CN-116622468-B - Antifouling micro-bubble for biological orthogonal capture and preparation and application thereof

Abstract

The invention discloses an anti-fouling microbubble for biological orthogonal capture, which comprises hollow glass microspheres, an anti-fouling coating layer and a capture layer, wherein the anti-fouling coating layer is arranged on the surfaces of the hollow glass microspheres, the anti-fouling coating layer is phase-change BSA, and the capture layer is formed by arranging N-hydroxysuccinimide derivatives on the anti-fouling coating layer. The invention also discloses a preparation method and application of the antifouling micro-bubbles for biological orthogonal capture. The antifouling micro-bubble for bioorthogonal capture can be used for specifically capturing a target object subjected to bioorthogonal modification by means of the bioorthogonal groups on the surface, has high speed and high efficiency, and has wide application system and application prospect.

Inventors

• YANG FAN
• XIANG YUANHANG
• Wei Binqi
• LI XINCHUN
• LU HAO

Assignees

• 广西医科大学

Dates

Publication Date

20260505

Application Date

20230427

Claims (9)

1. 1. An anti-fouling microbubble for bio-orthogonal capture, comprising: hollow glass microspheres; the anti-fouling coating is arranged on the surface of the hollow glass microsphere, and is phase-change BSA, wherein a BSA solution and a TCEP solution are mixed, and the pH value is regulated to 5.0 to obtain phase-change BSA; a trapping layer derived from an N-hydroxysuccinimide derivative disposed on the anti-fouling coating.
2. 2. The anti-fouling micro-bubble for bio-orthogonal capture of claim 1, wherein the hollow glass microsphere has a diameter of 10-100 μm.
3. 3. An anti-fouling microbubble for bio-orthogonal capture according to claim 1, characterized in that the N-hydroxysuccinimide derivative is an N-hydroxysuccinimide derivative with azide, alkyne, dibenzocyclooctyne, trans-cyclooctene or 1,2,4, 5-tetrazine.
4. 4. The method for preparing anti-fouling microbubbles for bio-orthogonal capture of claim 1, comprising the steps of: 1) Adding BSA solution and TCEP solution into hollow glass microspheres, adjusting the pH value to 5.0, and mixing for 0.5-12h; 2) Centrifuging the mixture obtained in the step 1), removing the lower layer solution, adding ultrapure water for washing, repeating the centrifugation for 1-5 times, and vacuum drying to obtain glass microbubbles with an antifouling function; 3) Reacting the glass microbubbles with the antifouling function obtained in the step 2) with N-hydroxysuccinimide derivative, centrifuging, removing the lower solution, adding ultrapure water for washing, repeating the centrifuging for 1-5 times, and vacuum drying to obtain the antifouling microbubbles with biological orthogonal capturing.
5. 5. The method for producing anti-fouling microbubbles for bio-orthogonal capture according to claim 4, wherein in step 1), the hollow glass microsphere has a mass of 0.5 to 5g, the concentration of the BSA solution is 1 to 5mg/mL, the volume is 10 to 30mL, the concentration of the TCEP solution is 10 to 50mM, and the volume is 10 to 50mL.
6. 6. The method for producing an antifouling micro-bubble for bio-orthogonal trapping according to claim 4, wherein in the step 2), the mass of the glass micro-bubble having an antifouling function is 50 to 500mg, the concentration of the N-hydroxysuccinimide derivative is 0.5 to 2mM, and the volume is 0.5 to 5mL.
7. 7. Use of anti-fouling microbubbles for bio-orthogonal capture according to claim 1, for the separation detection of cells, exosomes, proteins for non-disease diagnostic purposes.
8. 8. The use of anti-fouling microbubbles for bio-orthogonal capture of claim 7, wherein the cells are tumor cells or endothelial cells.
9. 9. The use of anti-fouling microbubbles for bio-orthogonal capture of claim 7, wherein the exosomes are derived from healthy cells or exosomes of lung cancer, liver cancer, cervical cancer, breast cancer, colon cancer, rectal cancer, bladder cancer, ovarian cancer, breast tissue cancer, pancreatic cancer, skin cancer, gastric cancer, thyroid cancer, prostate cancer, head and neck cancer, esophageal cancer, uterine cancer, brain cancer cells.

Description

Antifouling micro-bubble for biological orthogonal capture and preparation and application thereof Technical Field The invention belongs to the field of biomedical engineering. More particularly, the invention relates to an antifouling micro-bubble for bio-orthogonal capture, and preparation and application thereof. Background Bio-orthogonal chemistry is a class of chemical reactions that can occur in biological systems. They have high specificity, endogenous biomolecules do not interfere with the reaction, and have good biocompatibility, usually in water or PBS, and the reaction is rapid, meeting the real-time research demands, and drawing a great deal of attention. The patent publication No. CN113092784A discloses a functionalized magnetic bead and a one-step capturing method of a macromolecule by adopting bio-orthogonal chemistry, wherein the method for capturing the macromolecule by utilizing the magnetic bead modified by unconjugated antibody small molecules does not involve antigen-antibody reaction, adopts the azido alkyne cycloaddition reaction principle in click chemistry, avoids the generation of nonspecific binding protein, captures the protein biological macromolecule by adopting a one-step method, is more direct, efficient and lower in background, is suitable for capturing the protein under a denaturation condition, and has wider applicability. Compared with the magnetic beads, the hollow glass microsphere is a hollow micron silicon microsphere with low cost, light weight, stability and high strength. Due to its low density, self-aggregation properties, it can spontaneously separate from solution without external force. Hollow glass microspheres have great biological analysis application potential as a self-driven and self-separating material. Hollow glass microspheres are usually in micron size and larger in particle size, so that the specific surface area is small, the contact area with a target object is small, and because of the large surface area, a large amount of target probes are often required to be modified, so that the utilization rate is low in practical application, and the biological recognition probes are difficult to preserve for a long time after being modified. In addition, the surface of the hollow glass microsphere has a large amount of negative charges, and is easy to nonspecifically absorb pollutants with positive charges, so that the specificity is low, and the hollow glass microsphere is not applied to bio-orthogonal chemistry. Disclosure of Invention It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later. The invention aims to provide an antifouling microbubble for bioorthogonal capture, which can be used for efficiently and specifically capturing substances containing corresponding bioorthogonal molecule modification, and solves the problems of strong nonspecificity and low efficiency of hollow glass microspheres. To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided an anti-fouling micro-bubble for bio-orthogonal capture, comprising: hollow glass microspheres; An anti-fouling coating layer which is arranged on the surface of the hollow glass microsphere, wherein the anti-fouling coating layer is phase-change BSA; a trapping layer derived from an N-hydroxysuccinimide derivative disposed on the anti-fouling coating. Preferably, the hollow glass microspheres have a diameter of 10-100 μm. Preferably, the BSA (bovine serum albumin) solution and the TCEP solution are mixed and the pH is adjusted to 5.0 to obtain phase-changed BSA. Preferably, the N-hydroxysuccinimide derivative is an N-hydroxysuccinimide derivative with azide, alkyne, dibenzocyclooctyne, trans-cyclooctene or 1,2,4, 5-tetrazine. The preparation method of the antifouling micro-bubbles for bio-orthogonal capture comprises the following steps: 1) Adding BSA solution and TCEP solution into hollow glass microspheres, adjusting the pH value to 5.0, and mixing for 0.5-12h; 2) Centrifuging the mixture obtained in the step 1), removing the lower layer solution, adding ultrapure water for washing, repeating the centrifugation for 1-5 times, and vacuum drying to obtain glass microbubbles with an antifouling function; 3) Reacting the glass microbubbles with the antifouling function obtained in the step 2) with N-hydroxysuccinimide derivative, centrifuging, removing the lower solution, adding ultrapure water for washing, repeating the centrifuging for 1-5 times, and vacuum drying to obtain the antifouling microbubbles with biological orthogonal capturing. Preferably, in step 1), the hollow glass microspheres have a mass of 0.5-5g, a concentration of 1-5mg/mL of BSA solution, a volume of 10-30mL, a concentration of 10-50mM of TCEP solution, and a volume of 10-50mL. Preferably, in the step 2), the glass microbubbles having an antifouling function have a mass of 50 to 500mg, a co