Antifouling microbubbles for bio-orthogonal capture and preparation and use thereof

Abstract

This invention discloses an antifouling microbubble for bioorthogonal capture, comprising: hollow glass microspheres; an antifouling coating disposed on the surface of the hollow glass microspheres, wherein the antifouling coating is a phase-change BSA; and a capture layer formed by depositing an N-hydroxysuccinimide derivative on the antifouling coating. This invention also discloses a method for preparing the antifouling microbubble for bioorthogonal capture and its applications. The antifouling microbubble for bioorthogonal capture of this invention, utilizing surface bioorthogonal groups, can be used for the specific capture of bioorthogonally modified target substances, exhibiting high speed and efficiency, and possessing broad application systems and prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering. More specifically, this invention relates to an antifouling microbubble for bioorthogonal capture, its preparation, and its application. Background Technology

[0002] Bioorthogonal chemistry is a class of chemical reactions that can occur in biological systems. They exhibit high specificity, are unaffected by endogenous biomolecules, and possess good biocompatibility. Typically carried out in water or PBS, these reactions are rapid, meeting the needs of real-time research and thus attracting widespread attention.

[0003] Patent publication number CN113092784A discloses functionalized magnetic beads and a one-step macromolecule capture method using bioorthogonal chemistry. This method, utilizing magnetic beads modified with uncoupled antibody small molecules to capture macromolecules, avoids antigen-antibody reactions. It employs the principle of azidoyne cycloaddition reaction in click chemistry, preventing the generation of non-specific binding proteins. This one-step capture method for protein biomolecules is more direct, efficient, and has lower background, and is applicable to protein capture under denaturing conditions, thus having wider applicability. Compared to magnetic beads, hollow glass microspheres are inexpensive, lightweight, stable, and high-strength hollow micron-sized silica spheres. Due to their low density and self-aggregating properties, they can spontaneously separate from solution without external force. Hollow glass microspheres, as a self-driven and self-separating material, have enormous potential for bioanalytical applications. However, hollow glass microspheres are typically micrometers in size, with a relatively large particle size, resulting in a small specific surface area and a small contact area with the target analyte. Furthermore, due to their large surface area, they often require modification with a large number of target probes, leading to low utilization rates in practical applications, and the modified biorecognition probes are difficult to preserve long-term. In addition, the surface of hollow glass microspheres has a large number of negative charges, which makes them easy to non-specifically adsorb positively charged pollutants. This low specificity has prevented hollow glass microspheres from being used in bioorthogonal chemistry. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0005] One objective of this invention is to provide a fouling-resistant microbubble for bioorthogonal capture, which can be used to efficiently and specifically capture substances containing corresponding bioorthogonal molecular modifications, thus solving the problems of high nonspecificity and low efficiency of hollow glass microspheres.

[0006] To achieve these objectives and other advantages of the present invention, an antifouling microbubble for bioorthogonal capture is provided, comprising:

[0007] Hollow glass microspheres;

[0008] An antifouling coating is disposed on the surface of the hollow glass microspheres, and the antifouling coating is phase-change BSA;

[0009] The trapping layer is formed by setting an N-hydroxysuccinimide derivative on the antifouling coating.

[0010] Preferably, the diameter of the hollow glass microspheres is 10-100 μm.

[0011] Preferably, BSA (bovine serum albumin) solution and TCEP solution are mixed and the pH is adjusted to 5.0 to obtain phase-transition BSA.

[0012] Preferably, the N-hydroxysuccinimide derivative is an N-hydroxysuccinimide derivative containing azide, alkyne, dibenzocyclooctylene, transcyclooctene, or 1,2,4,5-tetraazine.

[0013] A method for preparing antifouling microbubbles for bioorthogonal trapping includes the following steps:

[0014] 1) Add BSA solution and TCEP solution to hollow glass microspheres, adjust the pH value to 5.0, and mix for 0.5-12 hours;

[0015] 2) Centrifuge the mixture from step 1), remove the lower layer solution, wash with ultrapure water, repeat centrifugation 1-5 times, and vacuum dry to obtain glass microbubbles with antifouling function.

[0016] 3) React the glass microbubbles with antifouling function obtained in step 2) with N-hydroxysuccinimide derivative, centrifuge, remove the lower layer solution, add ultrapure water to wash, repeat centrifugation 1-5 times, and vacuum dry to obtain antifouling microbubbles with bioorthogonal trapping.

[0017] Preferably, in step 1), the mass of the hollow glass microspheres is 0.5-5g; the concentration of the BSA solution is 1-5mg / mL and the volume is 10-30mL; the concentration of the TCEP solution is 10-50mM and the volume is 10-50mL.

[0018] Preferably, in step 2), the mass of the glass microbubbles with antifouling function is 50-500 mg, the concentration of the N-hydroxysuccinimide derivative is 0.5-2 mM, and the volume is 0.5-5 mL.

[0019] Applications of antifouling microbubbles for bioorthogonal capture, used for the separation and detection of cells, exosomes, and proteins.

[0020] Preferably, the cells are tumor cells or endothelial cells.

[0021] Preferably, 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, stomach cancer, thyroid cancer, prostate cancer, head and neck cancer, esophageal cancer, uterine cancer, or brain cancer cells.

[0022] Preferably, the protein is CD45 (lymphocyte common antigen) protein, EpCAM (epidermal cell adhesion molecule) protein, EGFR (epidermal growth factor) protein, HER2 (human epidermal factor receptor 2) protein, PSA (prostate-specific antigen) protein, VEGFR (vascular endothelial growth factor receptor) protein, tumor marker-related protein, or total protein obtained from cell lysis.

[0023] The present invention has at least the following beneficial effects:

[0024] First, the antifouling microbubbles of the present invention for bioorthogonal capture have the characteristics of biofouling prevention and have a good antifouling effect on non-specific cells, nucleic acids and proteins.

[0025] Secondly, the antifouling microbubble preparation process of the present invention for bioorthogonal capture is simple, the raw materials are inexpensive, the stability is good, and it can be stored at low temperature for a long time, thus having the potential for application in on-site diagnosis.

[0026] Third, the antifouling microbubbles of the present invention for bioorthogonal capture can be used to specifically capture bioorthogonally modified target substances by means of bioorthogonal groups on the surface. They are fast and efficient, and have great application prospects in medical analysis, diagnosis and treatment.

[0027] Fourth, this invention utilizes phase change BSA (PTB) coating on hollow glass microspheres to form a biofouling-resistant coating on the hollow glass microspheres, creating a surface that makes it difficult for dirt and organisms to adhere, reducing the non-specific adsorption of nucleic acids, proteins, etc. by the hollow glass microspheres, and improving the sensitivity and specificity of the hollow glass microspheres in bioorthogonal chemical detection.

[0028] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the preparation process of the antifouling microbubbles for bioorthogonal capture according to the present invention.

[0030] Figure 2 The image shows a fluorescence characterization of the antifouling microbubble coating for bioorthogonal capture according to the present invention.

[0031] Figure 3The images show SEM and TEM characterization of the antifouling microbubble coating for bioorthogonal capture of the present invention.

[0032] Figure 4 This is a characterization diagram of the antifouling performance of the antifouling microbubbles for bioorthogonal capture of the present invention;

[0033] Figure 5 Fluorescence image of the antifouling microbubbles for cell capture using the present invention for bioorthogonal capture;

[0034] Figure 6 SEM image of the antifouling microbubbles for cell capture using the bioorthogonal capture method of the present invention, scale bar 2 μm;

[0035] Figure 7 The image shows a fluorescence pattern of the antifouling microbubbles used for bioorthogonal capture of exosomes according to the present invention.

[0036] Figure 8 SEM image of the antifouling microbubbles for exosome capture using the bioorthogonal capture method of the present invention;

[0037] Figure 9 This is a fluorescence image of the antifouling microbubbles used for protein capture in the present invention for bioorthogonal capture. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0039] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0040] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0041] An antifouling microbubble for bioorthogonal trapping, comprising:

[0042] Hollow glass microspheres;

[0043] An antifouling coating is disposed on the surface of the hollow glass microspheres, and the antifouling coating is phase-change BSA;

[0044] The trapping layer is formed by setting an N-hydroxysuccinimide derivative on the antifouling coating.

[0045] In another technical solution, the diameter of the hollow glass microspheres is 10-100 μm.

[0046] In another technical solution, a phosphate buffer solution of BSA (bovine serum albumin) and an aqueous solution of TCEP are mixed and the pH is adjusted to 5.0 to obtain phase-transition BSA.

[0047] In another technical solution, the N-hydroxysuccinimide derivative is an N-hydroxysuccinimide derivative containing azide, alkyne, dibenzocyclooctylene, transcyclooctene, or 1,2,4,5-tetraazine.

[0048] A method for preparing antifouling microbubbles for bioorthogonal trapping includes the following steps:

[0049] 1) Add BSA solution and TCEP solution to hollow glass microspheres, adjust the pH value to 5.0, and mix for 0.5-12 hours;

[0050] 2) Centrifuge the mixture from step 1), remove the lower layer solution, wash with ultrapure water, repeat centrifugation 1-5 times, and vacuum dry to obtain glass microbubbles with antifouling function.

[0051] 3) React the glass microbubbles with antifouling function obtained in step 2) with N-hydroxysuccinimide (NHS) derivative, centrifuge, remove the lower layer solution, add ultrapure water to wash, repeat centrifugation 1-5 times, and vacuum dry to obtain antifouling microbubbles with bioorthogonal trapping.

[0052] In another technical solution, in step 1), the mass of the hollow glass microspheres is 0.5-5g; the concentration of the BSA solution is 1-5mg / mL and the volume is 10-30mL; the concentration of the TCEP solution is 10-50mM and the volume is 10-50mL.

[0053] In another technical solution, in step 2), the mass of the glass microbubbles with antifouling function is 50-500 mg, the concentration of the N-hydroxysuccinimide derivative is 0.5-2 mM, and the volume is 0.5-5 mL.

[0054] Applications of antifouling microbubbles for bioorthogonal capture include cell separation and detection, exosome separation and detection, and protein separation and detection.

[0055] In another technical solution, the cells are tumor cells or endothelial cells.

[0056] In another technical solution, 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, stomach cancer, thyroid cancer, prostate cancer, head and neck cancer, esophageal cancer, uterine cancer, and brain cancer cells.

[0057] In another technical solution, the protein is CD45 (lymphocyte common antigen) protein, EpCAM (epithelial cell adhesion molecule) protein, EGFR (epidermal growth factor) protein, HER2 (human epidermal factor receptor 2) protein, PSA (prostate-specific antigen) protein, VEGFR (vascular endothelial growth factor receptor) protein, tumor marker-related protein, or total protein obtained from cell lysis.

[0058] Example 1

[0059] A method for preparing antifouling microbubbles for bioorthogonal trapping includes the following steps:

[0060] 1) Weigh 1g of hollow glass microspheres and add them to a 50mL centrifuge tube. Add 20mL of BSA (bovine serum albumin) with a concentration of 2mg / mL and 20mL of TCEP solution with a concentration of 25mM. Adjust the pH to 5.0 using sodium hydroxide solution (1M). Mix for 2 hours at room temperature using a rotary mixer.

[0061] 2) Centrifuge the mixture from step 1) at 1000 rpm for 1 minute to remove the lower layer solution; add ultrapure water to wash, repeat centrifugation 3 times, and vacuum dry at 37℃ to obtain glass microbubbles (GB@PTB) with antifouling function, and store at -20℃.

[0062] 3) Weigh 100 mg of GB@PTB and add it to 1 mL of 1 mM azidoacetic acid-N-hydroxysuccinimide PBS solution. React at room temperature for 2 hours, centrifuge at 4000 rpm for 10 s, remove the lower layer solution, wash with ultrapure water, repeat centrifugation 3 times, and then vacuum dry to obtain antifouling glass microbubbles (GB@PTB@N3) with bioorthogonal function.

[0063] Example 2

[0064] A method for preparing antifouling microbubbles for bioorthogonal trapping includes the following steps:

[0065] 1) Weigh 1g of hollow glass microspheres and add them to a 50mL centrifuge tube. Add 20mL of BSA with a concentration of 2mg / mL and 20mL of TCEP solution with a concentration of 25mM. Adjust the pH to 5.0 using sodium hydroxide solution (1M). Mix for 2 hours at room temperature using a rotary mixer.

[0066] 2) Centrifuge the mixture from step 1) at 1000 rpm for 1 minute to remove the lower layer solution; add ultrapure water to wash, repeat centrifugation 3 times, and vacuum dry at 37℃ to obtain glass microbubbles (GB@PTB) with antifouling function, and store at -20℃.

[0067] 3) Weigh 100 mg of GB@PTB and add it to 1 mL of 1 mM 5-hexynoic acid-NHS ester in PBS. React at room temperature for 2 hours, centrifuge at 4000 rpm for 10 s, remove the lower layer, wash with ultrapure water, repeat centrifugation 3 times, and then vacuum dry to obtain antifouling glass microbubbles (GB@PTB@Alkyne) with bioorthogonal function.

[0068] Example 3

[0069] A method for preparing antifouling microbubbles for bioorthogonal trapping includes the following steps:

[0070] 1) Weigh 1g of hollow glass microspheres and add them to a 50mL centrifuge tube. Add 20mL of BSA with a concentration of 2mg / mL and 20mL of TCEP solution with a concentration of 25mM. Adjust the pH to 5.0 using sodium hydroxide solution (1M). Mix for 2 hours at room temperature using a rotary mixer.

[0071] 2) Centrifuge the mixture from step 1) at 1000 rpm for 1 minute to remove the lower layer; wash with ultrapure water and repeat centrifugation 3 times. Vacuum dry at 37℃ to obtain glass microbubbles (GB@PTB) with antifouling function, and store at -20℃.

[0072] 3) Weigh 100 mg of GB@PTB and add it to 1 mL of 1 mM dibenzocyclooctylene-NHS ester in PBS. React at room temperature for 2 hours, centrifuge at 4000 rpm for 10 s, remove the lower layer, wash with ultrapure water, repeat centrifugation 3 times, and then vacuum dry to obtain antifouling glass microbubbles (GB@PTB@DBCO) with bioorthogonal function.

[0073] Example 4

[0074] A method for preparing antifouling microbubbles for bioorthogonal trapping includes the following steps:

[0075] 1) Weigh 1g of hollow glass microspheres and add them to a 50mL centrifuge tube. Add 20mL of BSA with a concentration of 2mg / mL and 20mL of TCEP solution with a concentration of 25mM. Adjust the pH to 5.0 using sodium hydroxide solution (1M). Mix for 2 hours at room temperature using a rotary mixer.

[0076] 2) Centrifuge the mixture from step 1) at 1000 rpm for 1 minute to remove the lower layer; wash with ultrapure water and repeat centrifugation 3 times. Vacuum dry at 37℃ to obtain glass microbubbles (GB@PTB) with antifouling function, and store at -20℃.

[0077] 3) Weigh 100 mg of GB@PTB and add it to 1 mL of 1 mM trans-cyclooctene (TCO)-NHS ester in PBS. React at room temperature for 2 hours, centrifuge at 4000 rpm for 10 s, remove the lower layer, wash with ultrapure water, repeat centrifugation 3 times, and then vacuum dry to obtain antifouling glass microbubbles (GB@PTB@TCO) with bioorthogonal function.

[0078] Example 5

[0079] like Figure 1 As shown, a method for preparing antifouling microbubbles for bioorthogonal trapping includes the following steps:

[0080] 1) Weigh 1g of hollow glass microspheres (GB) and add them to a 50mL centrifuge tube. Add 20mL of BSA with a concentration of 2mg / mL and 20mL of TCEP solution with a concentration of 25mM. Adjust the pH to 5.0 using sodium hydroxide solution (1M). Mix for 2 hours at room temperature using a rotary mixer.

[0081] 2) Centrifuge the mixture from step 1) at 1000 rpm for 1 minute to remove the lower layer; wash with ultrapure water and repeat centrifugation 3 times. Vacuum dry at 37℃ to obtain glass microbubbles (GB@PTB) with antifouling function, and store at -20℃.

[0082] 3) Weigh 100 mg of GB@PTB and add it to 1 mL of 1 mM 1,2,4,5-tetraazine (Tz)-NHS ester PBS solution. React at room temperature for 2 hours, centrifuge at 4000 rpm for 10 s, remove the lower layer solution, wash with ultrapure water, repeat centrifugation 3 times, and then vacuum dry to obtain antifouling glass microbubbles (GB@PTB@Tz) with bioorthogonal function.

[0083] Example 6

[0084] The hollow glass microspheres (GB), antifouling glass microbubbles (GB@PTB), and antifouling glass microbubbles (GB@PTB@Tz) with bioorthogonal function of Example 5 were characterized.

[0085] For fluorescence characterization, since phase transition BSA (PTB) contains β-amyloid protein, thiamine T (ThT) can be used to characterize the coating on the microbubble surface. 1 mg of GB, GB@PTB, and GB@PTB@Tz were weighed and reacted in PBS solution containing 1 mM ThT at 37°C for 2 hours. After washing five times with ultrapure water, the mixture was observed using a laser confocal microscope. To demonstrate the bioorthogonality, 1 mg of GB, GB@PTB, GB@PTB@Tz, and Cy5-labeled TCO dye (TCO-Cy5) (2 μM) were reacted in 200 μL of PBS at room temperature for 10 minutes. After washing five times with ultrapure water, the mixture was observed using a laser confocal microscope.

[0086] Fluorescence characterization results as follows Figure 2 As shown, GB@PTB and GB@PTB@Tz modified with PTB showed obvious fluorescence signals on the surface of the microbubbles after ThT staining, while the unmodified GB surface showed no signal. This proves that PTB was successfully formed on the microbubble surface. Furthermore, after the addition of TCO-Cy5, only the Tz-modified microbubble surface showed obvious fluorescence signals, demonstrating the bioorthogonal ability of the microbubbles.

[0087] To observe the morphology of the PTB coating on the microbubble surface, SEM and TEM were used. Figure 3 As shown, the surface of ordinary GB is relatively flat with almost no large particles. However, the surface of microbubbles modified with PTB has obvious nanoparticles, proving that PTB can form a nanofilm on the surface of microbubbles.

[0088] The characterization results of the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 are similar to those of GB@PTB@Tz prepared in Example 5.

[0089] Example 7

[0090] The antifouling performance of hollow glass microspheres (GB), antifouling glass microbubbles (GB@PTB), and antifouling glass microbubbles (GB@PTB@Tz) with bio-orthogonal function in Example 5 was examined.

[0091] Take 1 mg of GB, GB@PTB and GB@PTB@Tz and mix them with DiO-labeled A431 cells, MCF7 cells and Jurkat cells to investigate the antifouling ability of GB, GB@PTB and GB@PTB@Tz on cells.

[0092] Take 1 mg of GB, GB@PTB and GB@PTB@Tz, mix with 1 mg / mL FITC-labeled BSA and 10 μg / mL Alexa Fluor 488-labeled IgG, and examine the antifouling ability of GB, GB@PTB and GB@PTB@Tz against proteins.

[0093] Take 1 mg of GB, GB@PTB and GB@PTB@Tz, mix with 5 μM ssDNA, and examine the anticontamination ability of GB, GB@PTB and GB@PTB@Tz against nucleic acids.

[0094] The results are as follows Figure 4 As shown, GB exhibits non-specific adsorption for most biological contaminants. Hollow glass microspheres with a PTB coating demonstrate excellent antifouling capabilities for cells, proteins, and nucleic acids. This confirms the successful preparation of bioorthogonal antifouling microbubbles.

[0095] The antifouling performance of the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 is similar to that of GB@PTB@Tz prepared in Example 5.

[0096] Example 8

[0097] The capture of orthogonally modified monocytes from blood samples by the antifouling glass microbubbles (GB@PTB@Tz) with bioorthogonal function prepared in Example 5 was investigated.

[0098] Take 1 mL of whole blood cells and process them with commercially available erythrocyte lysis buffer to obtain monocytes. Suspend the cells in 100 μL of TCO-NHS ester containing 1 mM and incubate for 10 minutes to obtain orthogonally modified cells. Mix the orthogonally modified cells with bioorthogonal antifouling microbubbles to capture monocytes from the blood sample.

[0099] The capture results of orthogonally modified mononuclear cells from blood samples by the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 were similar to those of orthogonally modified mononuclear cells from blood samples by GB@PTB@Tz prepared in Example 5.

[0100] Example 9

[0101] The study investigated the capture of orthogonally modified liver cancer cells by the antifouling glass microbubbles (GB@PTB@Tz) with bioorthogonal function prepared in Example 5.

[0102] Take 10 5HepG2 (human liver cancer) cells were pre-stained with 10 μM DiO in serum-free DMEM for 20 minutes. After centrifugation at 300g for 3 minutes, the supernatant was removed, and the cells were incubated in 100 μL of 1 mM TCO-NHS ester for 10 minutes to obtain orthogonally modified cells. HepG2 cells were then captured by mixing the orthogonally modified cells with bio-orthogonal antifouling microbubbles.

[0103] The capture results of orthogonally modified hepatocellular carcinoma cells by the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 were similar to those of orthogonally modified hepatocellular carcinoma cells by GB@PTB@Tz prepared in Example 5.

[0104] Example 10

[0105] The study investigated the capture of orthogonally modified skin cancer cells by the antifouling glass microbubbles (GB@PTB@Tz) with bioorthogonal function prepared in Example 5.

[0106] Take 10 5 A431 (human squamous cell carcinoma) cells were pre-stained with 10 μM DiO in serum-free DMEM for 10 minutes. After centrifugation at 300g for 3 minutes, the supernatant was removed, and the cells were incubated in 100 μL of TCO-NHS ester containing 1 mM for 10 minutes to obtain orthogonally modified cells. The orthogonally modified cells were then mixed with bioorthogonal antifouling microbubbles to achieve cell capture.

[0107] like Figure 5 As shown, DiO-stained cells were successfully captured on the surface of the microbubbles. The captured cells were fixed with glutaraldehyde and observed using SEM, as shown below. Figure 6 As shown, the cells adhere well to the surface of the microbubbles.

[0108] The results of the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 in capturing orthogonally modified skin cancer cells were similar to those of the GB@PTB@Tz prepared in Example 5 in capturing orthogonally modified skin cancer cells.

[0109] Example 11

[0110] The effects of hollow glass microspheres (GB), antifouling glass microbubbles (GB@PTB), and antifouling glass microbubbles (GB@PTB@Tz) prepared in Example 5 on the capture of orthogonally modified lung cancer exosomes were investigated.

[0111] Take 10 7Exosomes derived from A549 (non-small cell lung cancer) cells were reacted in 100 μL of PBS solution containing 5 μM TCO-NHS ester at room temperature for 30 minutes to obtain TCO-modified exosomes. The exosomes were then captured by mixing 0.4 mg of GB@PTB@Tz with the TCO-modified exosomes at room temperature for 10 minutes.

[0112] The results of the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 on capturing orthogonally modified lung cancer exosomes were similar to those of the GB@PTB@Tz prepared in Example 5 on capturing orthogonally modified lung cancer exosomes.

[0113] Example 12

[0114] The study investigated the capture of orthogonally modified hepatocellular carcinoma exosomes by the antifouling glass microbubbles (GB@PTB@Tz) with bioorthogonal function prepared in Example 5.

[0115] Take 10 7 Exosomes derived from HepG2 (human liver cancer) cells were reacted in 100 μL of PBS solution containing 5 μM TCO-NHS ester at room temperature for 30 minutes to obtain TCO-modified exosomes. The exosomes were then captured by mixing 0.4 mg of GB@PTB@Tz with the TCO-modified exosomes at room temperature for 10 minutes.

[0116] To observe the captured exosomes, DiI staining was used to stain them. For example... Figure 7 As shown, the microbubble surface exhibits a clear fluorescent signal. The captured exosomes were fixed using glutaraldehyde and observed using SEM. Figure 8 As shown, a distinct membrane structure is attached to the surface of the microbubbles, demonstrating the successful capture of exosomes.

[0117] The results of the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 on capturing orthogonally modified liver cancer exosomes were similar to those of the GB@PTB@Tz prepared in Example 5 on capturing orthogonally modified lung cancer exosomes.

[0118] Example 13

[0119] The effects of the bio-orthogonal-functional antifouling glass microbubbles (GB@PTB@Tz) prepared in Example 5 on the capture of orthogonally modified CD45 protein were investigated.

[0120] Take 2 ng of recombinant CD45 protein, add 1 μL of TCO-NHS ester (10 mM), and react in 100 μL of PBS at room temperature for 10 minutes to obtain TCO-modified protein. Mix 0.4 mg of GB@PTB@Tz with the TCO-modified protein at room temperature for 10 minutes to achieve CD45 protein capture.

[0121] The capture results of the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 on orthogonally modified CD45 protein were similar to those of the GB@PTB@Tz prepared in Example 5 on orthogonally modified CD45 protein.

[0122] Example 14

[0123] The study investigated the capture of orthogonally modified EGFR protein by the antifouling glass microbubbles (GB@PTB@Tz) with bioorthogonal function prepared in Example 5.

[0124] Take 2 ng of recombinant EGFR protein, add 1 μL of TCO-NHS ester (10 mM), and react in 100 μL of PBS at room temperature for 10 minutes to obtain TCO-modified protein. Mix 0.4 mg of GB@PTB@Tz with the TCO-modified protein at room temperature for 10 minutes to achieve EGFR protein capture.

[0125] The capture results of the orthogonally modified EGFR protein by the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 were similar to those of the orthogonally modified EGFR protein by the GB@PTB@Tz prepared in Example 5.

[0126] Example 15

[0127] The effects of the bio-orthogonal-functional antifouling glass microbubbles (GB@PTB@Tz) prepared in Example 5 on the capture of orthogonally modified total protein were investigated.

[0128] Take 10 5 Cells were lysed using 1 mL of non-denaturing cell lysis buffer, and the supernatant was collected after centrifugation to obtain total cellular protein. 100 μL of this supernatant was added to 1 μL of TCO-NHS ester (10 mM), and the mixture was reacted at room temperature for 10 minutes to obtain TCO-modified protein. 0.4 mg of GB@PTB@Tz was then mixed with the TCO-modified protein at room temperature for 10 minutes to achieve protein capture.

[0129] To detect the captured proteins, 100 nM Cy5-tagged DNA aptamers were used. Figure 9 As shown, the Cy5-labeled EpCAM aptamer can successfully recognize the EpCAM protein captured on the microbubbles, demonstrating the successful protein capture.

[0130] The total protein capture results of the antifouling glass microbubbles with bioorthogonal function prepared in Examples 1-4 were similar to those of the total protein capture results of the orthogonally modified GB@PTB@Tz prepared in Example 5.

[0131] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Antifouling microbubbles for bioorthogonal capture, characterized in that, include: Hollow glass microspheres; An antifouling coating is disposed on the surface of the hollow glass microspheres, and the antifouling coating is phase-change BSA; wherein, BSA solution and TCEP solution are mixed and the pH is adjusted to 5.0 to obtain phase-change BSA; The trapping layer is formed by setting an N-hydroxysuccinimide derivative on the antifouling coating.

2. The antifouling microbubbles for bioorthogonal capture according to claim 1, characterized in that, The diameter of the hollow glass microspheres is 10-100 μm.

3. The antifouling microbubbles for bioorthogonal capture according to claim 1, characterized in that, N-hydroxysuccinimide derivatives are N-hydroxysuccinimide derivatives containing azide, alkyne, dibenzocyclooctylene, transcyclooctene, or 1,2,4,5-tetraazine.

4. The method for preparing antifouling microbubbles for bioorthogonal trapping as described in claim 1, characterized in that, Includes the following steps: 1) Add BSA solution and TCEP solution to the hollow glass microspheres, adjust the pH value to 5.0, and mix for 0.5-12 hours; 2) Centrifuge the mixture from step 1), remove the lower layer solution, wash with ultrapure water, repeat centrifugation 1-5 times, and vacuum dry to obtain glass microbubbles with antifouling function. 3) React the glass microbubbles with antifouling function obtained in step 2) with N-hydroxysuccinimide derivative, centrifuge, remove the lower layer solution, add ultrapure water to wash, repeat centrifugation 1-5 times, and vacuum dry to obtain antifouling microbubbles with bioorthogonal trapping.

5. The method for preparing antifouling microbubbles for bioorthogonal capture according to claim 4, characterized in that, In step 1), the mass of the hollow glass microspheres is 0.5-5g; the concentration of the BSA solution is 1-5mg / mL and the volume is 10-30mL; the concentration of the TCEP solution is 10-50mM and the volume is 10-50mL.

6. The method for preparing antifouling microbubbles for bioorthogonal capture according to claim 4, characterized in that, In step 2), the mass of the antifouling glass microbubbles is 50-500 mg, the concentration of the N-hydroxysuccinimide derivative is 0.5-2 mM, and the volume is 0.5-5 mL.

7. The application of antifouling microbubbles for bioorthogonal capture as described in claim 1, characterized in that, Isolation and detection of cells, exosomes, and proteins for non-disease diagnostic purposes.

8. The antifouling microbubble application for bioorthogonal capture as described in claim 7, characterized in that, The cells are tumor cells or endothelial cells.

9. The antifouling microbubble application for bioorthogonal capture as described in claim 7, characterized in that, The exosomes are derived from healthy cells or from 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, stomach cancer, thyroid cancer, prostate cancer, head and neck cancer, esophageal cancer, uterine cancer, and brain cancer cells.

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