Photosensitive polyvinylidene fluoride film, method for producing the same, and use thereof

Abstract

The present invention relates to the field of biotechnology and materials, in particular to an improved Western blotting membrane, its method of preparation and use. Specifically, the present invention relates to a chemically modified polyvinylidene fluoride membrane, said membrane being surface modified with a group according to Formula I: Formula I. After transfer of a protein to the chemically modified polyvinylidene fluoride membrane of the present invention, 365 nm UV light can be used to covalently crosslink the protein to the membrane. The present invention also relates to a method of preparation of the chemically modified polyvinylidene fluoride membrane, and its use in Western blotting.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and materials, and particularly to an improved protein imprinting transfer membrane, its preparation method, and its uses. Background Technology

[0002] Western blotting is a method for detecting protein expression levels in cell or tissue extracts. This method involves multiple steps: first, total protein is extracted from a biological sample, and then the proteins are separated by polyacrylamide gel electrophoresis. The separated proteins are transferred to PVDF or nitrocellulose membranes or other solid supports, and subsequently bound to specific antibodies. Enzyme- or isotope-labeled antibodies bind to the target protein, and finally, appropriate markers are used for detection to determine the presence and relative abundance of the target protein. Western blotting has a wide range of applications, including biomedical research, drug development, and clinical diagnostics. Its advantages, such as high sensitivity, high specificity, and quantification, make it widely used in these fields.

[0003] Continuous detection of different proteins using Western blotting can save time and valuable samples. However, current Western blotting transfer membranes have shortcomings. Traditional protein-membrane binding is non-covalent, which is unstable. During antibody stripping and membrane regeneration, proteins easily detach from the membrane, making it difficult to reuse. Forced reuse often leads to inaccurate and unreliable results. On the one hand, this single-use model results in significant resource waste. In research and production, Western blotting experiments typically require multiple runs, each using a new transfer membrane, increasing costs and putting considerable pressure on the environment. On the other hand, for experiments requiring precious protein samples, single-use means consuming more protein samples. Obtaining precious protein samples is often difficult, potentially requiring complex extraction processes or expensive purchases. Moreover, due to limited sample volume, once used up, subsequent experiments may not be possible, severely limiting in-depth research. Furthermore, from a time perspective, each experiment requires preparing a new transfer membrane, including pretreatment and transfer, undoubtedly increasing the time cost. For some time-sensitive research projects or production tasks, this could affect the overall project schedule. In summary, existing protein blotting membranes have significant shortcomings in terms of reusability, and there is an urgent need for a new technology to achieve membrane regeneration, reuse, and repeated protein determination, in order to solve the problems of resource waste, high sample consumption, and high time costs.

[0004] Antibody stripping and membrane regeneration refers to the removal of primary and secondary antibodies from a Western blot membrane and the re-addition of primary antibody for assay. Ideally, the previously bound antibody can be removed and re-detected multiple times. Membrane regeneration is particularly useful when studying more than one protein on the same blot (e.g., target protein and loading control). When detecting multiple targets, electrophoresis and multiple gels are not required; instead, regeneration and re-detection are performed on a single membrane. Modern Western blot experiments typically allow for three membrane regenerations, enabling a total of four Western blot assays. Mild stripping buffers, such as low-pH buffers or buffers containing 0.1% SDS, are typically used for stripping at room temperature. Strong stripping buffers, containing 2% SDS, are reacted at 50°C for antibody stripping. However, this does not guarantee that only the primary and secondary antibodies will be eluted while the antigen (Ag) protein remains intact. In fact, after three stripping cycles, the ECL signal often becomes too weak to be detected due to significant protein loss on the membrane. The main reason is that the proteins transferred onto the membrane are non-covalently bonded to the membrane, making them easy to wash off.

[0005] Therefore, in view of the shortcomings of existing protein blot transfer membranes, such as non-reusability, resource waste, high sample consumption, and high time cost, it is still necessary to develop a membrane that can be peeled off and regenerated multiple times for continuous and repeated determination of signals of multiple target proteins in protein blots. Summary of the Invention

[0006] To address the drawbacks of existing protein blot transfer membranes, such as non-reusability leading to resource waste, high sample consumption, and high time costs, this invention innovatively modifies the surface of a polyvinylidene fluoride (PVDF) membrane with the photosensitive group bisacrididine, thereby preparing a photosensitive PVDF membrane, named PVDF-diazirine (PVDF-Dia). After transferring proteins onto the PVDF-diazirine membrane of this invention, irradiation with 365 nm ultraviolet light can covalently cross-link the proteins to the membrane. This allows antibodies to be repeatedly peeled off while the transferred proteins remain covalently bound to the membrane without separation. These cross-linked membrane-bound proteins can withstand at least 12 peels, making them suitable for repeated protein assays and analyses based on protein blotting.

[0007] I. Chemically modified polyvinylidene fluoride membrane

[0008] Therefore, in a first aspect, the present invention provides a chemically modified polyvinylidene fluoride membrane, the surface of which is modified with groups represented by Formula I.

[0009]

[0010] Formula I

[0011] R1 is selected from C1-C6 alkyl, fluorinated C1-C6 alkyl, and preferably methyl;

[0012] L1 is selected from Where m is selected from integers from 1 to 6; L1 is preferred. The position marked 1 indicates that it is connected to the ring carbon atom in Formula I, and the position marked 2 indicates that it is connected to L2;

[0013] L2 is a linking unit that may contain at least one siloxy group, wherein the silicon atom is optionally substituted with a C1-C6 alkyl or a C6-C12 aryl group; L2 is preferably... , where n is selected from an integer from 1 to 6, n is preferably an integer from 1 to 3, n is more preferably 2, where position 1 indicates connection with position 2 in L1, and the O atoms connected to Si are connected to the surface of the polyvinylidene fluoride film.

[0014] In some embodiments, the chemically modified membrane has the ability to bind to proteins under ultraviolet light irradiation.

[0015] In some embodiments, the group represented by Formula I undergoes a ring-opening reaction under ultraviolet irradiation to generate a highly reactive free radical or reactive intermediate.

[0016] In some embodiments, the free radical or active intermediate can undergo a covalent bonding reaction with groups on the protein (e.g., amino, carboxyl, thiol, etc.), thereby covalently linking the protein to the membrane.

[0017] In some embodiments, the wavelength of the ultraviolet light is selected from long wavelengths, such as 330 nm-390 nm, for example 365 nm.

[0018] As used in this article, the group represented by Formula I has two acridine rings in its molecular structure, which endows it with unique reactivity under specific light conditions.

[0019] In some embodiments, modifying the PVDF membrane surface with the groups represented by Formula I alters the original chemical properties and surface activity of the PVDF membrane.

[0020] In some implementations, based on the conventional protein blotting transfer process, after the protein is transferred onto the chemically modified membrane, ultraviolet (UV) irradiation is used to initiate a chemical reaction.

[0021] II. Preparation Method

[0022] In a second aspect, the present invention provides a method for preparing the chemically modified membrane described in the first aspect, comprising the following steps:

[0023] (i) The polyvinylidene fluoride membrane was treated with an alkaline solution to obtain an oxidized polyvinylidene fluoride membrane;

[0024] (ii) Treating the oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution to obtain an amino-modified polyvinylidene fluoride membrane; and

[0025] (iii) Treat the amino-modified polyvinylidene fluoride membrane with the compound shown in Formula I' to obtain a chemically modified polyvinylidene fluoride membrane (PVDF-diazirine).

[0026]

[0027] Formula I'

[0028] R1 is selected from C1-C6 alkyl and fluorinated C1-C6 alkyl, preferably methyl;

[0029] R2 is selected from Where m is selected from integers from 1 to 6, preferably ;

[0030] Preferably, the chemically modified membrane has the ability to bind to proteins under ultraviolet irradiation;

[0031] Preferably, the chemically modified film undergoes a ring-opening reaction under ultraviolet irradiation to generate highly reactive free radicals or active intermediates;

[0032] Preferably, the free radical or active intermediate can undergo a covalent bonding reaction with groups on the protein (such as amino, carboxyl, thiol, etc.), thereby covalently linking the protein to the membrane.

[0033] Preferably, the wavelength of the ultraviolet light is selected from long wavelengths, such as 330 nm-390 nm, for example 365 nm.

[0034] In some embodiments, the treatment of the polyvinylidene fluoride membrane with an alkaline solution in step (i) includes:

[0035] The polyvinylidene fluoride membrane is immersed in the alkaline solution at 20℃-60℃ (e.g., 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃) for 1-10 hours (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours).

[0036] In some embodiments, the polyvinylidene fluoride membrane is immersed in the alkaline solution at 25°C for 2 hours.

[0037] In some embodiments, the alkaline solution is selected from potassium hydroxide (KOH) and sodium hydroxide (NaOH); more preferably, the alkaline solution is selected from KOH.

[0038] In some embodiments, the concentration of the alkaline solution is 0.1M-1M, preferably 0.2M.

[0039] In some embodiments, the treatment of the oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution in step (ii) includes:

[0040] The oxidized polyvinylidene fluoride membrane is immersed in the siloxane coupling agent solution for 1-2 hours at 30°C-60°C; preferably, it is immersed for 1 hour at 40°C.

[0041] In some embodiments, the siloxane coupling agent is selected from 3-aminopropyltriethoxysilane (APTES), 3-aminopropyltrimethoxysilane (APTMS), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (DAMO), vinyltrimethoxysilane (VTMS), vinyltriethoxysilane (VTES), and 3-methacryloyloxypropyltrimethoxysilane.

[0042] In some embodiments, the APTES solution is a solution of APTES dissolved in toluene; preferably, the concentration of the APTES solution is 2% v / v (volume ratio).

[0043] In some embodiments, the treatment of the amino-modified polyvinylidene fluoride membrane with the compound of formula I' in step (iii) comprises: immersing the amino-modified polyvinylidene fluoride membrane in a solution containing the compound of formula I' and incubating it at 20°C-40°C for 10 min-60 min; preferably, incubating it at 25°C for 30 min.

[0044] In some embodiments, the final concentration of the compound represented by Formula I' is 0.5 mM.

[0045] In a third aspect, the present invention provides a chemically modified membrane prepared according to the method described in the second aspect.

[0046] III. Uses

[0047] In a fourth aspect, the present invention provides the use of the membrane described in the first aspect, or the chemically modified membrane described in the third aspect, in Western blotting.

[0048] In some implementations, based on the traditional protein blotting transfer process, after the protein is transferred onto the membrane described in the first aspect or the chemically modified membrane described in the third aspect, ultraviolet irradiation is used to initiate a chemical reaction, thereby covalently linking the protein to the chemically modified membrane.

[0049] In some embodiments, the wavelength of the ultraviolet light is selected from long wavelengths, such as 330 nm-390 nm, for example 365 nm.

[0050] In a fifth aspect, the present invention provides the use of the membrane described in the first aspect or the chemically modified membrane described in the third aspect in detecting the presence and / or relative abundance of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) target proteins in a sample.

[0051] In some embodiments, the membrane described in the first aspect, or the chemically modified membrane described in the third aspect, is covalently bound to the target protein, which can withstand at least 12 peels and is suitable for repeated protein determination and analysis based on protein blotting.

[0052] In a sixth aspect, the present invention provides a method for detecting the presence and / or relative content of a target protein in a sample by Western blotting, comprising the following steps:

[0053] (1) Extract total protein from the sample;

[0054] (2) Proteins are separated by gel electrophoresis (e.g., polyacrylamide gel electrophoresis);

[0055] (3) The separated protein is transferred onto the chemically modified membrane described in the first or third aspect to obtain a transfer membrane;

[0056] (4) The transfer film is subjected to ultraviolet irradiation to obtain a covalently cross-linked transfer film;

[0057] (5) Incubating the covalently cross-linked transfer membrane with the first group of specific antibodies, comprising:

[0058] The covalently cross-linked transfer membrane is incubated with a first antibody that specifically recognizes and binds to a first target protein, and further incubated with a labeled second antibody that specifically recognizes and binds to the first antibody, and the labeling provides a measurable signal; and

[0059] (6) Detect the signal and obtain a first protein imprint membrane to determine the presence and / or relative content of the first target protein;

[0060] Preferably, the wavelength of the ultraviolet light is selected from long wavelengths, such as 330 nm-390 nm, and more preferably, the wavelength of the ultraviolet light is 365 nm.

[0061] In some implementations, the method further includes the following steps:

[0062] (7) The first protein imprint membrane is regenerated to remove the first group of specific antibodies and obtain a regenerated covalently cross-linked transfer membrane;

[0063] (8) Incubating the regenerated covalently cross-linked transfer membrane with a second group of specific antibodies, comprising:

[0064] The regenerated covalently cross-linked transfer membrane is incubated with a first antibody that specifically recognizes and binds to a second target protein, and further incubated with a labeled second antibody that specifically recognizes and binds to the first antibody, and the labeling provides a measurable signal.

[0065] (9) Detect the signal and obtain a second protein imprint membrane to determine the presence and / or relative content of the second target protein;

[0066] (10) The (N-1) protein blot membrane is regenerated to remove the (N-1) group of specific antibodies, resulting in a regenerated covalently cross-linked transfer membrane;

[0067] (11) Incubating the regenerated covalently cross-linked transfer membrane with the Nth group of specific antibodies, comprising:

[0068] The regenerated covalently cross-linked transfer membrane is incubated with a first antibody that specifically recognizes and binds to the Nth target protein, and further incubated with a labeled second antibody that specifically recognizes and binds to the first antibody, and the labeling provides a measurable signal.

[0069] (12) Detect the signal and obtain the Nth protein imprint membrane to determine the presence and / or relative content of the Nth target protein;

[0070] Where N≥2, and N is an integer; for example, N is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0071] In some embodiments, the chemically modified membrane is covalently bound to the target protein, and the protein-blotted membrane can withstand at least 12 regeneration processes, making it suitable for repeated protein determination and analysis based on protein blotting.

[0072] In some embodiments, the regeneration process includes immersing the protein blot membrane in a stripping buffer selected from any of the following:

[0073] (a) A buffer solution containing 20% ​​hydrogen peroxide;

[0074] (b) Buffer containing 1%–5% sodium dodecyl sulfate (SDS).

[0075] In some embodiments, the stripping buffer is a buffer containing 5% sodium dodecyl sulfate (SDS).

[0076] In some embodiments, the regeneration treatment is performed at 50°C-70°C for 30 minutes.

[0077] In some embodiments, steps (4), (7) and / or (10) further include: blocking the covalently cross-linked transfer membrane or the regenerated covalently cross-linked transfer membrane to prevent the antibody from binding to nonspecific sites on the surface of the covalently cross-linked transfer membrane or the regenerated covalently cross-linked transfer membrane.

[0078] In some embodiments, nonspecific sites on the covalently cross-linked transfer membrane or the regenerated covalently cross-linked transfer membrane are blocked by incubating the membrane with a blocking buffer (e.g., 5% milk, 5% BSA).

[0079] In some implementations, the measurable signal is detected by visual inspection, chemiluminescence (e.g., horseradish peroxidase (HRP)-mediated chemiluminescence), or fluorescence.

[0080] In some embodiments, the chemically modified membrane of the present invention can be repeatedly stripped of antibodies, while the transferred proteins are covalently bound to the chemically modified membrane and are not easily washed away. The proteins transferred to the chemically modified membrane can withstand at least 12 stripping cycles of antibodies under strong conditions (e.g., 5% SDS in PBS, 70°C, 30 min) without significant attenuation of the protein assay signal.

[0081] IV. Reagent Kit

[0082] In a seventh aspect, the present invention provides a kit comprising a chemically modified membrane as described in the first or third aspect;

[0083] Preferably, the chemically modified membrane has the ability to bind to proteins under ultraviolet irradiation;

[0084] Preferably, the chemically modified film undergoes a ring-opening reaction under ultraviolet irradiation to generate highly reactive free radicals or active intermediates;

[0085] Preferably, the free radical or active intermediate can undergo a covalent bonding reaction with groups on the protein (such as amino, carboxyl, thiol, etc.), thereby covalently linking the protein to the membrane.

[0086] Preferably, the wavelength of the ultraviolet light is selected from long wavelengths, such as 330 nm-390 nm, for example 365 nm.

[0087] In some embodiments, the kit further includes other reagents required for Western blotting transfer, such as transfer solutions (e.g., Tris-Gly transfer solution, CAPS transfer solution) and transfer apparatus (e.g., transfer clamps, sponges, transfer filter paper).

[0088] In an eighth aspect, the present invention provides the use of the kit described in the seventh aspect in Western blotting.

[0089] In some implementations, based on the traditional protein blotting transfer process, after the protein is transferred onto the chemically modified membrane described in the first or third aspect, ultraviolet irradiation is used to initiate a chemical reaction, thereby covalently linking the protein to the chemically modified membrane.

[0090] In some embodiments, the wavelength of the ultraviolet light is selected from long wavelengths, such as 330 nm-390 nm, for example 365 nm.

[0091] In a ninth aspect, the present invention provides the use of the kit described in the seventh aspect for detecting the presence and / or relative abundance of multiple (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) target proteins in a sample.

[0092] In some embodiments, the chemically modified membrane described in the first or third aspect is covalently bound to the target protein, which can withstand at least 12 peels and is suitable for repeated protein determination and analysis based on protein blotting.

[0093] Terminology Definition

[0094] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the biochemical, molecular biological, cellular, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0095] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0096] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both singular and plural forms in the context of describing the invention (especially in the context of the claims).

[0097] The terms first, second, third, (a), (b), (c), and similar in the specification and claims are used to distinguish similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and that embodiments described in the invention can be implemented in a different order than that described or illustrated in the invention.

[0098] As used herein, the term "blotting" refers to a method for transferring biological samples from a gel to a membrane under the influence of an electric field. This method requires a membrane capable of immobilizing the biomolecular sample to ensure its stability in subsequent analyses. The membrane's surface area, porosity, and protein-binding capacity all significantly influence the experimental results.

[0099] The term "Western blotting," also known as "protein immunoblotting," is a type of blotting that uses an electric field to migrate proteins from a gel to a membrane, forming corresponding protein bands on the membrane. This involves immobilizing proteins on a membrane before detection using monoclonal or polyclonal antibodies. Before immobilization, sample proteins are separated into native or denatured proteins using SDS-PAGE. These proteins are then transferred or electroblotted onto the membrane, where they are detected using antibodies specific to the target proteins. Protein-blotted membranes are typically made of nitrocellulose (NC) or polyvinylidene fluoride (PVDF). The specificity of antibody-antigen interactions ensures the identification of individual proteins in complex protein mixtures.

[0100] As used herein, the term "conventional protein blotting" refers to protein transfer techniques well known to those skilled in the art, and the experimental procedures of conventional protein blotting typically include the following parts:

[0101] (1) Sample preparation: First, total protein was extracted from cells or tissues and dissolved in a lysis buffer (such as RIPA buffer). Then, the total protein concentration was quantified at 595 nm using Bradford reagent.

[0102] (2) Gel electrophoresis: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to separate protein samples according to their molecular weight. SDS is an anionic detergent that can destroy the higher-order structure of proteins, making them negatively charged, and thus allowing them to migrate towards the positive electrode under the action of an electric field.

[0103] (3) Protein transfer: Proteins on the gel are transferred to a solid support by electrophoresis or diffusion. Commonly used membrane materials include nitrocellulose (NC) and polyvinylidene fluoride (PVDF). PVDF membranes have high hydrophobicity and high binding force, making them suitable for applications that require multiple peeling and relabeling; while NC membranes are compatible with a variety of detection methods and have a strong binding capacity for proteins.

[0104] In protein transfer, an electroblotting apparatus is required, typically using a wet or semi-dry transfer device. In wet transfer, both the gel and membrane are immersed in transfer buffer, while in semi-dry transfer, the gel and membrane are sandwiched between filter paper soaked in buffer. During electroblotting, the gel and membrane are arranged in a "sandwich" structure, placed in the electroblotting chamber, and an electric current is applied. The direction of the current determines the direction of protein migration, usually from the gel to the membrane.

[0105] (4) Blocking: In order to prevent non-specific binding, the regions of the membrane where proteins are not bound need to be blocked with a blocking agent (such as 5% skim milk or BSA).

[0106] (5) Antibody detection: First, the membrane is incubated with a primary antibody targeting the target protein to allow the primary antibody to bind specifically to the target protein. Then, the membrane is washed to remove unbound primary antibody. Next, the membrane is incubated with a labeled secondary antibody, which typically targets the constant region of the primary antibody. Labeling (such as HRP or a fluorescent group) enhances detection sensitivity.

[0107] (6) Signal detection: Finally, the antigen-antibody complex is detected by chemiluminescence, fluorescence or colorimetry. For example, a chemiluminescent signal can be generated using horseradish peroxidase (HRP) labeled secondary antibody and corresponding substrate (such as ECL reagent), which can be detected by photographic film or an imager.

[0108] As described herein, this invention improves the membrane used for protein transfer in protein blotting. As used herein, the chemically modified polyvinylidene fluoride membrane of this invention, with its surface modified with groups represented by Formula I, is called a photosensitive polyvinylidene fluoride membrane, named "PVDF-diazirine," "PVDF-diazyrine," or "PVDF-Dia." The PVDF-diazirine membrane is constructed by modifying the surface of a PVDF membrane with the groups represented by Formula I, and then using ultraviolet (UV) light to covalently bond proteins to the membrane.

[0109] As described in this article, "antibody stripping" refers to the removal of primary and secondary antibodies from a protein blot membrane. In this process, the protein blot membrane is "membrane regeneration," and new primary antibodies can be added to measure other target proteins.

[0110] As used herein, the term "Tris-Gly transfer buffer" is known to those skilled in the art; it is a commonly used buffer for protein transfer in Western blot experiments. Its main components include tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), and methanol, which play crucial roles in the transfer process. Tris-Glycine transfer buffer is typically provided in concentrated 10× or 25× form and must be diluted according to experimental requirements before use. For example, the dilution ratio for 10× transfer buffer is 1:10, i.e., 100 ml of 10× transfer buffer is added to 200 ml of anhydrous methanol and 700 ml of water to prepare 1 L of 1× working solution. For example, a commonly used Tris-Glycine transfer buffer (1×) contains 25 mM Tris, 192 mM Glycine, 20% (v / v) methanol, and has a pH of approximately 8.3.

[0111] As used herein, the term "CAPS transfer buffer" is known to those skilled in the art; it is a commonly used buffer solution for protein transfer in Western blot experiments. Its main components include CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), DTT (dithiothreitol), and methanol. Depending on the experimental requirements, CAPS transfer buffer is available in 1× and 10× forms. For example, 10× CAPS transfer buffer contains: 22.13 g of CAPS, 900 mL of deionized water, approximately 20 mL of 2 mol / L NaOH (to adjust the pH to 11.0), brought to a final volume of 1 L, and stored at 4°C. Alternatively, 1× CAPS transfer buffer contains 200 mL of 10× CAPS, 200 mL of methanol, and 1600 mL of deionized water.

[0112] As used herein, the term “C1-C6 alkyl” refers to having 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5 or 6 carbon atoms; examples include, but are not limited to, methyl, ethyl, propyl, propyl, butyl and the like.

[0113] As used herein, the term "fluorinated C1-C6 alkyl" refers to a group obtained by substituting a C1-C6 alkyl group as defined above with one or more fluorine molecules, examples of which include, but are not limited to, CF3, CHF2, or CF2CF3.

[0114] As used herein, the term “C6-C12 aryl” refers to an unsaturated carbocyclic group consisting of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, or 12) carbon atoms having a conjugated π-electron system, with non-limiting examples including, but not limited to, phenyl.

[0115] As used herein, the term "siloxane coupling agent" refers to a class of bifunctional organosilicon compounds whose molecular structure simultaneously includes: a hydrolyzable silane group (e.g., -Si(OR)3, R=C1-C6 alkyl; -SiCl3) capable of forming chemical bonds (Si-OM, M=Si, Al, Fe, etc.) with the surface of inorganic materials (e.g., glass, metal oxides, fillers); and an organic active functional group (e.g., amino, epoxy, mercapto, vinyl, etc.) capable of chemically reacting or physically binding with organic polymers (e.g., resins, rubber, biomolecules). Non-limiting examples of "siloxane coupling agents" include, but are not limited to: 3-aminopropyltriethoxysilane (APTES), 3-aminopropyltrimethoxysilane (APTMS), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (DAMO), vinyltrimethoxysilane (VTMS), vinyltriethoxysilane (VTES), 3-methacryloyloxypropyltrimethoxysilane, etc.

[0116] Beneficial effects of the invention

[0117] This invention develops a chemically modified polyvinylidene fluoride membrane, the surface of which is modified with groups represented by Formula I.

[0118]

[0119] Formula I

[0120] It was named PVDF-diazyrine (PVDF-diazirine or PVDF-Dia), and UV light was used to induce the protein to form covalent bonds with the chemically modified membrane.

[0121] Compared to traditional protein blotting procedures, the protein blotting based on the PVDF-diazirine membrane of this invention requires irradiating the membrane with ultraviolet light after the protein transfer operation, so that the protein and the membrane can form covalent bonds.

[0122] The PVDF-diazyrine membrane of this invention enables stable protein binding on the transfer membrane, facilitating membrane regeneration and repeated protein assays. This covalent bonding significantly enhances the binding strength between the protein and the membrane. Compared to traditional physical adsorption or weaker non-covalent interactions, covalent bonds offer greater stability, withstanding various processing conditions during subsequent membrane regeneration, such as elution and rinsing, without easily causing protein detachment. Specifically, traditional detection membranes, after antibody elution and regeneration, can typically only be reused 3-4 times, while the PVDF-diazyrine membrane of this invention can be reused more than 12 times.

[0123] The PVDF-diazyrine membrane of this invention effectively solves the problem of non-reusable membranes in existing technologies, reducing resource waste and sample consumption, and provides a more efficient and economical solution for the practical application of protein blotting technology. Furthermore, for research projects requiring numerous repeated experiments to verify results, it can significantly reduce experimental costs and improve experimental efficiency.

[0124] The PVDF-diazyrine membrane of this invention can improve the accuracy and reliability of experimental results. In some experiments that monitor changes in protein expression over a long period of time, protein content at different time points can be measured multiple times on the same membrane, avoiding experimental errors that may be introduced by changing the membrane each time, thereby improving the consistency and reproducibility of experimental results. Attached Figure Description

[0125] Figure 1 Flowchart of PVDF-diazirine (PVDF-Dia) preparation.

[0126] Figure 2 A-2B: The amino groups on the surface of PVDF were determined by the ninhydrin reaction. Figure 2 A: Results of the reaction between the original PVDF membrane and ninhydrin solution; Figure 2 B: Results of the reaction between the PVDF-amine membrane and the ninhydrin solution.

[0127] Figure 2C: XPS spectra of PVDF and PVDF-Dia. The spectrum of PVDF-Dia shows new peaks at 956.62, 1088.65, and 1386.27 eV, corresponding to the binding energies of O1s, N1s, and Si2p. These findings confirm the presence of silicon (Si) and nitrogen (N) features in the PVDF-Dia film, indicating successful modification. Furthermore, the O1s band intensity (531 eV) increases due to the formation of silanol groups on the surface, while the CeF bond content decreases (800.88 eV) due to the defluorination process.

[0128] Figure 3 (A) 365 nm UV crosslinker; (B) Total energy parameters of 365 nm UV irradiation used for crosslinking. Most experiments used a total irradiation energy of 3.378 J / cm² (3 minutes of irradiation).

[0129] Figure 4 To investigate the tolerance of proteins of different molecular weights on PVDF membranes to SDS elution buffer. After transferring protein markers onto PVDF membranes, the membranes were treated with strong SDS elution buffer for 3, 6, and 12 cycles. The retention and quantification results of standard proteins of different molecular weights on the membranes were observed. Figure 4 A: Process 3 times; Figure 4 B: Processed 6 times; Figure 4 C: Process 12 times.

[0130] Figure 5 To investigate the tolerance of proteins of different molecular weights on PVDF-Dia membranes to SDS elution buffer. After transferring protein markers onto PVDF-Dia membranes, the membranes were treated with strong SDS elution buffer for 3, 6, and 12 cycles. The retention and quantification results of standard proteins of different molecular weights on the membranes were observed. Figure 5 A: Process 3 times; Figure 5 B: Processed 6 times; Figure 5 C: Process 12 times.

[0131] Figure 6 To investigate the tolerance of proteins of different molecular weights on PVDF-Dia membranes to SDS elution buffer after UV irradiation. Protein markers were transferred onto PVDF-Dia membranes, which were then irradiated with 365 nm UV light (total energy 3.378 J / cm², irradiation time 3 minutes). Subsequently, the membranes were treated with strong SDS elution buffer for 3, 6, and 12 cycles, and the retention and quantification results of standard proteins of different molecular weights on the membranes were observed. Figure 6 A: Process 3 times; Figure 6 B: Processed 6 times; Figure 6 C: Process 12 times.

[0132] Figure 7Multiple rounds of antibody elution and retesting were performed using Western blotting. BxPC3 protein from pancreatic cancer cells was transferred to PVDF and PVDF-Dia membranes. The PVDF-Dia membranes were divided into a non-UV-irradiated group (PVDF-DA) and an irradiated group (PVDF-DA + 365nm UV). A total of 12 elution cycles (3 + 3 + 6) were performed, and GAPDH expression was detected by Western blotting to complete multiple rounds of antibody elution and retesting. Figure 7 A: GAPDH expression was detected after a total of 0 elutions; Figure 7 B: GAPDH expression was detected after a total of 3 elutions; Figure 7 C: GAPDH expression was detected after a total of 6 elutions; Figure 7 D: GAPDH expression was detected after a total of 12 washes.

[0133] Figure 8 Multi-target antibody elution and retesting assay using Western blotting. BxPC3 protein from pancreatic cancer cells was transferred to PVDF and PVDF-Dia membranes. The PVDF-Dia membranes were divided into a non-UV-irradiated group (PVDF-DA) and an irradiated group (PVDF-DA + 365nm UV). The membranes were eluted a total of 12 times (3 + 3 + 6 times). GAPDH and Notch2 protein expression was detected by Western blotting, completing multiple rounds of antibody elution and retesting targeting Notch2 and GAPDH. Figure 8 A: Notch2 expression was detected after a total of 0 washes; Figure 8 B: GAPDH expression was detected after a total of 3 elutions; Figure 8 C: Notch2 expression was detected after a total of 6 washes; Figure 8 D: GAPDH expression was detected after a total of 12 washes. Detailed Implementation

[0134] Example 1: Preparation of diazirine-modified PVDF membrane

[0135] Step 1: Amine modification of PVDF membrane

[0136] PVDF membranes (0.45 μm pore size; prepared by our company) were cut into 7 × 8 cm pieces and immersed in 0.2 M KOH solution at 25°C for 12 h. After immersion, the membranes were rinsed with ultrapure water and then dried at room temperature. They were then immersed in methanol for 1 min. To modify surface properties, the PVDF membranes were immersed in a 2% v / v solution of 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich, catalog number 706493) in toluene at 40°C for 1 hour. The samples were then removed from the APTES solution, washed several times with toluene and isopropanol, and air-dried. Finally, they were baked at 60°C for 1 hour. The prepared membrane is called amino-modified PVDF (denoted as PVDF-amine).

[0137] Each group of membranes underwent a ninhydrin reaction to determine the aminoninhydrin reaction on the PVDF surface. The experimental procedure was as follows: A small piece of PVDF-amine membrane was immersed in 1.5 mL of ethanol, and then 30 μL of freshly prepared 1% ninhydrin ethanol solution was added to the membrane surface. The membrane and ninhydrin solution were heated at 100℃ for 5 minutes, and the color change was observed. A piece of original PVDF membrane was treated in the same way as a negative control.

[0138] The results are as follows Figure 2 As shown, this indicates that the PVDF-amine membrane surface was successfully modified with amino groups.

[0139] Step 2: Preparation of diazirine-modified PVDF membrane

[0140] 10 mM NHS-diazirine (C9H) was prepared using DMSO as a solvent. 11 N3O4; Sigma-Aldrich, catalog number 803413. NHS diazirine was added to PBS (Phosphate-buffered saline, 0.1 M sodium phosphate, 0.15 M sodium chloride; pH 7.2) to a final concentration of 0.5 mM. PVDF-amine (7 × 8 cm) was immersed in the PBS containing NHS-diazirine and incubated at room temperature for 30 min. Then, Quenching Buffer (1 M Tris•HCl, pH 8.0) was added to a final Tris•HCl concentration of 10 mM. The membrane was then washed three times with 10 ml of PBS buffer. After drying, the membrane was stored away from light. The prepared membrane is called PVDF-diazirine (PVDF-Dia). Figure 1 ).

[0141] XPS measurements were performed on PVDF and PVDF-Dia films using a PHI VersaProbe III X-ray photoelectron spectroscopy system. The results are as follows: Figure 2 As shown in Figure C, the modified membrane has a lower fluorine content and new peaks appear, such as those for N, Si, and O.

[0142] Example 2: Western blotting analysis of membrane proteins

[0143] 2-1: Cell Culture

[0144] Human pancreatic BxPC3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia) and cultured in DMEM medium containing 10% fetal bovine serum (Life Technologies, Gaithersburg, Maryland). Cells were placed in 75 cm² culture flasks and cultured in a humidified incubator at 37°C with 5% CO2. Cells were passaged when they reached 80% confluence.

[0145] 2-2: Western blotting analysis of membrane proteins

[0146] After processing, remove the culture medium from BxPC3 cells and wash twice with pre-cooled PBS. Add RIPA lysis buffer (0.05 mM Tris-HCl, 50 mM β-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) to evenly cover the cells. Incubate on ice for 5 minutes, then scrape the cells from the culture plate using a cell scraper and transfer them to 1.5 mL EP tubes. Incubate the EP tubes on ice for 30 minutes, shaking every 5 minutes to ensure complete cell lysis. Centrifuge at 12000 g for 20 minutes at 4°C, and transfer the supernatant to a new EP tube to obtain total cellular protein. The extracted total cellular protein can be stored at -80°C. Protein concentration was determined using the BCA method. After adding 50 μg of protein sample to an EP tube, add 5×SDS Loading Buffer, boil in a 95°C metal bath for 5 minutes, cool, and store at -20°C or perform SDS-PAGE experiments.

[0147] 50 μg of protein sample was loaded onto an SDS-PAGE gel for electrophoresis separation. The sample was then transferred to PVDF or surface-modified PVDF and blocked with 5% skim milk for 1 hour. The membrane was incubated overnight at 4°C with anti-GAPDH antibody (1:5000 dilution, Sigma-Aldrich, St. Louis, Missouri) or mouse anti-human Notch2 antibody (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, California). The membrane was then washed four times at room temperature (5 minutes each time) with TBST buffer, followed by incubation for 60 minutes at room temperature with horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG secondary antibody (1:5000 dilution, Zymed, San Francisco, California). Finally, after four washes with TBST buffer (10 mM Tris.HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5), the protein bands were visualized using an enhanced chemiluminescence detection system (GeneSafeSci-tech, Beijing, China). Chemiluminescence detection was performed using an AI600 chemiluminescence imager (GE, USA).

[0148] Example 3: Covalent crosslinking stability experiment

[0149] Before the transfer, the transfer buffer needs to be prepared in advance (this product is compatible with various transfer buffers such as Tris-Gly transfer buffer and CAPS transfer buffer), and methanol needs to be prepared in advance; commonly used transfer equipment in protein blotting (transfer clips, sponges, transfer filter paper, etc.) are required.

[0150] Place a sponge and transfer filter paper on the transfer clamp, completely saturate them with transfer buffer, and remove any air bubbles from the sponge and filter paper. Carefully remove the electrophoretically extracted polyacrylamide gel and place it on the transfer filter paper (ensuring no air bubbles between the gel and the filter paper). Remove the transfer membrane (cut to the appropriate size as needed) and immerse it in methanol for 1 minute. Remove the membrane and place it on the polyacrylamide gel (ensuring no air bubbles between the membrane and the gel). Cover the transfer membrane with transfer filter paper and a sponge (pre-wetted with transfer buffer), and close the transfer clamp. Place the transfer clamp into the transfer electrophoresis tank (Bio-Rad, Miniprotean, catalog number: 1658005; to maintain a low temperature, it is recommended to cover the outside of the transfer electrophoresis tank with ice). Fill with transfer buffer and perform the transfer. Recommended transfer conditions: 250mA, 110 minutes.

[0151] After the transfer is complete, the covalently transferred film is removed and placed in a crosslinker (JYA UV cross-linker, UV365-80W, Beijing, China). Figure 3Place the metal separator (as shown in A) on the upper layer of the crosslinking instrument (5cm away from the UV lamp), adjust the power to 40% (32W), and set the time to 3 minutes. Figure 3 A). After crosslinking is complete, remove the covalent transfer film and soak it in methanol for 1 minute.

[0152] To observe whether photosensitive modification covalently binds to transferred proteins, experiments were conducted using PVDF membranes and PVDF-Dia membranes. For ease of observation, the protein marker was transferred to the membrane and then irradiated with UV 365 (the control group was not irradiated). Generally, membrane regeneration is performed using 20% ​​hydrogen peroxide or 1%-2% SDS. This invention employed more intense conditions: antibody stripping and membrane regeneration were performed at 70°C for 30 minutes using SDS stripping buffer (5% SDS in PBS buffer). The membranes were subjected to 1-12 regeneration cycles, and the degree of protein reduction from the membrane was observed.

[0153] To investigate the stability of proteins transported to membranes, protein markers were transferred onto both standard PVDF and PVDF-Dia membranes. For the PVDF-Dia membranes, both non-irradiated and irradiated treatments were performed. The standard PVDF membrane group, the unirradiated PVDF-Dia membrane group, and the irradiated PVDF-Dia membrane group were designated as Group A, Group B, and Group C, respectively. The following procedure constituted one stripping reaction: protein stripping experiments were performed using 5% SDS solution (or SDS stripping buffer), with each stripping condition set at 70°C for 30 minutes, followed by washing with TBST for 5 minutes each time, three times. The retention and quantification results of standard proteins of different molecular weights on the membranes were observed.

[0154] Experimental results:

[0155] (1) Results of protein stability on ordinary PVDF membranes are as follows Figure 4 As shown:

[0156] (i) If Figure 4 Results A show that after three stripping treatments, the proteins on the PVDF membrane were almost completely eluted. Approximately 20% of proteins above 100kD and 15% of proteins above 130kD were not stripped. 95% of proteins below 70kD were stripped.

[0157] (ii) To further investigate the stability of different protein binding processes, these membranes were further peeled three more times using SDS stripping solution, for a total of six peeling operations. For example... Figure 4Results B showed that after six elutions, the elution of proteins on the membrane was similar to that after three elutions.

[0158] (iii) Based on this, an additional 6 stripping processes were performed using SDS stripping solution, for a total of 12 processes. For example... Figure 4 The results showed that after 12 treatments, almost all the proteins on the ordinary PVDF membrane were removed, with only about 10% of the 100kD proteins remaining.

[0159] (2) The stability results of proteins on PVDF-Dia membranes without 365 nm irradiation are as follows: Figure 5 As shown:

[0160] (i) If Figure 5 Results A showed that after three stripping cycles, approximately 65% ​​of proteins with molecular weights between 100kD and 130kD were stripped, while 35% remained on the membrane. After three treatments with SDS stripping buffer, 80%, 90%, and 98% of proteins with molecular weights of 70kD, 50kD, and 35kD were stripped, respectively.

[0161] (ii) To further investigate the stability of different protein binding processes, these membranes were further peeled three more times using SDS stripping solution, for a total of six peeling operations. For example... Figure 5 The results showed that after six elutions, the elution of proteins on the membrane was similar to that after three elutions.

[0162] (iii) Based on this, an additional 6 stripping processes were performed using SDS stripping solution, for a total of 12 processes. For example... Figure 5 The results show that, without 365 nm irradiation, approximately 20% and 15% of proteins with molecular weights of 100 kD and 130 kD remained on the PVDF-Dia membrane after 12 stripping cycles. For proteins with molecular weights below 70 kD, over 90% were stripped after 12 SDS stripping cycles, leaving less than 10% of the protein on the membrane.

[0163] (3) The stability results of proteins on the PVDF-Dia membrane irradiated at 365 nm are as follows: Figure 6 As shown:

[0164] (i) If Figure 6 As shown in result A, after three stripping operations, no significant changes were observed in the elution of proteins of molecular weights from 15kD to 130kD in the PVDF-Dia membrane irradiated with 365 nm UV, with less than 5% of the proteins being stripped.

[0165] (ii) To further investigate the stability of different protein binding, these membranes were peeled three more times with SDS stripping solution, for a total of six peeling operations. Figure 6 Results B showed that after six elutions, the elution of proteins on the membrane was similar to that after three elutions.

[0166] (iii) Based on this, an additional 6 stripping processes were performed using SDS stripping solution, for a total of 12 processes. For example... Figure 6 The results (C) show that after 12 treatments, over 90% of the proteins on the PVDF-Dia membrane irradiated with 365 nm UV were not stripped and remained on the membrane. This indicates that most proteins, after 365 nm irradiation, are covalently bound to the PVDF membrane and can withstand 12 SDS stripping treatments. This binding mode is resistant to SDS stripping elution.

[0167] The results show that the binding affinity of PVDF membranes, PVDF-Dia membranes, and proteins of different molecular weights varies. On ordinary PVDF membranes, proteins with molecular weights above 100 kDa bind more strongly than those below 100 kDa. On PVDF-Dia membranes without 365 nm irradiation, approximately 33% of proteins with molecular weights between 30 kDa and 130 kDa remain on the membrane after three elutions, while proteins smaller than 30 kDa bind very little to PVDF-Dia. On PVDF-Dia membranes irradiated with 365 nm, proteins with molecular weights from 15 kDa to 130 kDa are covalently bound to the membrane and are resistant to elution. However, most proteins on ordinary PVDF membranes or PVDF-Dia membranes without 365 nm UV irradiation can only withstand about three elutions. Therefore, it is necessary to regenerate and repeatedly measure most proteins using 365 nm UV irradiation after transferring proteins onto PVDF-Dia membranes.

[0168] Example 4: Repeated determination of GAPDH protein using PVDF-Dia membrane and protein blotting

[0169] Before the transfer, the transfer buffer needs to be prepared in advance (this product is compatible with various transfer buffers such as Tris-Gly transfer buffer and CAPS transfer buffer), and methanol needs to be prepared in advance; commonly used transfer equipment in protein blotting (transfer clips, sponges, transfer filter paper, etc.) are required.

[0170] Place a sponge and transfer filter paper on the transfer clamp, completely wet them with transfer buffer, and remove any air bubbles from the sponge and filter paper. Carefully remove the electrophoretically extracted polyacrylamide gel and place it on the transfer filter paper (ensuring no air bubbles between the gel and the filter paper). Remove the transfer membrane (cut to the appropriate size as needed) and immerse it in methanol for 1 minute. Remove the membrane and place it on the polyacrylamide gel (ensuring no air bubbles between the membrane and the gel). Cover the transfer membrane with transfer filter paper and a sponge (pre-wetted with transfer buffer), and close the transfer clamp. Place the transfer clamp into the transfer electrophoresis tank (to maintain a low temperature, it is recommended to cover the outside of the transfer electrophoresis tank with ice), fill with transfer buffer, and perform the transfer (recommended transfer conditions: 250mA, 110 minutes).

[0171] After the transfer was complete, the covalent transfer membrane was removed and placed on the metal separator of a crosslinker (JYA UV cross-linker, UV365-80W, Beijing, China). The metal separator was placed on the upper layer of the crosslinker (5 cm away from the UV lamp), the power was adjusted to 40%, and the time was 3 minutes. After crosslinking, the covalent transfer membrane was removed and soaked in methanol for 1 minute. The covalent transfer membrane was then removed and can be used for subsequent blocking and GAPDH antibody incubation. Precautions: When performing crosslinking of the transfer membrane, the protein-containing side of the membrane should face upwards; please wear a lab coat and disposable gloves during the operation. In this example, both ordinary PVDF and PVDF-Dia without UV irradiation were used for Western blotting.

[0172] After transfer, wash the membrane three times with 10 ml of 1×TBST at room temperature for 5 minutes each time. Add 5 ml of milk powder blocking buffer (5% skim milk powder, TBST) and incubate at room temperature for 1 h. Then wash the membrane three times with 10 ml of TBST for 5 minutes each time. Dissolve the primary antibody in TBST containing 5% skim milk powder (GAPDH, 1:2000). Add 5 ml of primary antibody buffer and incubate at room temperature for 1 h (GAPDH). Wash the membrane three times with 10 ml of TBST for 5 minutes each time. Add secondary antibody containing HRP (1:2000 diluted in TBST containing 5% skim milk powder), and gently shake at room temperature for 1 h. Wash the membrane three times with 10 ml of TBST for 5 minutes each time. After adding the ultrasensitive luminescent solution, perform the assay using a chemiluminescence analyzer.

[0173] The membrane was then regenerated 3-12 times and incubated again with anti-GAPDH primary antibody and HRP-labeled secondary antibody to observe the degree of reduction of GAPDH protein from the membrane. The following procedure was considered a single stripping reaction: protein stripping experiments were performed using 5% SDS solution (or SDS stripping buffer), with each stripping condition set at 70°C for 30 minutes, followed by washing with TBST for 5 minutes each time, 3 times. The retention of GAPDH protein on the membrane was then observed.

[0174] Experimental results are as follows Figure 7 As shown:

[0175] After three elutions with SDS stripping solution, 90% of the 34kD GAPDH protein on the ordinary PVDF membrane was stripped, 80% of the GAPDH on the PVDF-Dia membrane without 365nm irradiation was stripped, and almost no GAPDH was stripped on the PVDF-Dia membrane irradiated with 365nm irradiation. Figure 7 B).

[0176] After six treatments with SDS stripping solution, 95% and 90% of the 34kD GAPDH protein on the ordinary PVDF membrane and PVDF-Dia membrane, respectively, were stripped away, almost completely eluted. However, the GAPDH on the PVDF-Dia membrane irradiated at 365 nm was hardly stripped away. Figure 7 C).

[0177] After a total of 12 SDS elutions, the protein markers on the ordinary PVDF membrane were almost completely lost. The markers on the PVDF-Dia membrane without 365 nm irradiation also disappeared, and the GAPDH band decreased to 10% of its original size. However, the markers on the PVDF-Dia membrane after 365 nm irradiation remained clear, and the GAPDH band showed no significant attenuation. Figure 7 D).

[0178] The results show that the PVDF-Dia membrane irradiated with 365 nm can withstand at least 12 SDS stripping solutions without significant signal attenuation, and can be used for multiple regeneration measurements.

[0179] Example 5: Repeated determination of GAPDH and Notch proteins using PVDF-Dia membrane and protein blotting.

[0180] Before the transfer, the transfer buffer needs to be prepared in advance (this product is compatible with various transfer buffers such as Tris-Gly transfer buffer and CAPS transfer buffer), and methanol needs to be prepared in advance; commonly used transfer equipment in protein blotting (transfer clips, sponges, transfer filter paper, etc.) are required.

[0181] Place a sponge and transfer filter paper on the transfer clamp, completely wet them with transfer buffer, and remove any air bubbles from the sponge and filter paper. Carefully remove the electrophoretically extracted polyacrylamide gel and place it on the transfer filter paper (ensuring no air bubbles between the gel and the filter paper). Remove the transfer membrane (cut to the appropriate size as needed) and immerse it in methanol for 1 minute. Remove the membrane and place it on the polyacrylamide gel (ensuring no air bubbles between the membrane and the gel). Cover the transfer membrane with transfer filter paper and a sponge (pre-wetted with transfer buffer), and close the transfer clamp. Place the transfer clamp into the transfer electrophoresis tank (to maintain a low temperature, it is recommended to cover the outside of the transfer electrophoresis tank with ice), fill with transfer buffer, and perform the transfer (recommended transfer conditions: 250mA, 110 minutes).

[0182] After the transfer was complete, the covalent transfer membrane was removed and placed on the metal separator of a crosslinker (JYA UV cross-linker, UV365-80W, Beijing, China). The metal separator was placed on the upper layer of the crosslinker (5 cm away from the UV lamp), the power was adjusted to 40%, and the time was 3 minutes. After crosslinking, the covalent transfer membrane was removed and soaked in methanol for 1 minute. The covalent transfer membrane was then removed and can be used for subsequent blocking and GAPDH antibody incubation. Precautions: When performing crosslinking of the transfer membrane, the protein-containing side of the membrane should face upwards; please wear a lab coat and disposable gloves during the operation. In this example, both ordinary PVDF and PVDF-Dia without UV irradiation were used for Western blotting.

[0183] After transfer, wash the membrane three times with 10 ml of 1×TBST for 5 minutes each time at room temperature. Add 5 ml of milk powder blocking buffer (5% skim milk powder, TBST) and incubate at room temperature for 1 h. Then wash the membrane three times with 10 ml of TBST for 5 minutes each time. Dissolve the primary antibody in TBST containing 5% skim milk powder (anti-GAPDH, 1:2000; anti-Notch2, 1:1000). Add 5 ml of primary antibody buffer and incubate at room temperature for 1 h (anti-GAPDH antibody) or at 4°C with gentle shaking overnight (anti-Notch2 antibody). Wash the membrane three times with 10 ml of TBST for 5 minutes each time. Add the secondary antibody containing HRP (1:2000 diluted in TBST containing 5% skim milk powder) and gently shake at room temperature for 1 h. Wash the membrane three times with 10 ml of TBST for 5 minutes each time. After adding the ultrasensitive luminescent solution, perform the assay using a chemiluminescence analyzer.

[0184] The membrane was then regenerated 3-12 times, and incubated with primary antibody against anti-GAPDH and secondary antibody with HRP, and primary antibody against anti-Notch2 and secondary antibody with HRP, respectively. The degree of reduction of GAPDH and Notch2 proteins from the membrane was observed. The following procedure was considered as one stripping reaction: protein stripping experiments were performed using 5% SDS solution (or SDS stripping buffer), with each stripping condition set at 70°C for 30 minutes, followed by washing the membrane with TBST for 5 minutes each time, 3 times. The retention of GAPDH and Notch2 proteins on the membrane was observed.

[0185] Experimental results are as follows Figure 8 As shown:

[0186] After three elutions with SDS stripping solution, 90% of the 34kD GAPDH protein on the ordinary PVDF membrane was stripped, 80% of the GAPDH on the PVDF-Dia membrane without 365nm irradiation was stripped, while almost no GAPDH was stripped from the PVDF-Dia membrane irradiated with 365nm irradiation. Figure 8 B).

[0187] After six treatments with SDS stripping solution, 95% and 90% of the 34kD GAPDH protein on the ordinary PVDF membrane and PVDF-Dia membrane, respectively, were stripped off, almost completely eluted. However, Notch2 on the PVDF-Dia membrane irradiated at 365 nm was hardly stripped off. Figure 8 C).

[0188] After a total of 12 SDS elutions, the protein markers on the ordinary PVDF membrane were almost completely lost. The markers on the PVDF-Dia membrane without 365 nm irradiation also disappeared, and the GAPDH band decreased to 10% of its original size. However, the markers on the PVDF-Dia membrane after 365 nm irradiation remained clear, and the GAPDH band showed no significant attenuation. Figure 8 D).

[0189] The results show that the PVDF-Dia membrane irradiated with 365 nm can withstand at least 12 SDS stripping solutions without significant signal attenuation, and can be used for multiple regeneration measurements.

Claims

1. A chemically modified polyvinylidene fluoride membrane, said membrane being surface modified with a group represented by Formula I, Formula I wherein R1is selected from the group consisting of C1-C6 alkyl, fluoro C1-C6 alkyl; L1is selected from wherein m is selected from an integer from 1 to 6, wherein the position marked 1 indicates the attachment to the ring carbon atom in formula I, and the position marked 2 indicates the attachment to L2; L2is selected from wherein n is selected from an integer from 1 to 6, wherein the index 1 position indicates attachment to the index 2 position in L1, and the O atom to which Si is attached is attached to the surface of the polyvinylidene fluoride membrane.

2. The chemically modified polyvinylidene fluoride membrane of claim 1, wherein, n in said L2is selected from an integer from 1 to 3.

3. The chemically modified polyvinylidene fluoride membrane of claim 1, wherein, n in said L2is selected from 2.

4. The chemically modified polyvinylidene fluoride membrane of any one of claims 1-3, wherein, said chemically modified polyvinylidene fluoride membrane is selected from one or more of the following: (a) R1is selected from the group consisting of methyl; or (b) L1is selected from .

5. A method of preparing the chemically modified polyvinylidene fluoride membrane of any one of claims 1-4, comprising the steps of: (i) treating a polyvinylidene fluoride membrane with a basic solution to obtain an oxidized polyvinylidene fluoride membrane; (ii) treating said oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution to obtain an amino-modified polyvinylidene fluoride membrane; and (iii) treating said amino-modified polyvinylidene fluoride membrane with a compound represented by Formula I’ to obtain a chemically modified polyvinylidene fluoride membrane; wherein R1is selected from the group consisting of C1-C6 alkyl, fluoro C1-C6 alkyl; Formula I' said compound represented by Formula I’ is selected from one or more of the following: R2is selected from wherein m is selected from an integer from 1-6.

6. The method of claim 5, wherein, (a) R1is selected from the group consisting of methyl; or said treating a polyvinylidene fluoride membrane with a basic solution in step (i) is selected from the following: (b) R2is selected from .

7. The method of claim 5, wherein, immersing said polyvinylidene fluoride membrane into said basic solution and soaking at 20 °C-60 °C for 1-10 h. said treating a polyvinylidene fluoride membrane with a basic solution in step (i) is selected from the following:

8. The method of claim 5, wherein, immersing said polyvinylidene fluoride membrane into said basic solution and soaking at 25 °C for 2 h. said basic solution in step (i) is selected from potassium hydroxide or sodium hydroxide.

9. The method of claim 5, wherein, said basic solution in step (i) is selected from KOH.

10. The method of claim 5, wherein, the concentration of said basic solution in step (i) is 0.1 M-1 M.

11. The method of claim 5, wherein, the concentration of said basic solution in step (i) is 0.2 M.

12. The method of claim 5, wherein, said treating said oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution in step (ii) is selected from the following:

13. The method of claim 5, wherein, immersing said oxidized polyvinylidene fluoride membrane into said siloxane coupling agent solution and soaking at 30 °C-60 °C for 1-2 h. said treating said oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution in step (ii) is selected from the following:

14. The method of claim 5, wherein, immersing said oxidized polyvinylidene fluoride membrane into said siloxane coupling agent solution and soaking at 40 °C for 1 h. said treating said oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution in step (ii) is selected from the following:

15. The method of claim 5, wherein, said siloxane coupling agent is selected from 3-aminopropyl triethoxysilane, 3- aminopropyl trimethoxysilane, N-(2-aminoethyl)-3-aminopropyl trimethoxysilane. said treating said oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution in step (ii) is selected from the following:

16. The method of claim 5, wherein, said siloxane coupling agent is selected from 3-aminopropyl triethoxysilane, said 3- aminopropyl triethoxysilane being dissolved in toluene. said treating said oxidized polyvinylidene fluoride membrane with a siloxane coupling agent solution in step (ii) is selected from the following:

17. The method of claim 5, wherein, ​ The siloxane coupling agent is selected from 3-aminopropyltriethoxysilane, the concentration of the solution of the 3-aminopropyltriethoxysilane is 2% v / v.

18. The method of claim 5, wherein, The treating the amino-modified polyvinylidene fluoride membrane with a compound of Formula I’ in step (iii) is selected from the following: immersing the amino-modified polyvinylidene fluoride membrane into a solution comprising the compound of Formula I’, incubating at 20-40 °C for 10-60 min.

19. The method of claim 5, wherein, The treating the amino-modified polyvinylidene fluoride membrane with a compound of Formula I’ in step (iii) is selected from the following: immersing the amino-modified polyvinylidene fluoride membrane into a solution comprising the compound of Formula I’, incubating at 25 °C for 30 min.

20. The method of claim 5, wherein, The treating the amino-modified polyvinylidene fluoride membrane with a compound of Formula I’ in step (iii) is selected from the following: the final concentration of the compound of Formula I’ is 0.5 mM.

21. A chemically modified polyvinylidene fluoride membrane prepared according to the method of any one of claims 5-20.

22. Use of the chemically modified polyvinylidene fluoride membrane of any one of claims 1-4 or 21 in Western blotting.

23. Use of the chemically modified polyvinylidene fluoride membrane of any one of claims 1-4 or 21 in detecting the presence and / or relative amount of one or more target proteins in a sample.

24. A method of detecting the presence and / or relative amount of a target protein in a sample by Western blotting, comprising the following steps: (1) extracting total proteins from the sample; (2) separating the proteins by gel electrophoresis; (3) transferring the separated proteins onto the chemically modified polyvinylidene fluoride membrane of any one of claims 1-4 or 21 to obtain a transferred membrane; (4) subjecting the transferred membrane to UV irradiation to obtain a covalently crosslinked transferred membrane; (5) incubating the covalently crosslinked transferred membrane with a first set of specific antibodies, comprising: incubating the covalently crosslinked transferred membrane with a first antibody that specifically recognizes and binds to a first target protein, and further incubating with a labeled second antibody that specifically recognizes and binds to the first antibody, and the label provides a measurable signal; and (6) detecting the signal and obtaining a first Western blot membrane, thereby determining the presence and / or relative amount of the first target protein.

25. The method of claim 24, wherein the wavelength of the UV is selected from long wavelength.

26. The method of claim 24, wherein the wavelength of the UV is selected from 330-390 nm.

27. The method of claim 24, wherein the wavelength of the UV is 365 nm. The gel electrophoresis is selected from polyacrylamide gel electrophoresis.

28. The method of claim 24, wherein, Further comprising the following steps:

29. The method of claim 24, wherein, (7) regenerating the first Western blot membrane to remove the first set of specific antibodies, to obtain a regenerated covalently crosslinked transferred membrane; (8) incubating the regenerated covalently crosslinked transferred membrane with a second set of specific antibodies, comprising: incubating the regenerated covalently crosslinked transferred membrane with a second antibody that specifically recognizes and binds to a second target protein, and further incubating with a labeled third antibody that specifically recognizes and binds to the second antibody, and the label provides a measurable signal; and (9) detecting the signal and obtaining a second Western blot membrane, thereby determining the presence and / or relative amount of the second target protein. incubating the regenerated covalently cross-linked transfer membrane with a first antibody that specifically recognizes and binds to the second target protein, further incubating with a labeled second antibody that is capable of specifically recognizing and binding to the first antibody, and the label is capable of providing a measurable signal; (9) detecting the signal and obtaining the second Western blot membrane, thereby determining the presence and / or relative amount of the second target protein; (10) regenerating the (N-1)th Western blot membrane to remove the (N-1)th set of specific antibodies, thereby obtaining a regenerated covalently cross-linked transfer membrane; (11) incubating the regenerated covalently cross-linked transfer membrane with an Nth set of specific antibodies, which comprises: incubating the regenerated covalently cross-linked transfer membrane with a first antibody that specifically recognizes and binds to the Nth target protein, further incubating with a labeled second antibody that is capable of specifically recognizing and binding to the first antibody, and the label is capable of providing a measurable signal; and (12) detecting the signal and obtaining the Nth Western blot membrane, thereby determining the presence and / or relative amount of the Nth target protein; wherein N is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

30. The method of claim 29, wherein, The regeneration process comprises:

31. The method of claim 29, wherein, immersing the Western blot membrane into a stripping buffer selected from a buffer comprising 20% hydrogen peroxide, or a buffer comprising 1-5% sodium dodecyl sulfate. The regeneration process comprises: the regeneration process is performed at 50°C-70°C for 30 minutes.

32. The method of claim 29, wherein, Step (4), step (7), and / or step (10) further comprises a step selected from the group consisting of blocking the covalently cross-linked transfer membrane or the regenerated covalently cross-linked transfer membrane.

33. The method of claim 29, wherein, Step (4), step (7), and / or step (10) further comprises a step selected from the group consisting of incubating the covalently cross-linked transfer membrane or the regenerated covalently cross-linked transfer membrane with a blocking buffer.

34. The method of claim 29, wherein, The measurable signal is detected by visual inspection, chemiluminescence, or fluorescence.

35. The method of claim 24, wherein, The measurable signal is detected by horseradish peroxidase-mediated chemiluminescence.

36. The method of claim 24, wherein, 37. A kit comprising: the chemically modified polyvinylidene fluoride membrane of any one of claims 1-4 or 21.

38. The kit of claim 37, further comprising additional reagents required for Western blotting, said additional reagents selected from the group consisting of: blotting solution, or blotting apparatus.

39. The kit of claim 37, further comprising additional reagents required for Western blotting, said additional reagents selected from the group consisting of: Tris-Gly blotting solution, CAPS blotting solution, blotting clamp, sponge, or blotting filter paper.

40. Use of the kit of any one of claims 37-39 in Western blotting.

41. Use of the kit of any one of claims 37-39 for detecting the presence and / or relative amount of more than one target protein in a sample. ​

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