Preparation of a streptavidin-coated quartz capillary and its use

Abstract

The application discloses a preparation method of a streptavidin quartz capillary and application thereof. The method uses a fused quartz capillary as a carrier, and after the capillary is cleaned, activated and pretreated, streptavidin is modified on the inner wall of the capillary through a chemical covalent bonding method, and can be applied to enrichment of ultramicro biotinylated samples. The application has the following advantages: (1) the preparation method is good in universality, and is mild in operation condition, and quartz capillaries with different performances and inner diameters can be modified by the method; (2) the streptavidin quartz capillary obtained by the method is good in enrichment performance of biotinylated samples, and the required sample amount is extremely low.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical chemistry technology, specifically relating to a method for preparing a functionalized capillary. Specifically, it involves chemically covalently binding streptavidin to the inner wall of a capillary and using it to specifically enrich biotin-labeled biomolecules (such as nucleic acids, peptides, proteins, etc.). Background Technology

[0002] In life sciences, clinical diagnostics, and drug development research, highly sensitive detection of target biomolecules with low abundance is crucial. However, these targets are often present in extremely low concentrations in complex biological samples, making direct detection very difficult. Therefore, developing efficient sample pretreatment techniques, especially enrichment techniques, has become a key step in improving detection sensitivity.

[0003] The biotin-streptavidin system is widely used for the immobilization, detection, and purification of biomolecules due to its extremely high affinity and specificity. Currently, commonly used enrichment methods based on this system often employ streptavidin-modified magnetic beads or agarose microbeads. However, these methods still suffer from problems such as high sample requirements and losses, poor reproducibility, and poor integration, making them unsuitable for the enrichment and analysis of ultra-micro samples. Capillaries, with their large specific surface area and ease of miniaturization and automation, are ideal carriers for microreactors. If their inner walls can be specifically functionally modified, it is hoped that the above problems can be solved, enabling rapid, efficient, and easily combinable enrichment of target analytes with downstream analytical techniques. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing streptavidin-based quartz capillaries and their application in the enrichment of ultra-micro biotinylated samples, so as to solve the problem of insufficient sample volume in the current enrichment of ultra-micro biotinylated samples.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing streptavidin-based quartz capillaries.

[0007] The method for preparing streptavidin-based silica capillaries provided by this invention includes the following steps: (1) Pretreatment of capillary: The inner wall of the capillary is cleaned and activated with strong acid, strong alkali and methanol solution to expose the silanol groups; (2) Silanization treatment: The silanizing agent is introduced into the pretreated capillary, and the inner wall of the capillary is silanized after the reaction. (3) Polymerization treatment: The polymerization reagent is introduced into the capillary to make the inner wall of the silanized capillary covered with epoxy groups; (4) Amination treatment: The amination reagent is introduced into the capillary to aminate the inner wall of the capillary with epoxy groups. (5) Iodine acetylation treatment: Iodine acetylation reagent is introduced into the capillary to achieve iodine acetylation modification of the aminated capillary inner wall; (6) Bonding of streptavidin: The thiolized streptavidin reagent is introduced into the capillary to achieve the bonding of streptavidin to the inner wall of the capillary; (7) Blocking: A thiol-based small molecule blocking agent is introduced into the capillary to block the remaining active sites in the capillary, thereby reducing non-specific adsorption and obtaining streptavidin-based quartz capillary.

[0008] Furthermore, the method also includes step (8) washing and storage: washing the streptavidin-based quartz capillary with a suitable buffer solution (such as phosphate buffer solution) and storing it in a 4°C refrigerator for later use.

[0009] In step (1) of the above method, the capillary can be a quartz capillary with different outer coatings and inner diameters, such as a deep ultraviolet transparent coating elastic fused silica capillary.

[0010] In some embodiments, the inner diameter of the capillary may be 50-150 μm.

[0011] The silanization process in step (2) of the above method is as follows: Prepare a suitable concentration of silanizing reagent, pump it into the pretreated capillary tube, seal the capillary tube, and incubate at 40-60℃ for 12-36 hours. The silanizing reagent may be 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS, 98%); the concentration of the silanizing reagent may be 50% (v / v), and the solvent may be methanol.

[0012] The polymerization treatment in step (3) of the above method is as follows: Prepare monomers and initiators of appropriate concentrations, pump them into the silanized capillary, seal the capillary, and incubate at 50-70°C for 4-10 hours. In the polymer treatment, the monomer used can be glycidyl methacrylate (GMA), and the initiator can be 2,2'-azobisisobutyronitrile (AIBN); the concentration of the monomer can be 10% (m / m), and its solvent is n-propanol; the concentration of the initiator can be 0.1% (m / m), and its solvent is n-propanol.

[0013] The amination treatment in step (4) of the above method is as follows: Prepare an amination reagent of appropriate concentration, pump it into the capillary tube after polymerization treatment, seal the capillary tube, and incubate at 40-60℃ for 3-10 hours. The amination reagent is specifically polyethyleneimine; the concentration of the amination reagent is 10 mg / mL, and its solvent is water.

[0014] The specific method for iodine acetylation treatment in step (5) of the above method is as follows: Prepare an iodine acetylation reagent of appropriate concentration, pump it into the capillary tube after amination treatment, seal the capillary tube, and incubate at 25-45℃ for 12-36 hours. The iodine acetylation reagent is specifically such as N-hydroxysuccinimide iodoacetate; the concentration of the iodine acetylation reagent can be 10 mg / mL.

[0015] The specific method for binding streptavidin in step (6) of the above method is as follows: Prepare a streptavidin-mercapto solution of appropriate concentration, pump it into a capillary tube after iodine acetylation treatment, seal the capillary tube, and incubate at room temperature for 2 hours or at 4°C overnight. The concentration of the streptavidin-mercapto solution can be 1-2 mg / mL, and its solvent is a 10 mM phosphate buffer solution with pH = 7.4.

[0016] The streptavidin-based quartz capillaries prepared by the method described in the first aspect of this invention are also within the scope of protection of this invention.

[0017] Secondly, the present invention provides a method for enriching biotinylated samples.

[0018] The method for enriching biotinylated samples provided by the present invention includes the step of enriching biotinylated samples using the streptavidin-quartz capillary as described in the second aspect of the present invention.

[0019] Furthermore, the biotinylated sample may be a biotin-labeled biomolecule (such as nucleic acid, polypeptide, protein, etc.).

[0020] Furthermore, the method specifically includes the following steps: (1) Enrichment stage: The sample solution containing biotinylated target molecules is introduced into the capillary, so that the biotinylated target molecules specifically bind to streptavidin bonded to the inner wall of the capillary, are captured and enriched on the inner wall of the capillary, and then the capillary is rinsed with washing buffer to remove unbound excess impurities. (2) Release stage: Select an appropriate method to release the enriched target molecules for subsequent analysis based on their characteristics.

[0021] In step (1) of the above method, the total amount of biotinylated target molecules in the sample solution can be as low as 50 ng.

[0022] In step (1) of the above method, the sample solution containing the biotinylated target molecule is introduced into the capillary at a certain flow rate, which can be 1-10 μL / min.

[0023] In step (1) of the above method, the washing buffer solution can be a phosphate buffer solution.

[0024] To address the shortcomings of existing technologies, this invention proposes a strategy for the efficient enrichment of ultra-micro biotinylated samples by using chemical covalent modification to bond streptavidin to the inner wall of a quartz capillary, thereby obtaining streptavidin-bonded quartz capillary.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation method of the present invention has good universality and mild operating conditions. Quartz capillaries with different outer coatings and inner diameters can be modified by this method. (2) The streptavidin-derived quartz capillary obtained by this method has good performance in enriching and releasing biotinylated samples, and requires very little sample amount. Attached Figure Description

[0026] Figure 1 A schematic diagram of the preparation process of streptavidin-based silica capillary tubes; Figure 2 Immunoblot image of streptavidin-based silica capillary for enriching biotinylated photocleavable green fluorescent protein; Figure 3 Immunoblot images of streptavidin agarose microbeads and streptavidin magnetic beads used to enrich biotinylated photocleavable green fluorescent protein. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Example 1: Preparation of streptavidin-based silica capillaries The preparation process of streptavidin-based silica capillaries is as follows: Figure 1 As shown.

[0030] (1) Pretreatment of capillary: Take a 30 cm long elastic fused silica capillary with an inner diameter of 100 μm and an outer diameter of 360 μm and a deep ultraviolet transparent coating. Rinse it sequentially with 1 mol / L hydrochloric acid solution, deionized water, 1 mol / L sodium hydroxide solution, deionized water, and 99.9% methanol for 1 hour at a flow rate of 5 μL / min. Finally, dry the capillary with nitrogen gas.

[0031] (2) Silanization and polymerization treatment of quartz capillary: ① Silanization treatment: Prepare a 50% (v / v) methanol solution of 3-(trimethoxysilyl) propyl methacrylate (γ-MAPS, 98%), take 30 μL and pump it into the pretreated quartz capillary. Seal both ends of the capillary with a sealed connector, incubate in a water bath at 50℃ for 24 h, and then wash the capillary with 99.9% methanol for 1 h. The pumping and washing flow rates are both 5 μL / min. Finally, blow the capillary dry with nitrogen. ②Polymerization treatment: Prepare a solution of 0.1% (m / m) 2,2'-azobisisobutyronitrile (AIBN, 99%) and 10% (m / m) glycidyl methacrylate (GMA, >95%) in n-propanol. Take 30 μL and pump it into a silanized quartz capillary. After sealing the capillary, incubate it in a water bath at 60°C for 6 h. Then wash the capillary with 99.9% methanol for 1 h. The pumping and washing flow rates are both 5 μL / min. Finally, blow the capillary dry with nitrogen.

[0032] (3) Aminoation treatment of silane-based quartz capillary: Prepare a 10 mg / mL aqueous solution of polyethyleneimine, take 30 μL and pump it into the silanized and polymerized quartz capillary. After sealing the capillary, incubate it in a water bath at 50°C for 5 h. Then wash the capillary with deionized water until neutral, and then wash the capillary with 99.9% methanol for 1 h. The pumping and washing flow rates are both 5 μL / min. Finally, blow the capillary dry with nitrogen.

[0033] (4) Iodine acetylation treatment of aminated quartz capillaries: Prepare a 10 mg / mL solution of iodoacetic acid N-hydroxysuccinimide (IAA-NHS, >95%) and sonicate it in methanol containing 16% (v / v) phosphate buffer (50 mM, pH = 7.4). Then, pump 30 μL into the aminated quartz capillary, seal it, and incubate it in a water bath at 40°C for 24 h. Then wash the capillary with 99.9% methanol for 1 h. The pumping and washing flow rates are both 5 μL / min. Finally, blow the capillary dry with nitrogen.

[0034] (5) Streptavidinization of iodinated quartz capillaries: Prepare a 2 mg / mL streptavidin-mercaptosol solution (dissolved in 10 mM, pH = 7.4 phosphate buffer solution), take 30 μL and pump it into the iodinated quartz capillary, seal it and incubate it at room temperature overnight, then rinse the capillary with phosphate buffer solution for 30 min. The pumping and washing flow rates are both 5 μL / min. Finally, blow the capillary dry with nitrogen gas.

[0035] (6) Blocking of streptavidinized quartz capillaries: Prepare a 10 mM solution of 6-mercaptohex-1-ol (dissolved in 10 mM, pH = 7.4 phosphate buffer solution), take 30 μL and pump it into streptavidinized quartz capillaries, seal and incubate at room temperature for 2 h, then rinse the capillaries with phosphate buffer solution and deionized water for 30 min respectively, with the pumping and washing flow rates being 5 μL / min.

[0036] Example 2: Streptavidin-based silica capillary tubes for the enrichment and release of biotinylated protein samples (1) Enrichment of biotinylated protein samples: Prepare a stock solution of 50 ng / μL (dissolved in phosphate buffer) of green fluorescent protein (GFP-biotin) labeled with photo-cleavable biotin groups, and pump it into streptavidin-based quartz capillary prepared in Example 1 at a flow rate of 1 μL / min, and incubate at room temperature for 2 hours or at 4°C overnight.

[0037] The green fluorescent protein sample labeled with a photosensitive biotinylated group was prepared in-house. The preparation method was as follows: 2 mg of purified green fluorescent protein (GFP) expressed in *E. coli* was taken, and 10 μM of eosin probe and 100 μM of propyneamine probe (HC≡C) were added. CH2 After mixing with NH2, irradiate with green light for 15 min, precipitate with methanol overnight, and wash to remove excess probe. Take an appropriate concentration and perform a click reaction with the azide-photocrackable-biotin probe (N3-PC-biotin), precipitate with methanol overnight, and you can obtain the biotin-labeled green fluorescent protein (GFP-biotin) sample.

[0038] (2) Washing: Rinse the capillary with phosphate buffer solution at a flow rate of 5 μL / min for 5 min to remove unbound protein samples.

[0039] (3) Release and detection: The capillary was placed under a 365nm UV lamp for 20 minutes, and the photo-cut protein sample was pumped out of the capillary for immunoblotting detection.

[0040] Immunoblot image of streptavidin-based silica capillary for enriching biotinylated green fluorescent protein, as shown below. Figure 2 As shown. By Figure 2 It can be seen that GFP-biotin with an injection volume of only 50 ng was enriched in streptavidin-modified quartz capillaries and could be released by ultraviolet light.

[0041] The enrichment effects of streptavidin agarose microbeads and streptavidin magnetic beads on GFP-biotin were compared under the same conditions. Figure 3As shown, the two commercially available beads were not as effective as streptavidin-based silica capillary beads in enriching and releasing GFP-biotin with an injection volume of only 50 ng.

[0042] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing streptavidin-based quartz capillaries, comprising the following steps: (1) Pretreatment of capillary: The inner wall of the capillary is cleaned and activated with strong acid, strong alkali and methanol solution to expose the silanol groups; (2) Silanization treatment: The silanizing agent is introduced into the pretreated capillary, and the inner wall of the capillary is silanized after the reaction. (3) Polymerization treatment: The polymerization reagent is introduced into the capillary to make the inner wall of the silanized capillary covered with epoxy groups; (4) Amination treatment: The amination reagent is introduced into the capillary to aminate the inner wall of the capillary with epoxy groups. (5) Iodine acetylation treatment: Iodine acetylation reagent is introduced into the capillary to achieve iodine acetylation modification of the aminated capillary inner wall; (6) Bonding of streptavidin: The thiolized streptavidin reagent is introduced into the capillary to achieve the bonding of streptavidin to the inner wall of the capillary; (7) Blocking: A thiol-based small molecule blocking agent is introduced into the capillary to block the remaining active sites in the capillary, thereby reducing non-specific adsorption and obtaining streptavidin-based quartz capillary.

2. The preparation method according to claim 1, characterized in that: The method also includes step (8) washing and storage: washing the streptavidin-based quartz capillary with a suitable buffer solution and storing it in a 4°C refrigerator for later use.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), the capillary is a quartz capillary with different outer coatings and inner diameters.

4. The preparation method according to any one of claims 1-3, characterized in that: The specific method of silanization treatment in step (2) is as follows: prepare a suitable concentration of silanization reagent, pump it into the pretreated capillary tube, seal the capillary tube, and incubate at 40-60℃ for 12-36 hours. And / or, the polymerization treatment method in step (3) is as follows: prepare monomers and initiators of appropriate concentrations, pump them into the capillary after silanization treatment, seal the capillary, and incubate at 50-70°C for 4-10 hours; in the polymer treatment, the monomer used is glycidyl methacrylate (GMA), and the initiator is 2,2'-azobisisobutyronitrile (AIBN).

5. The preparation method according to any one of claims 1-4, characterized in that: The amination treatment method in step (4) is as follows: prepare an amination reagent of appropriate concentration, pump it into the capillary after polymerization treatment, seal the capillary, and incubate at 40-60℃ for 3-10 hours; the amination reagent is polyethyleneimine. And / or, the method of iodine acetylation treatment in step (5) is as follows: prepare an iodine acetylation reagent of appropriate concentration, pump it into the capillary after amination treatment, seal the capillary, and incubate at 25-45℃ for 12-36 hours; the iodine acetylation reagent is N-hydroxysuccinimide acetate.

6. The preparation method according to any one of claims 1-5, characterized in that: The specific method for binding streptavidin in step (6) is as follows: prepare a streptavidin-thiol solution of appropriate concentration, pump it into the capillary tube after iodine acetylation treatment, seal the capillary tube, and incubate at room temperature for 2 hours or at 4°C overnight.

7. Streptoavidin-based silica capillary tubes prepared by the method according to any one of claims 1-6.

8. A method for enriching and releasing biotinylated samples, comprising: The step of enriching biotinylated samples using the streptavidin-based quartz capillary as described in claim 7.

9. The method according to claim 8, characterized in that: The method specifically includes the following steps: (1) Enrichment stage: The sample solution containing biotinylated target molecules is introduced into the capillary, so that the biotinylated target molecules specifically bind to streptavidin bonded to the inner wall of the capillary, are captured and enriched on the inner wall of the capillary, and then the capillary is rinsed with washing buffer to remove unbound excess impurities. (2) Release stage: Select an appropriate method to release the enriched target molecules for subsequent analysis based on their characteristics.

10. The method according to claim 8 or 9, characterized in that: The biotinylated sample is a biotin-labeled biomolecule, including but not limited to nucleic acids, peptides, and proteins; And / or, the total amount of biotinylated target molecules in the sample solution may be as low as 50 ng; And / or, the sample solution containing the biotinylated target molecule is introduced into the capillary at a certain flow rate, the flow... And / or, the washing buffer is a phosphate buffer solution.

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