Antibody-nanodiamond conjugate and preparation method and application thereof
By coupling the antibody with nanodiamond modified with active hydrogen groups, antibody-nanodiamond conjugates were prepared, which solved the problem of insufficient detection sensitivity in traditional flow cytometry, achieved high specificity and high sensitivity detection of low-abundance biomarkers, and significantly improved the accuracy and reliability of the detection results.
Patent Information
- Application Number
- CN202411937715.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-04-08
AI Technical Summary
In traditional flow cytometry, the instability, toxicity and excitation wavelength limitation of fluorescent dyes lead to insufficient detection sensitivity, especially in low-abundance biomarker detection, which makes it difficult to achieve high specificity and high sensitivity.
The nanodiamond is coupled with the antibody by modifying active hydrogen groups, and an antibody-nanodiamond conjugate is prepared as a luminescent substance to achieve high specificity and high sensitivity detection of antigens.
This method significantly improves the accuracy and reliability of the detection results, reduces the detection limit, and maintains excellent chemical and thermal stability under various environmental conditions.
Smart Images
Figure CN119780436A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomedical materials and detection technology, and specifically relates to an antibody-nanodiamond conjugate and a preparation method and application thereof. Background Art
[0002] Flow cytometry is an important quantitative analysis and sorting technology in the biological field. Its principle is to analyze the size, shape and surface markers of cells in real time by laser excitation of fluorescent markers in cells. Cells pass through the laser beam one by one in a flow state, generating scattered light and fluorescent signals, and the data is analyzed by a computer. The main steps of flow cytometry detection include: first, fluorescent labeling on antibodies, then labeling samples with fluorescent antibodies, and samples (such as blood, tissue homogenate, etc.) are processed into single cell suspensions to ensure that cells pass through the laser beam one by one in a flow state; the cell suspension passes through the flow chamber, and the cells pass through the laser beam one by one in the flow, and the laser excites the fluorescent dyes in the cells to generate fluorescent signals; the excited fluorescent signals and scattered light are captured by detectors (such as photomultiplier tubes). Flow cytometers are usually equipped with multiple detectors to simultaneously detect fluorescent signals of different wavelengths. The collected data are analyzed by computer software to generate quantitative and qualitative information of cell characteristics, such as cell size, shape, and surface marker expression.
[0003] Traditional flow cytometry uses fluorescent dyes as luminescent substances. Known fluorescent dyes include cyanine dyes, rhodamine dyes, etc., and their wavelength range can cover 350-800nm. However, some fluorescent dyes may show instability under different experimental conditions (such as pH, temperature, ionic strength, etc.), resulting in changes in fluorescence intensity. This instability may affect the repeatability and reliability of the experiment. Moreover, some fluorescent dyes may have certain toxicity to cells, affecting the physiological state and function of cells, which is particularly important in the analysis of living cells and may lead to deviations in results. In addition, different fluorescent dyes require specific excitation wavelengths, which limits the available laser sources and the configuration of flow cytometers. Some dyes may not be compatible with existing equipment, limiting the flexibility of the experiment. Most importantly, flow cytometry using fluorescent dyes as luminescent substances is not sensitive enough for the detection of low-abundance markers. In some cases, especially in early-stage diseases, the marker concentration may be too low, resulting in false negative results.
[0004] In general, the problem of insufficient sensitivity is becoming increasingly prominent in current in vitro detection and diagnostic methodologies, especially when detecting low-abundance biomarkers. The sensitivity issue is particularly critical. Traditional detection techniques such as flow cytometry often rely on high sample concentrations, but in the early stages of many diseases, the concentration of related biomarkers may be extremely weak, resulting in ineffective identification; at the same time, the specificity and affinity of existing antibodies and probes are limited, which may lead to false negative results. Therefore, the development of more sensitive and specific detection methods to improve the accuracy and reliability of test results is an urgent problem to be solved in this field. Summary of the invention
[0005] In view of the deficiencies of the prior art, the present invention aims to provide an antibody-nanodiamond conjugate and its preparation method and application, and realizes the coupling of nanodiamond and antibody by a specific method, so that the prepared antibody-nanodiamond conjugate has excellent sensitivity and stability as a luminescent substance and good biocompatibility. The antibody-nanodiamond conjugate can realize high specificity and high sensitivity detection of antigens, greatly improving the accuracy and reliability of the test results.
[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:
[0007] In a first aspect, the present invention provides a method for preparing an antibody-nanodiamond conjugate, the preparation method comprising: modifying one of a nanodiamond and an antibody with an active hydrogen group, activating the nanodiamond or the antibody with an activating reagent, and then performing a coupling reaction with the other to obtain the antibody-nanodiamond conjugate; the active hydrogen group comprises any one of a carboxyl group, an amino group, and a thiol group, or a combination of at least two of them.
[0008] Fluorescent Nanodiamonds (FND) with nitrogen-vacancy (NV) color centers are a new type of carbon-based nanomaterial with unique optical properties, high photostability, long-life fluorescence, excellent biocompatibility and low toxicity, and have broad application prospects in the biomedical field. The present invention uses active hydrogen groups to modify nanodiamonds, and effectively couples them with biomolecular antibodies through a specific method to obtain antibody-nanodiamond conjugates. The antibody-nanodiamond conjugate can specifically recognize and bind antigens to achieve targeted recognition of specific cells or tissues.
[0009] In the antibody-nanodiamond conjugate, nanodiamond is used as a fluorescent substance, has good biocompatibility, can interact with cells and tissues in the body without causing obvious toxic reactions, so that it can be safely used for marking and detecting biological samples in in vitro diagnosis and biological detection. At the same time, the antibody-nanodiamond conjugate shows excellent chemical and thermal stability under various environmental conditions (such as temperature, pH, ionic strength, etc.), and this stability enables it to maintain its luminescent properties during long-term experiments and storage, reducing the variability of experimental results. Compared with existing fluorescent dyes, the antibody-nanodiamond conjugate prepared by the present invention has higher sensitivity, excellent chemical stability and thermal stability, good biocompatibility, and can achieve high specificity and high sensitivity detection of antigens, effectively improving the accuracy and reliability of test results.
[0010] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. Through the following preferred technical solutions, the objectives and beneficial effects of the present invention can be better achieved and realized.
[0011] In a preferred technical solution, the active hydrogen group-modified nanodiamond is carboxyl-modified nanodiamond and / or amino-modified nanodiamond, and the preparation method comprises the following steps:
[0012] subjecting the active hydrogen group-modified nanodiamond to an activation reaction with an activation reagent to obtain an activated nanodiamond;
[0013] After the activated nanodiamond and the antibody undergo coupling reaction, a terminator is added to terminate the reaction to obtain the antibody-nanodiamond conjugate.
[0014] Preferably, the activation reagent comprises any one of N,N'-disuccinimidyl carbonate (DSC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS), or a combination of at least two thereof, preferably a combination of N,N'-disuccinimidyl carbonate or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.
[0015] Preferably, based on the mass of the active hydrogen group-modified nanodiamond as 1 mg, the mass of the activation reagent is 10-160 μg, for example, it can be 20 μg, 30 μg, 50 μg, 60 μg, 80 μg, 100 μg, 120 μg, 140 μg or 150 μg, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0016] Preferably, the activation reagent is N,N'-disuccinimidyl carbonate. Based on the mass of the active hydrogen group-modified nanodiamond as 1 mg, the mass of the N,N'-disuccinimidyl carbonate is 10-100 μg, for example, it can be 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg or 90 μg, as well as specific values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 20-80 μg is further preferred.
[0017] Preferably, the activation reagent is a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. Based on the mass of the active hydrogen group-modified nanodiamond as 1 mg, the masses of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are each independently 10-80 μg, for example, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg or 75 μg, as well as specific values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 20-60 μg is further preferred.
[0018] As a preferred technical solution of the present invention, N,N'-disuccinimidyl carbonate (DSC) is used to activate carboxyl-modified nanodiamonds and / or amino-modified nanodiamonds, so that the obtained antibody-nanodiamond conjugate has a higher signal-to-noise ratio in detection, which is more conducive to improving the accuracy and reliability of detection.
[0019] Preferably, the activation reaction is carried out under stirring conditions.
[0020] Preferably, the temperature of the activation reaction is 15-40°C, for example, it can be 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 35°C or 38°C, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range. 18-30°C is further preferred, and room temperature / normal temperature is more preferred.
[0021] Preferably, the activation reaction time is 0.2-3h, for example, it can be 0.5h, 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h or 2.8h, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 0.5-2h is further preferred.
[0022] Preferably, the activation reaction further includes a post-treatment step, wherein the post-treatment includes centrifugation and / or washing to remove unreacted activation reagent.
[0023] Preferably, the coupling reaction is carried out in a buffer solution.
[0024] Preferably, the buffer solution includes any one of HEPES buffer, PBS buffer or MES buffer, and HEPES buffer is more preferred.
[0025] Preferably, the concentrations of the HEPES buffer, PBS buffer and MES buffer are each independently 10-60 mM, for example, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM or 55 mM, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0026] Preferably, the pH value of the buffer solution is 7.0-8.0, for example, it can be 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0027] Preferably, based on the mass of the active hydrogen group-modified nanodiamond as 1 mg, the mass of the antibody is 5-50 μg, for example, it can be 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg or 45 μg, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 10-40 μg is further preferred.
[0028] Preferably, the coupling reaction method comprises: mixing the activated nanodiamond with a buffer solution to obtain a nanodiamond suspension; and mixing the nanodiamond suspension with an antibody solution to perform a coupling reaction.
[0029] Preferably, the concentration of activated nanodiamonds in the nanodiamond suspension is 5-20 mg / mL, for example, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL or 18 mg / mL, as well as specific values between the above points. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific points included in the range.
[0030] Preferably, the concentration of the antibody in the antibody solution is 0.5-2 mg / mL, for example, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL or 1.8 mg / mL, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0031] Preferably, the coupling reaction is carried out on a shaker.
[0032] Preferably, the rotation speed of the shaking table is 100-400rpm, for example, it can be 120rpm, 150rpm, 180rpm, 200rpm, 220rpm, 250rpm, 300rpm, 350rpm or 380rpm, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 150-250rpm is further preferred.
[0033] Preferably, the temperature of the coupling reaction is 15-40°C, for example, it can be 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 35°C or 38°C, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively enumerates the specific point values included in the range, and 18-30°C is further preferred, and room temperature / normal temperature is more preferred.
[0034] Preferably, the coupling reaction time is 0.5-5h, for example, it can be 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h, 3.8h, 4h, 4.2h, 4.5h or 4.8h, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively enumerates the specific point values included in the range, and 1-4h is further preferred.
[0035] Preferably, the terminator comprises any one of ethanolamine, bovine serum albumin (BSA), and tris(hydroxymethyl)aminomethane (Tris).
[0036] As a preferred technical solution of the present invention, a terminator is used to terminate the reaction after the coupling reaction, so that the unreacted active groups are terminated, and the antibody-nanodiamond conjugate obtained thereby is more conducive to achieving high sensitivity and high specificity detection of antigens; if no terminator is used, the antibody reaction titer will be low.
[0037] Preferably, the terminator is added to the system after the coupling reaction in the form of a terminator solution.
[0038] Preferably, the solvent in the terminator solution is a buffer solution, more preferably HEPES buffer, PBS buffer or MES buffer.
[0039] Preferably, the mass percentage of the terminator in the terminator solution is 0.5%-2%, for example, it can be 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.5%, 1.6% or 1.8%, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0040] Preferably, based on the volume of the system after the coupling reaction as 100%, the volume of the terminator solution is 0.5%-2%, for example, it can be 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.5%, 1.6% or 1.8%, as well as specific point values between the above points. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0041] Preferably, the termination reaction is performed on a shaker.
[0042] Preferably, the rotation speed of the shaking table is 100-400rpm, for example, it can be 120rpm, 150rpm, 180rpm, 200rpm, 220rpm, 250rpm, 300rpm, 350rpm or 380rpm, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 150-250rpm is further preferred.
[0043] Preferably, the termination reaction temperature is 15-40°C, for example, it can be 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 35°C or 38°C, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range. 18-30°C is further preferred, and room temperature / normal temperature is more preferred.
[0044] Preferably, the termination reaction time is 1-30 min, for example, it can be 2 min, 5 min, 8 min, 10 min, 12 min, 15 min, 18 min, 20 min, 22 min, 25 min or 28 min, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 5-20 min is further preferred.
[0045] Preferably, the termination reaction further includes a post-treatment step.
[0046] Preferably, the post-treatment method comprises: centrifuging the system after the reaction is terminated, discarding the supernatant, and washing the precipitate to obtain the antibody-nanodiamond conjugate.
[0047] Preferably, the centrifugal rotation speed is 5000-12000 rpm, for example, it can be 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, 10000 rpm or 11000 rpm, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0048] Preferably, the centrifugation time is 5-20 min, for example, it can be 6 min, 8 min, 10 min, 12 min, 15 min or 18 min, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0049] Preferably, the washing is performed 1-5 times, for example, 2 times, 3 times, 4 times, etc.; after each washing, centrifugation is performed and the supernatant is discarded to obtain a pure antibody-nanodiamond conjugate.
[0050] Preferably, the cleaning agent comprises PBS buffer.
[0051] Preferably, the active hydrogen group-modified nanodiamond is carboxyl-modified nanodiamond and / or amino-modified nanodiamond, and the preparation method comprises the following steps:
[0052] The active hydrogen group modified nanodiamond is subjected to an activation reaction with an activation reagent to obtain an activated nanodiamond; the activation reagent is N,N'-disuccinimidyl carbonate (DSC) or a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC and NHS); the activation reaction temperature is 15-40°C and the time is 0.2-3h;
[0053] The activated nanodiamond and the antibody are subjected to coupling reaction in a buffer solution, and then a terminator is added to terminate the reaction to obtain the antibody-nanodiamond conjugate; based on the mass of the active hydrogen group-modified nanodiamond being 1 mg, the mass of the antibody is 5-50 μg; the temperature of the coupling reaction is 15-40° C., and the time is 0.5-5 h; the terminator includes any one of ethanolamine, bovine serum albumin, and tris(hydroxymethyl)aminomethane; the temperature of the termination reaction is 15-40° C., and the time is 1-30 min.
[0054] In another preferred technical solution, the active hydrogen group-modified nanodiamond is thiol-modified nanodiamond, and the preparation method comprises the following steps:
[0055] Carrying out an activation reaction between the antibody and an activation reagent to obtain an activated antibody;
[0056] The activated antibody is coupled with the thiol-modified nanodiamond to obtain the antibody-nanodiamond conjugate.
[0057] Preferably, the activating agent comprises maleimide-PEG-N-hydroxysuccinimide ester (NHS-PEG-Maleimide).
[0058] Preferably, based on the mass of the antibody as 1 mg, the mass of the maleimide-PEG-N-hydroxysuccinimide ester is 10-100 μg, for example, it can be 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg or 90 μg, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range, and 20-60 μg is further preferred.
[0059] Preferably, the activation reaction is carried out in a buffer solution.
[0060] Preferably, the buffer solution includes any one of HEPES buffer, PBS buffer or MES buffer, and PBS buffer is more preferred.
[0061] Preferably, the concentrations of the HEPES buffer, PBS buffer and MES buffer are each independently 10-60 mM, for example, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM or 55 mM, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0062] Preferably, the pH value of the buffer solution is 7.0-8.0, for example, it can be 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0063] Preferably, the method for preparing the activated antibody comprises: mixing the antibody with a buffer solution to obtain an antibody solution; and subjecting the antibody solution to an activation reaction with maleimide-PEG-N-hydroxysuccinimide ester to obtain the activated antibody.
[0064] Preferably, the concentration of the antibody in the antibody solution is 0.5-2 mg / mL, for example, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL or 1.8 mg / mL, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0065] Preferably, the temperature of the activation reaction is 15-40°C, for example, it can be 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 35°C or 38°C, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range. 18-30°C is further preferred, and room temperature / normal temperature is more preferred.
[0066] Preferably, the activation reaction time is 10-60 min, for example, it can be 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min or 55 min, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.
[0067] Preferably, the activation reaction further includes a purification step to remove unreacted activation reagent.
[0068] Preferably, the purification method comprises using a desalting column to remove unreacted activation reagent.
[0069] Preferably, based on the mass of the thiol-modified nanodiamond as 1 mg, the mass of the activated antibody is 5-50 μg, for example, it can be 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg or 45 μg, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively enumerates the specific point values included in the range, and 10-40 μg is further preferred.
[0070] Preferably, the temperature of the coupling reaction is 15-40°C, for example, it can be 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 35°C or 38°C, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively enumerates the specific point values included in the range, and 18-30°C is further preferred, and room temperature / normal temperature is more preferred.
[0071] Preferably, the coupling reaction time is 0.5-5h, for example, it can be 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h, 3.8h, 4h, 4.2h, 4.5h or 4.8h, as well as specific point values between the above point values. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively enumerates the specific point values included in the range, and 1-4h is further preferred.
[0072] Preferably, the coupling reaction further includes a post-treatment step.
[0073] Preferably, the post-treatment method comprises: centrifuging the system after the coupling reaction, discarding the supernatant, and washing the precipitate to obtain the antibody-nanodiamond conjugate.
[0074] Preferably, the washing is performed 1-5 times, for example, 2 times, 3 times, 4 times, etc.; after each washing, centrifugation is performed and the supernatant is discarded to obtain a pure antibody-nanodiamond conjugate.
[0075] Preferably, the cleaning agent comprises PBS buffer.
[0076] Preferably, the active hydrogen group-modified nanodiamond is thiol-modified nanodiamond, and the preparation method comprises the following steps:
[0077] The antibody and maleimide-PEG-N-hydroxysuccinimide ester are activated in a buffer solution to obtain an activated antibody; based on the mass of the antibody being 1 mg, the mass of the maleimide-PEG-N-hydroxysuccinimide ester is 10-100 μg; the temperature of the activation reaction is 15-40° C., and the time is 10-60 min;
[0078] The activated antibody is coupled with the thiol-modified nanodiamond to obtain the antibody-nanodiamond conjugate; based on the mass of the thiol-modified nanodiamond being 1 mg, the mass of the activated antibody is 5-50 μg; the temperature of the coupling reaction is 15-40° C., and the time is 0.5-5 h.
[0079] In a second aspect, the present invention provides an antibody-nanodiamond conjugate, wherein the antibody-nanodiamond conjugate is prepared by the preparation method described in the first aspect.
[0080] In a third aspect, the present invention provides a use of the antibody-nanodiamond conjugate as described in the second aspect in a biological probe, a biological marker or a biological detection.
[0081] Preferably, the antibody-nanodiamond conjugate is applied to a flow cytometry platform.
[0082] Preferably, the method of applying the antibody-nanodiamond conjugate to a flow cytometric platform comprises: combining the antibody-encoded microsphere conjugate with an antigen to obtain a complex; and performing fluorescence detection on the complex by a flow cytometer to obtain a detection result.
[0083] Preferably, the antibody-encoded microsphere conjugate is used as the detection antibody, and the antibody-encoded microsphere conjugate, the capture antibody and the antigen are combined to obtain a complex.
[0084] In a fourth aspect, the present invention provides a method for detecting an antigen, the method comprising: combining the antigen to be detected with a capture antibody-encoding microsphere conjugate to obtain a first complex;
[0085] Mixing and incubating the antibody-nanodiamond conjugate provided by the second aspect with the first complex to obtain a second complex;
[0086] The second complex is subjected to fluorescence detection using a flow cytometer to obtain the detection result of the antigen.
[0087] Preferably, the excitation light wavelength of the flow cytometer is 532 nm.
[0088] The antibody-nanodiamond conjugate provided by the present invention is applied to flow cytometry and can be used as a detection antibody. A 532nm laser is preferably modulated to emit laser light to excite the NV color center, so that the NV color center emits fluorescence, and the intensity of the fluorescence signal is detected. As a luminescent substance, the antibody-nanodiamond conjugate can greatly improve the sensitivity of in vitro detection and in vitro diagnostic reagents and reduce the detection limit.
[0089] Compared with the prior art, the present invention has the following beneficial effects:
[0090] In the preparation method of the antibody-nanodiamond conjugate provided by the present invention, active hydrogen groups are used to modify nanodiamonds, which are effectively coupled with biological molecule antibodies by a specific method, so that the obtained antibody-nanodiamond conjugate has higher sensitivity as a luminescent substance, and at the same time has excellent chemical stability and thermal stability, good biocompatibility, can achieve high-specificity and high-sensitivity detection of antigens, reduce the detection limit, greatly improve the sensitivity of in vitro detection and in vitro diagnostic reagents, and enhance the accuracy and reliability of test results. BRIEF DESCRIPTION OF THE DRAWINGS
[0091] Figure 1 A schematic diagram of the process of the preparation method provided in Example 1;
[0092] Figure 2 This is a standard curve diagram of the antibody-nanodiamond conjugate prepared in Example 2 during detection;
[0093] Figure 3 This is the detection standard curve diagram of Comparative Example 1. DETAILED DESCRIPTION
[0094] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.
[0095] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
[0096] "Optionally" or "either" means that the subsequently described matter or event can or cannot occur, and that the description includes instances where the event occurs and instances where it does not.
[0097] In the following specific embodiments of the present invention, the materials used can be purchased through market channels, and the specific information is as follows:
[0098]
[0099] The antibody-nanodiamond conjugate and the preparation method thereof of the present invention are described in detail below by taking multiple embodiments as examples, but the antibody-nanodiamond conjugate and the preparation method thereof of the present invention are not limited to these embodiments.
[0100] Example 1
[0101] An antibody-nanodiamond conjugate and a preparation method thereof, the process schematic diagram of the preparation method is as follows Figure 1 As shown, the following steps are included:
[0102] (1) using DSC as an activation reagent, mixing carboxyl FND, DSC and a solvent (PBS buffer) to obtain a mixed solution; based on the mass of carboxyl FND being 1 mg, the mass of DSC being 20 μg, and the PBS buffer being 1 mL; the mixed solution is stirred at room temperature for reaction for 0.5 h; then centrifuged, the supernatant is discarded, and washed 3 times to obtain activated nanodiamonds;
[0103] (2) diluting the activated nanodiamond obtained in step (1) to 10 mg / mL with HEPES buffer to obtain a nanodiamond suspension; diluting the IL-6 antibody to 1 mg / mL with HEPES buffer, and then adding it to the nanodiamond suspension, with the mass of the IL-6 antibody being 10 μg based on 1 mg of carboxyl FND; placing the mixed system on a shaker at 200 rpm and room temperature for a coupling reaction for 1 hour to covalently bind the antibody to the activated nanodiamond;
[0104] (3) After the coupling reaction, a terminator (1% ethanolamine solution) was added to the mixture obtained in step (2), and the volume of the terminator was 1% based on the volume of the mixture obtained in step (2) as 100%; the mixture was mixed on a shaker for 10 minutes to terminate the unreacted active groups; the system was then centrifuged at 7000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed three times with PBS buffer, and the supernatant was discarded after each washing and centrifugation to ensure that a pure antibody-nanodiamond conjugate (IL-6-FND) was obtained.
[0105] Example 2-13
[0106] An antibody-nanodiamond conjugate and a preparation method thereof, wherein the preparation method is different from that in Example 1 in that at least one of the materials, dosages, and process parameters is different, as shown in Table 1; in Table 1, the mass (μg) of DSC is based on the mass of carboxyl FND as 1 mg, and the mass (μg) of the antibody is based on the mass of nanodiamond as 1 mg; items not shown in Table 1 are the same as those in Example 1.
[0107] Table 1
[0108]
[0109] Embodiment 14
[0110] An antibody-nanodiamond conjugate and a preparation method thereof. The preparation method is different from that in Example 1 only in that EDC and NHS are used as activation reagents, and based on the mass of carboxyl FND being 1 mg, the masses of EDC and NHS are both 40 μg; other materials, amounts, process steps and parameters are the same as those in Example 1, to obtain an antibody-nanodiamond conjugate.
[0111] Embodiment 15
[0112] An antibody-nanodiamond conjugate and a preparation method thereof, wherein the preparation method differs from Example 2 only in that carboxyl FND is replaced with amino FND of equal mass; other materials, amounts, process steps and parameters are the same as those in Example 1, to obtain an antibody-nanodiamond conjugate.
[0113] Example 16
[0114] An antibody-nanodiamond conjugate and a preparation method thereof, the preparation method comprising the following steps:
[0115] (1) diluting the IL-6 antibody to 1 mg / mL with PBS buffer, and then adding the activation reagent NHS-PEG-Maleimide, where the mass of the IL-6 antibody is 1 mg and the mass of the NHS-PEG-Maleimide is 40 μg; incubating the above mixture at room temperature for 30 minutes to perform an activation reaction, and then using a desalting column to remove excess NHS-PEG-Maleimide to obtain an activated antibody;
[0116] (2) The activated antibody obtained in step (1) was mixed evenly with the thiol FND, with the mass of the activated antibody being 20 μg based on 1 mg of thiol FND; the mixed system was subjected to coupling reaction at room temperature for 2 h, and the mixed system was gently shaken during the reaction to ensure a uniform reaction; after the coupling reaction was completed, the mixed system was centrifuged, the supernatant was discarded, and the precipitate was washed three times with PBS buffer, and the supernatant was discarded after each washing-centrifugation to obtain a pure antibody-nanodiamond conjugate.
[0117] The following will take application examples as an example to describe in detail the application of the antibody-nanodiamond conjugate of the present invention in detection, but the application of the antibody-nanodiamond conjugate of the present invention is not limited to these application examples.
[0118] Application Example 1-16
[0119] Application Examples 1-16 are respectively the antibody-nanodiamond conjugates provided in Examples 1-16 for the detection of IL-6 antigen, and the specific detection method is as follows:
[0120] (1) 50 μL of different concentrations of IL-6 antigen (solvent: PBS buffer) were added into 1.5 mL centrifuge tubes. The concentrations of IL-6 antigen were 0, 0.02 pg / mL, 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 2.5 pg / mL, 12.5 pg / mL, and 62.5 pg / mL, respectively;
[0121] (2) Add 5 μL of diluted IL-6 capture antibody-encoded microsphere conjugate to each antigen, gently mix the sample and microsphere to ensure full contact, and incubate at room temperature for 1 h to promote the binding of microspheres to antigens;
[0122] (3) Centrifuge at 300 g for 5 min, discard the supernatant, add 50 μL PBS to resuspend, then add 5 μL of the diluted antibody-nanodiamond conjugate (i.e., IL-6 detection antibody-nanodiamond conjugate, prepared by Examples 1-16, respectively), mix well, and incubate at room temperature for 30 min;
[0123] (4) Centrifuge at 300 g for 5 min, discard the supernatant, and add 50 μL PBS to resuspend;
[0124] (5) Using a flow cytometer with 532 nm excitation light modulated, the fluorescence intensity at each antigen concentration was recorded;
[0125] The fluorescence intensity data at each antigen concentration was used to draw a standard curve and obtain the determination coefficient r 2 ; The specific results are shown in Table 2; Among them, the standard curve of the antibody-nanodiamond conjugate prepared in Example 2 in the detection is as follows Figure 2 shown.
[0126] Comparative Example 1
[0127] The detection method of IL-6 antigen is different from the application example only in that the antibody-nanodiamond conjugate is replaced with the IL-6 antibody-Biotin conjugate and the dye SA-PE, and the flow cytometer is used for detection, and the fluorescence intensity of the PE channel is recorded. The standard curve is drawn as shown in Figure 3 The data are shown in Table 2.
[0128] Table 2
[0129]
[0130]
[0131] According to the data in Table 2, the present invention uses antibody-nanodiamond conjugates for antigen detection. By comparing the sensitivity of nanodiamond as a luminescent substance with that of comparative example 1 using PE as a luminescent substance, the sensitivity of PE as a luminescent substance is about 1pg / mL, and the sensitivity of the antibody-nanodiamond conjugate provided by the present invention reaches 0.1pg / mL, which is much higher than the sensitivity of fluorescent dyes as luminescent substances. Considering the signal-to-noise ratio, reaction time and experimental cost, the preparation method of Example 2 has the best technical effect. In summary, the present invention uses nanodiamond as a luminescent substance, and uses the antibody-nanodiamond conjugate for biological detection, which can greatly improve the sensitivity of in vitro diagnostic reagents.
[0132] The applicant declares that the present invention illustrates the antibody-nanodiamond conjugate and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A method for preparing an antibody-nanodiamond conjugate, characterized in that: The preparation method comprises: After activating one of the active hydrogen group-modified nanodiamond and the antibody with an activation reagent, a coupling reaction is performed with the other to obtain the antibody-nanodiamond conjugate; The active hydrogen group includes any one of a carboxyl group, an amino group, and a thiol group, or a combination of at least two of them.
2. The preparation method according to claim 1, characterized in that: The active hydrogen group modified nanodiamond is carboxyl modified nanodiamond and / or amino modified nanodiamond, and the preparation method comprises the following steps: subjecting the active hydrogen group-modified nanodiamond to an activation reaction with an activation reagent to obtain an activated nanodiamond; After the activated nanodiamond and the antibody undergo coupling reaction, a terminator is added to terminate the reaction to obtain the antibody-nanodiamond conjugate.
3. The preparation method according to claim 2, characterized in that: The activation reagent includes any one of N,N'-disuccinimidyl carbonate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide, or a combination of at least two thereof, preferably a combination of N,N'-disuccinimidyl carbonate or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; Preferably, the activation reagent is N,N'-disuccinimidyl carbonate, and based on the mass of the active hydrogen group-modified nanodiamond being 1 mg, the mass of the N,N'-disuccinimidyl carbonate is 10-100 μg, and more preferably 20-80 μg; Preferably, the activation reagent is a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and based on the mass of the active hydrogen group-modified nanodiamond being 1 mg, the masses of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are independently 10-80 μg, and more preferably 20-60 μg; Preferably, the temperature of the activation reaction is 15-40°C, more preferably 18-30°C; Preferably, the activation reaction time is 0.2-3 h, more preferably 0.5-2 h.
4. The preparation method according to claim 2, characterized in that: The coupling reaction is carried out in a buffer solution; Preferably, the buffer solution comprises any one of HEPES buffer, PBS buffer or MES buffer; Preferably, based on the mass of the active hydrogen group-modified nanodiamond being 1 mg, the mass of the antibody is 5-50 μg, more preferably 10-40 μg; Preferably, the coupling reaction temperature is 15-40°C, more preferably 18-30°C; Preferably, the coupling reaction time is 0.5-5 h, more preferably 1-4 h.
5. The preparation method according to claim 2, characterized in that: The terminator includes any one of ethanolamine, bovine serum albumin, and tris(hydroxymethyl)aminomethane; Preferably, the terminator is added to the system after the coupling reaction in the form of a terminator solution; Preferably, the mass percentage of the terminator in the terminator solution is 0.5%-2%; Preferably, based on the volume of the system after the coupling reaction as 100%, the volume of the terminator solution is 0.5%-2%; Preferably, the temperature of the termination reaction is 15-40°C, more preferably 18-30°C; Preferably, the termination reaction time is 1-30 min, more preferably 5-20 min; Preferably, the termination reaction further includes a post-treatment step.
6. The preparation method according to claim 1, characterized in that: The active hydrogen group modified nanodiamond is thiol modified nanodiamond, and the preparation method comprises the following steps: Carrying out an activation reaction between the antibody and an activation reagent to obtain an activated antibody; The activated antibody is coupled with the thiol-modified nanodiamond to obtain the antibody-nanodiamond conjugate.
7. The preparation method according to claim 6, characterized in that: The activation reagent includes maleimide-PEG-N-hydroxysuccinimide ester; Preferably, based on the mass of the antibody being 1 mg, the mass of the maleimide-PEG-N-hydroxysuccinimide ester is 10-100 μg, more preferably 20-60 μg; Preferably, the temperature of the activation reaction is 15-40°C, more preferably 18-30°C; Preferably, the activation reaction time is 10-60 min; Preferably, based on the mass of the thiol-modified nanodiamond being 1 mg, the mass of the activated antibody is 5-50 μg, more preferably 10-40 μg; Preferably, the coupling reaction temperature is 15-40°C, more preferably 18-30°C; Preferably, the coupling reaction time is 0.5-5h, more preferably 1-4h; Preferably, the coupling reaction further includes a post-treatment step.
8. An antibody-nanodiamond conjugate, characterized in that: The antibody-nanodiamond conjugate is prepared by the preparation method according to any one of claims 1 to 7.
9. Use of the antibody-nanodiamond conjugate according to claim 8 in biological probes, biological markers or biological detection; Preferably, the antibody-nanodiamond conjugate is applied to a flow cytometry platform.
10. A method for detecting an antigen, characterized in that: The detection method comprises: combining the antigen to be detected with the capture antibody-encoding microsphere conjugate to obtain a first complex; Mixing and incubating the antibody-nanodiamond conjugate according to claim 8 with the first complex to obtain a second complex; The second complex is subjected to fluorescence detection using a flow cytometer to obtain the detection result of the antigen; preferably, the excitation light wavelength of the flow cytometer is 532 nm.