A method for preparing a hydroxyl-modified protein chip

Abstract

The invention provides a method for preparing a hydroxyl-modified protein chip, and belongs to the technical field of protein chip preparation. The protein chip provided by the invention uses a glass slide as a main raw material, and uses a pre-prepared etching solution to perform periodic array etching on the slide; uses cyclosiloxane and epoxide as polymerization raw materials, and uses radiation polymerization to form a covalent connection between the polymerization raw materials and the slide to obtain a protein chip matrix; uses the protein chip matrix to adsorb proteins under physiological conditions, and the protein chip matrix has two different regions, and the region after etching and radiation polymerization not only provides a good water phase environment for the protein, but also can generate weak interactions such as hydrogen bonds with groups on the surface of the protein, while the untreated region is hydrophobic, so the contrast is huge, so that the prepared protein chip has a high signal-to-noise ratio and high sensitivity.

Description

Technical Field

[0001] The present invention is a method for preparing a hydroxyl-modified protein chip, belonging to the technical field of protein chip preparation. Background Art

[0002] Different protein fixation methods are used according to the different uses of protein chips. General protein fixation methods include: passive adsorption, covalent coupling, and affinity binding. Among them, passive adsorption is the simplest method for preparing protein chips: the protein is directly spotted on the surface of a hydrophobic or charged medium, and the protein will be adsorbed on the surface of the medium.

[0003] Lueking et al. used a spotting robot to spot protein solutions onto PVDF filter membranes at high density, and were able to detect specific proteins on the filter membranes with high sensitivity. In the process of analyzing the serum of autoimmune patients for quantitative analysis of autoantibodies, Joos et al. used nitrocellulose membranes and polylysine-coated glass slides to make chips. In addition, nitrocellulose membranes can also be placed on glass slides for chip preparation and subsequent detection and analysis.

[0004] Although polylysine-modified glass slides do not fix proteins through covalent bonds, many protein microarrays still use polylysine-modified glass slides as carriers because polylysine-modified glass slides are widely used as carriers of gene chips, are easy to make, have high signal-to-noise ratios, and have more uniform spot morphology. Angenendt et al. compared a variety of carriers and their surface modification methods and found that the morphology of the spots on polylysine-modified glass slides was more uniform, and the coefficient of variation between spots (11%) was smaller than that of other carriers. However, polylysine-modified glass slides, like other two-dimensional carrier surfaces, have poor sensitivity and a higher minimum detection limit. Haab et al. reported an antibody microarray using polylysine-modified glass slides as carriers to study the interaction between antigens and antibodies, but the tailing phenomenon was obvious when the antibodies were fixed on the glass slide surface, and the distribution of antibodies inside the spots was also uneven, affecting the correctness and accuracy of the results.

[0005] Polyacrylamide gel (3-dimensional polyacrylamide gel) is a three-dimensional surface medium that can provide higher sensitivity, dynamic range and natural conception compared to two-dimensional surfaces. It is based on the principle of polyacrylamide gel electrophoresis. After the acrylamide gel is polymerized, the formed gel can be placed on the surface of a slide. Miller et al. spotted 184 antibodies on a hydrogel-coated slide and a poly-L-lysine-coated slide (with a cross-linking layer). The hydrogel surface has a higher signal-to-noise ratio.

[0006] Passive adsorption is suitable for sensitive protein expression and antibody specificity screening, but because the protein is randomly adsorbed to the surface of the medium, the protein will be partially denatured, quantitative analysis cannot be performed, and the direction of protein binding to the matrix is ​​difficult to control. Different proteins have different adsorption capacities and low signal-to-noise ratios. Due to the weak adsorption capacity of some proteins, some proteins may fall off during the elution process. Due to the different locations and directions of protein fixation on the matrix surface, their biological activities will be different, making the experimental repeatability poor.

[0007] In order to solve the problems caused by the above passive adsorption method, we first designed the array. First, the glass slide was etched. One of the raw materials for radiation polymerization, polysiloxane, the polymerization product of cyclosiloxane, has good biocompatibility and moisture retention, so it can provide a good hydrophilic environment for proteins; and the other raw material, epoxide, forms a large number of hydroxyl groups after radiation polymerization, which forms a dot array with protein affinity. Utilizing the adsorption characteristics of the dot array, the protein adsorption process of the dot array is a spotting process, so that the protein to be adsorbed is adsorbed in a physiological environment, greatly reducing the possibility of denaturation. Summary of the invention

[0008] The purpose of the present invention is to provide a method for preparing a hydroxyl-modified protein chip. The substrate is a glass slide, which has hydrophobic properties, while the radiation-polymerized dot array has good hydrophilic properties, which forms a strong contrast with the substrate, thereby greatly increasing the signal-to-noise ratio. The hydroxyl groups introduced by polysiloxane and epoxide provide a large number of hydrophilic groups, have good biocompatibility and moisture retention, and provide a guarantee for the adsorbed protein to maintain good physiological activity; the introduced hydroxyl groups can interact with the groups on the surface of the protein to produce hydrogen bonds, thereby improving the adsorption of the matrix and the protein.

[0009] The method for preparing the hydroxyl-modified protein chip for dressing of the present invention comprises the following steps:

[0010] 1) Slide pretreatment:

[0011] Place a glass slide on a flat surface and perform periodic array etching on the glass slide using a pre-prepared etching solution. After etching for a period of time, rinse the etching solution with water and place the glass slide in a vacuum drying oven to dry to a constant weight for later use.

[0012] The glass slide is made of silicon dioxide; the etching solution is 40% hydrofluoric acid, 40% hydrofluoric acid-sulfuric acid mixture, and 48% sodium hydroxide aqueous solution; the amount of etching solution added to each etching point is 300-2000pL; and the spacing between etching points is 0.5-2.0mm.

[0013] 2) Preparation of protein chip matrix:

[0014] Under heating conditions, cyclic siloxane is placed in the etched array of the glass slide obtained in step 1), and after cooling to room temperature, a certain amount of epoxy compound is added to the etched array after the cyclic siloxane is completely solidified; the treated glass slide is placed on an irradiation table, and irradiated and cross-linked under a certain absorbed dose of high-energy rays to obtain a protein chip matrix.

[0015] Wherein, the cyclic siloxane is one of hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane and dodecamethylcyclohexasiloxane; the groups on both sides of the epoxy group of the epoxy compound are: The molar ratio of epoxy compound to cyclosiloxane is 1:0.5-1.5. The high-energy ray type used for the high-energy ray irradiation crosslinking is one or more of gamma ray, electron beam, ion beam and ultraviolet ray; the total absorbed dose is 25kGy-100kGy.

[0016] 3) Preparation of protein chip:

[0017] The protein chip matrix obtained in step 2) is placed in the protein solution to be tested, the protein solution to be tested is sealed, and placed in a constant temperature incubator at 37°C for adsorption reaction for a period of time. Then, it is taken out and washed with PBS / 1% Tween20 and PBS three times, each time for 5 minutes, and blown dry under nitrogen flow to obtain a protein chip.

[0018] The advantages and beneficial effects of the present invention are:

[0019] (1) The chip substrate used in the present invention is an ordinary glass slide, which is inexpensive. The selected polymerization method forms a covalent connection between the polymerized raw material and the glass slide, thereby preventing the loaded substance from being peeled off from the substrate. Polysiloxane itself can be used as a dressing for skin preparations, and has good biocompatibility.

[0020] (2) Since the protein chip matrix provided by the present invention has undergone a radiation polymerization process, it does not need to be sterilized before protein adsorption experiments can be performed, which greatly simplifies the experimental process.

[0021] (3) The protein chip matrix provided by the present invention can adsorb proteins under physiological conditions, thus avoiding the occurrence of protein denaturation.

[0022] (4) The protein chip matrix provided by the present invention has two different regions. The region after etching and radiation polymerization not only provides a good aqueous environment for proteins, but also can form weak interactions such as hydrogen bonds with groups on the surface of proteins, while the untreated region is hydrophobic. Therefore, the contrast is huge, so that the prepared protein chip has a high signal-to-noise ratio and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the preparation principle of the protein chip of the present invention.

[0024] Figure 2 This is a schematic diagram of the principle of producing hydroxyl groups by etching a glass slide in Example 1 of the present invention.

[0025] Figure 3 It is a schematic diagram of the radiation cross-linking reaction of Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to the accompanying drawings and specific implementation methods:

[0027] The present invention relates to a method for preparing a hydroxyl-modified protein chip, which is further described in conjunction with examples, but these examples should not be regarded as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] 1) Slide pretreatment:

[0030] Place the glass slide on a flat surface and perform periodic array etching on the glass slide using a pre-prepared etching solution. The etching solution is 40% hydrofluoric acid, and the amount added is 500 pL. After etching for 30 minutes, rinse the etching solution with water, and place the glass slide in a vacuum drying oven to dry to constant weight for later use.

[0031] 2) Preparation of protein chip matrix:

[0032] Under heating conditions, hexamethylcyclotrisiloxane is placed in the etched array of the glass slide obtained in step 1), and after cooling to room temperature, a certain amount of glycidol is added to the etched array after the cyclosiloxane is completely solidified; the molar ratio of glycidol to hexamethylcyclotrisiloxane is 1:1. The treated glass slide is placed on an irradiation table and irradiated with a Co-60 source (total absorbed dose of 25 kGy) for cross-linking reaction to obtain a protein chip matrix.

[0033] 3) Preparation of protein chips:

[0034] The protein chip matrix obtained in step 2) was placed in the metal β-lactamase aqueous solution, the metal β-lactamase aqueous solution was sealed, and placed in a constant temperature incubator at 37°C for adsorption reaction for 30 minutes. Then, it was taken out and washed with PBS / 1% Tween20 and PBS three times, each time for 5 minutes, and dried under nitrogen flow to obtain a metal β-lactamase chip.

[0035] Example 2

[0036] 1) Slide pretreatment:

[0037] Place the glass slide on a flat surface and perform periodic array etching on the glass slide using a pre-prepared etching solution. The etching solution is a 40% hydrofluoric acid-sulfuric acid mixture with a drop amount of 1000 pL. After etching for 20 minutes, rinse the etching solution with water and place the glass slide in a vacuum drying oven to dry to constant weight for later use.

[0038] 2) Preparation of protein chip matrix:

[0039] Under heating conditions, octamethylcyclotetrasiloxane is placed in the etched array of the glass slide obtained in step 1), and after cooling to room temperature, after the cyclic siloxane is completely solidified, a certain amount of ethylene oxide is added to the etched array; the molar ratio of ethylene oxide to octamethylcyclotetrasiloxane is 1:0.5. The treated glass slide is placed on an irradiation table and irradiated with an electron accelerator (total absorbed dose of 50 kGy) for cross-linking reaction to obtain a protein chip matrix.

[0040] 3) Preparation of protein chip:

[0041] The protein chip matrix obtained in step 2) was placed in the aflatoxin antibody aqueous solution, the aflatoxin antibody aqueous solution was sealed, and placed in a constant temperature incubator at 37°C for adsorption reaction for 30 minutes. Then, it was taken out and washed with PBS / 1% Tween20 and PBS three times, each time for 5 minutes, and dried under nitrogen flow to obtain the aflatoxin antibody chip.

[0042] Example 3

[0043] 1) Slide pretreatment:

[0044] Place the glass slide on a flat surface and perform periodic array etching on the glass slide using a pre-prepared etching solution. The etching solution is a 48% sodium hydroxide aqueous solution with a drop amount of 1500 pL. After etching for 40 minutes, rinse the etching solution with water and place the glass slide in a vacuum drying oven to dry to constant weight for later use.

[0045] 2) Preparation of protein chip matrix:

[0046] Under heating conditions, decamethylcyclopentasiloxane is placed in the etched array of the glass slide obtained in step 1), and after cooling to room temperature, after the cyclosiloxane is completely solidified, a certain amount of ethylene oxide-2,3-dimethanol is added to the etched array; the molar ratio of ethylene oxide-2,3-dimethanol to decamethylcyclopentasiloxane is 1:1.5. The treated glass slide is placed on an irradiation table and irradiated with a Co-60 source (total absorbed dose of 100 kGy) for cross-linking reaction to obtain a protein chip matrix.

[0047] 3) Preparation of protein chip:

[0048] The protein chip matrix obtained in step 2) was placed in the human immunoglobulin G aqueous solution, which was sealed and placed in a constant temperature incubator at 37°C for adsorption reaction for 30 minutes. The matrix was then taken out and washed with PBS / 1% Tween20 and PBS three times, each time for 5 minutes, and dried under a nitrogen flow to obtain a human immunoglobulin G chip.

Claims

1. A method for preparing a hydroxyl-modified protein chip, characterized in that: The following steps are involved: Step 1, pretreatment of the glass slide: place the glass slide on a flat surface, perform periodic array etching on the glass slide using a pre-prepared etching solution, rinse the etching solution with water after etching, and then place the glass slide in a vacuum drying oven to dry to constant weight for later use; Step 2, preparation of protein chip matrix: under heating conditions, cyclic siloxane is placed in the etched array of the glass slide, cooled to room temperature, and after the cyclic siloxane is completely solidified, epoxy compound is added dropwise to the etched array of the glass slide, and then placed on an irradiation table, irradiated and cross-linked under a certain absorbed dose of high-energy rays to obtain a protein chip matrix; Step 3, preparation of protein chip: placing the obtained protein chip matrix into the protein solution to be tested, sealing the protein solution to be tested, placing it in a constant temperature incubator at 37°C, adsorbing and reacting for a period of time, then taking it out and washing it with washing solution A and washing solution B for 3 times, each time for 5 min to 10 min, and drying it under nitrogen flow to obtain a protein chip; The glass slide is made of silicon dioxide, the amount of etching solution added to each etching point in the etching array is 300-2000 pL, and the spacing between the etching points is 0.5-2.0 mm; The etching solution is any one of 40% hydrofluoric acid, 40% hydrofluoric acid-sulfuric acid mixture or 48% sodium hydroxide aqueous solution; The general structural formula of the cyclic siloxane is as follows: ； The cyclic siloxane is one of hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane and dodecamethylcyclohexasiloxane; The general structural formula of the epoxy compound is as follows: ； Where R1 and R2 are: ; n is equal to 0, 1 or 2, and the molar ratio of the epoxy compound to the cyclic siloxane is 1:0.5-1.

5.

2. The method for preparing a hydroxyl-modified protein chip according to claim 1, characterized in that: The high-energy ray type used for the irradiation cross-linking under high-energy ray is one or more of gamma ray, electron beam, ion beam and ultraviolet ray, and the total absorbed dose is 25 kGy to 100 kGy.

3. The method for preparing a hydroxyl-modified protein chip according to claim 1, characterized in that: The protein chip obtained in step 3 includes proteins having affinity sites for interacting with substrates; and the adsorption reaction time is 30 min-45 min.

4. The method for preparing a hydroxyl-modified protein chip according to claim 1, characterized in that: The washing solution A is a 1% PBS-Tween20 solution at pH 7.0-pH 7.5, and the washing solution B is a PBS solution at pH 7.0-pH 7.5.

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