Fixed carrier based on bovine serum albumin-biotin as well as preparation method and application of fixed carrier

Abstract

The invention provides an immobilized carrier based on bovine serum albumin-biotin as well as a preparation method and application of the immobilized carrier. The biological sample fixing carrier comprises a substrate and a composite layer of bovine serum albumin coupled biotin and neutral avidin, wherein the composite layer is fixed on the surface of the substrate; the bovine serum albumin coupled biotin and neutral avidin composite layer is connected to the surface of the substrate after being co-incubated with the substrate through bovine serum albumin coupled biotin, and then the neutral avidin is combined with biotin in the bovine serum albumin coupled biotin through co-incubation with the neutral avidin; the bovine serum albumin coupled biotin and neutral avidin composite layer is fixed on the surface of the substrate. The biomolecules on the surface of the carrier are high in fixing efficiency, good in uniformity, high in adaptability, high in signal-to-noise ratio and easy to operate.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically provides a bovine serum albumin-biotin-based fixed carrier, a preparation method thereof, and an application thereof. Background Art

[0002] Fixed biological samples can be used in many fields. High-quality fixed biological samples can not only ensure the stability and reproducibility of biological sample signals, but also effectively reduce background interference and improve the signal-to-noise ratio. However, existing immobilization methods still have many shortcomings in terms of operational complexity, sample loss, nonspecific adsorption, and background fluorescence. Especially when performing high-sensitivity detection, these problems will significantly affect the accuracy and interpretability of experimental results. Therefore, there is an urgent need to develop a single-molecule sample immobilization strategy that is efficient, low-background, simple to operate, and has good biocompatibility to meet the growing demand for single-molecule biological sample immobilization. Summary of the Invention

[0003] Develop a biological sample immobilization strategy that is efficient, low-background, simple to operate, and has good biocompatibility to meet the growing demand for fixation of biological samples.

[0004] In one aspect, the present invention provides a fixing carrier for fixing a biological sample, wherein the fixing carrier for fixing a biological sample comprises a substrate, and a composite layer of bovine serum albumin-conjugated biotin (BSA-Biotin) and neutravidin (NeutrAvidin) fixed on the surface of the substrate;

[0005] The composite layer of bovine serum albumin-coupled biotin and neutravidin is connected to the surface of the substrate after the bovine serum albumin-coupled biotin and the substrate are co-incubated, and then the neutravidin is co-incubated with neutravidin to combine the biotin in the bovine serum albumin-coupled biotin with the neutravidin, thereby obtaining the composite layer of bovine serum albumin-coupled biotin and neutravidin fixed on the surface of the substrate.

[0006] Furthermore, the substrate is selected from glass, silicon wafer, metal, metal oxide, and polymer. Furthermore, the metal is selected from gold and silver; the metal oxide is selected from titanium oxide and iron oxide; and the polymer is selected from polydimethylsiloxane (PDMS), polystyrene (PS), and polymethyl methacrylate (PMMA).

[0007] Furthermore, the biological sample is DNA, RNA, protein, polypeptide, polysaccharide, glycoprotein, or vesicle modified with biotin.

[0008] Furthermore, a semi-open cavity is provided on the substrate, or the substrate is provided at the bottom of the cavity.

[0009] Further, the chamber is a rubber ring, a closed structure constructed by the base and the empty cover glass, a microfluidic chip, a PDMS molded cavity, a fluid pool.

[0010] Another aspect of the present application provides a method for preparing the above-mentioned fixed carrier, comprising the following steps:

[0011] S1) washing the surface of the base;

[0012] S2) surface modification of bovine serum albumin conjugated biotin: co-incubating a solution containing bovine serum albumin conjugated biotin with the base, and then washing to remove the unconnected bovine serum albumin conjugated biotin, to obtain a base connected with bovine serum albumin conjugated biotin;

[0013] S3) conjugation establishment: co-incubating the base connected with bovine serum albumin conjugated biotin with a solution containing neutravidin, to form a complex layer of bovine serum albumin conjugated biotin and neutravidin, and then washing to remove the unconnected neutravidin, to obtain a carrier for fixing a biological sample.

[0014] Further, the method for washing in step S1) is to use any one or several of a surfactant, an organic solvent, and an acid solvent for washing, and then washing with water and drying.

[0015] Further, the concentration of the solution containing bovine serum albumin conjugated biotin in step S2) is 0.001-10 g / L.

[0016] Further, the incubation time in step S2) is 5-15 minutes.

[0017] Further, the washing in step S2) is to sequentially wash with a buffer solution and water. Further, the buffer solution is PBS buffer, Tris-HCl buffer, or HEPES buffer.

[0018] Further, the incubation time in step S3) is 8-15 minutes.

[0019] Further, the washing in step S3) is to use a buffer solution for flushing replacement, or to use a sample solution of the biological sample to be fixed for flushing replacement.

[0020] Another aspect of the present application provides the use of the above-mentioned fixed carrier, which comprises use for fixing a biological sample.

[0021] Another aspect of the present application provides a method for fixing a biological sample by using the above-mentioned fixed carrier, comprising the following steps:

[0022] S01) obtaining a biological sample modified with biotin;

[0023] S02) contacting the above-mentioned biotin-modified biological sample with the above-mentioned immobilization carrier, and the biotin-modified biological sample is immobilized on the immobilization carrier;

[0024] S03) washing the non-immobilized biotin-modified biological sample with a displacement protection solution to obtain the immobilization carrier with the immobilized biological sample.

[0025] Further, the biological sample in step S01) is selected from DNA, RNA, protein, polypeptide, polysaccharide, glycoprotein, and vesicle.

[0026] Further, the displacement protection solution comprises one or more of the following components: protocatechuic acid (PCA), a combination of protocatechuate 3,4-dioxygenase (PCD) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), glucose-glucose oxidase-hydrogen peroxidase combination, mercaptoethylamine (MEA), thioctic acid, β-mercaptoethanol (BME), ascorbic acid (vitamin C), cyclooctatetraene (COT), and nitrophenyl glycerol (NPG).

[0027] Further, the displacement protection solution is composed of protocatechuic acid (PCA), protocatechuate 3,4-dioxygenase (PCD), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox).

[0028] In another aspect of the present application, an experimental carrier containing a biological sample is provided, wherein the experimental carrier comprises the above-mentioned immobilization carrier and the biological sample immobilized on the immobilization carrier.

[0029] Further, the experimental carrier containing a biological sample is prepared by the above-mentioned method.

[0030] In another aspect of the present application, the use of the above-mentioned experimental carrier is provided, wherein the use comprises single-molecule imaging experiments, smFRET detection, microscope imaging, and preparation of biosensors.

[0031] Further, the microscope imaging is super-resolution microscope imaging, such as total internal reflection fluorescence microscopy (TIRF) and single-molecule time-resolved multi-channel protein interaction analysis system.

[0032] Advantages

[0033] 1. Efficient surface modification: the present application significantly improves the efficiency of biological molecule immobilization on the surface of the slide through the combination of BSA-Biotin and NeutrAvidin.

[0034] 2. Good surface uniformity: multi-step cleaning and buffer flushing ensure uniform modification of the slide surface and reduce the risk of contamination.

[0035] 3. Strong applicability: the fixed carrier and experimental carrier of the application can be widely used in single molecule experiments, protein interaction research, DNA analysis and other fields.

[0036] 4. Good stability: the use of NeutrAvidin enhances the connection and ensures the stability of the fixed biological sample in subsequent experiments.

[0037] 5. High signal-to-noise ratio: the fixed carrier of the application can effectively prevent other non-target molecules from attaching to the substrate surface, ensuring the specificity and signal-to-noise ratio during detection.

[0038] 6. Simple operation: the fixed carrier of the application does not require complex chemical coupling reactions or special equipment, and has the advantages of simple operation, low cost and good repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Structure diagram of BSA-Biotin slide for biological sample fixation, which can connect Biotin-modified nucleic acids (left), proteins (middle) and vesicles (right) and other biological samples

[0040] Figure 2 Imaging comparison of DNA-Atto532 through PEG-Biotin slide (left) and BSA-Biotin slide (right) in single-molecule time-resolved multi-channel protein interaction analysis system. DETAILED DESCRIPTION

[0041] The content of the application will be described in detail by specific examples. It should be noted that these examples are only used to illustrate the application and do not limit the scope of the application. In order to make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be described clearly and completely. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market. The features and performance of the application are described in detail below in combination with the examples.

[0042] The application provides a preparation method of a fixed carrier for biological samples, Figure 1The structure diagram of the fixed carrier for biological sample experiment. The method ensures uniform modification of the slide surface by optimizing the steps of cleaning, chamber preparation, BSA-Biotin solution incubation, etc., and realizes efficient target molecule fixation by effective binding of NeutrAvidin and Biotin-modified biological samples, maintains the activity of biological molecules, reduces background noise, and provides stable and reliable experimental data.

[0043] The following best mode is provided to better further understand the present application and is not limited to the best mode, does not constitute a limitation on the content and protection scope of the present application, and any person who obtains any product same or similar to the present application under the inspiration of the present application or combines other existing technical features of the present application belongs to the protection scope of the present application. In the following examples, three repeated experiments are set to reduce the influence of accidental errors.

[0044] Example 1: Preparation of BSA-Biotin slide

[0045] Step 1: Clean the slide

[0046] 1-1. Ultrasonically clean the slide with surfactant for 20 minutes to remove impurities on the surface of the slide.

[0047] 1-2. Ultrasonically clean with deionized water for 10 minutes, repeat this step 3 times to ensure that all detergent residues are removed.

[0048] 1-3. Blow dry the slide surface with nitrogen to prevent water stains and contamination.

[0049] Step 2: Make a chamber

[0050] 2-1. Take a rubber ring and coat the outer edge with vacuum glue.

[0051] 2-2. Stick the rubber ring to the surface of the slide cleaned in step 1 to form a semi-open circular chamber for containing subsequent solutions.

[0052] Step 3: BSA-Biotin modification

[0053] 3-1. Take the BSA-Biotin solution and add it to the chamber to ensure that the solution completely covers the surface of the slide. BSA-Biotin is a conjugate obtained by chemical cross-linking of Biotin and carrier protein-BSA, which can be obtained by commercial products or synthesized by oneself. The concentration of BSA-Biotin solution is 0.001-10 g / L.

[0054] 3-2. Incubate for 10 minutes at room temperature to ensure BSA-Biotin is fully reacted and bound to the surface of the slide.

[0055] Step 4: Wash the slide

[0056] 4-1. Rinse the chamber 2-3 times with deionized water to remove unbound BSA-Biotin.

[0057] 4-2. Rinse the chamber 2-3 times with PBS buffer to remove residual impurities, ensuring that impurities in the solution do not interfere with subsequent experiments.

[0058] Step 5: NeutrAvidin connection

[0059] Add NeutrAvidin solution, incubate at 20-25°C for 10 minutes. NeutrAvidin completes the connection with Biotin on the surface of the slide, enhancing the stability and binding capacity of the slide surface. Then wash to remove unbound NeutrAvidin. Use the "rice" type injection method, inject buffer solution or sample solution to be coupled into the chamber each time, and simultaneously suck out an equal amount of NeutrAvidin solution, repeat multiple times to fully replace the NeutrAvidin not fixed on the slide, ensure thorough cleaning, and reduce experimental interference.

[0060] Example 2: Fixation of biological sample with Atto532 labeled DNA strand (DNA-Atto532)

[0061] Step 1. Biological sample connection

[0062] Add Biotin-modified DNA-Atto532 to the chamber, allowing the biological sample to be fixed to the surface of the slide prepared in Example 1 through the connection of Biotin and NeutrAvidin. Through this connection step, the biological sample is fixed on the slide, ensuring that the molecules can be stably attached during the experiment.

[0063] Step 2: Prepare displacement protection solution and clean the chamber

[0064] The displacement protection solution contains the following components: PCA (protocatechuic acid), PCD (protocatechuate 3,4-dioxygenase), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Used to synergistically reduce fluorescence bleaching and phototoxicity, stabilize the fluorescence signal, and improve the quality and duration of fluorescence imaging. Use the "rice" type injection method, inject 10-20% of the chamber capacity of protection solution each time, simultaneously suck out an equal amount of solution, repeat until the total displacement volume reaches 5-10 times the chamber capacity. Fully replace the free sample not fixed on the slide, ensure thorough cleaning, and reduce experimental interference.

[0065] Step 3: Close the chamber

[0066] Put a clean cover glass (not functionalized) on the chamber to prepare for the following single molecule experiment.

[0067] Comparative Example 1

[0068] The biological sample was fixed with Atto532-labeled DNA strands (DNA-Atto532) according to the method of Example 2, with the only difference being that the glass slide prepared in Example 1 was replaced with a PEG-Biotin glass slide (Patent Publication No. CN107101981A), the main difference between the PEG-Biotin glass slide and the glass slide prepared in Example 1 of the present application being:

[0069] 1) The Biotin on the PEG-Biotin glass slide is not connected to the glass slide through BSA, but is prepared by dissolving Biotin-PEG and mPEG in a 0.1 mol / L aqueous sodium bicarbonate solution and removing air bubbles to obtain a PEGylation reagent, which is incubated on the glass slide overnight to prepare a carrier with a biological marker fixed, i.e. a PEG-Biotin glass slide.

[0070] 2) The PEG-Biotin glass slide preparation process is complicated and different from the BSA-Biotin glass slide.

[0071] 3) The PEG-Biotin glass slide uses streptavidin when connecting samples, and the non-specific binding is high during the experiment, which may cause background signals and easily cause experimental errors. The BSA-Biotin glass slide uses NeutrAvidin when connecting samples, and the non-specific binding is lower, which is commonly used in experiments requiring high sensitivity and low background, and the experimental error is small. Moreover, NeutrAvidin is more neutral, with an isoelectric point (pI) of about 6.3, which is closer to physiological conditions and thus better protects the natural state of the biological sample.

[0072] Example 3: Detection of Atto532-labeled DNA (DNA-Atto532) of a biological sample

[0073] The single molecule time-resolved multi-channel protein interaction analysis system device was used to detect whether the DNA-Atto532 was successfully connected to the surface of the glass slide.

[0074] Based on Figure 2 The result shows that the DNA-Atto532 has been successfully connected to the surface of the glass slide.

[0075] Compare the signal-to-noise ratio of DNA-Atto532 on the BSA-biotin glass slide and the PEG-Biotin glass slide.

[0076] Based on the experimental data of Table 1, by comparing the fixing performance of BSA-Biotin glass and PEG-Biotin glass on DNA-Atto532, it is found that BSA-Biotin glass shows significant advantages: Figure 2

[0077] Signal specificity analysis: Under the same imaging conditions (excitation wavelength 532 nm, each pixel scanning time 50 us, total scanning time 10 minutes), the signal-to-noise ratio (SNR = target signal intensity mean value / background signal intensity mean value) of the target area was quantified using Fiji software, and the results showed that:

[0078] (1) PEG-Biotin glass: SNR = 16.01 ± 0.85 (n = 100)

[0079] (2) BSA-Biotin glass: SNR = 44.38 ± 1.12 (n = 100)

[0080] Anti-adsorption mechanism: The BSA protein layer inhibits non-specific adsorption through steric hindrance effect, reducing the background signal by about 64% and improving the signal-to-noise ratio by 277%;

[0081] Table 1: Comparison of signal-to-noise ratio of DNA-Atto532 on two kinds of glass

[0082] Glass slide Average signal of sample Average signal of background Signal-to-noise ratio (SNR) Number of single molecules collected PEG-Biotin glass slide 124.08 7.75 16.01 100 BSA-Biotin glass slide 122.94 2.77 44.38 100

[0083] In summary, the present application provides an efficient, stable and simple-to-operate biomolecule fixation technology, which significantly improves the biomolecule fixation efficiency on the surface of the glass by specific binding of BSA-Biotin and NeutrAvidin. The technology uses a multi-step cleaning and buffer flushing process to ensure uniform surface modification and no contamination, providing a high-reproducible substrate platform for subsequent experiments. Based on the high-affinity connection system of NeutrAvidin, the fixation carrier shows excellent stability and can effectively maintain the activity of the fixed biomolecules. Its outstanding surface blocking property can greatly reduce non-specific adsorption, significantly improve the signal-to-noise ratio and specificity. The operation process of the technology is simple, without the need for complex chemical coupling or special equipment, and has the advantages of low cost and good reproducibility, which can be widely applied to single molecule imaging, protein interaction research, nucleic acid detection and other biological technology fields.​

Claims

1. A fixed carrier for fixing a biological sample, characterized in that: The fixing carrier for fixing the biological sample comprises a substrate and a composite layer of bovine serum albumin-coupled biotin and neutravidin fixed on the surface of the substrate; the composite layer of bovine serum albumin-coupled biotin and neutravidin is connected to the surface of the substrate after being co-incubated with the bovine serum albumin-coupled biotin and the substrate, and then is co-incubated with neutravidin to combine the neutravidin with the biotin in the bovine serum albumin-coupled biotin, thereby obtaining the composite layer of bovine serum albumin-coupled biotin and neutravidin fixed on the surface of the substrate.

2. The fixed carrier according to claim 1, characterized in that The substrate is selected from glass, silicon wafer, metal, metal oxide, polymer; preferably, the metal is selected from gold and silver; the metal oxide is selected from titanium oxide and iron oxide; the polymer is selected from polydimethylsiloxane (PDMS), polystyrene (PS), and polymethyl methacrylate (PMMA).

3. The fixed carrier according to claim 1, characterized in that Biological samples include DNA, RNA, proteins, peptides, polysaccharides, glycoproteins, and vesicles modified with biotin.

4. The method for preparing a fixed carrier according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1) cleaning the substrate surface; S2) surface modification with bovine serum albumin-coupled biotin: incubating a solution containing bovine serum albumin-coupled biotin with the substrate, and then washing to remove unbound bovine serum albumin-coupled biotin to obtain a substrate coupled with bovine serum albumin-coupled biotin; S3) Coupling establishment: The substrate coupled with bovine serum albumin-coupled biotin is incubated with a solution containing neutravidin to form a composite layer of bovine serum albumin-coupled biotin and neutravidin, and the uncoupled neutravidin is removed by washing to obtain a carrier for immobilizing a biological sample.

5. The preparation method according to claim 4, characterized in that The incubation time in step S2) is 5 minutes to 15 minutes; Preferably, the washing in step S2) is performed sequentially with a buffer solution and water; More preferably, the buffer solution is PBS buffer, Tris-HCl buffer or HEPES buffer.

6. The preparation method according to claim 4, characterized in that The incubation time in step S3) is 8 minutes to 15 minutes; Preferably, the washing in step S3) is performed by flushing and replacing with a buffer solvent, or directly flushing and replacing with a sample solution of the biological sample to be fixed.

7. Use of the fixed carrier according to any one of claims 1 to 3, characterized in that: The uses include use for fixing biological samples.

8. The method for fixing a biological sample using a fixed carrier according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S01) obtaining a biological sample modified with biotin; S02) contacting the biological sample modified with biotin with the fixed carrier according to any one of claims 1 to 3, wherein the biological sample modified with biotin is fixed on the fixed carrier; S03) washing the unfixed biological sample modified with biotin with a replacement protection solution to obtain a fixed carrier with the biological sample fixed thereon; Preferably, the biological sample in step S01) is selected from DNA, RNA, protein, polypeptide, polysaccharide, glycoprotein, and vesicle; Preferably, the replacement protective solution comprises one or more of the following ingredients: protocatechuic acid (PCA); a combination of protocatechuic acid 3,4-dioxygenase (PCD) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), a glucose-glucose oxidase-catalase combination, thioethylamine (MEA), lipoic acid, β-mercaptoethanol (BME), ascorbic acid (vitamin C), cyclooctatetraene (COT) and nitrophenyl glycerol (NPG).

9. An experimental carrier containing a biological sample, characterized in that: The experimental carrier comprises the fixed carrier according to any one of claims 1 to 3, and a biological sample fixed on the fixed carrier; Preferably, the experimental carrier containing the biological sample is prepared by the method according to claim 8.

10. The use of the experimental carrier according to claim 9, characterized in that: The applications include single molecule imaging experiments, fluorescence resonance energy transfer (smFRET) detection, microscopy imaging, and preparation of biosensors.

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