Multifunctional glass microbubble and high-throughput rapid immunoassay system and application thereof

Abstract

The invention discloses a multifunctional glass microbubble, a high-flux rapid immunoassay system and application thereof, and the multifunctional glass microbubble is prepared by the following steps: S1, adding a bovine serum albumin solution into hollow glass microbubbles to obtain a bovine serum albumin coating, centrifuging, removing a lower-layer solution, cleaning and drying to obtain a hollow glass microbubble solution; the antifouling hollow glass microbubbles are obtained; s2, mixing the antifouling hollow glass microbubbles with tetrazine-active ester, centrifuging, removing a lower solution, washing and drying to obtain the antifouling hollow glass microbubbles with the biological orthogonal function. The anti-fouling hollow glass microbubble has the beneficial effects that the anti-fouling hollow glass microbubble with a biological orthogonal function, the self-suspension performance, the anti-fouling function and the bovine serum albumin rich in tyrosine residues not only can realize high-flux array detection in a 96-well plate, but also can realize the effects of anti-fouling and signal amplification, and can be applied to the field of biosensor detection. Therefore, the defects of low flux, low sensitivity, long detection time and the like of a traditional detection method are overcome.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and more particularly to a multifunctional glass microbubble and high-throughput rapid immunoassay system and applications thereof. Background Art

[0002] Immunoassay technology plays an important role in disease diagnosis, biomarker detection, and basic medical research. Enzyme-linked immunosorbent assay (ELISA) is widely used due to its high specificity and standardized operation. However, existing technologies still face many limitations, especially in terms of high-throughput detection, sensitivity, and operational efficiency, which make it difficult to meet clinical and scientific research needs.

[0003] The throughput of traditional ELISA technology is limited by the physical structure of the microplate and the detection mode. Conventional 96-well plate assays require well-by-well processing and multiple manual washes during the reaction step, making true high-throughput difficult to achieve. Furthermore, traditional methods require pre-coating the capture antibody onto the microplate surface to form a solid-phase carrier. This process is time-consuming and suffers from poor batch-to-batch stability, resulting in extended testing cycles and increased costs.

[0004] Insufficient sensitivity is another significant drawback of existing technologies. The detection of low-abundance proteins (such as circulating tumor markers) is often limited by low signal amplification efficiency and background noise caused by nonspecific adsorption. Traditional signal amplification methods (such as enzyme-catalyzed colorimetry) rely on single enzyme labels, which have limited catalytic efficiency and are difficult to achieve effective signal accumulation. At the same time, nonspecific protein adsorption on the surface of microspheres or carriers will interfere with the specific binding of target molecules, further reducing the detection signal-to-noise ratio.

[0005] Operational complexity is also a bottleneck of existing technologies. In traditional methods, microspheres or carriers need to be separated from the solid and liquid by centrifugation or magnetic separation, which is a cumbersome and time-consuming process. For example, magnetic bead separation relies on an external magnetic field, while centrifugation has high equipment requirements and is difficult to adapt to high-throughput scenarios. In addition, existing microplate designs fail to fully utilize the self-suspending properties of hollow glass microbubbles, resulting in low cleaning efficiency and difficulty in completely removing residual liquid, affecting the accuracy of subsequent reaction steps.

[0006] The root cause of these problems lies in the functional limitations of existing carrier materials and inadequate detection system design. Traditional microsphere surface modification methods, such as polystyrene microspheres, rely on a single method, making it difficult to simultaneously achieve antifouling properties and efficient coupling of bioactive groups. For example, while conventional bovine serum albumin (BSA) blocking can reduce nonspecific adsorption, it masks active sites on the microsphere surface, reducing the capture efficiency of target molecules.

[0007] At the system design level, existing microplates lack optimized fluid dynamics, making it impossible to leverage the self-suspending properties of microbubbles for rapid separation. For example, traditional horizontally placed microplates require multiple pipetting operations for cleaning, and microbubbles are easily dissipated with the liquid flow, resulting in sample loss and poor reproducibility.

[0008] To address these challenges, the field urgently needs to develop novel vectors for capturing tumor target proteins and novel immunoassay systems that can achieve high-throughput detection while maintaining high sensitivity, while streamlining operational procedures through optimized vector functionality and system design. However, key challenges in achieving this technology remain: balancing the antifouling properties of the microbubble surface with the exposure of bioactive groups, designing efficient and stable bioorthogonal reaction systems, and leveraging the physical properties of microbubbles to optimize separation efficiency. Summary of the Invention

[0009] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0010] Another object of the present invention is to provide a multifunctional glass microbubble and a high-throughput rapid immunoassay system and their uses. The multifunctional glass microbubble has bioorthogonal and antifouling functions. At the same time, its inherent self-suspending properties (hollowness) and bovine serum albumin rich in tyrosine residues not only enable high-throughput array detection in a 96-well plate, but also achieve antifouling and signal amplification effects. In addition, when using a high-throughput rapid immunoassay containing the bioorthogonal antifouling hollow glass microbubbles to detect target proteins, compared with traditional ELISA detection methods, it does not require the advance preparation of enzyme-labeled test plates. The operation is simple, rapid, and efficient, and protein detection can be achieved, providing a basis for guiding the early, rapid, and high-throughput diagnosis of protein marker-related diseases and tumor diseases.

[0011] In order to achieve these purposes and other advantages according to the present invention, a multifunctional glass microbubble is provided, which is prepared by the following preparation steps:

[0012] S1, adding a bovine serum albumin solution to the hollow glass microbubbles to obtain a bovine serum albumin coating, centrifuging, removing the lower layer of solution, washing with ultrapure water, and drying to obtain antifouling hollow glass microbubbles;

[0013] S2. Mixing the antifouling hollow glass microbubbles obtained in S1 with tetrazine-active ester, centrifuging, removing the lower layer solution, washing with ultrapure water, and drying to obtain antifouling hollow glass microbubbles with bioorthogonal function, i.e., the multifunctional glass microbubbles.

[0014] A high-throughput rapid immunoassay system comprises the multifunctional glass microbubbles.

[0015] Preferably, biotin-tyramide, hydrogen peroxide, and HRP-labeled streptavidin are also included.

[0016] Preferably, TMB color developing solution and TMB stop solution are also included.

[0017] Preferably, a 96-well microplate is also included.

[0018] Preferably, it further includes a bracket, the bracket includes a vertical L-shaped block, an inclined block provided on the top of the L-shaped block, the inclined block is inclined toward the horizontal block side of the L-shaped block, and the high end of the inclined block is higher than the L-shaped block, the angle between the inclined block and the vertical plane is 45 to 60 degrees, and the top surface of the microplate is provided with a slot adapted to the inclined block, so that when the inclined block is inserted into the slot, the microplate is inclined.

[0019] Preferably, the method further comprises a cleaning solution, which is a 0.1% Tween-20 / PBS solution.

[0020] Preferably, the particle size of the multifunctional glass microbubbles is 10-50 microns.

[0021] Preferably, the horizontal block of the L-shaped block has a size of 2 cm in length, 2 cm in width and 0.5 cm in height, the vertical block of the L-shaped block has a size of 1.2 cm in length, 0.3 cm in width and 7 cm in height, and the material of the bracket is PLA or ABS resin material.

[0022] A multifunctional anti-fouling hollow glass microbubble and a high-throughput rapid immunoassay system and their use in detecting a target protein, wherein the target protein is EpCAM protein or PD-L1 protein.

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

[0024] The present invention provides a multifunctional glass microbubble, which is an antifouling hollow glass microbubble with bioorthogonal function. Specifically, first, the surface of the hollow glass microbubble is modified with bovine serum albumin. The hydrophilicity and abundant lysine residues of bovine serum albumin can form a "shielding layer" to cover the hydrophobic area on the surface of the microbubble, effectively blocking the nonspecific binding of foreign proteins and enhancing the binding ability with specific proteins (i.e., target proteins), thereby achieving antifouling effect; secondly, the antifouling hollow glass microbubble is modified with a specific chemical group (tetrazine, Tz) on the surface, so that it can bind to the target protein in the biological system. Target protein molecules carrying complementary groups (trans-cyclooctene, TCO) undergo efficient and specific click chemistry reactions without the need for catalysts and without interfering with natural biological processes, achieving efficient immobilization of target molecules and avoiding the damage to protein activity caused by traditional coupling methods (such as EDC / NHS). Simultaneously, multiple signal molecules are densely labeled at the target site through bioorthogonal reactions, significantly improving detection sensitivity. Finally, the anti-fouling hollow glass microbubbles, due to their hollow structure, have suspension properties. When in a reaction liquid system, they can quickly float to the top of the liquid surface, facilitating the removal of waste liquid and thus improving cleaning efficiency.

[0025] The overall anti-fouling hollow glass microbubbles with bio-orthogonal functions can not only realize high-throughput array detection in 96-well plates, but also achieve anti-fouling and signal amplification effects;

[0026] The present invention also provides a multifunctional antifouling hollow glass microbubble and a high-throughput rapid immunoassay system and uses thereof, which include the antifouling hollow glass microbubble with bioorthogonal function. In the presence of hydrogen peroxide, the HRP in the HRP (horseradish peroxidase)-labeled antibody IgG-HRP can catalyze the conversion of biotin-tyramine into a highly reactive free radical form, which then covalently binds to the tyrosine residue in the target molecule (i.e., the target protein). Because tyramide molecules can bind to multiple tyrosine residues, the streptavidin in the added HRP-labeled streptavidin can bind to a large amount of deposited biotin, thereby forming a high-density label on the target molecule. The large amount of HRP attached to the target molecule can quickly develop color in the TMB colorimetric solution, thereby enhancing the signal and thus improving the detection sensitivity. At the same time, the surface of the anti-fouling hollow glass microbubbles modified with bovine serum albumin also carries a large number of tyrosine residues. Therefore, in the presence of hydrogen peroxide, HRP catalyzes the conversion of biotin-tyramine into a highly reactive free radical form, which can then bind to the tyrosine residues on the surface of the hollow glass microbubbles, further amplifying the signal.

[0027] In addition, the Qualcomm Rapid Immunoassay System also includes a 96-well microplate and a holder. The microplate enables high-throughput testing. When the microplate is mounted on the holder, the microplate is tilted. In this state, the hollow glass microbubbles in the microplate wells quickly float to the top of the liquid surface and adhere to one side of the well wall. This makes it easier for operators to remove waste liquid from the other side, improving cleaning efficiency and shortening testing time.

[0028] In general, the high-throughput rapid immunoassay system containing multifunctional glass microbubbles, in the process of detecting the target protein, firstly, the multifunctional glass microbubbles bind to the trans-cyclooctene modified on the surface of the target protein through the surface-modified tetrazine, thereby capturing the target protein, and then the detection antibody (primary antibody, such as EpCAM / CD326 polyclonal antibody used to detect EpCAM protein, PD-L1 / CD274 polyclonal antibody used to detect PD-L1 protein) specifically recognizes the target protein and further binds to the target protein, and then, under the action of hydrogen peroxide, the HRP in the secondary antibody (derived from the HRP-labeled antibody) catalyzes the After the tyramine molecules in biotin-tyramide form a highly reactive free radical form, they bind to the tyrosine residues in the target molecule and the tyrosine residues modified on the surface of the multifunctional glass microbubbles, thereby enhancing the signal. In addition, the streptavidin in the HRP-labeled streptavidin binds to the biotin in the biotin-tyramide molecule to form a high-density label, further enhancing the signal. The HRP in the HRP-labeled streptavidin can then quickly color the TMB color developing solution, thereby improving the detection sensitivity. Overall, these steps are closely linked, improving the shortcomings of traditional detection methods such as low throughput, low sensitivity, and long detection time, and have good application prospects.

[0029] The present invention also provides an application of a high-throughput rapid immunoassay system in detecting a target protein, wherein the target protein is EpCAM protein or PD-L1 protein, which can provide a basis for early, rapid, and high-throughput diagnosis of protein marker-related diseases and tumor diseases.

[0030] Other advantages, objectives and features of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the preparation process of anti-fouling glass microbubbles with bio-orthogonal functions (i.e., multifunctional glass microbubbles) according to one of the technical solutions of the present invention;

[0032] Figure 2 Elemental characterization results of GB, GB@BSA, and GB@BSA@Tz of the present invention;

[0033] Figure 3Figure 2 is the fluorescence characterization result of GB, GB@BSA, and GB@BSA@Tz of the present invention;

[0034] Figure 4 Schematic diagram of a microplate and a microplate mounted on a bracket according to one of the technical solutions of the present invention, wherein the left figure is a schematic diagram of the microplate, and the right figure is a schematic diagram of the microplate and the bracket;

[0035] Figure 5 This is a schematic diagram of the process of installing a microplate according to one of the technical solutions of the present invention;

[0036] Figure 6 This is a schematic structural diagram of a microplate and a bracket according to one of the technical solutions of the present invention;

[0037] Figure 7 Schematic diagram of the process of detecting tumor target proteins according to the present invention; wherein, NHS-PEG4-TCO represents trans-cyclooctene-tetraethylene glycol-active ester, Click represents click reaction, 1gG-HRP represents HRP (horseradish peroxidase)-labeled antibody, Detection antibody represents detection antibody, Activated biotin-tyramide represents activated biotin-tyramide, Streptavidin-HRP represents HRP-labeled streptavidin, 96-well plat represents 96 microplate, Microplate holder represents microplate holder, bubble fast wash step represents the step of quickly washing hollow glass microbubbles, self-floating represents self-suspension, Liquid leave represents waste liquid removal, and Bubble keep represents that the multifunctional glass microbubbles are retained in the wells;

[0038] Figure 8 The left figure (a) shows the absorbance detection results of biotin-tyramide at different concentrations, and the right figure (b) shows the absorbance detection results of biotin-tyramide at different reaction times. Figure 8 Where Biotin-tyramine concentration indicates biotin-tyramine concentration, Absorbance indicates absorbance, control indicates control group, and blank indicates blank group.

[0039] Figure 9 This is a test result diagram of the signal enhancement effect of the present invention;

[0040] Figure 10 This is a graph showing the results of detecting EpCAM protein in lung cancer cells according to the present invention;

[0041] Figure 11 This is a graph showing the results of detecting PD-L1 protein in lung cancer cells according to the present invention;

[0042] Figure 12 This is a graph showing the results of the present invention detecting EpCAM protein in clinical blood samples of lung cancer patients;

[0043] Figure 13 This is a graph showing the results of detecting PD-L1 protein in clinical blood samples of lung cancer patients according to the present invention;

[0044] Figure 14 This is a schematic diagram of the structure in which hollow glass microbubbles in a microporous plate float to the liquid surface and adhere to the pore wall according to one of the technical solutions of the present invention.

[0045] Reference numerals: 1-multifunctional glass microbubble; 2-waste liquid; 3-support; 301-horizontal block; 302-vertical block; 303-oblique block; 4-microplate. DETAILED DESCRIPTION

[0046] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0047] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0048] like Figure 1-14 As shown, the present invention provides a multifunctional glass microbubble, which is prepared by the following preparation steps:

[0049] S1, adding a bovine serum albumin solution to the hollow glass microbubbles to obtain a bovine serum albumin coating, centrifuging, removing the lower layer of solution, washing with ultrapure water, and drying to obtain antifouling hollow glass microbubbles;

[0050] S2. Mixing the antifouling hollow glass microbubbles obtained in S1 with tetrazine-active ester, centrifuging, removing the lower layer solution, washing with ultrapure water, and drying to obtain antifouling hollow glass microbubbles with bioorthogonal function, namely, the multifunctional glass microbubbles 1.

[0051] The beneficial effect of adopting this technical solution is that by first modifying bovine serum albumin on the surface of hollow glass microbubbles, hollow glass microbubbles with anti-fouling function are obtained, which reduces the nonspecific adsorption of non-target molecules (such as miscellaneous proteins, lipids, etc.) by the hollow glass microbubbles, thereby reducing the detection background noise and improving the detection specificity; by further modifying the surface of the hollow glass microbubbles with anti-fouling function with specific chemical groups (tetrazine, Tz), it can enable it to undergo efficient and specific click chemistry reactions with target protein molecules carrying complementary groups (trans-cyclooctene, TCO) in biological systems without the need for catalysts and without interfering with natural biological processes, thereby improving the detection sensitivity.

[0052] The present invention also provides a high-throughput rapid immunoassay system, comprising the multifunctional glass microbubbles 1, which has the advantages of high detection sensitivity and high detection efficiency when detecting target proteins.

[0053] In another technical solution, the high-throughput rapid immunoassay system also includes biotin-tyramide, hydrogen peroxide, and HRP-labeled streptavidin; using this technical solution, the beneficial effect is that by designing biotin-tyramide, hydrogen peroxide, and HRP-labeled streptavidin, when used, in the presence of hydrogen peroxide, the HRP in the HRP-labeled antibody IgG-HRP can catalyze the conversion of biotin-tyramide into a highly reactive free radical form, which then covalently binds to the tyrosine residue in the target molecule (i.e., the target protein). Since tyramide molecules can bind to multiple tyrosine residues, the streptavidin in the added HRP-labeled streptavidin can bind to a large amount of deposited biotin, thereby forming a high-density label on the target molecule. The large amount of HRP attached to the target molecule can quickly develop color in the TMB colorimetric solution, thereby enhancing the signal and thereby improving the detection sensitivity. At the same time, the surface of the hollow glass microbubbles modified with bovine serum albumin also carries a large number of tyrosine residues. Therefore, in the presence of hydrogen peroxide, the HRP in the HRP-labeled streptavidin catalyzes the conversion of biotin-tyramine into a highly reactive free radical form, which can then bind to the tyrosine residues on the surface of the hollow glass microbubbles, further amplifying the signal. Overall, by designing biotin-tyramide, hydrogen peroxide, and HRP-labeled streptavidin, an efficient signal amplification system is constructed, which is convenient for operators to use directly without additional preparation, thereby improving ease of use. In actual use, the high-throughput rapid immunoassay system can further include an HRP-labeled antibody, which is the antibody used for detection of the target protein, to further facilitate detection by the tester.

[0054] In another technical solution, the high-throughput rapid immunoassay system also includes a TMB colorimetric solution and a TMB stop solution. Specifically, the TMB colorimetric solution serves as a signal output method and as a substrate for horseradish peroxidase (HRP). Under the catalysis of HRP, it is oxidized to generate a blue soluble product, the color depth of which is proportional to the concentration of the target molecule, thereby achieving signal visualization and quantitative detection. A TMB stop solution is preferably a TMB stop solution that does not contain sulfuric acid. This stops the enzymatic reaction under acidic conditions, stably converts the blue product to yellow, and avoids precipitation or light absorption interference that may be caused by traditional sulfuric acid stop solutions, ensuring accurate reading of absorbance values ​​at a wavelength of 450nm. The use of this technical solution has the beneficial effect of being easy for operators to use directly without the need for additional preparation, thereby improving ease of use.

[0055] In another technical solution, a 96-well microplate 4 is also included; adopting this technical solution has the beneficial effect that, by designing the 96-well microplate 4, high-throughput rapid detection can be achieved, thereby improving detection efficiency.

[0056] In another technical solution, a bracket 3 is further included, wherein the bracket 3 includes a vertical L-shaped block and an inclined block 303 provided on the top of the L-shaped block. The inclined block 303 is inclined toward the horizontal block side of the L-shaped block, and the high end of the inclined block 303 is higher than the L-shaped block. The angle between the inclined block 303 and the vertical plane is 45°. The top surface of the microplate 4 is provided with a slot adapted to the inclined block 303, so that when the inclined block 303 is inserted into the slot, the microplate 303 is tilted.

[0057] In the above technical solution, the bracket 3 includes an L-shaped block and an oblique block 303. The L-shaped block includes a horizontal block 301 and a vertical block 302 provided at one end of the top of the horizontal block 301. The bottom of the oblique block 303 is transitionally connected to the top of the vertical block 302. The angle between the oblique block 303 and the vertical plane is 45 degrees. The oblique block 303 is inclined toward the side of the horizontal block 301, and the high end of the oblique block 303 is higher than the vertical block 302. The top of the microplate 4 is provided with a slot (this slot is provided). The top refers to the top of the microplate 4 when the microplate 4 is vertical), and the slot is adapted to the inclined block 303 so that when the inclined block 303 is inserted into the slot, the microplate 4 is mounted on the bracket 3 and tilted. In actual design, the angle between the inclined block 303 and the vertical plane is preferably 45 degrees. In actual use, the FPT010 model microplate 4 produced by Biyuntian Biotechnology Co., Ltd. can be selected. This microplate 4 has a slot on the top that can be connected to the inclined block 303;

[0058] In this technical solution, during use: when the multifunctional glass microbubbles 1 in the microplate 4 need to be cleaned or the waste liquid 2 in the microplate 4 needs to be sucked out, the inclined block 303 of the bracket 3 is inserted into the slot of the microplate 4. At this time, the microplate 4 is tilted. Due to the overall tilt of the microplate 4 and the suspension characteristics of the multifunctional glass microbubbles 1 themselves (density of 0.6 g / cm 3 About, smaller than the solution in the hole), so the multifunctional glass microbubble 1 can quickly float to the top of the microplate 4 and adhere to one side of the hole wall (such as Figure 14 As shown, Figure 14 The reference 2 represents the waste liquid 2 inside the well. At this point, an 8-channel or 12-channel pipette is used to aspirate the liquid from the other side of the microplate's four wells. The waste liquid 2, after being washed with 0.1% Tween-20 / PBS solution, is then separated from the multifunctional glass microbubbles 1 using the same procedure. Compared to existing centrifugal separation methods, this greatly shortens the time required for washing and separation, significantly improves washing efficiency, and significantly reduces overall detection time.

[0059] In another technical solution, a cleaning solution is further included, which is a 0.1% Tween-20 / PBS solution. Specifically, the cleaning solution is prepared as follows: 1 ml of 100% Tween-20 is added to 1000 ml of PBS to obtain a 0.1% Tween-20 / PBS cleaning solution;

[0060] The beneficial effect of adopting this technical solution is that, by designing the cleaning liquid, the operator can directly use the cleaning liquid for cleaning without having to configure additional cleaning liquid.

[0061] It should be noted that the high-throughput rapid immunoassay system of the present invention can be manufactured into various forms of commercial products such as test kits during actual production.

[0062] In another technical solution, the multifunctional glass microbubbles 1 have a particle size of 10-50 microns. This technical solution has the beneficial effect that the low density of the multifunctional glass microbubbles 1 combined with the particle size range of 10-50 microns ensures that the multifunctional glass microbubbles 1 have sufficient buoyancy in the liquid, allowing them to quickly float to the top of the microplate 4, facilitating the use of a multichannel pipette to aspirate and discard the liquid at the bottom. In addition, this particle size range prevents the multifunctional glass microbubbles from being unstable in suspension due to being too small, or from settling slowly due to being too large, thereby significantly shortening the washing step time and improving separation efficiency.

[0063] In another technical solution, the dimensions of the horizontal block 301 of the L-shaped block are 2 cm long, 2 cm wide, and 0.5 cm high, the dimensions of the vertical block 302 of the L-shaped block are 1.2 cm long, 0.3 cm wide, and 7 cm high, and the material of the bracket 3 is PLA or ABS resin material.

[0064] In this technical solution, the dimensions of the horizontal block 301 of the L-shaped block are 2 cm in length, 2 cm in width, and 0.5 cm in height. The dimensions of the vertical block 302 of the L-shaped block are 1.2 cm in length, 0.3 cm in width, and 7 cm in height. The oblique block 303 can be regarded as a square block with a size of 1.2 x 1.2 cm and a thickness of 0.2 cm. A 45-degree angle is formed between the oblique block 303 and the vertical block 302 (i.e., the angle between the oblique block 303 and the vertical plane is 45 degrees). It should be noted that the bottom of the oblique block 303 is transitionally connected to the top of the vertical block 302 (leaving no gap). In actual manufacturing, the bracket 3 can be printed using 3D printing technology.

[0065] The beneficial effect of adopting this technical solution is that a bracket 3 of a specific size is provided, which is convenient for 3D printing and simple to manufacture.

[0066] The application of a high-throughput rapid immunoassay system in detecting target proteins, wherein the target proteins are EpCAM proteins or PD-L1 proteins, can provide a basis for early, rapid, and high-throughput diagnosis of protein marker-related diseases and tumor diseases.

[0067] <Preparation of Antifouling Hollow Glass Microbubbles (GB@BSA) and Antifouling Hollow Glass Microbubbles with Bioorthogonal Functionality (GB@BSA@Tz, the Multifunctional Glass Microbubbles)>

[0068] like Figure 1 The preparation of the antifouling hollow glass microbubbles and the antifouling hollow glass microbubbles with bioorthogonal function is as follows:

[0069] 1) Weigh 0.5 g of hollow glass microbubbles (GB) and add them to a 50 ml centrifuge tube. Add 10 ml of anhydrous ethanol containing 0.5 ml of 3-(2,3-epoxypropoxy)propyltriethoxysilane (analytical grade). Mix on a rotary mixer at room temperature for 10 h. After the reaction is complete, centrifuge at 1000 rpm for 1 minute to remove the lower layer solution. Wash with ultrapure water, centrifuge again, repeat the washing three times, and dry under vacuum at 37° C. to obtain silanized hollow glass microbubbles GB@GPTES. The hollow glass microbubbles are iM30K hollow glass microbubbles produced by 3M.

[0070] 2) Weigh 0.5 g of the silanized hollow glass microbubbles from step 1) into a 15 ml centrifuge tube, add 5 ml of 2M NaCl solution, 4.5 ml of ultrapure water, and 0.5 ml of 100 mg / ml BSA solution, and mix on a rotary mixer at room temperature for 10 h. After the reaction is complete, centrifuge at 1000 rpm for 1 minute, and remove the lower layer of solution. Wash with ultrapure water, centrifuge again, and repeat the wash three times. Dry under vacuum at 37°C to obtain antifouling hollow glass microbubbles (GB@BSA), which are then stored at -20°C.

[0071] 3) Weigh 20 mg of GB@BSA and dissolve it in 1 ml of 1 mM Tz-PEG4-NHS (tetrazine-active ester, purchased from Xi'an Kangfuno Biotechnology Co., Ltd.) in PBS. Mix the mixture on a rotary mixer at room temperature for 10 hours, centrifuge at 1000 rpm for 1 minute, and remove the lower layer. Wash the mixture with ultrapure water, centrifuge again, and repeat the wash three times. Dry the mixture under vacuum at 37°C to obtain bioorthogonal antifouling hollow glass microbubbles (GB@BSA@Tz), i.e., the multifunctional glass microbubbles, which are stored at -20°C.

[0072] <Elemental Characterization of Glass Microbubbles>

[0073] Methods: Surface chemical composition was determined using X-ray photoelectron spectroscopy (XPS). Hollow glass microbubbles were dried overnight under vacuum at room temperature and then transferred to an XPS analysis system. XPS spectra of GB, GB@BSA, and GB@BSA@Tz were measured from 0 to 1000 eV, and high-resolution XPS spectra of N 1s, C 1s, and O 1s were collected. Energy calibration was performed using the carbon C-C peak (C 1s = 284.8 eV).

[0074] Samples: Elemental characterization of insulating glass microbubbles (GB), antifouling insulating glass microbubbles (GB@BSA), and antifouling insulating glass microbubbles with bioorthogonal function (GB@BSA@Tz).

[0075] Results: As Figure 2 As shown in the figure, ordinary GB has only three peaks of carbon, oxygen and silicon. However, after modification with BSA coating (bovine serum albumin), the antifouling hollow glass microbubbles (GB@BSA) can be observed at 400 cm- 1 The nitrogen peak appeared at the position of , proving that BSA was successfully covered on the GB surface. Similarly, the antifouling hollow glass microbubbles with bioorthogonal function (GB@BSA@Tz) after modification of Tz (tetrazine) coating can also be observed at 400cm- 1 A nitrogen peak appeared at the position of , proving that Tz was successfully covered on the GB surface. Figure 2 In the table, BingdingEnergy represents binding energy and Counts represents peak value.

[0076] <Fluorescence Characterization of Glass Microbubbles>

[0077] Methods: Since Cy5-labeled trans-cyclooctene (TCO-Cy5) reacts with tetrazines, Cy5-labeled trans-cyclooctene was used to characterize the coating on the glass microbubble surface. The following steps were performed: 0.5 mg of GB, GB@BSA, or GB@BSA@Tz was weighed and reacted in 100 μl of PBS containing 1 mM TCO-Cy5 (purchased from Xi'an Kangfuno Biotechnology Co., Ltd.) at 37°C for 30 minutes. The samples were then washed five times with ultrapure water and observed using an inverted fluorescence microscope.

[0078] Samples: Fluorescence characterization of hollow glass microbubbles (GB), antifouling hollow glass microbubbles (GB@BSA), and antifouling hollow glass microbubbles with bioorthogonal function (GB@BSA@Tz).

[0079] The fluorescence characterization results are as follows Figure 3 As shown, after GB@BSA@Tz modified with Tz was stained with TCO-Cy5, an obvious fluorescence signal appeared on the surface of GB@BSA@Tz ( Figure 3 The bottom of the third column from left to right in the figure), while the unmodified GB and GB@BSA surfaces do not have any signal ( Figure 3 The top two in the third column from left to right) prove that Tz is successfully formed on the surface of microbubbles in anti-fouling hollow glass. Figure 3 In the figure, TCO-cy5 represents the fluorescence imaging image after staining with TCO-Cy5, and Brightfield represents the bright field of the fluorescence image.

[0080] <Production of Microplate Holder>

[0081] The bottom of the 3D-printed microplate 4 support is an L-shaped horizontal block 301, which is a square block with a size of 2×2 cm and a height of 0.5 cm. One end of the square block is an L-shaped vertical block 302, and the vertical block 302 is a rectangular support block with a size of 1.2×7 cm and a thickness of 0.3 cm. The top of the rectangular support block is provided with an oblique block 303, which can be regarded as a square block with a thickness of 1.2×1.2 cm and a thickness of 0.2 cm. A 45-degree angle is formed between the oblique block 303 and the rectangular support block (that is, the angle between the oblique block and the vertical plane is 45 degrees). It should be noted that the bottom of the oblique block 303 is transitionally connected to the top of the rectangular support block (leaving no gap);

[0082] like Figure 5 As shown in the assembly process, when in use, after the inclined block 303 is inserted into the slot on the microplate 4, the microplate 4 forms a certain angle with the horizontal plane, thereby making the microplate 4 tilted, and the result is as shown in FIG. Figure 4 、 Figure 6 The microplate high-throughput detection device shown can now perform corresponding operations in the microplate 4.

[0083] <Optimization of biotin-tyramide concentration and time>

[0084] Optimization of biotin-tyramide concentration and time. The specific method is as follows:

[0085] 1) GB@BSA@Tz was placed in a centrifuge tube and PBS was added to make a 10 mg / ml GB@BSA@Tz solution. 2 μl of 200 μg / ml EpCAM protein was placed in a 600 μl centrifuge tube. 1 μl of 10 mM trans-cyclooctene active lipid and 1 mM sodium bicarbonate solution were added. The mixture was vortexed at 1000 rpm for 20 minutes to obtain a 10 μg / ml trans-cyclooctene-labeled protein. EpCAM protein was purchased from Ibotek Biotechnology Co., Ltd.

[0086] 2) Take 50 μl of the GB@BSA@Tz solution from step 1 and dilute the prepared protein to 50 ng / ml with PBS. For the control group, take 50 μl of the diluted protein and GB@BSA@Tz in a 96-well microplate and react on a microplate shaker for 40 min. The blank group is treated with 50 μl of PBS solution. After 40 minutes of reaction on a microplate shaker, the bracket's bevel was inserted into the microplate slot. The microplate was tilted. Due to the overall tilt of the microplate and the buoyancy of the multifunctional glass microbubbles GB@BSA@Tz, the bubbles quickly floated to the top of the microplate and adhered to one side of the well wall. An 8- or 12-channel pipette was then used to aspirate the liquid from the other side of the well. The waste liquid from the 0.1% Tween-20 / PBS wash was then separated from the multifunctional glass microbubbles using the same procedure. This centrifugation method significantly shortened the time required for cleaning and separation compared to existing centrifugation methods, greatly improving cleaning efficiency (the following cleaning procedures are similar).

[0087] 3) Dilute EpCAM / CD326 polyclonal antibody (purchased from Wuhan Tri-Ting Biotechnology Co., Ltd.) at a ratio of 1:1000 with PBS. Add 100 μl of the diluted EpCAM / CD326 polyclonal antibody to the microplate and incubate on a microplate shaker for 40 min. Repeat the procedure in step 2). Place the microplate on a stand and wash twice with 0.1% Tween-20 / PBS.

[0088] Then, HRP (horseradish peroxidase)-labeled antibody IgG-HRP (purchased from Beijing Solebow Technology Co., Ltd.) was diluted with PBS at a ratio of 1:1000, and 100 μl of the diluted antibody IgG-HRP was added to the microplate. The plate was reacted on a microplate shaker for 20 minutes and washed twice.

[0089] 100 μl of 0.5 M Tris·HCl, 100 μl of 1 M NaCl, 100 μl of 0.05 M KCl, 80 μl of 12.5 mM MgCl2, 10 μl of biotin-tyramide (purchased from Merck KGaA, Darmstadt, Germany) at different concentrations (2, 5, 10, 15, 20, 25 μg / ml), 100 μl of 3% hydrogen peroxide solution, and 510 μl of ultrapure water were mixed. 100 μl of the mixed solution was added to the microplate to which the antibodies had been added twice, and the mixture was reacted on a microplate shaker for 10 min. The plate was then washed twice.

[0090] Then, HRP-labeled streptavidin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with PBS at a ratio of 1:2000, and 100 μl of the diluted HRP-labeled streptavidin solution was added to react on a microplate shaker for 5 minutes, and the plate was washed twice.

[0091] 4) After the multifunctional glass microbubble in step 3) is cleaned and reacted, 100 μl of TMB colorimetric solution is added to the microplate shaker for 10 minutes, and then TMB stop solution is added to stop the colorimetric reaction. At this time, the colorimetric method can be observed by using a microplate reader. Figure 8 Left (a), by comparing the colorimetric results of different concentrations of biotin-tyramide, 15 μg / ml biotin-tyramide was selected as the optimal condition;

[0092] 5) After the optimization of the above conditions, the same steps were followed by changing the reaction time of biotin-tyramide (2, 5, 10, 20, 30 min) and observing the colorimetric method using an enzyme-labeled instrument. Figure 8 Right (Fig. b), by comparing the colorimetric results of biotin-tyramine at different concentrations, 10 min was selected as the optimal reaction time.

[0093] <Signal enhancement effect test>

[0094] The test steps for the signal enhancement group are as follows:

[0095] 1) GB@BSA@Tz was placed in a centrifuge tube and PBS was added to make a 10 mg / ml GB@BSA@Tz solution. 2 μl of 200 μg / ml EpCAM protein was placed in a 600 μl centrifuge tube. 1 μl of 10 mM trans-cyclooctene active lipid and 1 mM sodium bicarbonate solution were added. The mixture was vortexed at 1000 rpm for 20 minutes to obtain a 10 μg / ml trans-cyclooctene-labeled protein. EpCAM protein was purchased from Ibotek Biotechnology Co., Ltd.

[0096] 2) Take 50 μl of the GB@BSA@Tz solution from step 1, dilute the prepared protein to 50 ng / ml with PBS solution, and then take 50 μl of the diluted protein and GB@BSA@Tz in a 96-well microplate. The control group takes 50 μl of the diluted protein and GB@BSA@Tz, and the blank group takes 50 μl of PBS solution. The reaction is carried out in a microplate shaker for 40 minutes. After reacting in the microplate shaker for 40 minutes, the inclined block of the bracket is inserted into the slot of the microplate. At this time, the microplate is tilted. Due to the overall tilt of the microplate and the buoyancy of the hollow glass microbubbles GB@BSA@Tz themselves, the multifunctional glass microbubbles can quickly float to the top of the microplate and adhere to one side of the hole wall (such as Figure 14At this point, use an 8-channel or 12-channel pipette to aspirate the liquid in the well from the other side. Use the same procedure to separate the waste liquid from the multifunctional glass microbubbles after washing with 0.1% Tween-20 / PBS. Compared with the existing centrifugal analysis, the time required for washing and separation is greatly shortened, and the washing efficiency is greatly improved.

[0097] 3) Dilute EpCAM / CD326 polyclonal antibody at a ratio of 1:1000 with PBS, add 100 μl of the diluted antibody to the microplate, and react on a microplate shaker for 40 min. Repeat the procedure in step 2), then place the microplate on a stand and wash twice with 0.1% Tween-20 / PBS.

[0098] Then, biotin-labeled IgG antibody (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with PBS at a ratio of 1:1000, and 100 μl of the diluted antibody was added to the microplate. The plate was reacted on a microplate shaker for 20 minutes and washed twice.

[0099] Then, HRP-labeled streptavidin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with PBS at a ratio of 1:2000, and 100 μl of the diluted HRP-labeled streptavidin solution was added to react on a microplate shaker for 5 minutes, and the plate was washed twice.

[0100] Mix 100 μl of 0.5 M Tris·HCl, 100 μl of 1 M NaCl, 100 μl of 0.05 M KCl, 80 μl of 12.5 mM MgCl2, 10 μl of 15 μg / ml biotin-tyramine, 100 μl of 3% hydrogen peroxide solution, and 510 μl of ultrapure water. Add 100 μl of the mixed solution and react on a microplate shaker for 10 minutes. Wash twice.

[0101] Next, streptavidin-coupled Cy5 dye (purchased from Beijing Solebau Technology Co., Ltd.) was diluted 1:100 with PBS. 100 μl of the diluted streptavidin-coupled Cy5 dye was added and reacted on a microplate shaker for 10 minutes. The plate was then washed twice. The cleaned hollow glass microbubbles were then dissolved in 1000 ml of PBS solution. After mixing thoroughly, 10 μl of the solution was dropped onto a glass slide, and fluorescence imaging was performed using an inverted fluorescence microscope.

[0102] The test steps without signal enhancement group are as follows:

[0103] Step 1): Same as step 1 of the signal enhancement group;

[0104] Step 2): Same as step 2 of the signal enhancement group;

[0105] Step 3): Dilute EpCAM / CD326 polyclonal antibody with PBS at a ratio of 1:1000, add 100 μl of the diluted antibody to the microplate, and react on a microplate shaker for 40 minutes. Repeat the steps in step 2). Place the microplate on a stand and wash twice with 0.1% Tween-20 / PBS.

[0106] Then, the biotin-labeled IgG antibody was diluted with PBS at a ratio of 1:1000, and 100 μl of the diluted antibody was added to the microplate. The plate was reacted on a microplate shaker for 20 minutes and washed twice.

[0107] Next, streptavidin-coupled Cy5 dye (purchased from Beijing Solebau Technology Co., Ltd.) was diluted 1:100 with PBS. 100 μl of the diluted streptavidin-coupled Cy5 dye was added and reacted on a microplate shaker for 10 minutes. The plate was then washed twice. The cleaned hollow glass microbubbles were then dissolved in 1000 ml of PBS solution. After mixing thoroughly, 10 μl of the solution was dropped onto a glass slide, and fluorescence imaging was performed using an inverted fluorescence microscope.

[0108] Specific results can be found in Figure 9 . Figure 9 -EpCAM indicates blank group, +EpCAM indicates control group, FI indicates fluorescence intensity, No Amplication indicates group without signal enhancement, and Amplication indicates group with signal enhancement;

[0109] from Figure 9 It can be seen that in the signal enhanced group, the fluorescence signal of the control group is much greater than that in the signal non-enhanced group, and in the signal enhanced group, the difference between the control group and the negative control group is much greater than that in the signal non-enhanced group.

[0110] <Detection of EpCAM protein in lung cancer cells>

[0111] The high-throughput rapid immunoassay system of the present invention was used to detect EpCAM in lung cancer cells. The specific steps are as follows:

[0112] 1) Protein lysates were obtained from human non-small cell lung cancer cell line (A549) and human normal lung epithelial cell line (BEAS-2B) in T75 culture dishes using commercially available protein lysis buffer (RIPA). Protein lysates from non-small cell lung cancer cell line (A549) served as a control group, and protein lysates from human normal lung epithelial cell line (BEAS-2B) served as a negative control group.

[0113] 2) GB@BSA@Tz was placed in a centrifuge tube and PBS was added to prepare a 10 mg / ml GB@BSA@Tz solution. 1 mg / ml of the two different cell protein lysates from step 1) were placed in separate 600 μl centrifuge tubes, and 1 μl of 10 mM trans-cyclooctene active lipid and 1 mM sodium bicarbonate solution were added to each tube. The mixture was vortexed at 1000 rpm for 20 min to obtain two groups of trans-cyclooctene-labeled proteins with a protein concentration of 1 mg / ml. The two groups of proteins corresponded to the two GB@BSA@Tz solutions.

[0114] 3) For each group in step 2), take 50 μl of the GB@BSA@Tz solution from step 2) and add 50 μl of the prepared protein to the corresponding 50 μl GB@BSA@Tz solution in a 96-well microplate. Incubate on a microplate shaker for 40 minutes. After 40 minutes of reaction, insert the bevel of the bracket into the slot of the microplate. The microplate will tilt as a whole. Due to the buoyancy of the multifunctional glass microbubbles GB@BSA@Tz, the multifunctional glass microbubbles will quickly float to the top of the microplate and adhere to one side of the well. At this point, use an 8-channel or 12-channel pipette to aspirate the liquid from the other side of the well. Use the same procedure to separate the waste liquid from the multifunctional glass microbubbles after washing with 0.1% Tween-20 / PBS. Compared with existing centrifugation analysis, this method greatly shortens the washing and separation time and greatly improves the washing efficiency.

[0115] 4) Dilute EpCAM / CD326 polyclonal antibody at a ratio of 1:1000 with PBS, add 100 μl of the diluted antibody to the microplate, and react on a microplate shaker for 40 min. Repeat the procedure in step 2), place the microplate on a stand, and wash twice with 0.1% Tween-20 / PBS.

[0116] Next, horseradish peroxidase (HRP)-labeled IgG antibody (purchased from Beijing Solaibao Technology Co., Ltd.) was diluted with PBS at a ratio of 1:1000, and 100 μl of the diluted antibody was added to the microplate. The plate was reacted on a microplate shaker for 20 minutes and washed twice. 100 μl of 0.5 M Tris·HCl, 100 μl of 1 M NaCl, 100 μl of 0.05 M KCl, 80 μl of 12.5 mM MgCl2, 10 μl of 15 μg / ml biotin-tyramide (purchased from Merck KGaA, Darmstadt, Germany), 100 μl of 3% hydrogen peroxide solution, and 510 μl of ultrapure water were mixed. 100 μl of the mixed solution was added and reacted on a microplate shaker for 10 minutes and washed twice.

[0117] Then, HRP-labeled streptavidin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with PBS at a ratio of 1:2000, and 100 μl of the diluted HRP-labeled streptavidin solution was added to react on a microplate shaker for 5 minutes, and the plate was washed twice.

[0118] 5) After the multifunctional glass microbubble in step 3 is cleaned and reacted, 100 μl of TMB is added to the microplate shaker for 10 minutes, and then TMB stop solution is added to stop the color development. At this time, the colorimetric method can be observed by using a microplate reader. Figure 10 .

[0119] like Figure 10 As shown, compared with the negative control group (BEAS-2B), the EpCAM protein content in the control group (A549 group) was higher than that in the BEAS-2B group. This is because BEAS-2B is a human normal lung epithelial cell line, in which the expression content of EpCAM protein is low, while A549 is a human non-small cell lung cancer cell line, in which the expression content of EpCAM protein is high, indicating that a bubble-enhanced microplate high-throughput rapid immunoassay system can be used for the detection of protein markers in lung cancer cells.

[0120] <Detection of PD-L1 protein in lung cancer cells>

[0121] The high-throughput rapid immunoassay system of the present invention was used to detect PD-L1 in lung cancer cells. The specific steps are as follows:

[0122] 1) Protein lysates were obtained from human non-small cell lung cancer cell line (A549) and human normal lung epithelial cell line (BEAS-2B) in T75 culture dishes using commercially available protein lysis buffer (RIPA). Protein lysates from non-small cell lung cancer cell line (A549) served as a control group, and protein lysates from human normal lung epithelial cell line (BEAS-2B) served as a negative control group.

[0123] 2) GB@BSA@Tz was placed in a centrifuge tube and PBS was added to prepare a 10 mg / ml GB@BSA@Tz solution. 1 mg / ml of the two different cell protein lysates from step 1) were reacted in separate 600 μl centrifuge tubes. 1 μl of 10 mM trans-cyclooctene active lipid and 1 mM sodium bicarbonate solution were added to each tube. The mixture was vortexed at 1000 rpm for 20 min to obtain two groups of trans-cyclooctene-labeled proteins at a protein concentration of 1 mg / ml. The two groups of proteins corresponded one to one with the two GB@BSA@Tz solutions.

[0124] 3) For each group in step 2), take 50 μl of the GB@BSA@Tz solution from step 2) and add 50 μl of the prepared protein to the corresponding 50 μl GB@BSA@Tz solution in a 96-well microplate. Incubate on a microplate shaker for 40 minutes. After 40 minutes of reaction, insert the inclined block of the stand into the slot of the microplate. The microplate will tilt as a whole, and the buoyancy of the multifunctional glass microbubbles GB@BSA@Tz will cause them to quickly float to the top of the microplate and adhere to one side of the well. At this point, use an 8-channel or 12-channel pipette to aspirate the liquid from the other side of the well. Use the waste liquid from the 0.1% Tween-20 / PBS wash to separate the wash solution from the multifunctional glass microbubbles using the same procedure. Compared to existing centrifugation analysis, this method significantly shortens the time required for washing and separation, greatly improving washing efficiency.

[0125] 4) Dilute PD-L1 / CD274 polyclonal antibody (purchased from Wuhan Tri-Take Biotechnology Co., Ltd.) with PBS at a ratio of 1:1000. Add 100 μL of the diluted antibody to a microplate and react on a microplate shaker for 40 min. Repeat the procedure in step 2). Place the microplate on a stand and wash twice with 0.1% Tween-20 / PBS.

[0126] Then, horseradish peroxidase-labeled IgG antibody (purchased from Beijing Solebow Technology Co., Ltd.) was diluted with PBS at a ratio of 1:1000, and 100 μl of the diluted antibody was added to the microplate. The plate was reacted on a microplate shaker for 20 minutes and washed twice.

[0127] 100 μl of 0.5 M Tris·HCl, 100 μl of 1 M NaCl, 100 μl of 0.05 M KCl, 80 μl of 12.5 mM MgCl2, 10 μl of 15 μg / ml biotin-tyramide (purchased from Merck KGaA, Darmstadt, Germany), 100 μl of 3% hydrogen peroxide solution and 510 μl of ultrapure water were mixed, 100 μl of the mixed solution was added and reacted on a microplate shaker for 10 min, and the plate was washed twice.

[0128] Then, HRP-labeled streptavidin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with PBS at a ratio of 1:2000, and 100 μl of the diluted HRP-labeled streptavidin solution was added to react on a microplate shaker for 5 minutes, and the plate was washed twice.

[0129] 5) After the hollow glass microbubbles in step 4) are reacted and cleaned, 100 μl of TMB is added to the microplate shaker for 10 minutes of color development, and then TMB stop solution is added to stop the color development. At this time, the colorimetric method can be observed by using an enzyme reader. The results are as follows. Figure 11 shown.

[0130] from Figure 11 It can be seen that compared with the negative control group (BEAS-2B), the PD-L1 protein content in the control group (A549 group) was higher than that in the BEAS-2B group. This is because BEAS-2B is a human normal lung epithelial cell line, in which the expression content of PD-L1 protein is low, while A549 is a human non-small cell lung cancer cell line, in which the expression content of PD-L1 protein is high, indicating that a bubble-enhanced microplate high-throughput rapid immunoassay system can be used for protein marker detection in lung cancer cells.

[0131] <Detection of EpCAM protein in clinical blood samples of lung cancer patients>

[0132] The high-throughput rapid immunoassay system of the present invention was used to detect EpCAM in clinical blood samples from lung cancer patients. The specific steps are as follows:

[0133] 1) Clinical blood samples from lung cancer patients were centrifuged at 3000 g for 10 min at room temperature, and plasma was collected into 1.5 ml centrifuge tubes.

[0134] 2) GB@BSA@Tz was placed in a centrifuge tube and PBS was added to prepare a 10 mg / ml GB@BSA@Tz solution. 100 μl of clinical plasma from a lung cancer patient treated in step 1) was placed in a 600 μl centrifuge tube, 10 μl of 10 mM trans-cyclooctene active lipid was added, and the mixture was vortexed at 1000 rpm for 20 minutes to obtain the prepared trans-cyclooctene-labeled plasma.

[0135] 3) Take 50 μl of the GB@BSA@Tz solution from step 2) and dilute the plasma prepared in step 2) with 1000 ml of PBS. Place 50 μl of the diluted plasma and GB@BSA@Tz solution in a 96-well microplate and incubate on a microplate shaker for 40 minutes. After 40 minutes of incubation on a microplate shaker, insert the tilting block of the stand into the slot of the microplate. The microplate is tilted, and the buoyancy of the multifunctional glass microbubbles GB@BSA@Tz allows them to quickly float to the top of the microplate and adhere to one side of the well. At this point, use an 8- or 12-channel pipette to aspirate the liquid from the other side of the well. Use the same procedure to separate the wash solution from the multifunctional glass microbubbles using the wastewater from the 0.1% Tween-20 / PBS wash. Compared to existing centrifugation analysis, this significantly shortens the time required for washing and separation, greatly improving washing efficiency.

[0136] 4) Dilute EpCAM / CD326 polyclonal antibody (purchased from Wuhan Tri-Ting Biotechnology Co., Ltd.) at a ratio of 1:1000 with PBS. Add 100 μL of the diluted antibody to a microplate and incubate on a microplate shaker for 40 min. Repeat the procedure in step 3). Place the microplate on a stand and wash twice with 0.1% Tween-20 / PBS.

[0137] Then, horseradish peroxidase-labeled IgG antibody (purchased from Beijing Solebow Technology Co., Ltd.) was diluted with PBS at a ratio of 1:1000, and 100 μl of the diluted antibody was added to the microplate. The plate was reacted on a microplate shaker for 20 minutes and washed twice.

[0138] 100 μl of 0.5 M Tris·HCl, 100 μl of 1 M NaCl, 100 μl of 0.05 M KCl, 80 μl of 12.5 mM MgCl2, 10 μl of 15 μg / ml biotin-tyramide (purchased from Merck KGaA, Darmstadt, Germany), 100 μl of 3% hydrogen peroxide solution, and 510 μl of ultrapure water were mixed. 100 μl of the mixed solution was added and reacted on a microplate shaker for 10 min, and the plate was washed twice.

[0139] Then, HRP-labeled streptavidin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with PBS at a ratio of 1:2000, and 100 μl of the diluted HRP-labeled streptavidin solution was added to react on a microplate shaker for 5 minutes, and the plate was washed twice.

[0140] 5) After the multifunctional glass microbubble in step 4) is cleaned and reacted, 100 μl of TMB is added to the microplate shaker for 10 minutes of color development. Then, TMB stop solution is added to stop the color development. At this time, colorimetric observation can be performed using a microplate reader. The results are as follows. Figure 12 As shown, the EpCAM protein content in the lung cancer group was higher than that in the healthy group, indicating that a high-throughput rapid immunoassay system of the present invention can be used for the detection of protein markers in the plasma of lung cancer patients.

[0141] in, Figure 12 In the figure, HC (n=16) represents 16 healthy human samples, Early (n=5) represents 5 early patient samples, Adv (n=14) represents 14 late patient samples, and Normalized signal represents the signal value. It should be noted that the healthy human samples were obtained from the plasma of healthy persons, the early patient samples were obtained from the plasma of early patients, and the late patient samples were obtained from the plasma of late patients. Only the samples are different, and the detection methods are the same.

[0142] <Detection of PD-L1 protein in clinical blood samples of lung cancer patients>

[0143] The high-throughput rapid immunoassay system of the present invention was used to detect PD-L1 in clinical blood samples of lung cancer patients.

[0144] 1) Clinical blood samples from lung cancer patients were centrifuged at 3000 g for 10 min at room temperature, and plasma was collected into 1.5 ml centrifuge tubes.

[0145] 2) GB@BSA@Tz was placed in a centrifuge tube and PBS was added to prepare a 10 mg / ml GB@BSA@Tz solution. 100 μl of clinical plasma from a lung cancer patient treated in step 1) was placed in a 600 μl centrifuge tube, 10 μl of 10 mM trans-cyclooctene active lipid was added, and the mixture was vortexed at 1000 rpm for 20 minutes to obtain the prepared trans-cyclooctene-labeled plasma.

[0146] 3) Take 50 μl of the GB@BSA@Tz solution from step 2) and dilute the plasma prepared in step 2) with 1000 ml of PBS. Place 50 μl of the diluted plasma and GB@BSA@Tz solution in a 96-well microplate and incubate on a microplate shaker for 40 minutes. After 40 minutes of incubation on a microplate shaker, insert the tilting block of the stand into the slot of the microplate. This tilts the microplate as a whole, and the buoyancy of the multifunctional glass microbubbles GB@BSA@Tz allows them to quickly float to the top of the microplate and adhere to one side of the well. At this point, use an 8- or 12-channel pipette to aspirate the liquid from the other side of the well. Use the same procedure to separate the wash solution from the multifunctional glass microbubbles using the wastewater from the 0.1% Tween-20 / PBS wash. Compared to existing centrifugation analysis, this significantly shortens the washing and separation time and greatly improves washing efficiency.

[0147] 4) Dilute PD-L1 / CD2746 polyclonal antibody (purchased from Wuhan Tri-Ting Biotechnology Co., Ltd.) with PBS at a ratio of 1:1000. Add 100 μL of the diluted antibody to a microplate and react on a microplate shaker for 40 min. Repeat the procedure in step 2). Place the microplate on a stand and wash twice with 0.1% Tween-20 / PBS.

[0148] Then, horseradish peroxidase-labeled IgG antibody (purchased from Beijing Solebow Technology Co., Ltd.) was diluted with PBS at a ratio of 1:1000, and 100 μl of the diluted antibody was added to the microplate. The plate was reacted on a microplate shaker for 20 minutes and washed twice.

[0149] 100 μl of 0.5 M Tris·HCl, 100 μl of 1 M NaCl, 100 μl of 0.05 M KCl, 80 μl of 12.5 mM MgCl2, 10 μl of 15 μg / ml biotin-tyramide (purchased from Merck KGaA, Darmstadt, Germany), 100 μl of 3% hydrogen peroxide solution and 510 μl of ultrapure water were mixed, 100 μl of the mixed solution was added and reacted on a microplate shaker for 10 min, and the plate was washed twice.

[0150] Then, HRP-labeled streptavidin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with PBS at a ratio of 1:2000, and 100 μl of the diluted HRP-labeled streptavidin solution was added to react on a microplate shaker for 5 minutes, and the plate was washed twice.

[0151] 5) After the multifunctional glass microbubble in step 4) is cleaned and reacted, 100 μl of TMB is added to the microplate shaker for 10 minutes of color development. Then, TMB stop solution is added to stop the color development. At this time, colorimetric observation can be performed using a microplate reader. The results are as follows. Figure 13 As shown, the PD-L1 protein content in the lung cancer group was higher than that in the healthy group, indicating that a high-throughput rapid immunoassay system of the present invention can be used for the detection of protein markers in the plasma of lung cancer patients.

[0152] in, Figure 13 In the figure, HC (n=16) represents 16 healthy human samples, Early (n=5) represents 5 early patient samples, Adv (n=14) represents 14 late patient samples, and Normalized signal represents the signal value. It should be noted that the healthy human samples were obtained from the plasma of healthy persons, the early patient samples were obtained from the plasma of early patients, and the late patient samples were obtained from the plasma of late patients. Only the samples are different, and the detection methods are the same.

[0153] The number of devices and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the bubble-enhanced microplate high-throughput rapid immunoassay system of the present invention will be apparent to those skilled in the art.

[0154] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Multifunctional glass microbubbles, characterized by: Prepared by the following preparation steps: S1, adding a bovine serum albumin solution to the hollow glass microbubbles to obtain a bovine serum albumin coating, centrifuging, removing the lower layer of solution, washing with ultrapure water, and drying to obtain antifouling hollow glass microbubbles; S2. Mixing the antifouling hollow glass microbubbles obtained in S1 with tetrazine-active ester, centrifuging, removing the lower layer solution, washing with ultrapure water, and drying to obtain antifouling hollow glass microbubbles with bioorthogonal function, i.e., the multifunctional glass microbubbles.

2. High-throughput rapid immunoassay system, characterized in that: The invention comprises the multifunctional glass microbubbles as claimed in claim 1.

3. The high-throughput rapid immunoassay system according to claim 2, wherein: Also included are biotin-tyramide, hydrogen peroxide, and HRP-labeled streptavidin.

4. The high-throughput rapid immunoassay system according to claim 3, wherein: Also includes TMB color development solution and TMB stop solution.

5. The high-throughput rapid immunoassay system according to claim 3, wherein: Also includes 96-well microplates.

6. The high-throughput rapid immunoassay system according to claim 5, wherein: It also includes a bracket, which includes a vertical L-shaped block and an inclined block arranged on the top of the L-shaped block. The inclined block is inclined toward the horizontal block side of the L-shaped block, and the high end of the inclined block is higher than the L-shaped block. The angle between the inclined block and the vertical plane is 45°. The top surface of the microporous plate is provided with a slot adapted to the inclined block, so that when the inclined block is inserted into the slot, the microporous plate is tilted.

7. A high-throughput rapid immunoassay system according to claim 6, characterized in that: The invention also includes a washing solution, which is a 0.1% Tween-20 / PBS solution.

8. The high-throughput rapid immunoassay system according to claim 6, wherein: The particle size of the multifunctional glass microbubbles is 10-50 microns.

9. The high-throughput rapid immunoassay system according to claim 6, wherein: The horizontal block of the L-shaped block has a size of 2 cm in length, 2 cm in width, and 0.5 cm in height. The vertical block of the L-shaped block has a size of 1.2 cm in length, 0.3 cm in width, and 7 cm in height. The bracket is made of PLA or ABS resin.

10. The high-throughput rapid immunoassay system according to any one of claims 2 to 9, characterized in that: The target protein is EpCAM protein or PD-L1 protein.

Images