Method for adsorbing protein, method for quantifying protein using the adsorption method, and method for evaluating proteolytic ability using the quantification method

Abstract

To provide a method for adsorbing dropped protein to a base material having a titanium oxide layer on the surface at a high rate.SOLUTION: A method for adsorbing a protein to a substrate having a titanium oxide layer on a surface thereof, the method comprising: a protein solution dropping step of dropping a protein solution containing a protein onto the titanium oxide layer of the substrate; and a protein adsorption step of adsorbing the protein contained in the dropped protein solution onto the titanium oxide layer, wherein the protein solution contains 0.1 mg to 5 mg / mL of bovine serum albumin, and the substrate onto which the protein solution has been dropped is held for 85 minutes to 480 minutes in the protein adsorption step.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The disclosure of the present application relates to a method for adsorbing proteins onto a substrate having a titanium oxide layer on its surface, a method for quantifying proteins adsorbed onto a substrate having a titanium oxide layer on its surface, and a method for evaluating the protein-decomposing ability of a substrate having a titanium oxide layer on its surface. [Background technology]

[0002] In recent years, infections caused by the novel coronavirus SARS-CoV-2 have had a major impact on our lives. SARS-CoV-2 can be transmitted through droplets or contact. Therefore, to prevent the spread of infection, there is a need to provide material surfaces with antiviral functionality. Titanium oxide, which exhibits high photocatalytic activity, is known to have antiviral properties (see Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 7018154 Summary of the Invention [Problem to be solved by the invention]

[0004] Titanium oxide is relatively inexpensive, has excellent chemical stability, is harmless to the human body, and exhibits properties such as the ability to decompose organic compounds and antibacterial (antiviral) properties. Therefore, it is expected that research and development into titanium oxide will continue to progress. To efficiently evaluate the antibacterial (antiviral) properties of prepared titanium oxide, in other words, its ability to decompose proteins, it is desirable to be able to directly quantify the degree of decomposition of proteins adsorbed to titanium oxide. Furthermore, to facilitate efficient evaluation, it is preferable that most of the proteins dropped onto the prepared titanium oxide are adsorbed, in other words, that the amount of proteins that are not adsorbed onto titanium oxide is small. However, no method is known that can adsorb a high percentage of proteins contained in a protein solution dropped onto titanium oxide.

[0005] The present invention has been disclosed to solve the above-mentioned problems. As a result of extensive research, the present inventors have newly discovered that a high percentage of protein can be adsorbed onto a titanium oxide layer by (1) setting the concentration of bovine serum albumin contained in a protein solution to be dropped onto a titanium oxide layer within a specific range, and (2) maintaining the substrate containing the titanium oxide layer onto which the protein solution has been dropped for a predetermined period of time.

[0006] That is, an object of the disclosure of the present application is to provide a method by which a high percentage of dropped protein can be adsorbed onto a substrate having a titanium oxide layer on its surface. [Means for solving the problem]

[0007] (1) A method for adsorbing a protein onto a substrate having a titanium oxide layer on its surface, comprising: The adsorption method comprises: a protein solution dropping step of dropping a protein solution containing a protein onto the titanium oxide layer of the substrate; a protein adsorption step of adsorbing the protein contained in the dropped protein solution onto the titanium oxide layer; Including, The protein solution contains 0.1 mg to 5 mg / mL of bovine serum albumin, In the protein adsorption step, the substrate onto which the protein solution has been dropped is maintained for 85 to 480 minutes. Adsorption method. (2) The titanium oxide layer contains carbon atoms. The adsorption method according to (1) above. (3) A method for quantifying a protein adsorbed on a substrate having a titanium oxide layer on its surface, comprising: The quantification method comprises: a protein labeling step of reacting the protein with a labeling reagent for detecting the protein adsorbed on the titanium oxide layer; a quantification step of quantifying the labeled protein; Including, The protein adsorbed on the titanium oxide layer is adsorbed by the adsorption method described in (1) or (2) above. Quantification method. (4) before the protein labeling step, a protein recovery step is carried out to recover proteins that are not adsorbed to the titanium oxide layer, The protein labeling step is carried out after the protein recovery step is carried out. The quantification method described in (3) above. (5) an unadsorbed protein labeling step of reacting the unadsorbed protein recovered in the protein recovery step with a labeling reagent for detecting the unadsorbed protein; a quantification step of quantifying the labeled unadsorbed protein; Contains The quantification method described in (4) above. (6) A method for evaluating the protein degradation ability of a substrate having a titanium oxide layer on its surface, comprising: The evaluation method is a first quantification step of performing a first visible light irradiation step of irradiating a substrate having a protein adsorbed thereon with visible light under first conditions before performing the quantification method described in (3) to (5) above, and performing the quantification method described in (3) to (5) above on the substrate that has been subjected to the first visible light irradiation step; a second quantification step of performing a second visible light irradiation step of irradiating the substrate having the protein adsorbed thereon with visible light under second conditions before performing the quantification method described in (3) to (5) above, and performing the quantification method described in (3) to (5) above on the substrate that has been subjected to the second visible light irradiation step; an evaluation step of evaluating the protein-decomposing ability of titanium oxide due to differences in irradiation conditions when performing the visible light irradiation step by comparing the quantification results of the first quantification step and the second quantification step; Contains Evaluation method. (7) The difference between the first condition and the second condition is at least one selected from the group consisting of visible light irradiation time, visible light irradiation intensity, visible light wavelength, and base material temperature during visible light irradiation. The evaluation method described in (6) above. (8) The difference between the first condition and the second condition is the visible light irradiation intensity, One of the first condition and the second condition is a condition in which visible light of a predetermined intensity is irradiated, and the other is a condition in which the sample is kept in a dark place where no visible light is irradiated. The evaluation method described in (7) above. [Effects of the Invention]

[0008] The production and preparation of proteins used in experiments is time-consuming and costly. The protein adsorption method disclosed in this application allows a high percentage of the protein contained in a protein solution dropped onto titanium oxide to be adsorbed onto titanium oxide. Therefore, when evaluating the produced titanium oxide, less protein is wasted in experiments. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram showing an outline of the protein adsorption step in the protein adsorption method of Example 1. As shown in FIG. [Figure 2] FIG. 2 is a graph showing the recovery rate of the fusion protein in the recovery solution in the protein adsorption method of Example 1. [Figure 3] FIG. 3 is a graph showing the recovery rate of the fusion protein in the recovery solution in the protein adsorption method of Example 2. [Figure 4] FIG. 4 is a schematic diagram showing the experimental procedures for the protein quantification method and substrate evaluation method of Example 3. [Figure 5] FIG. 5 is a graph showing the results of the protein quantification method of Example 3. [Figure 6] FIG. 6 is a graph showing the results of examining the protein adsorption capacity of various substrates carried out in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0010] Below, we will explain in detail the method for adsorbing a protein onto a substrate having a titanium oxide layer on its surface (hereinafter, sometimes simply referred to as the "adsorption method"), the method for quantifying a protein adsorbed onto a substrate having a titanium oxide layer on its surface (hereinafter, sometimes simply referred to as the "quantification method"), and the method for evaluating the protein-decomposing ability of a substrate having a titanium oxide layer on its surface (hereinafter, sometimes simply referred to as the "evaluation method"), all of which are disclosed in the present application.

[0011] In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. Furthermore, in this specification, numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") are to be interpreted as indicating numerical values, numerical ranges, and properties that include errors generally accepted in the technical field.

[0012] (Embodiment of Adsorption Method) The adsorption method according to the embodiment is a method for adsorbing a protein onto a substrate having a titanium oxide layer on its surface. a protein solution dropping step of dropping a protein solution containing a protein onto the titanium oxide layer on the surface of the substrate; a protein adsorption step of adsorbing proteins contained in the dropped protein solution onto the titanium oxide layer; Includes:

[0013] It is known that titanium oxide has proteolytic properties such as antiviral and antibacterial properties due to its photocatalytic function. The titanium oxide layer disclosed in the present application is not particularly limited as long as it is titanium oxide with known proteolytic properties. The titanium oxide layer may be formed solely from titanium oxide, or may be doped with a metal element having antiviral properties as needed. Examples of such metal elements include, but are not limited to, copper, silver, gold, iron, aluminum, zinc, and zirconium. Furthermore, as shown in the examples below, the titanium oxide layer may be doped with carbon atoms, nitrogen atoms, sulfur, and the like.

[0014] The substrate is not particularly limited as long as a titanium oxide layer can be formed on its surface. Examples include, but are not limited to, titanium, glass, copper, aluminum, stainless steel, and other metal substrates. When titanium is used as the substrate, a titanium oxide layer can be formed on the titanium surface by oxidation treatment. When a substrate other than titanium is used, a titanium layer can be first formed on the substrate surface and then oxidized to form a titanium oxide layer. A titanium oxide layer can also be directly formed on any substrate by a film formation process such as chemical vapor deposition (CVD) or physical vapor deposition (PVD). Titanium oxide can also be powdered and used as a white paint or white colorant. Therefore, the titanium oxide to be evaluated can be powdered and then coated onto a substrate with a solvent. In this case, the substrate is not particularly limited as long as it can be coated with titanium oxide powder. Examples include, in addition to the above substrates, fibers, cloth, and resin films. The surface of titanium oxide powder or titanium oxide bulk can also be used.

[0015] The protein is not particularly limited as long as it can be adsorbed to titanium oxide. Examples include spike proteins and nucleocapsid proteins of coronaviruses, and hemagglutinin and neuraminidase of influenza viruses. The protein is not limited to proteins derived from viruses or pathogenic microorganisms, and may be modified proteins such as bone collagen proteins and extracellular matrix glycoproteins such as fibronectin. Furthermore, the protein may be a naturally occurring protein or a fragment thereof, or a protein not found in nature, such as a synthetic protein or a fusion protein formed by fusing any protein.

[0016] The protein solution dripped in the protein solution dripping step contains bovine serum albumin (BSA). BSA is used to stabilize the protein in the protein solution. Therefore, if the BSA concentration in the protein solution is too low, the protein in the protein solution will be difficult to stabilize. The lower limit of the BSA concentration in the protein solution is not limited, but examples include 0.1 mg or more, 0.2 mg or more, 0.3 mg or more, 0.4 mg or more, 0.5 mg or more, 0.6 mg or more, 0.7 mg or more, 0.8 mg or more, 0.9 mg or more, 1.0 mg or more, 1.1 mg or more, 1.2 mg or more, 1.3 mg or more, 1.4 mg or more, and 1.5 mg or more per mL. On the other hand, if the BSA concentration is too high, a large amount of protein will not be adsorbed to the titanium oxide layer, which may result in waste of the prepared protein. Examples of upper limits for the BSA concentration in a protein solution include, but are not limited to, 5.0 mg or less, 4.9 mg or less, 4.8 mg or less, 4.7 mg or less, 4.6 mg or less, 4.5 mg or less, 4.4 mg or less, 4.3 mg or less, 4.2 mg or less, 4.1 mg or less, 4.0 mg or less, 3.9 mg or less, 3.8 mg or less, 3.7 mg or less, 3.6 mg or less, and 3.5 mg or less per mL. The above values ​​are shown to one decimal point. Alternatively, values ​​to two decimal points may be used. Values ​​between 1.5 mg and 3.5 mg and values ​​to two decimal points are omitted, but when specifying the upper and lower limits, values ​​within the omitted range may be selected. Furthermore, using exemplified numerical values ​​and omitted ranges, the upper and lower limits may be expressed as ranges, for example, 0.1 mg to 5.0 mg / mL, 0.2 mg to 3.05 mg / mL, etc.

[0017] The protein adsorption step is a step in which the substrate onto which the protein solution has been dropped is held in place to allow more protein to be adsorbed onto the substrate. If the time for the protein adsorption step is too short, the dropped protein is less likely to be adsorbed onto the titanium oxide layer. While not limited to, the lower limit of the holding time may be 85 minutes or more, 90 minutes or more, 95 minutes or more, 100 minutes or more, 105 minutes or more, 110 minutes or more, 115 minutes or more, 120 minutes or more, 125 minutes or more, 130 minutes or more, 135 minutes or more, 140 minutes or more, 145 minutes or more, 150 minutes or more, 155 minutes or more, 160 minutes or more, 165 minutes or more, 170 minutes or more, 175 minutes or more, 180 minutes or more, or 185 minutes or more. On the other hand, as shown in the examples below, it has been found that if the holding time is too long, the amount of protein that is not adsorbed onto the titanium oxide layer increases. Therefore, although not limited, the upper limit of the retention time is 480 minutes or less, 475 minutes or less, 470 minutes or less, 465 minutes or less, 460 minutes or less, 455 minutes or less, 450 minutes or less, 445 minutes or less, 440 minutes or less, 435 minutes or less, 430 minutes or less, 425 minutes or less, 420 minutes or less, 415 minutes or less, 410 minutes or less, 405 minutes or less, 400 minutes or less, 395 minutes or less, 390 minutes or less. Examples of such values ​​include 385 minutes or less, 380 minutes or less, 375 minutes or less, 370 minutes or less, 365 minutes or less, 360 minutes or less, 355 minutes or less, 350 minutes or less, 345 minutes or less, 340 minutes or less, 335 minutes or less, 330 minutes or less, 325 minutes or less, 320 minutes or less, 315 minutes or less, 310 minutes or less, 305 minutes or less, 300 minutes or less, 295 minutes or less, 290 minutes or less, 285 minutes or less, and 280 minutes or less. While the above values ​​are listed in 5-minute increments for illustrative purposes, the placement and retention time may also be listed in 1-minute increments or to one decimal point. Numerical values ​​between 185 minutes and 280 minutes and values ​​in 1-minute increments and to one decimal point are omitted, but when listing the lower and upper limits, values ​​within the omitted range may be selected. Furthermore, using exemplified numerical values ​​and omitted ranges, the upper and lower limits may be expressed as ranges, for example, 85 to 480 minutes, 100 to 260 minutes, etc. The protein adsorption step may be performed by retaining the substrate onto which the protein solution has been dropped for a predetermined period of time, but may also be performed optionally in an environment less susceptible to the photocatalytic activity of the titanium oxide layer (an environment with low light intensity).

[0018] The adsorption method according to the embodiment has the following advantages. By combining (1) setting the BSA concentration in the protein solution dropped in the protein dropping step to 0.1 mg to 5 mg / mL and (2) holding the substrate containing the titanium oxide layer onto which the protein solution has been dropped for 85 to 480 minutes in the protein adsorption step, a high percentage of the dropped protein can be adsorbed onto the titanium oxide layer. This reduces the amount of protein that does not adsorb to the titanium oxide layer, reducing waste of prepared protein and enabling efficient experiments. (2) The adsorption method according to the embodiment can efficiently adsorb protein-related viruses, cells, and bacteria onto a titanium oxide layer. Therefore, the adsorption method disclosed in the present application is expected to be used as a removal method for viruses, cells, and bacteria from a solution.

[0019] (Embodiment of Quantitation Method) The quantitative determination method according to the embodiment is a method for quantifying a protein adsorbed on a substrate having a titanium oxide layer on its surface. a protein labeling step of reacting a labeling reagent for detecting the protein adsorbed on the titanium oxide layer with the protein; a quantification step of quantifying the labeled protein; Includes: The protein adsorbed on the titanium oxide layer was adsorbed by the adsorption method according to the embodiment described above.

[0020] The quantification method is not particularly limited as long as it can quantify the protein adsorbed to the titanium oxide layer. It is possible, but not limited to, to use an enzyme-labeled antibody that specifically recognizes the protein adsorbed to the titanium oxide layer as a labeling reagent in the protein labeling step, add a substrate that emits fluorescence upon reacting with the labeled enzyme in the quantification step, and quantify the amount of protein adsorbed to the titanium oxide layer based on the fluorescence intensity. Alternatively, instead of using an enzyme-labeled antibody that specifically recognizes the protein adsorbed to the titanium oxide layer, a primary antibody that specifically recognizes the protein adsorbed to the titanium oxide layer may first be adsorbed to the protein, and then a secondary antibody that specifically recognizes the primary antibody may be enzyme-labeled. Alternatively, an organic compound-type fluorescent label may be used instead of the enzyme label.

[0021] The quantification method according to the embodiment has the following advantages. (1) As explained in the embodiment of the adsorption method, the adsorption method disclosed in the present application reduces waste of prepared protein. Therefore, when carrying out a quantification method, waste of prepared samples can also be reduced. (2) Proteins adsorbed to the titanium oxide layer can be quantified in the adsorbed state. In principle, it is possible to detach the adsorbed protein from the titanium oxide layer and quantify the detached protein, but this requires additional steps for quantification. The quantification method disclosed in this application simplifies the quantification process because it can directly quantify the amount of protein in the state where the protein is adsorbed to the titanium oxide layer. (3) Proteins present in a liquid and viruses, cells, and bacteria containing the proteins can be recovered and quantified at a high rate. Therefore, the quantification method disclosed in the present application is expected to be used to confirm whether or not proteins and viruses, cells, and bacteria containing the proteins are present in a solution.

[0022] (Optional additional configuration example of quantification method) (Configuration example 1) Next, optional configuration examples in which the quantification method according to the embodiment can be employed will be described. In configuration example 1, the quantification method may include a protein recovery step of recovering proteins not adsorbed to the titanium oxide layer before the protein labeling step, and the protein labeling step may be performed after the protein recovery step. The protein recovery step may involve, for example, immersing the substrate after the protein adsorption step in a buffer solution or the like and stirring the solution to separate unadsorbed proteins not adsorbed to the titanium oxide layer.

[0023] When the quantification method employs Configuration Example 1, unadsorbed proteins remaining on the substrate are removed, thereby reducing errors when carrying out the quantification step.

[0024] (Configuration example 2) In the second configuration example, the quantification method is as follows: an unadsorbed protein labeling step in which a labeling reagent for detecting the unadsorbed protein recovered in the protein recovery step described in Configuration Example 1 is reacted with the unadsorbed protein; a quantification step of quantifying the labeled unadsorbed protein; In Configuration Example 2, the unadsorbed protein may be quantified using the same procedure as in the quantification method according to the embodiment, except that the recovered unadsorbed protein is used instead of the protein adsorbed to the titanium oxide layer.

[0025] As shown in the Examples below, the adsorption method disclosed in the present application allows a high percentage of dropped protein to be adsorbed onto the titanium oxide layer. Therefore, the evaluation method described below can evaluate the decomposition ability of protein adsorbed onto the titanium oxide layer without quantifying unadsorbed protein. However, by also quantifying unadsorbed protein as shown in Configuration Example 2, the protein decomposition ability of the titanium oxide layer can be evaluated over a wide range of conditions, as shown in the Examples below. Note that Configuration Example 2 is an optional configuration, as described above. It is, of course, possible to quantify only the protein adsorbed onto the titanium oxide layer without adopting Configuration Example 2. In this case, the effect of simplifying the evaluation method described below is achieved.

[0026] (Embodiment of Evaluation Method) Next, an evaluation method according to an embodiment will be described. The evaluation method according to an embodiment is a method for evaluating the protein-decomposing ability of a substrate having a titanium oxide layer on its surface. The evaluation method includes the following steps: a first quantification step of performing a first visible light irradiation step of irradiating a substrate having a protein adsorbed thereon with visible light under first conditions before performing the quantification method according to any of the above-described embodiments, and performing the quantification method according to any of the above-described embodiments on the substrate that has been subjected to the first visible light irradiation step; a second quantification step of performing a second visible light irradiation step of irradiating the substrate to which the protein has been adsorbed with visible light under second conditions before performing the quantification method according to any of the above-described embodiments, and performing the quantification method according to any of the above-described embodiments on the substrate that has been subjected to the second visible light irradiation step; an evaluation step of evaluating the protein-decomposing ability of titanium oxide due to differences in irradiation conditions when performing the visible light irradiation step by comparing the quantification results of the first quantification step and the second quantification step; Includes:

[0027] The visible light to be irradiated has a wavelength of 400 nm or longer. The difference between the first and second conditions is not particularly limited as long as it is assumed that the quantification results of the quantification step will differ depending on the irradiation conditions, even for the same substrate. Examples of the difference include, but are not limited to, the visible light irradiation time, the visible light irradiation intensity, the visible light wavelength, and the temperature of the substrate during visible light irradiation.

[0028] When changing the visible light irradiation time, the irradiation time under the second condition can be made longer or shorter than that under the first condition. By changing the visible light irradiation time, the protein degradation ability depending on the length of the visible light irradiation time on the titanium oxide layer can be evaluated.

[0029] When changing the visible light irradiation intensity, the irradiation intensity under the second condition can be made stronger or weaker than the irradiation intensity set under the first condition. Note that weakening the visible light irradiation intensity includes setting the visible light irradiation intensity to zero, in other words, holding the substrate in a dark place where visible light is blocked. By changing the visible light irradiation intensity, the protein degradation ability depending on the intensity of visible light irradiated onto the titanium oxide layer can be evaluated.

[0030] When changing the wavelength of visible light, the wavelength under the second condition can be set longer or shorter than that under the first condition. By changing the wavelength of visible light, the protein degradation ability depending on the wavelength of visible light irradiated onto the titanium oxide layer can be evaluated.

[0031] When changing the temperature of the substrate during visible light irradiation, the temperature under the second condition can be set higher or lower than the temperature set under the first condition. The temperature of the substrate is not particularly limited as long as it can be adjusted; for example, the substrate can be heated by placing it on a hot plate or the like. Alternatively, the substrate can be cooled using a cooling device. By changing the temperature of the substrate during visible light irradiation, the protein degradation ability due to differences in the temperature of the substrate when visible light is irradiated onto the titanium oxide layer can be evaluated.

[0032] The evaluation method according to the embodiment has the following advantages. (1) By carrying out the above-mentioned evaluation method using the adsorption method and quantification method disclosed in the present application, the protein-decomposing ability of the titanium oxide layer can be directly evaluated while the protein is adsorbed on the titanium oxide layer, thereby improving the convenience of evaluating the photocatalytic function of titanium oxide materials.

[0033] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or represent a limitation on the scope of the invention disclosed in the present application. [Example]

[0034] [Protein adsorption method] Example 1 In carrying out the protein adsorption method, we first investigated the BSA concentration in the protein solution. The specific experimental method is shown below.

[0035] [Preparation of a substrate having a titanium oxide layer on its surface] The substrate having the titanium oxide layer (doped with carbon atoms) on its surface, which was used in Example 1, was prepared by the following procedure. <Material> The substrate used was commercially pure (CP) Ti (diameter = 12 mm, thickness = 1 mm, manufactured by UEX Co., Ltd.) The purchased commercially pure (CP) Ti was mirror-polished on one side at Shotoku Seisakusho Co., Ltd. <Production procedure> (1) A Ti(C,O) layer was formed on the surface of the Ti substrate by treating the Ti surface at 800°C for 1 hour in an argon atmosphere containing 1% CO. (2) The substrate prepared in (1) above was treated in an air atmosphere at 400°C for 3 hours to form a titanium oxide layer doped with carbon atoms on the Ti substrate. (3) Analysis of the prepared titanium oxide layer revealed that the film thickness was 0.5 μm and the crystal structure was predominantly anatase type.

[0036] [Protein regulation] The protein used was a fusion protein (RBD-Fc) of the SARS-CoV-2 spike protein (RBD) and mouse immunoglobulin (Fc region) (Spike S1RBD mouse Fc-fusion (SARS-CoV-2) manufactured by BPS).

[0037] [Preparation of protein solution] Bovine serum albumin (BSA, 011-27055, Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted with phosphate-buffered saline (PBS) to concentrations of 0 mg, 1 mg, 5 mg, and 15 mg per mL (BSA-PBS). The fusion protein solution was prepared by adding BSA to each of the BSA-PBS solutions containing 50 ng of the fusion protein (RBD-Fc) to be applied to the titanium oxide layer.

[0038] [Method for adsorbing fusion proteins onto titanium oxide layers] Five microliters of the prepared fusion protein solution was dropped onto the titanium oxide layer on the substrate surface, and a cover glass (borosilicate glass, 10 mm diameter x 0.13-0.17 mm thickness, Matsunami Glass Industry Co., Ltd.) was placed on top. A filter paper soaked with 50 μL of sterilized water for moisture retention, a SiO2 glass spacer, a sample support, and the sample were placed in this order in a Petri dish (see Figure 1), and the sample was subjected to an adsorption treatment (protein adsorption process) in a dark place for 240 minutes.

[0039] [Measurement of recovery rate of fusion protein in recovery solution] After the adsorption treatment, the substrate together with the cover glass was immersed in 2 mL of 10 mg / mL BSA-PBS (hereinafter referred to as the "recovery solution") and stirred with a vortex mixer to peel the cover glass from the substrate. A culture tube (2332-012S, WATSON) was used as the recovery container. The method for quantifying the fusion protein in the recovery solution is as follows.

[0040] [Method for quantifying fusion protein in recovered solution]

[0041] <Reagents for quantification of fusion proteins> Detection antibody: Anti-mIgG-HRP (Peroxidase AffiniPure F(ab')2 Fragment Donkey Anti-Mouse IgG (H+L), 715-036-151, Jackson Immuno Research Laboratories Inc.), an antibody against mouse IgG conjugated with horseradish peroxidase (HRP). ·Substrate: TMB (3,3',5,5'-Tetramethyl benzidine, 1×TMB Substrate Solution, Invitrogen) Capture antibody against RBD: Anti-RBD (Anti-SARS-CoV2 (COVID-19) spike RBD, Rabbit-Mono, GTX635793, Gene Tex)

[0042] <Quantitative method> A 96-well plate (9018, Corning Inc.) was used as the ELISA plate. The capture antibody was diluted to 2.5 μg / mL in PBS, and 50 μL of the solution was added to the well plate and allowed to stand overnight at 4°C for immobilization. The plate was then washed five times with phosphate buffered saline (PBS-T), a surfactant (Tween 20, Polyoxyethylene(20) sorbitan monolaurate) solution used as the washing buffer for the ELISA plate. 200 μL of PBS-T was used per well to wash the plate. Next, 50 mg / mL BSA-PBS was added dropwise to a 200 mL well plate, and blocking was performed at room temperature for 60 minutes. After blocking, the plate was washed five times with PBS-T. RBD-Fc was then diluted to a concentration of 0-0.25 μg / mL with 1 mg / mL BSA-PBS, and 50 μL was added to the well plate. The capture antibody and RBD-Fc were reacted at room temperature for 60 minutes. After the capture antibody reaction, the plate was washed five times with PBS-T. 50 μL of the detection antibody, diluted 5000-fold with PBS, was added dropwise to the well plate, and the RBD-Fc and detection antibody were reacted at room temperature for 60 minutes. After the detection antibody reaction, the plate was washed seven times with PBS-T. Finally, 50 μL of TMB was added dropwise and left to stand for the specified time to induce luminescence, and the luminescence was stopped with 50 μL of 2N H2SO4. Thereafter, absorbance was measured using a microplate reader (Model 680, BIO-RAD).

[0043] The recovery rate of the fusion protein in the recovery solution (calculated as follows: amount of fusion protein not adsorbed to the titanium oxide layer, quantified by the above [Method for quantification of fusion protein in the recovery solution] / 50 ng (amount of fusion protein dropped) × 100) when the BSA concentration in the fusion protein solution was changed is shown in Table 1 and Figure 2. Note that the n number for each BSA concentration was 3. [Table 1]

[0044] As shown in Table 1 and Figure 2, at BSA concentrations of 1 mg / mL or less, the transfer of the fusion protein into the recovery solution was low (less than 2%), whereas at BSA concentrations of 5 mg / mL or more, the recovery rate increased to more than 30%. We confirmed that BSA concentrations of 1 mg / mL or less were effective for adsorption of RBD-Fc to the titanium oxide layer, while BSA concentrations of 5 mg / mL or more were not expected to effectively adsorb the fusion protein. Furthermore, we found that BSA, which functions as a carrier protein without interfering with the 3D structure, is essential for stabilizing the fusion protein in solution and maintaining its antigenicity. Furthermore, considering that a BSA concentration of 0.1 mg / mL or more is required to not interfere with the adsorption of the fusion protein to the substrate, we confirmed that a BSA concentration of approximately 0.1 mg / mL to 5 mg / mL is preferable in the fusion protein solution.

[0045] <Example 2> Next, the influence of the time (adsorption time) during which the adsorption treatment (protein adsorption step) is carried out on the recovery rate of the fusion protein was examined. The recovery rate was measured in the same manner as in Example 1, except that the BSA concentration was 1 mg / mL, the mass of the fusion protein (RBD-Fc) dropped onto the titanium oxide layer was adjusted to 25 ng, 50 ng, or 100 ng, and the adsorption time kept in the dark was changed as shown in Table 2 below. The results are shown in Table 2 and Figure 3. The n value for each experiment was 3.

[0046] [Table 2]

[0047] As shown in Table 2 and Figure 3, when the adsorption time was 85 to 480 minutes, the recovery rate was 10% or less, regardless of the mass of the fusion protein dropped onto the titanium oxide layer. On the other hand, when the adsorption time was extremely short, such as 0 minutes, or extremely long, such as 720 minutes, the recovery rate of the fusion protein exceeded 10%. Therefore, it was confirmed that the adsorption time is preferably approximately 85 to 480 minutes, and more preferably 240 to 480 minutes.

[0048] Furthermore, the results of Examples 1 and 2 lead to the following considerations. (a) From the results shown in Example 1, it can be said that a low BSA concentration of 5 mg / mL or less is preferable for the fusion protein solution. On the other hand, in Example 2, even though the BSA concentration in the fusion protein solution was set to a relatively low concentration of 1 mg / mL, the recovery rate of the fusion protein exceeded 10% when the adsorption time was extremely short, such as 0 minutes, or extremely long, such as 720 minutes. (b) From the results shown in Example 2, it can be said that an adsorption time of 240 minutes is a preferable adsorption time regardless of the concentration of the fusion protein in the fusion protein solution. On the other hand, in Example 1, even though the adsorption time was 240 minutes, the recovery rate of the fusion protein increased as the BSA concentration in the fusion protein solution increased. (c) From the results described in (a) and (b) above, the adsorption method disclosed in the present application requires a combination of (1) setting the concentration of BSA contained in the protein solution dropped onto the titanium oxide layer within a specific range, and (2) setting the protein adsorption time within a specific range.

[0049] [Method for quantifying proteins and evaluating substrates] Example 3 Next, a method for quantifying the amount of fusion protein adsorbed to the titanium oxide layer by the adsorption method described in Example 1 and a method for evaluating the protein degradation ability of the titanium oxide layer were carried out. Figure 4 shows an outline of the experimental procedure, and the details of the experimental procedure are described below. In Example 3, the BSA concentration in the fusion protein solution was 1 mg / mL, the adsorption time was 240 minutes, and the mass of fusion protein dropped onto the titanium oxide layer was 50 ng.

[0050] [Method for quantifying RBD-Fc adsorbed to titanium oxide layer (first quantification step) and (second quantification step)] The reagents used in the first and second quantification steps were the same as the detection antibody and substrate in the <Reagents for Protein Quantification> described in Example 1. After the adsorption treatment described in Example 1, the sample was irradiated with visible light (visible light irradiation intensity I = 1.15 mW / cm 2The samples were then exposed to visible light (irradiation time t = 25-480 min, wavelength > 400 nm) (first quantification step) and kept in the dark (second quantification step). After the visible light irradiation and dark storage, the substrate with the cover glass was immersed in 2 mL of 10 mg / mL BSA-PBS and stirred with a vortex mixer to remove the cover glass from the substrate. Culture tubes (2332-012S, WATSON) were used as collection containers. The substrates were removed from the immersion solution and placed in each well of a 12-well plate (353043, Corning Inc.). The substrates were washed three times with PBS. The substrates were washed with 2 mL of PBS per well. The substrates were then placed in each well of a separate 12-well plate, and blocked with 2 mL of 10 mg / mL BSA-PBS at room temperature for 60 minutes. After blocking, the substrates were washed again with PBS. After washing, 7 μL of the detection antibody (Anti-mIgG-HRP) was directly applied to the substrate surface, and a cover glass (borosilicate glass, 15 mm diameter x 0.13-0.17 mm thickness, Matsunami Glass Industry Co., Ltd.) was placed on top. The substrates were then incubated in the dark at room temperature for 60 minutes. The substrates were then washed to wash away any excess detection antibody that was not bound to the RBD-Fc adsorbed on the substrate. After washing, the substrate was placed in each well of a 24-well plate (353047, Corning Inc.), and 250 μL of TMB was added dropwise. After the reaction time, 50 μL of TMB was transferred to a 96-well plate, and luminescence was quenched with 50 μL of 2N H2SO4. The luminescent solution was then transferred to an ELISA plate, and the absorbance was measured using a microplate reader (Model 680, BIO-RAD). Each experiment had an n of 3.

[0051] "Method for quantifying RBD-Fc in recovered solution" Since this is the same as the [Method for quantifying fusion protein in recovered solution] in Example 1, detailed description will be omitted.

[0052] The experimental results are shown in Figure 5. The vertical axis of Figure 5 shows the amount of fusion protein (RBD-Fc) quantified in Example 3, and the lower the amount below the dotted line (Dropped amount), the more decomposed the fusion protein. As shown in a to c, even when the substrate with the fusion protein adsorbed was kept in the dark for 25, 100, or 240 minutes, almost no decomposition of the fusion protein occurred. On the other hand, as shown in a and b, when the visible light intensity was 15 mW / cm 2 It was confirmed that the fusion protein was decomposed more with increasing irradiation time, even when the visible light intensity was the same as that of the fusion protein. 2 Even though the results were the same as those in Example 1 and 2, we confirmed that the longer the irradiation time, the greater the decomposition of the fusion protein. Note that the 480-minute period in Example d represents an example in which the sample was subjected to 240 minutes of protein adsorption in the dark, followed by an additional 480 minutes of dark storage for comparison with visible light irradiation. In other words, the amount of fusion protein dropped (50 ng), the adsorption time (720 minutes), and the recovery rate (15.02%) in Example 2 are essentially the same. Therefore, in Example d, a considerable amount of fusion protein was also detected in the recovered solution. In Example d, where the sample was kept in the dark, the total amount of fusion protein was calculated by summing the amount of fusion protein on the substrate and the amount of fusion protein in the recovered solution. In Example c, where the sample was kept in the dark, the amount of fusion protein in the recovered solution was also analyzed, but was found to be zero. These results confirm that the protein-decomposing ability of a titanium oxide layer can be evaluated by irradiating the titanium oxide layer with visible light and storing it in the dark, and then directly quantifying the protein adsorbed on the titanium oxide layer. Furthermore, since the adsorption method disclosed in the present application allows most of the dropped protein to be adsorbed onto the titanium oxide layer, if the dark holding time is short, it is not necessary to quantify the protein that has migrated to the recovery solution, and it is sufficient to quantify only the protein that has adsorbed onto the titanium oxide layer, thereby achieving the effect of simplifying the evaluation method.

[0053] Example 4 Next, the protein adsorption ability of the titanium oxide layer, which does not contain carbon atoms, was investigated. <Material> (1) A substrate having a titanium oxide layer (not doped with carbon atoms) on its surface. To fabricate a titanium oxide layer without carbon doping, reactive sputtering was used to achieve high purity. The fabrication method is as follows: A 2-inch dual-target sputtering system (HMS2 300; Hybridge Corporation) was used to fabricate a titanium oxide layer by reactive sputtering. Metallic Ti (purity 99.999%, diameter 50.8 mm, thickness 3 mm; ADVANTEC Corporation) was used as the target. A quartz plate (SiO2, 10 × 10 × 0.5 T The substrate was a 3×10 mm thick (Sendai Quartz Glass Manufacturing Co., Ltd.). The substrate temperature during film formation was 500°C. Ultra-high purity Ar (G1) and ultra-high purity O2 (G1) were used as sputtering gases. The chamber was heated to 3×10 -4 After the pressure was reduced to below 1 Pa, the following operations were performed. Ultra-high purity Ar gas was used to remove dirt and oxide films on the target surface, and pre-sputtering was performed under the conditions of a flow rate of 15 sccm, a total pressure of 2 Pa, an output of 50 W, and a sputtering time of 10 minutes. Ultra-high purity Ar gas and ultra-high purity O2 gas were used to remove O 22 A titanium oxide layer was produced to a thickness of 0.5 μm under the conditions of a flow rate of 0.9 sccm, an Ar flow rate of 14.1 sccm (O2 / O2+Ar=0.06), a total pressure of 0.5 Pa, and an output of 200 W. The crystalline structure was anatase type. (2) For comparison, commercially pure (CP) Ti, which was used as the material in Example 1, and SiO2 (manufactured by Sendai Quartz Glass Manufacturing Co., Ltd.) were used.

[0054] <Experimental Procedure> The fusion protein was adsorbed using the same procedure as in Example 1, except that the BSA concentration in the fusion protein solution was 1 mg / mL and the mass of the fusion protein dropped was 50 ng and 100 nm. The results are shown in Figure 6. The vertical axis in Figure 6 indicates the amount of fusion protein recovered in the recovered solution, and the lower the value below the dotted line (Dropped amount) in Figure 6, the greater the amount of protein adsorbed to the titanium oxide layer. As is clear from Figure 6, with commercially pure titanium and SiO2, more protein migrated into the recovered solution without adsorbing to the titanium oxide layer compared to titanium oxide.

[0055] From the above results, from the viewpoint of evaluating the protein degradation ability of titanium oxide due to its photocatalytic function, the adsorption method disclosed in the present application can adsorb most of the dropped protein onto the titanium oxide layer. Furthermore, since the protein degradation ability of the titanium oxide layer can be evaluated in a state in which the protein is adsorbed, the adsorption method, quantification method, and evaluation method disclosed in the present application are very useful methods from the viewpoint of evaluating the protein degradation ability of titanium oxide. [Industrial Applicability]

[0056] The adsorption, quantification, and evaluation methods disclosed in the present application enable evaluation of the protein-degrading ability of a titanium oxide layer, and are therefore useful in the medical industry.

Claims

1. A method for adsorbing a protein onto a substrate having a titanium oxide layer on its surface, comprising: The adsorption method comprises: a protein solution dropping step of dropping a protein solution containing a protein onto the titanium oxide layer of the substrate; a protein adsorption step of adsorbing the protein contained in the dropped protein solution onto the titanium oxide layer; Including, the protein solution contains 0.1 mg to 5 mg / mL of bovine serum albumin; The protein adsorption step involves holding the substrate onto which the protein solution has been dropped for 85 to 480 minutes. Adsorption method.

2. The titanium oxide layer contains carbon atoms. The adsorption method according to claim 1 .

3. A method for quantifying a protein adsorbed on a substrate having a titanium oxide layer on its surface, comprising: The quantification method comprises: a protein labeling step of reacting the protein with a labeling reagent for detecting the protein adsorbed on the titanium oxide layer; a quantification step of quantifying the labeled protein; Including, The protein adsorbed on the titanium oxide layer is adsorbed by the adsorption method according to claim 1 or 2. Quantification method.

4. a protein recovery step of recovering proteins not adsorbed to the titanium oxide layer is carried out before the protein labeling step; The protein labeling step is carried out after the protein recovery step is carried out. The quantitative method according to claim 3.

5. an unadsorbed protein labeling step of reacting the unadsorbed protein recovered in the protein recovery step with a labeling reagent for detecting the unadsorbed protein; a quantification step of quantifying the labeled unadsorbed protein; Contains The quantitative method according to claim 4.

6. 1. A method for evaluating the protein degradation ability of a substrate having a titanium oxide layer on its surface, comprising: The evaluation method is a first quantification step of irradiating a substrate having adsorbed proteins with visible light under first conditions with a first visible light irradiation step before carrying out the quantification method according to claim 3, and carrying out the quantification method according to claim 3 on the substrate that has been subjected to the first visible light irradiation step; a second quantification step of irradiating a substrate having adsorbed proteins with visible light under second conditions with a second visible light irradiation step before carrying out the quantification method of claim 3, and carrying out the quantification method of claim 3 on the substrate that has been subjected to the second visible light irradiation step; an evaluation step of evaluating the protein-decomposing ability of titanium oxide due to differences in irradiation conditions when performing the visible light irradiation step by comparing the quantification results of the first quantification step and the second quantification step; Contains Evaluation method.

7. The difference between the first condition and the second condition is at least one selected from the group consisting of visible light irradiation time, visible light irradiation intensity, visible light wavelength, and base material temperature during visible light irradiation. The evaluation method according to claim 6.

8. the difference between the first condition and the second condition is the visible light irradiation intensity, One of the first condition and the second condition is a condition in which visible light of a predetermined intensity is irradiated, and the other is a condition in which the sample is kept in a dark place where no visible light is irradiated. The evaluation method according to claim 7.

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