Universal functional coating material and preparation method thereof

Abstract

The invention relates to an industrial method, in particular to a method for preparing a universal functional protein nano-film on the surface of a base material, which comprises the following steps: (1) mixing a protein solution, an optional functional monomer and a photoinitiator solution to prepare a mixed solution; (2) soaking a base material in the mixed solution; and (3) irradiating with ultraviolet rays to obtain the protein nano-film on the surface of the base material. According to the universal method for continuously preparing the functional protein nano-film on the surface of the base material, rapid self-assembly aggregation and monomer polymerization of the protein can be carried out at the same time under ultraviolet induction, and the protein-functional polymer two-dimensional nano-film prepared on the surface of the base material is compact, low in roughness, high in transparency and good in application prospect. The biocompatibility is good.

Description

Technical Field

[0001] The present invention relates to the fields of surface modification technology and supramolecular self-assembly, and particularly to a method with industrial continuous production capacity for preparing protein nanofilms on the surface of a substrate. Background Art

[0002] In recent years, bio-inspired surface modification technologies have gradually become a hot topic in scientific research. In 2007, American scientists first reported the application of the polydopamine system in surface modification; subsequently in 2013, Australian scientists proposed the tannic acid-iron complex system. These two methods have been widely used in the field of surface modification. However, these methods also have some inevitable deficiencies. First, the polydopamine system and the tannic acid system have caused controversy due to their colors; more importantly, the preparation process of the polydopamine system is relatively complex, and the surface of the coating is rough, especially its stability is poor under alkaline conditions. In addition, the preparation time of both methods requires several hours or longer, which is time-consuming, and the raw materials required in the industrial production process are numerous, and the operation is cumbersome, restricting the feasibility of large-scale production.

[0003] Based on traditional surface modification methods, the importance of surface functionalization technology in industry and daily life has become increasingly prominent. Surface functionalization can not only effectively prevent the invasion of external pollution to materials, but also endow materials with new physical and chemical properties, greatly broadening their application scope. Currently, the most common surface functionalization methods usually include: first, the surface of the substrate is preliminarily treated by classical surface modification technologies (such as polydopamine modification), then active sites are fixed on the surface of the substrate through chemical modification, and finally the coupling or polymerization reaction of functional substances is initiated. However, this process has complex steps and is time-consuming, and has high requirements for energy consumption and reaction conditions (such as deoxygenation and dehydration), and there are still significant limitations in large-scale production.

[0004] In view of this, the present invention proposes an innovative method to address the challenges faced by the existing technologies. Summary of the Invention

[0005] To solve the existing technical problems, the present invention provides a method for preparing a protein nanofilm on the surface of a substrate. This method is simple to implement and has a rapid reaction, and is suitable for large-scale production. The present invention also relates to the protein nanofilm obtained by the above method.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a method for preparing a protein nanofilm on the surface of a substrate, comprising the following steps:

[0008] (1) Mix a protein solution, an optional functional monomer solution, and a photoinitiator solution to obtain a mixed solution;

[0009] (2) Immerse a substrate in the mixed solution;

[0010] (3) Irradiate with ultraviolet light to obtain a protein nanofilm on the surface of the substrate.

[0011] Preferably or optionally, the protein is selected from any one or more of proteins of plant origin and proteins of animal origin.

[0012] Preferably or optionally, the plant origin is:

[0013] Table 1 Plant origin

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] Preferably or optionally, the animal origin is:

[0027] Table 2 Animal origin

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] Preferably or optionally, the protein is selected from any one of lysozyme, bovine serum albumin, ovalbumin, lactoferrin, bovine fibrinogen, zein, collagen, chymotrypsin, mucin, hemoglobin, insulin, porcine chymotrypsin, concanavalin A, β-lactoglobulin, pepsin, and laccase.

[0037] Preferably or optionally, the concentration of the protein solution is 1 - 20 mg / mL, such as 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, and any value within the range composed of any two of the above values.

[0038] Preferably or optionally, the photoinitiator is selected from any one or more of free radical photoinitiators, cationic photoinitiators, and hybrid photoinitiators.

[0039] Preferably or optionally, the free radical photoinitiator is selected from any one or more of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, benzyl alcohol ethyl ether, acetophenone, benzyl alcohol, cyclopentenone, 2-hydroxy-2-methylpropiophenone, vinyldibenzylamine, isophorone, 4,4'-dichlorobenzophenone, triphenylmethyl peroxide, photoinitiator-184, triphenyl phosphate, 2,2-dimethyl-2-phenylpropene, epichlorohydrin, dicyano-2,4,6-trimethylbenzene, 1-hydroxycyclohexyl phenyl ketone, photoinitiator-819, 1,4-dichlorobenzophenone, 2-methyl-4-phenyl-2-butene.

[0040] Preferably or optionally, the cationic photoinitiator is selected from any one or more of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, toluenesulfonate, 1,2-diphenyliodonium, phenylphosphoryl chloride, tetrakis(phenyl)iodonium salt, triphenylsulfonic acid, 2,4,6-trimethylphenylbenzenesulfonyl chloride, 1,2,3-triphenylcyclohexane, 4-methylphenylmethane, methylstyrene, hydroxyethylphenylsilane, vinyl epoxy resin, 2-phenylphenyl triphenyl sulfate, 2-phenylphenyl triphenyl borate.

[0041] Preferably or optionally, the hybrid photoinitiator is selected from any one or more of the combination of benzyl alcohol ether and cyclopentenone, the combination of 2-hydroxy-2-methylpropiophenone and acetophenone, the combination of 2,4,6-trimethylphenylbenzenesulfonyl chloride and cyclopentenone, the combination of triphenylsulfate chloride and acetophenone, the combination of acetophenone and 2,2-dimethyl-2-phenylpropene, the combination of 1,4-dichloroacetophenone and acetophenone, the combination of acetophenone and benzyl alcohol ether, the combination of acetophenone and triphenyl phosphate, and the combination of acetophenone and 1,2-diphenyliodonium.

[0042] Preferably or optionally, the photoinitiator is selected from any one or more of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and (2,4,6-trimethylbenzoyl)diphenylphosphine oxide.

[0043] Preferably or optionally, the concentration of the photoinitiator solution is 1-20 mg / mL, such as 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, and any value within the range composed of any two of the above values.

[0044] Preferably or optionally, the functional monomer is selected from any one or more of olefin monomers, acrylic monomers, ester monomers, alcohol monomers, aldehyde monomers, ketone monomers, nitrogen-containing monomers, fluorine-containing monomers, aromatic olefin monomers, silane monomers, thioether monomers, silicon-containing monomers, cyclic monomers, zwitterionic monomers, carboxylic acid monomers, quaternary ammonium cations, quaternary phosphonium cations, pyridinium ions, imidazolium ions, sulfonate anions, carboxylate anions, and phosphate anions.

[0045] Preferably or optionally, the olefin monomers are selected from any one or more of ethylene, propylene, butadiene, styrene, vinylbenzene, and methylstyrene.

[0046] Preferably or optionally, the acrylic monomers are selected from any one or more of acrylic acid, methyl methacrylate, ethyl acrylate, butyl acrylate, 2-hydroxyethyl acrylate, isooctyl acrylate, and acrylic epoxy vinyl ester.

[0047] Preferably or optionally, the ester monomers are selected from any one or more of vinyl ether, methyl methacrylate, vinyl ethyl ether, phenyl methacrylate, and vinyl acetate.

[0048] Preferably or optionally, the alcohol monomers are selected from any one or more of vinyl alcohol, hydroxyethylene, and dimethylol methacrylate.

[0049] Preferably or optionally, the aldehyde monomer is selected from methacrolein and / or acrolein.

[0050] Preferably or optionally, the ketone monomer is selected from methyl vinyl ketone and / or cyclopentenone.

[0051] Preferably or optionally, the nitrogen-containing monomer is selected from any one or more of vinylimidazole, vinylpyridine, pyrrolidone, and picolinic acid.

[0052] Preferably or optionally, the fluorine-containing monomer is selected from any one or more of tetrafluoroethylene, hexafluoropropylene, and tetrafluoroethylene.

[0053] Preferably or optionally, the aromatic olefin monomer is selected from any one or more of styrene, vinylbenzene, and methylstyrene.

[0054] Preferably or optionally, the silane monomer is selected from vinyltrichlorosilane and / or methyltrichlorosilane.

[0055] Preferably or optionally, the thioether monomer is selected from vinyl thioether and / or methacrylic acid thioether.

[0056] Preferably or optionally, the silicon-containing monomer is selected from vinylsilane and / or methyl methacrylate silane.

[0057] Preferably or optionally, the cyclic monomer is selected from any one or more of cyclopentene, benzene ring, and tetrahydrofuran.

[0058] Preferably or optionally, the zwitterionic monomer is selected from 2-hydroxyethyl methacrylate and / or 2-hydroxyacrylic acid.

[0059] Preferably or optionally, the carboxylic acid monomer is selected from any one or more of acrylic acid, methacrylic acid, and 2-acryloyloxyacetic acid.

[0060] Preferably or optionally, the quaternary ammonium salt cation is selected from tetramethylammonium chloride and / or tetraphenyl quaternary ammonium salt.

[0061] Preferably or optionally, the quaternary phosphonium salt cation is tetraphenyl phosphonium salt.

[0062] Preferably or optionally, the pyridinium ion is pyridinium salt.

[0063] Preferably or optionally, the imidazolium ion is imidazolium salt.

[0064] Preferably or optionally, the sulfonate anion is methanesulfonic acid.

[0065] Preferably or optionally, the carboxylate anion is acetic acid.

[0066] Preferably or optionally, the phosphate anion is phosphoric acid.

[0067] Preferably or optionally, the functional monomer is selected from any one of acrylic monomers, acrylamide, zwitterionic monomers, quaternary ammonium salt monomers, cationic monomers, temperature-sensitive monomers, fluorine-containing monomers or polypeptides containing double bonds.

[0068] Preferably or optionally, the functional monomer is selected from any one of methacryloylethyl sulfobetaine, methacryloyloxyethyl dimethyl benzyl ammonium chloride, methacryloyloxyethyl trimethyl ammonium chloride and acrylic acid.

[0069] Preferably or optionally, the volume ratio of the protein solution, the functional monomer solution and the photoinitiator solution is 2:0 - 1:2.

[0070] Preferably or optionally, the substrate is selected from any one of polyethylene terephthalate substrates, silicon wafer substrates, quartz glass substrates and polymethyl methacrylate substrates.

[0071] Preferably or optionally, the ultraviolet wavelength used for the ultraviolet irradiation is 300 - 500 nm, and the intensity is 400 - 2000 mW / cm 2 。

[0072] Preferably or optionally, the ultraviolet irradiation time is 3 - 100 seconds, such as 3 seconds, 5 seconds, 10 seconds, 30 seconds, 50 seconds, 100 seconds, and any value within the range composed of any two of the above values.

[0073] The present invention also provides a protein nanofilm prepared according to the above method.

[0074] Beneficial effects

[0075] The present invention provides a method for preparing a protein nanofilm on the surface of a substrate. Under the induction of ultraviolet light, rapid self-assembly aggregation of proteins can be achieved, and the two-dimensional protein nanofilm prepared on the surface of the substrate is dense, has low roughness, high transparency and good biocompatibility. Description of the Drawings

[0076] Figure 1 is a schematic diagram of a large-scale (industrial) surface modification technology.

[0077] Figure 2(a) is a schematic diagram of the formation of a protein film; (b) is an atomic force microscope (AFM) image of the protein film (PTL-UV) coating prepared in Example 6; (c) is the optical transmittance of the protein film (PTL-UV) modified quartz glass prepared in Example 7; (d) is a fluorescence microscopy image of the protein film (PTL-UV) prepared in Example 8 stained with ThT; (e) is an image of the protein film (PTL-UV) prepared in Example 9 stained with Congo red; (f) is a microscope detection of the rapid aggregation process of proteins within 0 - 3 seconds under ultraviolet irradiation (Example 10).

[0078] Figure 3 are atomic force microscope (AFM) images of different protein nanofilms. Such as ovalbumin (OVA), lactoferrin, fibrinogen, bovine serum albumin (BSA), zein, collagen, chymotrypsin, mucin, hemoglobin, insulin, porcine chymotrypsin, concanavalin A, β-lactoglobulin, pepsin, laccase.

[0079] Figure 4 are contact angle images (WCA) of different protein nanofilms.

[0080] Figure 5 are AFM images of protein films under extreme conditions, including organic solvents, plasma treatment, extreme pH values, enzyme treatment, and 3M peeling. Inset: WCA images of protein films after treatment under extreme conditions.

[0081] Figure 6 (a) in is a schematic diagram of the anti-fouling performance of the protein anti-fouling film (PTL-pSBMA) against various pollutants; field emission scanning electron microscope (b, c) and laser confocal (d, e) images show the adhesion of Staphylococcus aureus and Escherichia coli on bare silicon wafers and PTL-pSBMA nanofilms.

[0082] Figure 7 (a) in is a schematic diagram of the antibacterial properties of the protein antibacterial film (PTL-pQAc) nanofilm; (b) is the in vitro bactericidal activity of PTL-pQAc; (c) is an agar plate photograph showing the bactericidal activity against Staphylococcus aureus and Escherichia coli.

[0083] Figure 8 In (a), it is a schematic diagram of glasses fogging up when entering a warm indoor environment from a cold outdoor environment; (b - d) are the optical and microscopic images of the glasses when entering an indoor environment (25°C) from a cold outdoor environment (-15°C).

[0084] Figure 9 In (a), it is the cytotoxicity experiment of the protein thin film (PTL - UV); (b) is the cytotoxicity experiment of the protein anti - fouling thin film (PTL - pSBMA). Detailed implementation manners

[0085] To facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings of the specification and preferred experimental examples. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0086] Unless otherwise defined, all the technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0087] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0088] Example 1

[0089] This example provides a method for industrially and continuously preparing a protein nanometer thin film on the surface of a substrate.

[0090] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0091] Mix the above two aqueous solutions in equal volumes to obtain a mixed solution, and prepare a rectangular polyethylene terephthalate (PET) substrate with a specification of 10×2 m.

[0092] Place the flat substrate on the production line at a speed of 3 m / min. Start the sprayer, ultraviolet light curing machine, cleaning machine, and dryer in sequence. The working pressure of the sprayer is 0.6 MPa. The ultraviolet light curing machine uses an LED light source with UV 365 nm and an intensity of 400 mW / cm 2 ², and the irradiation time is 10 seconds. The working temperature of the dryer is 70°C.

[0093] The PET substrate continuously passes through from the sprayer to the dryer, and then is taken out to obtain a polyethylene terephthalate substrate coated with a protein nanometer thin film.

[0094] Example 2

[0095] This embodiment provides a method for industrially and continuously preparing a protein nanofilm on the surface of a substrate.

[0096] Prepare an aqueous solution of bovine serum albumin with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0097] Mix the above two aqueous solutions in equal volumes to obtain a mixed solution, and prepare a rectangular polyethylene terephthalate (PET) substrate with a specification of 10×2 m.

[0098] Place the flat substrate on the production line at a speed of 3 m / min. Start the sprayer, ultraviolet light curing machine, cleaning machine, and dryer in sequence. The working pressure of the sprayer is 0.6 MPa. The ultraviolet light curing machine uses an LED light source with UV 365 nm and an intensity of 400 mW / cm 2 , and the irradiation time is 10 seconds. The working temperature of the dryer is 70°C.

[0099] The PET substrate continuously passes through the sprayer to the dryer, and then is taken out to obtain a polyethylene terephthalate substrate coated with a protein nanofilm.

[0100] Example 3

[0101] This embodiment provides a method for industrially and continuously preparing a protein nanofilm on the surface of a substrate.

[0102] Prepare an aqueous solution of fibrinogen with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0103] Mix the above two aqueous solutions in equal volumes to obtain a mixed solution, and prepare a rectangular polyethylene terephthalate (PET) substrate with a specification of 10×2 m.

[0104] Place the flat substrate on the production line at a speed of 3 m / min. Start the sprayer, ultraviolet light curing machine, cleaning machine, and dryer in sequence. The working pressure of the sprayer is 0.6 MPa. The ultraviolet light curing machine uses an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 , and the irradiation time is 10 seconds. The working temperature of the dryer is 70°C.

[0105] The PET substrate continuously passes through the sprayer to the dryer, and then is taken out to obtain a polyethylene terephthalate substrate coated with a protein nanofilm.

[0106] Example 4

[0107] This embodiment provides a method for industrially and continuously preparing a protein nanofilm on the surface of a substrate.

[0108] Prepare an aqueous solution of ovalbumin with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0109] Mix the above two aqueous solutions in equal volumes to obtain a mixed solution, and prepare a rectangular polyethylene terephthalate (PET) substrate with a specification of 10×2 m.

[0110] Place the flat substrate on the production line at a speed of 3 m / min. Start the sprayer, ultraviolet light curing machine, cleaning machine, and dryer in sequence. The working pressure of the sprayer is 0.4 MPa. The ultraviolet light curing machine uses an LED light source with UV 365 nm and an intensity of 2000 mW / cm 2 , and the irradiation time is 10 seconds. The working temperature of the dryer is 70 °C.

[0111] The PET substrate continuously passes through the sprayer to the dryer, and then is taken out to obtain a polyethylene terephthalate substrate coated with a protein nanofilm.

[0112] Example 5

[0113] This embodiment provides a method for industrially and continuously preparing a protein nanofilm on the surface of a substrate.

[0114] Prepare an aqueous solution of lactoferrin with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0115] Mix the above two aqueous solutions in equal volumes to obtain a mixed solution, and prepare a rectangular polyethylene terephthalate (PET) substrate with a specification of 10×2 m.

[0116] Place the flat substrate on the production line at a speed of 3 m / min. Start the sprayer, ultraviolet light curing machine, cleaning machine, and dryer in sequence. The working pressure of the sprayer is 0.5 MPa. The ultraviolet light curing machine uses an LED light source with UV 365 nm and an intensity of 400 mW / cm 2 , and the irradiation time is 10 seconds. The working temperature of the dryer is 70 °C.

[0117] The PET substrate continuously passes through the sprayer to the dryer, and then is taken out to obtain a polyethylene terephthalate substrate coated with a protein nanofilm.

[0118] Effect Example 1

[0119] Figure 1A process for rapidly and continuously industrializing the preparation of large-scale substrates with protein nanofilms is shown. This process is adopted in Examples 1-5 and subsequent examples.

[0120] Example 6

[0121] This example provides a method for preparing a protein nanofilm on the surface of a Si substrate for surface roughness and morphology detection.

[0122] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0123] Mix the above two aqueous solutions in equal volumes. After obtaining the mixed solution, drop it onto the surface of the Si substrate, and use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 to irradiate for 3 seconds. Subsequently, after the solution turns into a slightly white emulsion, wash the surface solution clean, dry the silicon wafer, and prepare for AFM instrument testing. The results are shown in Figure 2 (b).

[0124] Example 7

[0125] This example provides a method for preparing a protein nanofilm on the surface of a quartz substrate for transmittance detection.

[0126] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0127] Mix the above two aqueous solutions in equal volumes. After obtaining the mixed solution, immerse the quartz substrate into the solution, and use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 to irradiate for 3 seconds. Subsequently, after the solution turns into a slightly white emulsion, wash the surface solution clean, dry the quartz wafer, and then use a UV spectrophotometer to measure the transmittance at wavelengths of 200 - 800 nm. The results are shown in Figure 2 (c).

[0128] Example 8

[0129] This example provides a method for preparing a protein nanofilm on the surface of a quartz substrate for ThT fluorescence-labeled laser confocal testing.

[0130] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0131] Mix the above two aqueous solutions in equal volumes. After preparing the mixed solution, immerse the quartz substrate into the solution, and use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 for 3 seconds. Subsequently, after the solution turns into a slightly white emulsion, wash the surface solution clean, dry the quartz wafer, soak it in a 1 mM ThT solution for 1 hour, rinse it with water, and then dry it. Use a laser confocal microscope to test the film staining under an excitation light of 488 nm. The results are shown in Figure 2 (d).

[0132] Example 9

[0133] This example provides a method for preparing a protein nanofilm on a substrate surface for Congo red staining.

[0134] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0135] Mix the above two aqueous solutions in equal volumes. After preparing the mixed solution, immerse the substrate into the solution, and use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 for 3 seconds. Subsequently, after the solution turns into a slightly white emulsion, wash the surface solution clean, dry the quartz wafer, soak it in a 10 mg / mL Congo red solution for 10 minutes, rinse it with water, and then dry it. Take a picture under an optical camera. The results are shown in Figure 2 (e).

[0136] Example 10

[0137] This example provides a method for detecting protein aggregation.

[0138] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0139] Mix the above two aqueous solutions in equal volumes to obtain a mixed solution.

[0140] Under an optical microscope, use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 for 0 - 3 seconds. Detect the formation process of its aggregated particles, as shown in Figure 2 (f).

[0141] Example 11

[0142] This example provides a method for preparing a protein nanofilm on the Si substrate surface for surface topography detection and contact angle measurement.

[0143] Prepare an aqueous protein solution with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0144] Mix the above two aqueous solutions in equal volumes. After preparing the mixed solution, drop it onto the surface of the Si substrate. Use an LED light source with UV365 nm and an intensity of 1000 mW / cm 2 to irradiate for 3 seconds. Subsequently, after the solution turns into a slightly white emulsion, wash the surface solution clean and dry the silicon wafer. Prepare for testing with an AFM instrument and an optical video contact angle test. The results are as Figure 3 and Figure 4 .

[0145] Example 12

[0146] This example provides a method for preparing a protein nanofilm on the surface of a Si substrate for stability detection.

[0147] Prepare an aqueous protein solution with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0148] Mix the above two aqueous solutions in equal volumes. After preparing the mixed solution, drop it onto the surface of the Si substrate. Use an LED light source with UV365 nm and an intensity of 1000 mW / cm 2 to irradiate for 3 seconds. Subsequently, after the solution turns into a slightly white emulsion, wash the surface solution clean and dry the silicon wafer. Subsequently, after the silicon wafer with the protein film is soaked in organic solvents, plasma treated, soaked in extreme pH values, enzyme treated, and peeled off with 3M, etc., use an AFM instrument and contact angle to detect the surface morphology and contact angle to determine whether the film still exists stably. The results are as Figure 5 .

[0149] Effect Example 2

[0150] Characterize the basic properties of the protein films prepared in Examples 6 - 12. The results are as Figures 2 - 5 shown.

[0151] After LAP breaks the intramolecular disulfide bonds of lysozyme, aggregates are formed during the phase transition process, and stable 2D lysozyme nanofilms (PTL - UV) are rapidly formed on various substrates, while larger aggregates (PTL - UV products) are formed in the bulk phase ( Figure 2(a)). During this aggregation process, the conformational transition of the protein from α-helix to β-sheet is reflected. The fluorescence spectrum of the ThT-stained PTL-UV coating and the optical image of the Congo red staining further confirm the presence of abundant β-sheet structures in the PTL-UV coating. Different from the polydopamine and tannic acid / Fe(III) complex coatings, the PTL-UV coating is colorless and exhibits nearly 100% optical transmittance on quartz, making it suitable for various applications without affecting the optical properties of the bulk material( Figure 2 ).

[0152] Traditional surface modification techniques rely on slow assembly in solution and require specific conditions (such as temperature, time, pH) to form nano-films at the interface, which limits industrial scalability and commercialization. In contrast, under ultraviolet irradiation, lysozyme mixed with LAP solution completes ultra-rapid surface modification within 1-3 seconds( Figure 2 (f)). This process includes nucleation and then rapid assembly at the interface, forming a dense protein nano-film and large particle aggregates in the bulk phase. This rapid assembly and aggregation enable continuous industrial production, which is the first case of continuous production of protein nano-films in the field of surface modification. In addition, this study can be extended to 15 other common proteins, including lactoferrin, fibrinogen, OVA, BSA, zein, collagen, chymotrypsin, mucin, hemoglobin, insulin, porcine chymotrypsin, concanavalin A, β-lactoglobulin, pepsin, and laccase, and the contact angles of the two-dimensional nano-films prepared from various proteins are all around 70 degrees( Figure 3 and Figure 4 ). In addition, the protein film has excellent biocompatibility and interfacial stability and is resistant to acids, bases, and organic reagents( Figure 5 ).

[0153] Example 13

[0154] This example provides a method for preparing an anti-fouling protein nano-film on the surface of a substrate.

[0155] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL, an aqueous solution of methacryloylethyl sulfobetaine with a concentration of 10 mg / mL, and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0156] Mix the above three solutions in a ratio of 2:1:2 to obtain a mixed solution. Place an 18 mm * 18 mm square glass or a 1 * 1 cm silicon wafer in the mixed solution, and use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 for 50 seconds. Subsequently, wash the solution on the surface of the glass slide and dry it for standby.

[0157] According to the method of this embodiment, a glass / silicon substrate coated with an anti-fouling protein nanofilm is obtained.

[0158] Example 14

[0159] This embodiment provides a method for preparing an antibacterial protein nanofilm on the surface of a substrate.

[0160] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL, an aqueous solution of methacryloyloxyethyl dimethyl benzyl ammonium chloride with a concentration of 20 mg / mL, and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0161] Mix the above three solutions in a ratio of 2:1:2 to obtain a mixed solution. Place an 18 mm × 18 mm square glass slide in the mixed solution, and use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 to irradiate for 100 seconds. Then wash the solution on the surface of the glass slide clean and dry it for standby.

[0162] According to the method of this embodiment, a glass substrate coated with an antibacterial protein nanofilm is obtained.

[0163] Example 15

[0164] This embodiment provides a method for preparing an anti-fog protein nanofilm on the surface of a substrate.

[0165] Prepare an aqueous solution of lysozyme with a concentration of 20 mg / mL, an aqueous solution of acrylic acid with a concentration of 2% (v / v), and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0166] Mix the above three solutions in a ratio of 2:1:2 to obtain a mixed solution. Place a 2 cm × 2 cm PC substrate sheet in the mixed solution, and use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 to irradiate for 20 seconds. Then wash the solution on the surface of the PC sheet clean and dry it for standby.

[0167] According to the method of this embodiment, a PC substrate coated with an anti-fog protein nanofilm is obtained.

[0168] Example 16

[0169] This embodiment provides a method for industrially and continuously preparing a functional protein nanofilm on the surface of a substrate.

[0170] For the industrial preparation formula of the anti-fouling coating, it follows Example 13; for the industrial preparation formula of the antibacterial coating, it follows Example 14; for the industrial preparation formula of the anti-fog coating, it follows Example 15.

[0171] Add the mixed solution in the above examples to the material barrel, and prepare a rectangular polyethylene terephthalate (PET) substrate with a specification of 10×2 m.

[0172] Place the flat substrate on the production line at a speed of 3 m / min. Start the spraying machine, ultraviolet light curing machine, cleaning machine, and drying machine in sequence. The working pressure of the spraying machine is 0.6 MPa. The ultraviolet light curing machine uses an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 The irradiation time is 50 s, 100 s, and 20 s respectively. The working temperature of the drying machine is 70 °C.

[0173] The PET substrate continuously passes through from the spraying machine to the drying machine, and then is taken out to obtain a polyethylene terephthalate substrate coated with a functional protein nanofilm.

[0174] Example 17

[0175] This example provides a test method for the cytotoxicity of preparing a protein nanofilm on the surface of a substrate.

[0176] Prepare an aqueous solution of lysozyme with a concentration of 10 mg / mL and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0177] Mix the above two solutions in equal volume to obtain a mixed solution. After immersing the silicone rubber in the mixed solution, use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 Irradiate for 3 seconds. Place the silicone rubber modified with the protein film in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and perform extraction at 37 °C for 24 h. Subsequently, dilute the silicone rubber extract and mix it with the cell culture medium, and then culture the cells to observe the cell proliferation. According to the MTT method, the results are as Figure 9 (a) shown. Taking the OD value of the untreated normal cell group as the control group (Control), it can be found that the cell survival rate of the PTL-UV nanofilm group is basically the same as that of the control group. Therefore, this coating has no obvious cytotoxicity.

[0178] Example 18

[0179] This example provides a test method for the cytotoxicity of preparing an anti-fouling protein nanofilm on the surface of a substrate.

[0180] Prepare an aqueous lysozyme solution with a concentration of 10 mg / mL, an aqueous solution of methacryloylethyl sulfobetaine with a concentration of 10 mg / mL, and an aqueous solution of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate with a concentration of 5 mg / mL.

[0181] Mix the above three solutions in a ratio of 2:1:2 to obtain a mixed solution. After immersing the silicone rubber in the mixed solution, use an LED light source with UV 365 nm and an intensity of 1000 mW / cm 2 to irradiate for 50 seconds. The silicone rubber modified with the anti-fouling protein film is placed in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and extracted at 37°C for 24 hours. Subsequently, the extract is diluted and mixed with the cell culture medium to culture cells and observe the cell proliferation. According to the MTT method, the results are as Figure 9 (b) shown. Taking the OD value of the untreated normal cell group as the control group (Control), it can be found that the cell survival rate of the PTL-pSBMA nanofilm group is basically the same as that of the control group. Therefore, this coating has no obvious cytotoxicity.

[0182] Effect Example 3

[0183] Take the substrates coated with functional protein nanofilms prepared in Examples 13-15, such as anti-fouling, antibacterial, and anti-fogging functions, and verify them by bacterial contamination, sterilization testing, and fogging experiments, etc., and observe with a scanning electron microscope and a microscope. The results are as Figures 6 - 8 shown. Among them, the laboratory test examples are Examples 13-15, and the industrialization example is Example 16.

[0184] Bacterial contamination test: Dilute the Escherichia coli and Staphylococcus aureus cultures in MHB to prepare a bacterial suspension. Immerse the substrate with the protein nanofilm prepared in Example 13 into the above bacterial suspension and culture for at least 12 hours. Then rinse each substrate with PBS buffer to remove planktonic bacteria, and use for staining, and observe the anti-adhesion situation using laser confocal microscopy; or chemically fix the bacteria adhered to the substrate surface with paraformaldehyde, and then analyze by field emission scanning electron microscopy. The results are as Figure 6 shown.

[0185] It can be seen from Figure 6 that the substrate with the anti-fouling protein nanofilm prepared in Example 13 can effectively prevent the adhesion of bacteria on the substrate surface.

[0186] Sterilization test: Take the glass substrate with the antibacterial protein nanofilm prepared in Example 14, evaluate the antibacterial activity of the PTL-pQAc coating, and conduct the experiment using the plate colony counting method. Staphylococcus aureus and Escherichia coli were selected as the test bacteria. The specific operations are as follows: First, take 1 mL of the bacterial suspension in the logarithmic growth phase, place it in a sterile centrifuge tube, centrifuge at 5000 rpm for 5 minutes, and then wash it 3 times repeatedly with PBS to completely remove the residual culture medium. Then, adjust the concentration of the test bacteria to 10 7 CFU / mL. Take 10 μL of the above bacterial suspension and drop it on the surface of the unmodified glass substrate or the substrate modified with the PTL-pQAc coating, and then cover it with another identical substrate to ensure that the bacteria are evenly distributed between the two glasses. Place it in a humid environment at 37 °C and incubate for 8 hours. After the incubation, carefully separate the glasses with tweezers and immerse them completely into a centrifuge tube containing 10 mL of sterile PBS. Ultrasonically process this centrifuge tube to completely transfer the bacteria attached to the material surface into the PBS solution. Subsequently, serially dilute the bacterial suspension and spread it on the MHA plate. After culturing in an incubator at 37 °C for 24 hours, record the number of colonies on the plate surface. The test results of the antibacterial rate are as Figure 7 shown.

[0187] It can be seen from Figure 7 that the substrate with the protein nanofilm prepared in Example 14 has good antibacterial effects.

[0188] Anti-fog experiment: Take the substrate with the anti-fog protein nanofilm prepared in Example 15. After the surface of the winter glasses encounters hot and cold air, it is found that there is a difference in the fogging effect between the side with the coating and the side without the coating on the glasses surface.

[0189] The test results of anti-fogging are as Figure 8 shown.

[0190] It can be seen from Figure 8 that the substrate with the anti-fog protein nanofilm prepared in Example 15 has good anti-fogging effects.

[0191] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a protein nanofilm on a substrate surface, characterized in that: The following steps are involved: (1) mixing a protein solution, an optional functional monomer solution and a photoinitiator solution to prepare a mixed solution; (2) immersing the substrate in the mixed solution; (3) Irradiate with ultraviolet light to obtain a protein nanofilm on the surface of the substrate.

2. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The protein is selected from any one or more of plant-derived proteins and animal-derived proteins; Preferably, the plant source is: Preferably, the animal source is: Preferably, the protein is selected from any one of lysozyme, bovine serum albumin, ovalbumin, lactoferrin, bovine fibrinogen, zein, collagen, chymotrypsin, mucin, hemoglobin, insulin, porcine chymotrypsin, concanavalin A, β-lactoglobulin, pepsin and laccase; Preferably, the concentration of the protein solution is 1-20 mg / mL.

3. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The photoinitiator is selected from any one or more of a free radical photoinitiator, a cationic photoinitiator and a mixed photoinitiator; Preferably, the free radical photoinitiator is selected from any one or more of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, benzyl alcohol ether, acetophenone, benzyl alcohol, cyclopentenone, 2-hydroxy-2-methylpropiophenone, vinyl dibenzhydrylamino, isophorone, 4,4'-dichloroacetophenone, triphenylmethyl peroxide, photoinitiator-184, triphenyl phosphate, 2,2-dimethyl-2-phenylpropylene, epichlorohydrin, dicyano-2,4,6-trimethylbenzene, 1-hydroxycyclohexyl phenyl ketone, photoinitiator-819, 1,4-dichloroacetophenone, and 2-methyl-4-phenyl-2-butene; Preferably, the cationic photoinitiator is selected from any one or more of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, toluene sulfonate, 1,2-diphenyl iodine, phenyl chlorine, tetrakis (phenyl) iodide cation salt, triphenyl sulfonic acid, 2,4,6-trimethylphenylbenzenesulfonic acid chloride, 1,2,3-triphenylcyclohexane, 4-methylphenylmethane, methyl styrene, hydroxyethylphenyl silane, vinyl epoxy resin, 2-phenylphenyl triphenyl sulfate, and 2-phenylphenyl triphenyl borate; Preferably, the mixed photoinitiator is selected from any one or more of a combination of benzyl alcohol ether and cyclopentenone, a combination of 2-hydroxy-2-methylpropionylbenzene and acetophenone, a combination of 2,4,6-trimethylphenylbenzenesulfonic acid chloride and cyclopentenone, a combination of triphenyl sulfate chloride and acetophenone, a combination of acetophenone and 2,2-dimethyl-2-phenylpropylene, a combination of 1,4-dichloroacetophenone and acetophenone, a combination of acetophenone and benzyl alcohol ether, a combination of acetophenone and triphenyl phosphate, and a combination of acetophenone and 1,2-diphenyl iodine; Preferably, the photoinitiator is selected from any one or more of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and (2,4,6-trimethylbenzoyl) diphenylphosphine oxide; Preferably, the concentration of the photoinitiator solution is 1-20 mg / mL.

4. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The functional monomer is selected from any one or more of olefin monomers, acrylic monomers, ester monomers, alcohol monomers, aldehyde monomers, ketone monomers, nitrogen-containing monomers, fluorine-containing monomers, aromatic olefin monomers, silane monomers, thioether monomers, silicon-containing monomers, cyclic monomers, zwitterionic monomers, carboxylic acid monomers, quaternary ammonium salt cations, quaternary phosphonium salt cations, pyridinium ions, imidazolium ions, sulfonate anions, carboxylate anions, and phosphate anions; Preferably, the olefin monomer is selected from any one or more of ethylene, propylene, butadiene, styrene, vinylbenzene, and methylstyrene; Preferably, the acrylic monomer is selected from any one or more of acrylic acid, methyl methacrylate, ethyl acrylate, butyl acrylate, hydroxyethyl acrylate, isooctyl acrylate, and epoxyethylene acrylate; Preferably, the ester monomer is selected from any one or more of vinyl ether, methyl methacrylate, vinyl ethyl ether, phenyl methacrylate, and vinyl acetate; Preferably, the alcohol monomer is selected from any one or more of vinyl alcohol, hydroxyethylene, and dimethyl methacrylate; Preferably, the aldehyde monomer is selected from methacrolein and / or acrolein; Preferably, the ketone monomer is selected from methyl acrylate ketone and / or cyclopentenone; Preferably, the nitrogen-containing monomer is selected from any one or more of vinyl imidazole, vinyl pyridine, pyrrolidone, and picolinic acid; Preferably, the fluorine-containing monomer is selected from any one or more of perfluoroethylene, hexafluoropropylene, and tetrafluoroethylene; Preferably, the aromatic vinyl monomer is selected from any one or more of styrene, vinylbenzene, and methylstyrene; Preferably, the silane monomer is selected from vinyl trichlorosilane and / or methyl trichlorosilane; Preferably, the thioether monomer is selected from vinyl thioether and / or methacrylic acid thioether; Preferably, the silicon-containing monomer is selected from vinyl silane and / or methyl methacrylate silane; Preferably, the cyclic monomer is selected from any one or more of cyclopentene, benzene ring, and tetrahydrofuran; Preferably, the zwitterionic monomer is selected from 2-hydroxyethyl methacrylate and / or 2-hydroxyacrylic acid; Preferably, the carboxylic acid monomer is selected from any one or more of acrylic acid, methacrylic acid, and 2-acryloyloxyacetic acid; Preferably, the quaternary ammonium salt cation is selected from tetramethylammonium chloride and / or tetraphenyl quaternary ammonium salt; Preferably, the quaternary phosphonium salt cation is a tetraphenyl quaternary phosphonium salt; Preferably, the pyridinium ion is a pyridinium salt; Preferably, the imidazolium ion is an imidazolium salt; Preferably, the sulfonate anion is methanesulfonic acid; Preferably, the carboxylate anion is acetic acid; Preferably, the phosphate anion is phosphoric acid; Preferably, the functional monomer is selected from any one of acrylic acid monomer, acrylamide, zwitterionic monomer, quaternary ammonium salt monomer, cationic monomer, thermosensitive monomer, fluorine-containing monomer or polypeptide containing double bonds.

5. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The functional monomer is selected from any one of methacryloylethyl sulfobetaine, methacryloyloxyethyl dimethylbenzyl ammonium chloride, methacryloyloxyethyl trimethylammonium chloride and acrylic acid.

6. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The volume ratio of the protein solution, the functional monomer solution and the photoinitiator solution is 2:0-1:

2.

7. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The substrate is selected from any one of a polyethylene terephthalate substrate, a silicon wafer substrate, a quartz glass substrate and a polymethyl methacrylate substrate.

8. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The ultraviolet light used in the ultraviolet irradiation has a wavelength of 300-500nm and an intensity of 400-2000mW / cm 2 .

9. The method for preparing a protein nanofilm on a substrate surface according to claim 1, characterized in that: The ultraviolet irradiation time is 3-100 seconds.

10. A protein nanofilm prepared according to the method of any one of claims 1-9.

Images