 、3.8454m·Pa·s -1 、1.9054m·Pa·s -1 、2.4689m·Pa·s -1 。
According to an embodiment of the invention, the photonic crystal ink has a viscosity of less than 3.5 mPa.s at 25 DEG C -1 Preferably, the photonic crystal ink has a viscosity of less than 2.5 mPa.s at 25 DEG C -1 For example, the photonic crystal ink has a viscosity of 1.5425 mPa.s at 25 DEG C -1 、1.5121m·Pa·s -1 、3.2564m·Pa·s -1 、3.3385m·Pa·s -1 、1.6604m·Pa·s -1 、2.1114m·Pa·s -1 。
[ method for producing Photonic Crystal film ]
The invention also provides a preparation method of the photonic crystal film, which comprises the following steps:
s1, configuring photonic crystal ink;
s2, forming the photonic crystal structure on at least one side surface of the substrate by the photonic crystal ink to obtain the photonic crystal film.
According to an embodiment of the invention, the photonic crystal ink, substrate has the definition as described above.
According to an embodiment of the present invention, in step S1, configuring photonic crystal ink includes the steps of: the photonic crystal, humectant, wetting agent and solvent are mixed.
According to an embodiment of the invention, the photonic crystal, humectant and wetting agent have the contents as described above.
According to an embodiment of the present invention, in step S2, the photonic crystal ink is sprayed, printed or printed on at least one side surface of the substrate to form the photonic crystal structure. Photonic crystal ink is printed onto at least one layer of the surface of the substrate, for example, by a roll printer or coater.
In particular, the photonic crystal structure has the definition described above.
[ Photonic Crystal type NC film ]
The invention also provides a photonic crystal type NC film, which comprises the NC film and a photonic crystal structure positioned in a photonic crystal region of the NC film, wherein the photonic crystal region comprises a region where a C line and a T line are positioned.
According to an embodiment of the invention, the photonic crystal structure is provided with a C-line and a T-line.
According to the embodiment of the invention, the photonic crystal region comprises a region where a C line and a T line are located, for example, the photonic crystal region comprises a C line region and a T line region, the width of the C line region is greater than or equal to the design width of the C line, and the width of the T line region is greater than or equal to the design width of the T line.
According to an embodiment of the present invention, the C line refers to a detection line, and the T line refers to a quality control line.
According to an embodiment of the invention, the photonic crystal structure has the definition described above.
In the invention, the photonic crystal structure does not influence the original capillary action of the NC film, namely the rapid chromatographic penetration, because the photonic crystal is a hydrophilic material.
According to an embodiment of the invention, the photonic crystal structure has a width of 1mm-2mm, preferably the photonic crystal structure has a width of 1.2mm-1.6mm, for example 1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm or 2mm.
According to an embodiment of the present invention, the C-line and T-line include a biomaterial having a group capable of reacting with the photonic crystal thereon, for example, the C-line and T-line are formed by connecting the biomaterial to the photonic crystal.
According to an embodiment of the invention, the biomaterial contains-NH 2 Functional groups.
According to an embodiment of the invention, the biomaterial is attached to the photonic crystal by chemical bonds, for example by amide bonds. Specifically, the biomaterial is connected to the photonic crystal to form a C line and a T line.
For example, the biomaterial is attached to a chemical bond on the surface of the photonic crystal through a coupling reaction.
According to an embodiment of the invention, the T-line forming biomaterial is a biomaterial that does not bind to the substance to be detected, for example, a goat anti-human IgG antibody.
According to an embodiment of the present invention, the C-line forming biomaterial is a material capable of specifically reacting with a target analyte.
According to an embodiment of the present invention, the specifically reactive material is selected from at least one of enzymes, DNA/RNA, antigens, antibodies, aptamers, biotin-streptavidin, protein receptors, etc.; further, when the target analyte is an antigen, the specifically reactive material is an antibody capable of specifically binding to the antigen, for example, goat anti-mouse IgG.
[ method for producing Photonic Crystal type NC film ]
The invention also provides a preparation method of the photonic crystal NC film, which comprises the following steps:
and transferring the photonic crystal structure on the photonic crystal film to a photonic crystal region of the NC film to obtain the photonic crystal NC film.
According to an embodiment of the invention, the transfer is performed by conventional transfer methods, for example by hot stamping.
According to the embodiment of the invention, the hot stamping method specifically refers to that the photonic crystal film is contacted with the NC film and is placed on a hot stamping machine for rapid transfer printing.
According to an embodiment of the invention, the temperature of the hot stamping is 80-120 ℃, and the time of the hot stamping is 5-30 s.
Preferably, the temperature of the hot stamping is 100-120 ℃, and the time of the hot stamping is 5-10 s.
According to an embodiment of the invention, the preparation method further comprises the steps of: a biological material is immobilized on the photonic crystal structure.
According to an embodiment of the present invention, before the biological material is immobilized on the photonic crystal structure, the method further comprises the steps of: the photonic crystal structure is activated to expose reactive groups on the photonic crystal, preferably groups capable of reacting with biological materials, such as carboxyl groups.
According to an embodiment of the invention, the activation comprises coating the photonic crystal structure with an activator, such as an EDC/NHS mixed solution.
As an example, the EDC/NHS mixed solution is firstly scribed on the photonic crystal structure by a scribing method of a scribing machine to activate, and then the biological material is scribed on the activated photonic crystal structure to form the photonic crystal structure with the biological material fixed thereon.
According to an embodiment of the present invention, the biological material is streaked at a streaking speed of 1 to 3. Mu.L/cm, for example, 1. Mu.L/cm, 2. Mu.L/cm or 3. Mu.L/cm.
According to an embodiment of the present invention, after scribing the biomaterial on the activated photonic crystal film, further comprising a step of sealing and incubating overnight at a temperature of 1-10 ℃, preferably, the incubation temperature is 1-5 ℃, for example, the incubation temperature is 2 ℃, 4 ℃, 5 ℃, 6 ℃, 8 ℃.
[ application of Photonic Crystal type NC film ]
The application of the photonic crystal NC film in biological detection, such as biomarker detection.
According to an embodiment of the invention, the biomarkers comprise protein and/or nucleic acid fragments, e.g. for detecting inflammatory four items (CRP\SAA\PCT\IL-6), cardiac six items (AST/CK/CKBB/LDH/CTNI), neocrown (COVID-19), etc.
According to an embodiment of the invention, the detection is a point-of-care detection, also known as POCT (Point-of-care testing)
[ method for detecting Photonic Crystal type NC film ]
The invention also provides a method for detecting the biomarker by adopting the photonic crystal NC film, which comprises the following steps: and enabling the sample to be detected to be in contact with the photonic crystal NC film through capillary chromatography.
According to an embodiment of the invention, the method comprises the steps of:
a) Measuring fluorescence values by contacting standard biomarker samples with different concentrations with the photonic crystal NC film through capillary chromatography;
b) And contacting the sample to be detected with the photonic crystal NC film through capillary chromatography, and detecting a fluorescence signal of a C/T line.
According to an embodiment of the present invention, before contacting the sample to be measured with the photonic crystal NC film by capillary chromatography, the method further comprises the steps of: mixing a sample to be tested with a detection antibody, wherein the detection antibody is connected with a receptor substance.
Preferably, the detection antibody, biological material is capable of forming a sandwich structure with the biomarker.
According to an embodiment of the present invention, the detection antibody has a receptor substance attached thereto, and the proximity of the receptor substance to the photonic crystal generates a fluorescence gain, and amplifies a fluorescence signal of the receptor substance.
According to an embodiment of the invention, the acceptor substance comprises at least one of nanocrystals and fluorescent dye-labeled molecules.
Preferably, the semiconductor nanocrystal is selected from at least one of silicon dioxide nanocrystal, titanium dioxide nanocrystal, zirconium oxide nanocrystal, zinc oxide nanocrystal, cadmium sulfide nanocrystal, cadmium telluride nanocrystal, gold nanoparticle, silver nanoparticle.
Preferably, the fluorescent dye labeling molecule is selected from at least one of Cy3, cy5, FITC, rhB, and the like.
Illustratively, the detection antibody is a Cy 3-labeled goat anti-rabbit IgG, a Cy 3-labeled nanobody primary antibody, or a Cy 5-labeled nanobody secondary antibody.
[ biological detection platform ]
The invention also provides a biological detection platform which contains the photonic crystal NC film, and the biological detection platform is a kit.
The photonic crystal NC film contacts with the target analyte through capillary chromatography, corresponding fluorescent signal capture is carried out through fluorescent detection, the fluorescent detection gain effect can be 10-100 times of the original effect, and preferably, the target analyte is marked by fluorescent molecules.
The compounds of the general formula of the present invention, as well as the methods for their preparation and use, will be described in further detail below in conjunction with the specific examples. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Example 1
The preparation method of the red photonic crystal industrial grade printing ink comprises the following steps:
(1) To 100mL of water were added 10.00mmol of methyl methacrylate, 13.89mmol of acrylic acid and 182.60mmol of styrene, followed by dissolution of sodium dodecylbenzenesulfonate (not more than 0.011mmol, which is a concentration lower than the critical micelle concentration) as an emulsifier and 6.30mmol of ammonium bicarbonate as a buffer to obtain a reaction solution, which was kept at 70℃for half an hour, and then an aqueous solution of 2.12mmol of ammonium persulfate was added, and polymerization was carried out at 80℃for 10 hours with continuous stirring to obtain monodisperse latex spheres which were usable without purification.
The particle size of the latex spheres was adjusted by adjusting the amount of styrene (182.6 mmol;121.73mmol;81.15mmol;54.1mmol;36.1mmol;24.04 mmol) to prepare latex spheres having particle sizes of 300nm, 280nm, 260nm, 220nm, 215nm and 180nm, respectively.
(2) Taking the latex ball with the particle size of 260nm (the color of the assembled photonic crystal structure is red) prepared in the step (1) as an example, diluting the photonic crystal stock solution (namely the aqueous solution of the latex ball after centrifugal washing) comprising the latex ball with the particle size of 260nm to 12 weight percent concentration, adding the humectant glycol (the mass fraction accounts for 10 percent) and the wetting agent BYK-3400 (the mass fraction accounts for 0.5 per mill) into the diluted photonic crystal solution to prepare the photonic crystal ink.
(3) And printing photonic crystal ink on the PET film by using the PET film as a base material through a roller printer, self-assembling the photonic crystals in the 260nm latex balls on the surface of the PET film to form a photonic crystal film, and drying and storing.
Referring to FIG. 1, the photonic crystal film formed by assembling the latex beads of 260nm of this example on a PET film was red.
Referring to fig. 2, which is an SEM image of a thin film of a photonic crystal (with a particle size of 260 nm) in this embodiment, it can be seen from fig. 2 that the monodisperse polystyrene latex microspheres self-assemble to form a photonic crystal morphology with periodically ordered arrangement.
Example 2
The preparation method of the green photonic crystal industrial grade printing ink comprises the following steps:
(1) To 100mL of water were added 10.00mmol of methyl methacrylate, 13.89mmol of acrylic acid and 65.23mmol of styrene, followed by dissolution of sodium dodecylbenzenesulfonate (not more than 0.011mmol, which is a concentration lower than the critical micelle concentration) as an emulsifier and 6.30mmol of ammonium bicarbonate as a buffer to give a reaction solution, which was kept at 70℃for half an hour, and then an aqueous solution of 2.12mmol of ammonium persulfate was added, and polymerization was carried out at 80℃for 10 hours with continuous stirring to give monodisperse latex spheres which were usable directly without purification.
(2) In this example, taking 230nm latex spheres (the color of the assembled photonic crystal structure is green), the photonic crystal stock solution (i.e. the aqueous solution of the latex spheres after centrifugal washing) comprising 230nm latex spheres is diluted to 8wt% concentration, and the humectant glycol (the mass fraction is 8%) and the wetting agent BYK-3400 (the mass fraction is 0.3%) are added into the diluted photonic crystal solution to prepare the photonic crystal ink.
(3) And (3) printing photonic crystal ink on a base material PET film by using the PET film as the base material through a coating machine, self-assembling photonic crystals in the 230nm latex balls on the surface of the PET film to form a photonic crystal film, and drying and storing.
Referring to fig. 3, the 230nm latex spheres were assembled on a PET film to form a photonic crystal film, and the crystal film appeared green.
Example 3
The photonic crystal NC film is prepared, the photonic crystal can be well printed on the NC film, and the good permeation chromatography effect of the original NC film is maintained.
(1) Taking a photonic crystal film material with the width of 1mm prepared in the embodiment 1, contacting the surface of the photonic crystal film in the film material with the NC film surface, placing the surface under a hot stamping machine for hot stamping, and transferring the photonic crystal on the photonic crystal film to a photonic crystal area on the NC film in a hot stamping mode, wherein the area is positioned at a C/T line position, the hot stamping temperature is 100 ℃, and the stamping time is 10s.
(2) And taking down the NC film, removing the base material of the photonic crystal film material, and cooling at room temperature to obtain the photonic crystal NC film.
Referring to fig. 4, a physical diagram of a photonic crystal NC film is shown, and it can be seen from the figure that the photonic crystal is located in the C/T line region.
Example 4
Preparation of antibody-containing Photonic Crystal type NC film
Cy 3-labeled goat anti-rabbit IgG and photonic crystal NC film prepared in example 3 (260 nm photonic crystal, PC for short) 260nm ) Constructing a photonic crystal NC film with biological functionality. The method comprises the following specific steps:
(1) 6mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 10mg/mL of N-hydroxysuccinimide (NHS) were prepared, and the volume ratio of the cross-linking reaction was 1:1 to obtain an EDC/NHS mixed solution for use.
(2) And (3) scribing the photonic crystal area by using EDC/NHS mixed solution through a scribing machine, and activating at room temperature for 15 min.
(3) After the strand was dried, antibody streaking was performed in situ on the activated photonic crystal region, with an antibody of 1mg/mL Cy 3-labeled goat anti-rabbit IgG, streaking speed of 2. Mu.L/cm, the petri dish was sealed, and incubated overnight in a refrigerator at 4 ℃. And then obtaining the functional photonic crystal NC film containing the biological antibody.
(4) And (3) placing the photonic crystal NC film containing the antibody prepared in the step (3) under a fluorescence microscope, selecting a filter with an excitation wave band of 512-552nm and an emission wave band of 565-615nm for observation and shooting fluorescent images.
(5) The photonic crystal NC film prepared in example 2 was taken and subjected to no coupling reaction, and Cy-3 labeled goat anti-rabbit IgG was directly added dropwise to the array, and a fluorescence image was captured.
Referring to FIG. 5, a schematic flow chart of the above steps with respect to a biofunctional photonic crystal type NC membrane (photonic crystal coupled Cy-3 labeled IgG antibody) is shown. As can be seen from the figure, since a large number of-COOH functional groups are exposed on the surface of the photonic crystal latex sphere, EDC/NHS is used as a coupling agent to form-NH in the antibody protein 2 And (3) reacting to form an amide bond, and fixing the biological material on the photonic crystal NC film.
In order to characterize the immobilization effect of the biofunctional photonic crystal type NC film, the coupling rate of antibody immobilization was calculated by the front-back variation of fluorescence intensity measured by a fluorescence microscope. FIG. 6 is a fluorescence microscope image of a photonic crystal NC film, wherein the left image represents a fluorescence imaging image of a photonic crystal uncoupled Cy-3 labeled IgG antibody in a direct dripping mode; the right panel represents a fluorescence imaging of the photonic crystal under the same parameters.
And calculating the fluorescence intensity value of each image by directly utilizing the self-contained software of the instrument by utilizing the fluorescence image shot by the confocal microscope, wherein the average fluorescence intensity value of the left image is 355, and the average fluorescence intensity value of the right image is 4044.
The test was repeated 20 times, and the coupling ratio of Cy-3 labeled IgG antibody was calculated to be 40-60% by the fluorescence intensity value obtained by the coupling method/direct dripping method, and the average antibody coupling efficiency was 52%.
Example 5
Preparation of aptamer-containing biophotonic crystal NC film
FITC aptamer and photonic crystal NC film (prepared by the method of example 3, 230nm photonic crystal, PC for short) 230nm ) The method comprises the following specific steps:
(1) 6mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 10mg/mL of N-hydroxysuccinimide (NHS) were prepared, and the volume ratio of the cross-linking reaction was 1:1 to obtain an EDC/NHS mixed solution for use.
(2) And (3) marking the photonic crystal area by using EDC/NHS mixed solution in a marking way by a marking machine, and activating at room temperature for 15 min.
(3) After drying, the activated photonic crystal region was streaked with 1mg/mL FITC aptamer in situ, the streaking rate was 2. Mu.L/cm, the petri dish was sealed, and incubated overnight at 4 ℃. And then, obtaining the functional photonic crystal NC film containing the biological aptamer.
The photonic crystal NC film containing the aptamer prepared by the embodiment is placed under a fluorescence microscope, a filter with an excitation wave band of 465-495nm and an emission wave band of 515-555nm is selected for observation and fluorescent image shooting.
Meanwhile, another photonic crystal NC film prepared in the embodiment 3 is taken, the coupling reaction treatment is not carried out, and the FITC aptamer is directly dripped on the lattice, so that a fluorescence image is shot.
And comparing the fluorescence intensity of the uncoupling photon crystal NC film with that of the aptamer-containing photon crystal NC film prepared in the embodiment, and calculating the average coupling rate of the aptamer fixation. After repeating 20 experiments, the average coupling rate of aptamer immobilization was calculated.
To characterize its immobilization effect, the rate of aptamer-immobilized coupling was calculated by the back and forth change in fluorescence intensity. Repeating the test for 20 times, and calculating the fluorescence intensity value obtained by a coupling method/a direct dripping method to obtain the fixed coupling rate range of the FITC aptamer, wherein the average coupling rate is about 80 percent.
Example 6
The fluorescence gain effect of the photonic crystal NC film containing the Cy 3-labeled IgG antibody is verified by the following method:
cy3 is often used as a fluorescent label to label antibodies or aptamers.
(1) Referring to example 4, PC was used 260 And simultaneously, taking a blank NC film as a comparison, respectively dripping 100ng/ml Cy 3-labeled IgG antibody into the blank NC film, wherein the dripping volume is 1 mu L, and obtaining the photonic crystal NC film containing the Cy 3-labeled IgG antibody.
(3) And after the liquid drops are completely dried, placing the pure NC film and the prepared photonic crystal NC film under a confocal fluorescence microscope to observe fluorescence, and shooting and counting fluorescence intensity values. After repeating the test 20 times, the average fluorescence intensity value was calculated.
Example 7
Establishing a rapid biological visualization detection standard curve of the photonic crystal NC film containing the FITC marked CK-MB detection antibody.
Taking an acute myocardial infarction CK-MB antigen-antibody system as an example, selecting a CK-MB antigen, a CK-MB capture antibody and a FITC labeled detection antibody, taking the fluorescence intensity of the target detection object CK-MB antigen (excitation wavelength: 488nm; emission wavelength: 490-530 nm) as a detection standard, and selecting a photon crystal NC film with the particle size of 230nm matched with FITC dye as an immobilization carrier (forbidden band range: 450-550 nm), wherein the specific steps are as follows:
(1) 6mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 30mg/mL of N-hydroxysuccinimide (NHS) were prepared, and the volume ratio of the cross-linking reaction was 1:1 to obtain an EDC/NHS mixed solution for use.
(2) And (3) marking the photonic crystal area by using EDC/NHS mixed solution through a marking method of a marking machine, and activating at room temperature for 15 min.
(3) After drying, carrying out in-situ antibody scribing on the activated photonic crystal region, wherein the antibody is a capture antibody of 1mg/mL CK-MB, and the C line of the scribing is used as a detection line; the goat IgG antibody streak T line with the antibody of 1mg/mL is used as a quality control line, the streaking speed is 2 mu L/cm, the culture dish is sealed, and the culture dish is placed in a refrigerator at 4 ℃ for incubation overnight.
(4) The CK-MB antigen standard sample (stock solution concentration is 100 mu g/mL) is diluted 10 times, 100 times and 10 times respectively 3 Multiple of 10 4 Multiple of 10 5 Multiple of 10 6 After doubling, CK-MB antigen standard samples (concentrations of 10. Mu.g/mL, 1. Mu.g/mL, 0.1. Mu.g/mL, 10 were obtained -2 μg/mL、10 -3 μg/mL、10 -4 mu.g/mL), 10. Mu.L of each CK-MB antigen standard sample was mixed with 2. Mu.L of FITC-labeled detection antibody (450. Mu.g/mL) and incubated for 10min at 37℃to obtain a mixed solution.
(5) And (3) dripping 10 mu L of the mixed solution in the step (4) to one end of the photonic crystal NC film, and performing recognition by capillary permeation chromatography of the NC film to a C/T line.
(6) And (3) placing the photonic crystal NC film which is identified in the step (5) under a confocal microscope, selecting 488nm excitation light for excitation, and observing and shooting fluorescent images.
Negative control group: a group of CK-MB antigen standard samples was set as a negative control group. Since interference of nonspecific adsorption cannot be excluded in the experiment, a control group of acute myocardial infarction CK-MB antigen was added as a background for each experiment. The preparation method of the control group is the same as that of the control group, except that the acute myocardial infarction CK-MB antigen is not coupled with FITC labeled detection antibody in the step (4).
Referring to FIG. 7, the photonic crystal NC membrane prepared in this example is used for identifying acute myocardial CK-MB antigen sandwich immunodetection (from left to right, the acute myocardial CK-MB antigen dilution gradients 10, 10 corresponding to the identified photonic crystal NC membrane respectively) 2 、10 3 、10 4 、10 5 、10 6 Fold and acute myocardial CK-MB antigen, initial antigen concentration of 100 μg/ml), the fluorescence intensity gradually increased with increasing CK-MB antigen concentration.
Test case
The tension and viscosity of photonic crystal ink with different proportions are tested, and the testing method is as follows:
measuring the tension of the photonic crystal ink by adopting a conventional surface tension tester; the viscosity of the photonic crystal ink was measured using a conventional viscometer, and the measurement was performed at normal temperature.
Referring to Table 1 (R is red photonic crystal corresponding to latex spheres with particle size of 260nm in Table 1), humectant and photonic crystal ink with different proportions are added, the surface tension of the photonic crystal ink is less than 35.5mN/m, and the average surface tension of the photonic crystal ink is more than 45mN/m.
TABLE 1 determination of surface tension value for Photonic Crystal ink formulation control
See Table 2 (wherein G isThe invention adds humectant and wetting agent, photon crystal ink with different proportion, the viscosity is less than 4 m.Pa.s at 20 DEG C -1 A viscosity at 25 ℃ of less than 3.5 mPa.s -1 。
TABLE 2 viscosity number determination for ink formulation control
| Sample name | viscosity/mPa.s at 20 DEG C -1 | viscosity/mPa.s at 25 DEG C -1 |
| 10％R+8％EG+1‰BYK | 1.7431 | 1.5425 |
| 10％R+8％EG+5‰BYK | 1.7280 | 1.5121 |
| 10％R+16％EG+1‰BYK | 3.7642 | 3.2564 |
| 10％R+16％EG+5‰BYK | 3.8454 | 3.3385 |
| 10％R+8％EG+1‰BYK | 1.9054 | 1.6604 |
| 20％G | 2.5151 | 2.2133 |
| 20％G+8％EG | 2.4689 | 2.1114 |
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The photonic crystal type NC film is characterized by comprising an NC film and a photonic crystal structure positioned in a photonic crystal area of the NC film, wherein the photonic crystal area comprises areas where a C line and a T line are positioned.
2. The photonic crystal NC film according to claim 1, wherein the photonic crystal structure is provided with C-lines and T-lines.
Preferably, the C-line and T-line include a biomaterial having a group capable of reacting with the photonic crystal thereon, and for example, the C-line and T-line are formed by connecting the biomaterial to the photonic crystal.
Preferably, the biomaterial is attached to the photonic crystal by chemical bonds.
Preferably, the biological material on the T-line is a biological material that does not bind to the substance to be detected.
Preferably, the biological material on the C-line is a material capable of specifically reacting with the target analyte.
Preferably, the specifically reactive material is selected from at least one of enzymes, DNA/RNA, antigens, antibodies, aptamers, biotin-streptavidin, protein receptors, and the like.
3. A method for producing the photonic crystal NC film according to claim 1 or 2, comprising the steps of:
and transferring the photonic crystal structure on the photonic crystal film to a photonic crystal region of the NC film to obtain the photonic crystal NC film.
4. The method for producing a photonic crystal NC film according to claim 3, wherein the transfer is performed by a conventional transfer method.
Preferably, the preparation method further comprises the steps of: a biological material is immobilized on the photonic crystal structure.
Preferably, before the immobilization of the biological material on the photonic crystal structure, the method further comprises the steps of: and activating the photonic crystal structure to expose a reactive group on the photonic crystal, wherein the reactive group is a group capable of reacting with biological materials.
5. The method of claim 4, wherein activating comprises coating an activator on the photonic crystal structure.
6. Use of a photonic crystal NC film according to claim 1 or 2 or prepared by the method according to any one of claims 3 to 5 in biological detection.
7. A method for detecting biomarkers using NC film of the photonic crystal type according to claim 1 or 2, characterized in that said method comprises the steps of: and enabling the sample to be detected to be in contact with the photonic crystal NC film through capillary chromatography.
8. The method of photonic crystal-based NC film detection biomarker according to claim 7, characterized in that the method comprises the steps of:
a) Measuring fluorescence values by contacting standard biomarker samples with different concentrations with the photonic crystal NC film through capillary chromatography;
b) And contacting the sample to be detected with the photonic crystal NC film through capillary chromatography, and detecting a fluorescence signal of a C/T line.
Preferably, in step b), before contacting the sample to be tested with the photonic crystal NC film by capillary chromatography, the method further comprises the steps of: mixing a sample to be tested with a detection antibody, wherein the detection antibody is connected with a receptor substance.
9. The method of photonic crystal-based NC film detection of biomarkers according to claim 8, wherein the acceptor species comprises at least one of nanocrystals and fluorescent dye-labeled molecules.
Preferably, the nanocrystalline is selected from at least one of silicon dioxide nanocrystalline, titanium dioxide nanocrystalline, zirconium oxide nanocrystalline, zinc oxide nanocrystalline, cadmium sulfide nanocrystalline, cadmium telluride nanocrystalline, gold nanoparticle, silver nanoparticle.
Preferably, the fluorescent dye labeling molecule is selected from at least one of Cy3, cy5, FITC, rhB, and the like.
10. A biological detection platform, characterized in that the biological detection platform comprises the photonic crystal NC film according to claim 1 or 2 or the photonic crystal NC film prepared by the method according to any one of claims 3 to 5.
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