A fluorescence gain type nc film and a preparation method and application thereof

Abstract

This invention belongs to the field of materials science, and specifically relates to a fluorescence-enhancing NC film, its preparation method, and its applications. The photonic crystal-type NC film comprises an NC film and a photonic crystal structure located in the photonic crystal region of the NC film. During detection on the NC film, when the detection antibody-target analyte-biomaterial form a sandwich structure on the photonic crystal, the distance between the receptor material connected to the detection antibody and the photonic crystal is reduced. Based on the original fluorescence of the receptor material, the gain effect of the photonic crystal can increase the fluorescence gain reported by the detection antibody by 10-100 times, lowering the detection limit by one to two orders of magnitude, thus enabling trace detection of low concentrations of the analyte.

Description

Technical Field

[0001] This invention belongs to the field of materials science, and in particular relates to a fluorescence-gaining NC film, its preparation method, and its application. Background Technology

[0002] Nitrocellulose filter membrane (NC membrane) is used as the carrier of the C / T line in colloidal gold test strips and is also the site where the immune reaction occurs. NC membrane is one of the most important consumables in biological experiments. Most current NC membranes identify the target by setting antibodies corresponding to the target on the C / T line and detecting the color change, but the sensitivity is relatively low.

[0003] Photonic crystals, due to their unique optical properties, hold significant research value in the field of highly sensitive detection of biomaterials such as low-concentration ions, DNA, proteins, and biological probes. By leveraging the inherent fluorescence enhancement and amplification properties of photonic crystals, the detection sensitivity of photonic crystal biosensors can be significantly improved, lowering the detection limit. However, current photonic crystals primarily rely on specific recognition of the analyte to form corresponding luminescent systems, such as resonant energy transfer recognition systems, for detection. These detection systems are complex, difficult to fabricate, and costly. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a photonic crystal type NC film, which includes an NC film and a photonic crystal structure located in the photonic crystal region of the NC film, wherein the photonic crystal region includes the region where the C-line and T-line are located.

[0005] According to an embodiment of the present invention, the photonic crystal structure is provided with C-lines and T-lines.

[0006] According to an embodiment of the present invention, the C-line and T-line comprise biomaterials having groups capable of reacting with the photonic crystal. For example, the C-line and T-line are formed by connecting the biomaterials to the photonic crystal.

[0007] According to an embodiment of the present invention, the biomaterial is chemically bonded to the photonic crystal.

[0008] According to an embodiment of the present invention, the biomaterial on the T-line is a biomaterial that does not bind to the substance to be detected.

[0009] According to an embodiment of the present invention, the biomaterial on line C is a material capable of specifically reacting with the target analyte.

[0010] According to an embodiment of the present invention, the material for the specific reaction is selected from at least one of enzymes, DNA / RNA, antigens, antibodies, aptamers, biotin-streptavidin, protein receptors, etc.

[0011] The present invention also provides a method for preparing the above-mentioned photonic crystal NC film, the method comprising the following steps:

[0012] The photonic crystal structure on the above-mentioned photonic crystal film is transferred to the photonic crystal region of the NC film to obtain the photonic crystal type NC film.

[0013] According to an embodiment of the present invention, the transfer is performed using a conventional transfer method.

[0014] According to an embodiment of the present invention, the preparation method further includes the following step: fixing biomaterials onto the photonic crystal structure.

[0015] According to an embodiment of the present invention, before immobilizing the biomaterial on the photonic crystal structure, the method further includes the following step: activating the photonic crystal structure to expose reactive groups on the photonic crystal, wherein the reactive groups are groups capable of reacting with the biomaterial.

[0016] According to an embodiment of the present invention, the activation includes coating the photonic crystal structure with an activator.

[0017] The present invention also provides an application of the above-described photonic crystal NC film or the photonic crystal NC film prepared by the above method in biological detection.

[0018] The present invention also provides a method for detecting biomarkers using the above-mentioned photonic crystal NC membrane, the method comprising the following steps: bringing the sample to be tested into capillary chromatographic contact with the photonic crystal NC membrane.

[0019] According to an embodiment of the present invention, the method includes the following steps:

[0020] a) The fluorescence value was measured by capillary chromatography contacting standard biomarker samples of different concentrations with the photonic crystal NC membrane;

[0021] b) The sample to be tested is brought into capillary chromatographic contact with the photonic crystal NC film, and the fluorescence signal of the C / T line is detected.

[0022] According to an embodiment of the present invention, in step b), before the sample to be tested is brought into capillary action chromatographic contact with the photonic crystal NC membrane, the following step is further included: mixing the sample to be tested with a detection antibody, wherein the detection antibody is attached with a receptor substance.

[0023] According to an embodiment of the present invention, the receptor material includes at least one of nanocrystals and fluorescent dye-labeled molecules.

[0024] According to an embodiment of the present invention, the nanocrystals are selected from at least one of silicon dioxide nanocrystals, titanium dioxide nanocrystals, zirconium oxide nanocrystals, zinc oxide nanocrystals, cadmium sulfide nanocrystals, cadmium telluride nanocrystals, gold quantum dots, and silver quantum dots.

[0025] 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.

[0026] The present invention also provides a biological detection platform, wherein the biological detection platform contains the above-described photonic crystal NC film or the photonic crystal NC film prepared by the above method.

[0027] Beneficial effects

[0028] 1. This invention regulates the viscosity and surface tension of photonic crystal (e.g., latex ball) ink using humectants and wetting agents. The average surface tension of the photonic crystal ink is less than 35.5 mN / m, and the viscosity at 20°C is less than 4 m·Pa·s. -1 The viscosity at 25°C is less than 3.5 mPa·s. -1 This effectively controls the spreading and wetting behavior of photonic crystal ink on the substrate surface, thereby enabling photonic crystals (such as latex balls) to be rapidly and orderly assembled on the substrate through a roll-to-roll printer and a coating machine to form a uniform photonic crystal film.

[0029] 2. The photonic crystal prepared in this invention has a fluorescence gain effect. The photonic crystal film is transferred onto the NC film by thermoimprinting to prepare a photonic crystal-type NC film. Due to the presence of the photonic crystal, when detection is performed on the NC film, when the detection antibody-target analyte-biomaterial form a sandwich structure on the photonic crystal, the distance between the receptor material connected to the detection antibody and the photonic crystal is shortened. On the basis of the original fluorescence of the receptor material, the gain effect of the photonic crystal can increase the fluorescence gain reported by the detection antibody by 10-100 times, reducing the detection limit by one to two orders of magnitude, and realizing trace detection of low concentrations of analytes.

[0030] 3. The biological platform constructed by this invention has universal applicability. It can immobilize biological materials (antibodies, aptamers, and peptides, etc.) through simple coupling reactions, and achieve visualized detection.

[0031] 4. The photonic crystal NC film preparation method of the present invention is simple, green and environmentally friendly, can be prepared on a large scale, and is easy to commercialize.

[0032] 5. The photonic crystal of the present invention can not only enhance the fluorescence of fluorescent markers, but also serve as a carrier for conjugating biological materials (antibodies, aptamers, and peptides, etc.), thus functioning as both a biological detection probe and a biological detection platform. Attached Figure Description

[0033] Figure 1 The photonic crystal thin film assembled from 260nm latex spheres in Example 1.

[0034] Figure 2 This is a partial scanning electron microscope (SEM) image of the photonic crystal thin film in Example 1.

[0035] Figure 3 The photonic crystal thin film assembled from 230nm latex spheres in Example 2.

[0036] Figure 4 This is a physical image of the photonic crystal NC film in Example 3.

[0037] Figure 5 This is a schematic diagram of the biofunctional photonic crystal NC membrane (photonic crystal coupled with Cy3-labeled IgG antibody) in Example 4.

[0038] Figure 6 The images shown are fluorescence micrographs of the photonic crystal NC film in Example 4. The left image represents the fluorescence imaging of the photonic crystal without Cy3-labeled IgG antibody, obtained by direct drop-on method; the right image represents the fluorescence imaging of the photonic crystal with Cy3-labeled IgG antibody.

[0039] Figure 7 The photonic crystal NC membrane prepared in Example 7 was used to identify different concentrations of the target analyte CK-MB antigen (the initial mass concentration of the target analyte CK-MB antigen was 100 μg / mL; from left to right in the figure, these represent the target analyte CK-MB antigen diluted to 10, 10, or 10 times its initial concentration, respectively). 2 10 3 10 4 10 5 10 6 The standard curve is (times). Detailed Implementation

[0040] [Photonic Crystal Film]

[0041] As described above, the present invention provides a photonic crystal film, the photonic crystal film comprising a substrate and a photonic crystal structure located on at least one side surface of the substrate.

[0042] According to an embodiment of the present invention, the photonic crystal structure is located on one or both surfaces of the substrate.

[0043] According to an embodiment of the present invention, the photonic crystal structure includes a lattice structure or a thin film structure, referred to as a photonic crystal lattice and a photonic crystal thin film, respectively.

[0044] According to an embodiment of the present invention, the lattice structure is, for example, a dot pattern, a line pattern, or a surface pattern, and is respectively referred to as a photonic crystal dot, a photonic crystal line, and a photonic crystal surface.

[0045] According to an embodiment of the present invention, the photonic crystal structure is formed on the surface of a substrate using photonic crystal ink. Specifically, the photonic crystal structure can be formed on the substrate surface using conventional spraying, printing, or coating methods. For example, the photonic crystal ink can be printed onto the substrate using a roller printer or coating machine to form the photonic crystal structure.

[0046] According to an embodiment of the present invention, the photonic crystal structure is hydrophilic.

[0047] According to an embodiment of the present invention, the photonic crystal structure is formed by the self-assembly of photonic crystals 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 and regular arrangement.

[0048] According to an embodiment of the present invention, the photonic crystal structure has a basic structure as follows: Figure 2 The scanning electron microscope image mentioned above.

[0049] According to an embodiment of the present invention, the substrate comprises a flexible material, preferably a flexible transparent material, selected from transparent plastics, such as PP plastic (Polypropylene), PS plastic (general purpose polystyrene), PVC plastic (Polyvinyl chloride) or PET plastic (Polyethylene terephthalate).

[0050] According to an embodiment of the present invention, the photonic crystal ink comprises a photonic crystal, a humectant, and a wetting agent.

[0051] According to an embodiment of the present invention, the photonic crystal in the photonic crystal ink is a photonic crystal with or without connecting groups.

[0052] Specifically, the linking group includes at least one of carboxyl, hydroxyl, mercapto, and amino groups, for example, the linking group is a carboxyl group.

[0053] According to an embodiment of the present invention, the photonic crystal is at least one of opal photonic crystal, inverse opal photonic crystal, and two-dimensional photonic crystal.

[0054] According to an embodiment of the present invention, the photonic crystal exists in the ink in the form of an emulsion.

[0055] According to an embodiment of the present invention, the photonic crystal exists in the ink in the form of latex spheres.

[0056] Preferably, the latex balls are monodisperse latex balls.

[0057] According to an embodiment of the present invention, the particle size of the monodisperse latex spheres is 150-320 nm, preferably 180-300 nm, and further, the particle size of the monodisperse latex spheres is 180-280 nm, exemplarily 180 nm, 190 nm, 200 nm, 205 nm, 215 nm, 220 nm, 230 nm, 250 nm, 260 nm, and 280 nm.

[0058] According to an embodiment of the present invention, the photonic crystal latex ball can be selected from at least one of photonic crystal-poly(methyl methacrylate-acrylic acid-styrene) latex balls, photonic crystal-silica microspheres, photonic crystal-polystyrene microspheres, etc., for example, photonic crystal-poly(methyl methacrylate-acrylic acid-styrene) latex balls.

[0059] According to an embodiment of the present invention, the surface of the monodisperse latex ball is equipped with carboxyl functional groups.

[0060] Preferably, the silica microspheres are carboxyl-modified silica microspheres, and the polystyrene microspheres are carboxyl-modified polystyrene microspheres.

[0061] According to an embodiment of the present invention, the photonic crystal ink has a photonic crystal mass concentration of 10-25%, preferably 10-20%, and further, the photonic crystal mass concentration is 14-18%, for example 10%, 12%, 14%, 16%, 18%, or 20%.

[0062] According to an embodiment of the present invention, the humectant may be selected from at least one of ethylene glycol, propylene glycol, glycerol, sorbitol, etc., for example, ethylene glycol.

[0063] According to an embodiment of the present invention, the mass concentration of the humectant in the photonic crystal ink is 8-15%, preferably 10-12%, and exemplary values ​​are 10%, 11%, and 12%.

[0064] According to an embodiment of the present invention, the wetting agent may be selected from at least one of BYK-3400 (polyether modified polydimethylsiloxane), Tween T-20, Triton X-100, etc., for example, BYK-3400.

[0065] According to an embodiment of the present invention, in the photonic crystal ink, the mass concentration of the wetting agent is 0.1-0.5‰, preferably 0.2-0.4‰, for example 0.5‰.

[0066] 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 wetting agent, wherein the solvent is selected from water, such as deionized water or ultrapure water.

[0067] As an example, the photonic crystal ink comprises monodisperse poly(methyl methacrylate-acrylic-styrene) latex balls, ethylene glycol, and BYK-3400.

[0068] According to an embodiment of the present invention, the average surface tension of the photonic crystal ink is less than 35.5 mN / m, preferably less than 25.5 mN / m. For example, the average surface tension of the photonic crystal ink is 35.158 mN / m, 23.658 mN / m, 20.892 mN / m, 26.086 mN / m, 21.492 mN / m, or 21.250 mN / m.

[0069] The average surface tension refers to the average value obtained after 5 tests under the same conditions.

[0070] According to an embodiment of the present invention, the viscosity of the photonic crystal ink at 20°C is less than 4 m·Pa·s. -1 Preferably, the viscosity of the photonic crystal ink at 20°C is less than 3 mPa·s. -1 For example, the viscosity of the photonic crystal ink at 20°C is 1.7431 mPa·s. -1 1.7280 m·Pa·s -1 3.7642 m·Pa·s -1 3.8454 m·Pa·s -1 1.9054 m·Pa·s -1 2.4689 mPa·s -1 .

[0071] According to an embodiment of the present invention, the viscosity of the photonic crystal ink at 25°C is less than 3.5 m·Pa·s. -1 Preferably, the viscosity of the photonic crystal ink at 25°C is less than 2.5 mPa·s. -1 For example, the viscosity of the photonic crystal ink at 25°C is 1.5425 mPa·s. -1 1.5121 m·Pa·s -1 3.2564 m·Pa·s -13.3385 m·Pa·s -1 1.6604 m·Pa·s -1 2.1114 m·Pa·s -1 .

[0072] [Preparation methods for photonic crystal films]

[0073] The present invention also provides a method for preparing the above-mentioned photonic crystal film, comprising the following steps:

[0074] S1. Configure photonic crystal ink;

[0075] S2. The photonic crystal ink forms the photonic crystal structure on at least one side of the substrate to obtain the photonic crystal film.

[0076] According to an embodiment of the present invention, the photonic crystal ink and the substrate have the definitions described above.

[0077] According to an embodiment of the present invention, in step S1, the preparation of photonic crystal ink includes the following steps: mixing photonic crystal, humectant, wetting agent and solvent.

[0078] According to an embodiment of the present invention, the photonic crystal, humectant, and wetting agent have the contents described above.

[0079] According to an embodiment of the present invention, in step S2, the photonic crystal structure is formed on at least one side surface of the substrate by spraying, printing, or coating. For example, the photonic crystal ink is printed onto at least one layer surface of the substrate using a roller printer or a coating machine.

[0080] Specifically, the photonic crystal structure has the definition described above.

[0081] [Photonic Crystal Type NC Film]

[0082] The present invention also provides a photonic crystal type NC film, wherein the photonic crystal type NC film comprises an NC film and a photonic crystal structure located in the photonic crystal region of the NC film, and the photonic crystal region includes the region where the C-line and T-line are located.

[0083] According to an embodiment of the present invention, the photonic crystal structure is provided with C-lines and T-lines.

[0084] According to an embodiment of the present invention, the photonic crystal region includes the region where the C-line and T-line are located. For example, the photonic crystal region includes a C-line region and a T-line region, wherein the width of the C-line region is greater than or equal to the designed width of the C-line, and the width of the T-line region is greater than or equal to the designed width of the T-line.

[0085] According to an embodiment of the present invention, the C-line refers to the testing line, and the T-line refers to the quality control line.

[0086] According to an embodiment of the present invention, the photonic crystal structure has the definition described above.

[0087] In this invention, the photonic crystal structure does not affect the original capillary action of the NC membrane, i.e., rapid chromatographic permeation, because the photonic crystal itself is a hydrophilic material.

[0088] According to an embodiment of the present invention, the width of the photonic crystal structure is 1mm-2mm, preferably 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.

[0089] According to an embodiment of the present invention, the C-line and T-line comprise biomaterials having groups capable of reacting with the photonic crystal. For example, the C-line and T-line are formed by connecting the biomaterials to the photonic crystal.

[0090] According to an embodiment of the present invention, the biomaterial contains a -NH2 functional group.

[0091] According to an embodiment of the present invention, the biomaterial is chemically bonded to the photonic crystal, for example, via amide bonds. Specifically, the biomaterial is bonded to the photonic crystal to form C-lines and T-lines.

[0092] For example, the biomaterial is attached to chemical bonds on the surface of a photonic crystal via a coupling reaction.

[0093] According to an embodiment of the present invention, the biomaterial forming the T-line is a biomaterial that does not bind to the substance to be detected, such as a goat anti-human IgG antibody.

[0094] According to an embodiment of the present invention, the biomaterial forming the C-line is a material capable of specifically reacting with the target analyte.

[0095] According to an embodiment of the present invention, the material for the specific reaction 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 material for the specific reaction is an antibody capable of specifically binding to the antigen, such as goat anti-mouse IgG.

[0096] [Preparation method of photonic crystal type NC film]

[0097] The present invention also provides a method for preparing the above-mentioned photonic crystal NC film, the method comprising the following steps:

[0098] The photonic crystal structure on the above-mentioned photonic crystal film is transferred to the photonic crystal region of the NC film to obtain the photonic crystal type NC film.

[0099] According to an embodiment of the present invention, the transfer is performed by a conventional transfer method, such as by heat embossing.

[0100] According to an embodiment of the present invention, the hot embossing method specifically refers to contacting the above-mentioned photonic crystal film and NC film together and placing them on a hot embossing machine for rapid transfer.

[0101] According to an embodiment of the present invention, the temperature of the hot embossing is 80°C to 120°C, and the hot embossing time is 5s to 30s.

[0102] Preferably, the temperature of the hot stamping is 100℃~120℃, and the hot stamping time is 5s~10s.

[0103] According to an embodiment of the present invention, the preparation method further includes the following step: fixing biomaterials onto the photonic crystal structure.

[0104] According to an embodiment of the present invention, before immobilizing the biomaterial on the photonic crystal structure, the method further includes the following step: activating the photonic crystal structure to expose reactive groups on the photonic crystal, preferably the reactive groups being groups capable of reacting with the biomaterial, such as carboxyl groups.

[0105] According to an embodiment of the present invention, the activation includes coating the photonic crystal structure with an activator, such as an EDC / NHS mixed solution.

[0106] As an example, the EDC / NHS mixed solution is first activated by scribing it onto the photonic crystal structure using a scribing machine, and then the biomaterial is scribed onto the activated photonic crystal structure to form a photonic crystal structure with the biomaterial immobilized.

[0107] According to an embodiment of the present invention, the scribing speed of the biomaterial is 1-3 μL / cm, for example, 1 μL / cm, 2 μL / cm or 3 μL / cm.

[0108] According to an embodiment of the present invention, after scribing the biomaterial onto the activated photonic crystal film, the process further includes a step of sealing and incubating overnight at a temperature of 1-10°C. Preferably, the incubation temperature is 1-5°C, for example, 2°C, 4°C, 5°C, 6°C, or 8°C.

[0109] Applications of photonic crystal-type NC films

[0110] An application of the above-mentioned photonic crystal NC film in biological detection, such as in the detection of biomarkers.

[0111] According to an embodiment of the present invention, the biomarker includes protein and / or nucleic acid fragments, such as those used to detect four inflammatory markers (CRP, SAA, PCT, IL-6), six cardiac markers (AST, CK, CKMB, LDH, CTNI), and COVID-19.

[0112] According to an embodiment of the present invention, the detection is a point-of-care test, also known as POCT (point-of-care testing).

[0113] [Methods for detecting photonic crystal-type NC films]

[0114] The present invention also provides a method for detecting biomarkers using the above-mentioned photonic crystal NC membrane, the method comprising the following steps: bringing the sample to be tested into capillary chromatographic contact with the photonic crystal NC membrane.

[0115] According to an embodiment of the present invention, the method includes the following steps:

[0116] a) The fluorescence value was measured by capillary chromatography contacting standard biomarker samples of different concentrations with the photonic crystal NC membrane;

[0117] b) The sample to be tested is brought into capillary chromatographic contact with the photonic crystal NC film, and the fluorescence signal of the C / T line is detected.

[0118] According to an embodiment of the present invention, before the sample to be tested is brought into capillary action chromatography contact with the photonic crystal NC membrane, the following step is further included: mixing the sample to be tested with a detection antibody, wherein the detection antibody is attached with a receptor substance.

[0119] Preferably, the detection antibody and biomaterial can form a sandwich structure with the biomarker.

[0120] According to an embodiment of the present invention, the detection antibody is attached to a receptor substance, and the receptor substance generates fluorescence gain when it is close to the photonic crystal, thereby amplifying the fluorescence signal of the receptor substance.

[0121] According to an embodiment of the present invention, the receptor material includes at least one of nanocrystals and fluorescent dye-labeled molecules.

[0122] Preferably, the semiconductor nanocrystals are selected from at least one of silicon dioxide nanocrystals, titanium dioxide nanocrystals, zirconium oxide nanocrystals, zinc oxide nanocrystals, cadmium sulfide nanocrystals, cadmium telluride nanocrystals, gold nanodots, and silver nanodots.

[0123] Preferably, the fluorescent dye labeling molecule is selected from at least one of Cy3, Cy5, FITC, RhB, etc.

[0124] For example, the detection antibody is Cy3-labeled goat anti-rabbit IgG, Cy3-labeled nanobody primary antibody, or Cy5-labeled nanobody secondary antibody.

[0125] [Biodetection Platform]

[0126] The present invention also provides a biological detection platform containing the above-mentioned photonic crystal NC film, wherein the biological detection platform is, for example, a reagent kit.

[0127] In this invention, the photonic crystal NC film comes into capillary chromatographic contact with the target analyte, and the corresponding fluorescence signal is captured by fluorescence detection. The fluorescence detection gain can be 10-100 times that of the original. Preferably, the target analyte is labeled with a fluorescent molecule.

[0128] The following detailed description, in conjunction with specific embodiments, illustrates the general formula compounds of the present invention, their preparation methods, and applications in further detail. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0129] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0130] Example 1

[0131] The steps for preparing red photonic crystal industrial-grade printing ink are as follows:

[0132] (1) Add 10.00 mmol of methyl methacrylate, 13.89 mmol of acrylic acid and 182.60 mmol of styrene to 100 mL of water, then dissolve sodium dodecylbenzenesulfonate (not exceeding 0.011 mmol, its concentration is lower than the critical micelle concentration) and ammonium bicarbonate 6.30 mmol of buffer to obtain a reaction solution. Keep the reaction solution at 70 °C for half an hour, then add 2.12 mmol of ammonium persulfate aqueous solution, and polymerize at 80 °C for 10 h with continuous stirring to obtain monodisperse latex balls that can be used directly without purification.

[0133] The particle size of latex balls was controlled by adjusting the amount of styrene (182.6 mmol; 121.73 mmol; 81.15 mmol; 54.1 mmol; 36.1 mmol; 24.04 mmol), and latex balls with particle sizes of 300 nm, 280 nm, 260 nm, 220 nm, 215 nm and 180 nm were prepared.

[0134] (2) Taking the latex balls with a particle size of 260nm prepared in step (1) as an example (the assembled photonic crystal structure is red), the photonic crystal stock solution (i.e., the aqueous solution of the latex balls after centrifugation and washing) containing the latex balls with a particle size of 260nm is diluted to a concentration of 12wt%. The humectant ethylene glycol (mass fraction of 10%) and the wetting agent BYK-3400 (mass fraction of 0.5‰) are added to the diluted photonic crystal solution to prepare the photonic crystal ink.

[0135] (3) PET film is selected as the substrate. Photonic crystal ink is printed onto the PET film using a roller printing machine. The photonic crystal in the 260nm latex ball self-assembles on the surface of the PET film to form a photonic crystal film, which is then dried and stored.

[0136] See Figure 1 As shown, this is a photonic crystal film formed by assembling 260nm latex spheres on a PET film in this embodiment. The photonic crystal film is red.

[0137] See Figure 2 The image shown is a partial scanning electron microscope (SEM) image of the photonic crystal (260 nm particle size) thin film in this embodiment. Figure 2 As can be seen, monodisperse polystyrene latex microspheres self-assemble to form a periodically and orderly arranged photonic crystal morphology.

[0138] Example 2

[0139] The steps for preparing green photonic crystal industrial-grade printing ink are as follows:

[0140] (1) Add 10.00 mmol of methyl methacrylate, 13.89 mmol of acrylic acid and 65.23 mmol of styrene to 100 mL of water, then dissolve sodium dodecylbenzenesulfonate (not exceeding 0.011 mmol, its concentration is lower than the critical micelle concentration) and ammonium bicarbonate 6.30 mmol of buffer to obtain a reaction solution. Keep the reaction solution at 70 °C for half an hour, then add 2.12 mmol of ammonium persulfate aqueous solution, and polymerize at 80 °C for 10 h with continuous stirring to obtain monodisperse latex balls that can be used directly without purification.

[0141] (2) In this embodiment, taking 230nm latex balls (assembled into a photonic crystal structure with a green color) as an example, the photonic crystal stock solution (i.e., the aqueous solution of the latex balls after centrifugation and washing) containing 230nm latex balls is diluted to a concentration of 8wt%. Ethylene glycol (mass fraction of 8%) and BYK-3400 (mass fraction of 0.3‰) are added to the diluted photonic crystal solution to prepare photonic crystal ink.

[0142] (3) PET film is selected as the substrate. Photonic crystal ink is printed onto the substrate PET film by a coating machine. The photonic crystal in the 230nm latex ball self-assembles on the surface of the PET film to form a photonic crystal film. After drying, it is stored.

[0143] See Figure 3 As shown, 230nm latex spheres are assembled on a PET film to form a photonic crystal film, which appears green.

[0144] Example 3

[0145] A photonic crystal-type NC membrane was prepared, in which the photonic crystal could be well printed onto the NC membrane while maintaining the good permeation chromatography effect of the original NC membrane.

[0146] (1) Take the photonic crystal thin film material with a width of 1 mm prepared in Example 1, and put the photonic crystal thin film in the thin film material into contact with the NC film surface. Place it under a hot press machine for hot pressing. The photonic crystal on the photonic crystal thin film is transferred to the photonic crystal area on the NC film by hot pressing. This area is located at the C / T line position. The hot pressing temperature is 100℃ and the pressing time is 10s.

[0147] (2) Remove the NC film, peel off the substrate of the photonic crystal thin film material, and cool at room temperature to obtain the photonic crystal type NC film.

[0148] See Figure 4 The image shown is a physical diagram of a photonic crystal-type NC film. As can be seen from the image, the photonic crystal is located in the C / T line region.

[0149] Example 4

[0150] Preparation of antibody-containing photonic crystal NC membrane

[0151] Cy3-labeled goat anti-rabbit IgG and the photonic crystal NC film prepared in Example 3 (using a 260nm photonic crystal, abbreviated as PC) were selected. 260nm Construct a biofunctional photonic crystal NC film. The specific steps are as follows:

[0152] (1) Prepare 6 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction is carried out at a volume ratio of 1:1 to obtain a mixed solution of EDC / NHS. Prepare and use immediately.

[0153] (2) Use the EDC / NHS mixed solution to scribble lines on the photonic crystal region using a scribing machine, and activate it for 15 minutes at room temperature.

[0154] (3) After the lines have dried, antibody lines are run in situ onto the activated photonic crystal region. The antibody is 1 mg / mL Lcy3-labeled goat anti-rabbit IgG. The running speed is 2 μL / cm. The culture dish is sealed and incubated overnight at 4°C. Subsequently, a functional photonic crystal NC film containing biological antibodies is obtained.

[0155] (4) Place the antibody-containing photonic crystal NC film prepared in step 3 under a fluorescence microscope, select a filter with an excitation band of 512-552nm and an emission band of 565-615nm for observation and capture fluorescence images.

[0156] (5) Take another photonic crystal NC film prepared in Example 2 and do not perform coupling reaction treatment. Add Cy-3 labeled goat anti-rabbit IgG directly to the matrix and take a fluorescence image.

[0157] See Figure 5 The diagram shows a flowchart illustrating the steps described above for the biofunctional photonic crystal NC membrane (photonic crystal coupled with Cy-3-labeled IgG antibody). As can be seen from the diagram, the surface of the photonic crystal latex spheres exposes a large number of -COOH functional groups. Using EDC / NHS as a coupling agent, these groups react with the -NH2 groups in the antibody protein to form amide bonds, thus immobilizing the biomaterial on the photonic crystal NC membrane.

[0158] To characterize the immobilization effect of the biofunctional photonic crystal NC membrane, the antibody immobilization coupling rate was calculated by measuring the change in fluorescence intensity before and after fluorescence microscopy. Figure 6 The images are fluorescence micrographs of a photonic crystal NC membrane. The left image represents the fluorescence imaging of the photonic crystal without being coupled with Cy-3-labeled IgG antibody, obtained by direct drop-on method; the right image represents the fluorescence imaging of the photonic crystal under the same parameters.

[0159] Fluorescence images were captured using a confocal microscope. The fluorescence intensity value of each image was calculated directly using the instrument's built-in software. The average fluorescence intensity value of the left image was 355, and the average fluorescence intensity value of the right image was 4044.

[0160] The experiment was repeated 20 times. The conjugation rate of Cy-3 labeled IgG antibody was calculated to be 40-60% based on the fluorescence intensity values ​​obtained by the conjugation method / direct drop method, with an average antibody conjugation efficiency of 52%.

[0161] Example 5

[0162] Preparation of biophotonic crystal NC films containing aptamers

[0163] The FITC aptamer and photonic crystal-type NC film were selected (prepared according to the method in Example 3, using a 230nm photonic crystal, abbreviated as PC). 230nm The specific steps are as follows:

[0164] (1) Prepare 6 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 10 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction is carried out at a volume ratio of 1:1 to obtain a mixed solution of EDC / NHS. Prepare and use immediately.

[0165] (2) Use the EDC / NHS mixed solution to scribble lines on the photonic crystal region using a scribing machine, and activate it at room temperature for 15 minutes.

[0166] (3) After drying, aptamers were streaked in situ onto the activated photonic crystal region. The antibody was 1 mg / mL FITC aptamer, and the streaking speed was 2 μL / cm. The culture dish was sealed and incubated overnight at 4°C. Subsequently, a functional photonic crystal NC film containing biological aptamers was obtained.

[0167] The NC film containing the aptamer prepared in this embodiment was placed under a fluorescence microscope, and a filter with an excitation band of 465-495nm and an emission band of 515-555nm was selected for observation and fluorescence image capture.

[0168] Meanwhile, another photonic crystal NC film prepared in Example 3 was used without coupling reaction treatment. FITC aptamers were directly dropped onto the lattice and fluorescence images were captured.

[0169] The fluorescence intensity of the uncoupled photonic crystal NC film and the aptamer-containing photonic crystal NC film prepared in this embodiment were compared to calculate the average coupling rate of aptamer fixation. After repeating the experiment 20 times, the average coupling rate of aptamer fixation was calculated.

[0170] To characterize its immobilization effect, the coupling rate of aptamer immobilization was calculated by measuring changes in fluorescence intensity before and after the process. The experiment was repeated 20 times, and the coupling rate of the FITC aptamer immobilization was calculated to be between 70% and 80% based on the fluorescence intensity values ​​obtained using the coupling method / direct drop method, with an average coupling rate of approximately 80%.

[0171] Example 6

[0172] The fluorescence gain effect of a photonic crystal NC membrane containing Cy3-labeled IgG antibody was verified using the following method:

[0173] Cy3 is often used as a fluorescent marker to label antibodies or aptamers.

[0174] (1) Referring to Example 4, PC is selected. 260A photonic crystal-type NC film was prepared for the photonic crystal. At the same time, a blank NC film was taken as a control. 100 ng / ml Cy3-labeled IgG antibody was dropped onto the film, and the dropping volume was 1 μL. The photonic crystal-type NC film containing Cy3-labeled IgG antibody prepared in this embodiment was obtained.

[0175] (3) After the droplets have dried completely, place the above-mentioned pure NC film and the prepared photonic crystal NC film under a confocal fluorescence microscope to observe the fluorescence, take pictures and count the fluorescence intensity values. After repeating the experiment 20 times, the average fluorescence intensity value is calculated.

[0176] Example 7

[0177] Establishment of a rapid bio-visualization detection standard curve for a photonic crystal NC membrane containing a FITC-labeled CK-MB detection antibody.

[0178] Taking the CK-MB antigen-antibody system for acute myocardial infarction as an example, CK-MB antigen, CK-MB capture antibody, and FITC-labeled detection antibody are selected. Since the target analyte CK-MB antigen is actually detected by the fluorescence intensity of the FITC-labeled detection antibody (excitation wavelength: 488nm; emission wavelength: 490-530nm), a 230nm photonic crystal NC film (bandgap range: 450-550nm) matching the FITC dye is selected as the immobilization carrier. The specific steps are as follows:

[0179] (1) Prepare 6 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 30 mg / mL of N-hydroxysuccinimide (NHS). The cross-linking reaction uses a volume ratio of 1:1 to obtain an EDC / NHS mixed solution, which should be prepared and used immediately.

[0180] (2) Use the EDC / NHS mixed solution to scribble lines on the photonic crystal region using a scribing machine, and activate it at room temperature for 15 minutes.

[0181] (3) After drying, antibody lines were run in situ on the activated photonic crystal region. The antibody was a capture antibody of 1 mg / mL CK-MB, and the C line was used as the detection line. The antibody was a goat IgG antibody of 1 mg / mL, and the T line was used as the quality control line. The running speed was 2 μL / cm. The culture dish was sealed and incubated overnight at 4°C.

[0182] (4) Dilute the CK-MB antigen standard sample (original solution concentration 100 μg / mL) 10 times, 100 times, and 10 times respectively. 3 times, 10 4 times, 10 5 times, 10 6After multiplication, CK-MB antigen standard samples were obtained (concentrations of 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 10 μg / mL, respectively). -2 μg / mL, 10 -3 μg / mL, 10 -4 Take 10 μL of each CK-MB antigen standard sample and 2 μL of FITC-labeled detection antibody (450 μg / mL), mix and incubate for 10 min at 37℃ to obtain a mixed solution.

[0183] (5) Take 10 μL of the mixed solution in step (4) and drop it onto one end of the photonic crystal NC membrane. The solution is then permeated through the capillary action of the NC membrane to the C / T line for identification.

[0184] (6) Place the photonic crystal NC film identified in step (5) under a confocal microscope, select 488nm excitation light for excitation, observe and capture fluorescence images.

[0185] Negative control group: A set of CK-MB antigen standard samples was set up as a negative control group. Because the interference of non-specific adsorption cannot be eliminated in the experiment, an acute myocardial infarction CK-MB antigen control group needs to be added as background for each experiment. The preparation method of the control group is the same as above, except that the acute myocardial infarction CK-MB antigen is not coupled with FITC-labeled detection antibody in step (4).

[0186] See Figure 7 As shown, this embodiment demonstrates a sandwich immunoassay using a photonic crystal NC membrane to recognize acute myocardial infarction (CK-MB) antigen (from left to right: CK-MB antigen dilution gradients of 10, 10, and 10 corresponding to the recognized photonic crystal NC membrane). 2 10 3 10 4 10 5 10 6 The standard curves for CK-MB antigen (initial antigen concentration of 100 μg / ml) were obtained. As shown in the figure, the fluorescence intensity gradually increased with the increase of CK-MB antigen concentration.

[0187] Test case

[0188] The surface tension and viscosity of photonic crystal inks with different formulations were tested using the following methods:

[0189] The surface tension of the photonic crystal ink was measured using a conventional surface tension meter; the viscosity of the photonic crystal ink was measured using a conventional viscosity meter. The measurements were performed at room temperature.

[0190] Referring to Table 1 (in Table 1, R is the red photonic crystal corresponding to latex spheres with a particle size of 260 nm), the photonic crystal inks of this invention with added humectants and wetting agents in different proportions have a surface tension of less than 35.5 mN / m, while the inks containing only photonic crystals have an average surface tension of greater than 45 mN / m.

[0191] Table 1. Measurement of surface tension values ​​due to photonic crystal ink ratio control.

[0192]

[0193] Referring to Table 2 (where G represents the green photonic crystal corresponding to latex spheres with a particle size of 230 nm), the photonic crystal inks of this invention, containing humectants and wetting agents in different proportions, have a viscosity of less than 4 mPa·s at 20°C. -1 The viscosity at 25°C is less than 3.5 mPa·s. -1 .

[0194] Table 2. Viscosity values ​​determined by ink mixing ratio.

[0195] 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

[0196] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A photonic crystal type NC film, characterized in that, The photonic crystal-type NC film includes an NC film and a photonic crystal structure located in the photonic crystal region of the NC film, wherein the photonic crystal region includes the region where the C-line and T-line are located; The photonic crystal structure is formed on the surface of a substrate by photonic crystal ink; the photonic crystal structure is formed on the surface of the substrate by spraying or printing. The photonic crystal ink comprises a photonic crystal, a humectant, and a wetting agent; In the photonic crystal ink, the mass concentration of the photonic crystal is 10-25%, the mass concentration of the humectant is 8-15%, and the mass concentration of the wetting agent is 0.1-0.5‰. The photonic crystal is an opal photonic crystal, and the photonic crystal in the photonic crystal ink is a carboxyl-containing photonic crystal. In the ink, the photonic crystal exists in the form of latex spheres; the latex spheres are monodisperse latex spheres; the particle size of the monodisperse latex spheres is 150-320 nm.

2. The photonic crystal NC film according to claim 1, characterized in that, The photonic crystal structure has C-lines and T-lines.

3. The photonic crystal NC film according to claim 2, characterized in that, The C-line and T-line comprise biomaterials having functional groups capable of reacting with photonic crystals.

4. The photonic crystal NC film according to claim 3, characterized in that, The C-line and T-line are formed on the photonic crystal by connecting them with biomaterials.

5. The photonic crystal NC film according to claim 4, characterized in that, The biomaterial is attached to the photonic crystal by chemical bonds.

6. The photonic crystal NC film according to claim 4, characterized in that, The biomaterial on the T-line is a biomaterial that does not bind to the substance to be detected.

7. The photonic crystal NC film according to claim 4, characterized in that, The biomaterial on line C is a material that can specifically react with the target analyte.

8. The photonic crystal NC film according to claim 7, characterized in that, The material for the specific reaction is selected from at least one of enzymes, DNA / RNA, antigens, antibodies, aptamers, biotin-streptavidin, and protein receptors.

9. A method for preparing a photonic crystal-type NC film according to any one of claims 1-8, characterized in that, The preparation method includes the following steps: First, a photonic crystal film is prepared, comprising a substrate and a photonic crystal structure located on at least one surface of the substrate. The photonic crystal structure is formed on at least one surface of the substrate by photonic crystal ink, which is applied by spraying or printing to form the photonic crystal structure on the substrate surface. The photonic crystal ink contains a photonic crystal, a humectant, and a wetting agent. In the photonic crystal ink, the mass concentration of the photonic crystal is 10-25%, the mass concentration of the humectant is 8-15%, and the mass concentration of the wetting agent is 0.1-0.5‰. Then, the photonic crystal structure on the above photonic crystal film is transferred to the photonic crystal region of the NC film to obtain the photonic crystal type NC film; The photonic crystal is an opal photonic crystal, and the photonic crystal in the photonic crystal ink is a carboxyl-containing photonic crystal. In the ink, the photonic crystal exists in the form of latex spheres; the latex spheres are monodisperse latex spheres; the particle size of the monodisperse latex spheres is 150-320 nm.

10. The method for preparing a photonic crystal-type NC film according to claim 9, characterized in that, The transfer is performed using conventional transfer methods.

11. The method for preparing a photonic crystal-type NC film according to claim 9, characterized in that, The preparation method further includes the following step: fixing biomaterials onto the photonic crystal structure.

12. The method for preparing a photonic crystal-type NC film according to claim 11, characterized in that, Before immobilizing the biomaterial onto the photonic crystal structure, the method further includes the following steps: activating the photonic crystal structure to expose reactive groups on the photonic crystal, wherein the reactive groups are groups capable of reacting with the biomaterial.

13. The method for preparing a photonic crystal-type NC film according to claim 12, characterized in that, The activation includes coating the photonic crystal structure with an activator.

14. The application of a photonic crystal NC film according to any one of claims 1-8 or a photonic crystal NC film prepared by the method according to any one of claims 10-13 in biological detection.

15. A method for detecting biomarkers using a photonic crystal-type NC membrane according to any one of claims 1-8, characterized in that, The method includes the following steps: bringing the sample to be tested into capillary contact with the photonic crystal NC film.

16. The method for detecting biomarkers using a photonic crystal NC membrane according to claim 15, characterized in that, The method includes the following steps: a) The fluorescence value was measured by capillary chromatography contacting standard biomarker samples of different concentrations with the photonic crystal NC membrane; b) The sample to be tested is brought into capillary chromatographic contact with the photonic crystal NC film, and the fluorescence signal of the C / T line is detected.

17. The method for detecting biomarkers using a photonic crystal NC membrane according to claim 16, characterized in that, In step b), before the sample to be tested is brought into contact with the photonic crystal NC membrane by capillary action chromatography, the following step is also included: mixing the sample to be tested with a detection antibody, wherein the detection antibody is attached with a receptor substance.

18. The method for detecting biomarkers using a photonic crystal NC membrane according to claim 17, characterized in that, The receptor material includes at least one of nanocrystals and fluorescent dye-labeled molecules.

19. The method for detecting biomarkers using a photonic crystal NC membrane according to claim 18, characterized in that, The nanocrystals are selected from at least one of silicon dioxide nanocrystals, titanium dioxide nanocrystals, zirconium oxide nanocrystals, zinc oxide nanocrystals, cadmium sulfide nanocrystals, cadmium telluride nanocrystals, gold nanodots, and silver nanodots.

20. The method for detecting biomarkers using a photonic crystal NC membrane according to claim 18, characterized in that, The fluorescent dye labeling molecule is selected from at least one of Cy3, Cy5, FITC, and RhB.

21. A biological detection platform, characterized in that, The biodetection platform contains the photonic crystal NC film according to any one of claims 1-8 or the photonic crystal NC film prepared by the method according to any one of claims 10-13.

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