Quantum dot labeled antibody, preparation method thereof and fluorescence immunochromatography test paper

Abstract

The embodiment of the invention provides a quantum dot labeled antibody, a preparation method thereof and fluorescence immunochromatography test paper, and relates to the field of detection. The quantum dot labeled antibody comprises a quantum dot particle and a monoclonal antibody, wherein the monoclonal antibody is grafted on the surface of the quantum dot particle through a grafting group; the grafting group comprises a modification group activated by an activating agent, and the modification group comprises at least one of a carboxyl group or an amino group. The quantum dot labeled antibody has the advantages of high fluorescence intensity, high detection sensitivity and high stability, and can significantly improve the detection sensitivity and stability of fluorescence immunochromatography test paper when being used in the fluorescence immunochromatography test paper.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection, in particular, to a quantum dot labeled antibody, a preparation method thereof and a fluorescent immunochromatography test paper. BACKGROUND

[0002] The immunochromatography technology is a new detection technology combining the immunolabeling technology and the chromatography technology. The qualitative, semi-quantitative and quantitative analysis of the analyte can be realized by using the color developing characteristics of the label. The micro-porous filter membrane is used as the solid carrier and the analyte is used as the mobile phase. The capillary action and siphon action of the micro-porous filter membrane are used to guide the forward flow of the analyte. At the same time, the related components in the analyte and the antigens or antibodies fixed on the filter membrane react to form the immune complex which is retained on the detection line. The detection result can be obtained by analyzing the color depth or the fluorescence optical density of the label. The colloidal gold immunochromatography test paper (using colloidal gold as the label) has low sensitivity and is difficult to detect trace drugs (such as low-concentration samples in hair and saliva). Therefore, the fluorescent immunochromatography test paper is widely used for detection.

[0003] However, the existing fluorescent immunochromatography test paper has the problems of poor stability and low coupling efficiency, resulting in unstable detection signal. Therefore, it is necessary to improve the stability of the fluorescent immunochromatography test paper. SUMMARY

[0004] The present application provides a quantum dot labeled antibody, a preparation method thereof and a fluorescent immunochromatography test paper. The fluorescent intensity of the quantum dot labeled antibody is high, the detection sensitivity is high, and the stability is high. When the quantum dot labeled antibody is used in the fluorescent immunochromatography test paper, the detection sensitivity and the stability of the fluorescent immunochromatography test paper can be significantly improved.

[0005] In a first aspect, the present application provides a quantum dot labeled antibody, which comprises quantum dot particles and a monoclonal antibody. The monoclonal antibody is grafted on the surface of the quantum dot particles through a grafting group. The grafting group comprises a modified group activated by an activating agent. The modified group comprises at least one of a carboxyl group or an amino group.

[0006] In the above technical solution, the activating agent can improve the activity of the modified group, so that the modified group can be combined with the monoclonal antigen. Through the activated modified group, the monoclonal antibody can be well grafted on the surface of the quantum dot particles, and the dissociation between the monoclonal antibody and the quantum dot particles is not easy to occur. Therefore, the stability of the entire quantum dot labeled antibody is high and decomposition is not easy to occur. In addition, due to the high specificity of the monoclonal antibody, the monoclonal antibody can be combined with a single antigen, and the fluorescent intensity of the quantum dot particles is high. Therefore, when the quantum dot labeled antibody is used in the fluorescent immunochromatography test paper, the sensitivity of the fluorescent immunochromatography test paper can be significantly improved.

[0007] In a possible implementation, the quantum dot-labeled antibody has an emission wavelength of 500-800 nm.

[0008] In the technical solution, the quantum dot-labeled antibody has a wide emission wavelength, and the quantum dot-labeled antibody has high stability. Therefore, quantum dot-labeled antibodies with different emission wavelengths can be combined with different antigens. In this way, the colors of the emitted light can be used to detect and determine multiple groups of antigens, thereby achieving detection of multiple antigens and avoiding cross interference.

[0009] In a possible implementation, the monoclonal antibody includes at least one of an anti-morphine antibody, an anti-methylamphetamine antibody, an anti-cocaine antibody, an anti-ethymal antibody, or an anti-methylamphetamine antibody.

[0010] In the technical solution, the quantum dot-labeled antibody can be used to detect multiple different drugs.

[0011] In a possible implementation, the activating agent includes at least one of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, or 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt.

[0012] In a possible implementation, the mass ratio of the quantum dot particles and the monoclonal antibody is 10:1-50:1.

[0013] In the technical solution, the mass ratio of the quantum dot particles and the monoclonal antibody in the above range can better improve the detection sensitivity.

[0014] In a second aspect, the embodiments of the present application provide a preparation method of a quantum dot-labeled antibody, which includes the following steps: treating quantum dot particles with a surface modifier to provide the quantum dot particles with a modified group, activating the modified group with an activating agent, and then coupling a monoclonal antibody with the activated modified group.

[0015] In the technical solution, the prepared quantum dot-labeled antibody has high stability and high detection sensitivity, and can be used in a fluorescent immunochromatography test paper to significantly improve the detection sensitivity and stability of the fluorescent immunochromatography test paper.

[0016] In a third aspect, the embodiments of the present application provide a fluorescent immunochromatography test paper, which includes a detection line, a quality control line, and the quantum dot-labeled antibody.

[0017] In the technical solution, the fluorescent immunochromatography test paper has high detection sensitivity and good stability because of the quantum dot-labeled antibody.

[0018] In a possible implementation, the detection line is sprayed by the coated antigen-BSA conjugate solution, and the quality control line is sprayed by the goat anti-mouse IgG antibody solution.

[0019] In a possible implementation, the concentration of the coated antigen-BSA conjugate solution is 0.2-3.0 mg / mL, and the concentration of the goat anti-mouse IgG antibody solution is 0.2-3.0 mg / mL. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments of the present application. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0021] Figure 1 A structure diagram of a fluorescent immunochromatographic test paper provided by the embodiments of the present application.

[0022] The reference signs are explained as follows: 1-bottom plate; 2-sample pad; 3-binding pad; 4-NC membrane; 5-filter paper. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not indicated, they are all conventional products that can be obtained by market purchase.

[0024] The immunochromatographic technology is a new detection technology combining the immunolabeling technology with the chromatographic technology. The qualitative, semi-quantitative and quantitative analysis of the to-be-detected substance can be realized by using the color developing characteristics of the label, including the colloidal gold immunochromatographic test paper and the fluorescent immunochromatographic test paper. Since the sensitivity of the colloidal gold immunochromatographic test paper is low, it is difficult to detect trace drugs, and therefore the fluorescent immunochromatographic test paper is usually used for trace detection. However, the existing fluorescent immunochromatographic test paper has the problems of poor stability and low coupling efficiency, which leads to unstable detection signal and easy detection failure.

[0025] Based on this, the present application provides a quantum dot labeled antibody, which has high fluorescence intensity, high detection sensitivity and high stability, and is used in the fluorescent immunochromatographic test paper, so that the detection sensitivity and stability of the fluorescent immunochromatographic test paper can be significantly improved.

[0026] The quantum dot labeled antibody and the preparation method thereof, and the fluorescent immunochromatographic test paper of the embodiments of the present application will be specifically described below.

[0027] The quantum dot labeled antibody of the present application comprises a quantum dot particle and a monoclonal antibody, the monoclonal antibody is grafted on the surface of the quantum dot particle through a grafting group; the grafting group comprises a modified group activated by an activating agent, the modified group comprises at least one of a carboxyl group or an amino group.

[0028] In the quantum dot labeled antibody of the present application, the quantum dot particle can emit fluorescence, and can play a role of labeling the monoclonal antibody; when the monoclonal antibody with high specificity binds with a specific antigen, whether the antigen exists can be judged through fluorescence. Since the fluorescence intensity of the quantum dot particle is high, the sensitivity of the quantum dot labeled antibody is high, and the quantum dot labeled antibody can be used for trace detection. In addition, since the monoclonal antibody is grafted on the quantum dot particle through the modified group after activation, one end of the modified group is grafted on the surface of the quantum dot particle, and the other end away from the quantum dot particle is activated and grafted with the monoclonal antibody, so that the monoclonal antibody is not only well grafted on the surface of the quantum dot particle, but also is not easy to dissociate from the quantum dot particle, and therefore the stability of the whole quantum dot labeled antibody is high, and decomposition is not easy to occur.

[0029] As an example, in some embodiments of the present application, the activating agent comprises at least one of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) or 4-(N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimidyl ester sodium salt (sulfo-SMCC); for example, EDC / NHS can play a role of activating a carboxyl group, and sulfo-SMCC can play a role of activating an amino group. Moreover, in some embodiments of the present application, the monoclonal antibody comprises at least one of an anti-morphine antibody, an anti-methylamphetamine antibody or an anti-cocaine antibody; in this way, the quantum dot labeled antibody can well detect trace drug molecules. In addition, the modified group in the present application is generally a polymer, such as a copolymer of maleic anhydride and styrene; the modified group being a polymer can form a protective layer outside the quantum dot particle, isolate the external environment, improve the chemical stability of the quantum dot particle; and can improve the biocompatibility, reduce the non-specific interaction of the quantum dot with biomolecules, and reduce the risk of immune response. The polymer modified group can introduce rich active groups (such as amino (-NH2), carboxyl (-COOH), thiol (-SH) and the like), so as to realize the coupling of antigens and antibodies, cells and the like.

[0030] In addition, in some embodiments of this application, to better improve detection sensitivity, the mass ratio of quantum dot particles to monoclonal antibodies is typically 10:1 to 50:1. If the mass ratio is too small, there will be fewer quantum dot particles in the quantum dot-labeled antibody, and its fluorescence intensity will easily weaken, which can easily reduce the detection density; if the mass ratio is too large, the content of monoclonal antibodies in the quantum dot-labeled antibody will be smaller, and its ability to bind antigens will also be weaker, which can also easily reduce detection sensitivity.

[0031] Furthermore, in some embodiments of this application, to achieve simultaneous detection of multiple antigens, the emission wavelength of quantum dot-labeled antibodies is generally 500-800 nm, which has a wide emission wavelength range. Therefore, quantum dot-labeled antibodies with different emission wavelengths can bind to different antigens, allowing for the detection and judgment of multiple antigens by the color of the emitted light. This achieves the detection of multiple antigens while avoiding cross-interference. Additionally, to ensure that the colors of the emission spectra do not easily overlap, in some embodiments of this application, the particle size of the quantum dot particles is generally 100 nm-300 nm, which is relatively uniform. This prevents the emission spectra of quantum dot particles of different materials from overlapping, further avoiding cross-interference.

[0032] This application does not impose any particular restrictions on the materials used for quantum dot particles, as long as they meet the objectives of this application. For example, in some other embodiments, the quantum dot particles may have a core-shell structure, wherein the shell structure of the quantum dot particles covers the surface of the core structure of the quantum dot particles. The material of the core structure includes, but is not limited to, at least one of CdSe, CdTe, or InP, and the material of the shell structure includes, for example, ZnS. In this way, the emission wavelength of the quantum dot particles can cover the 500-800 nm wavelength band well.

[0033] It should be noted that in order to achieve the detection of multiple antigens, the monoclonal monomers grafted onto quantum dot labeled antibodies with similar emission wavelengths (i.e., quantum dot particles with the same fluorescence color) should be of the same type; that is, usually the monoclonal antibodies on quantum dot particles of the same material and similar particle size are of the same type, so as to avoid cross-interference.

[0034] This application also provides a method for preparing the above-mentioned quantum dot-labeled antibody, which includes the following steps: S100, preparation of quantum dot particles.

[0035] This step can be used to prepare quantum dot particles via hydrothermal methods or microfluidic technology.

[0036] The hydrothermal method is a synthesis technique based on high-temperature, high-pressure liquid-phase reaction. It utilizes a closed reaction environment to promote the dissolution, nucleation, and crystallization of the precursor solution. In preparation, a solution is first prepared using metal salts, sulfur sources, and stabilizers. This solution is then sealed and heated to a set temperature for reaction. The reaction temperature and time are adjusted according to the type of materials. In microfluidic technology for preparing quantum dot particles, the precursor solution (such as metal salts and sulfur sources) converges through Y-type or T-type microchannels, achieving precise mixing through laminar flow. The flow rate ratio of the two phases is then adjusted to achieve uniform nucleation. After nucleation, heating is performed to achieve crystallization. Since the microfluidic and hydrothermal methods for preparing quantum dot particles are relatively mature, this application will not elaborate on the specific preparation methods of quantum dot particles.

[0037] Of course, quantum dot particles can also be purchased directly. As an example, the quantum dot particles used in this embodiment are all commercially available.

[0038] S200, Surface modification of quantum dot particles.

[0039] This step involves treating the resulting quantum dot particles with carboxylating and / or amination reagents to imbue their surfaces with modifying groups.

[0040] Specifically, quantum dot particles can be pretreated and uniformly dispersed in a solvent, then a polymer solution (the polymer can be introduced with carboxyl groups via maleic anhydride copolymerization, EG-COOH, chitosan-NH2, PMMA-SH, etc.) is added. The pH and temperature are adjusted appropriately to promote the reaction and achieve surface functionalization of the quantum dot particles. At this point, one end of the modifying group will be attached to the surface of the quantum dot particles, while the other end will have lower reactivity. The choice of reagents can be adjusted according to actual needs; this application does not impose any particular restrictions, as long as the purpose of this application is met.

[0041] S300, conjugation of monoclonal antibodies.

[0042] This step involves first activating the modified group with an activator, and then covalently coupling the monoclonal antibody with the activated modified group. This allows the monoclonal antibody to be grafted onto the quantum dot particles through the activated modified group. After blocking, the quantum dot-labeled antibody described above can be obtained.

[0043] It should be noted that in this application, "the monoclonal antibody is grafted onto the quantum dot particles through the activated modification group" means that the monoclonal antibody is first linked to the activated modification group and then grafted onto the surface of the quantum dot particles through the activated modification group. However, it does not mean that all the modification groups will be coupled with monoclonal antibodies at the ends away from the quantum dot particles.

[0044] This application also provides a fluorescent immunochromatographic test strip comprising the aforementioned quantum dot-labeled antibody. As an example, the structure of the fluorescent immunochromatographic test strip in this application is as follows: like Figure 1 As shown, the fluorescent immunochromatographic test strip of this application includes a base plate 1, on which a sample pad 2, a conjugate pad 3, an NC membrane 4 (i.e., a nitrocellulose membrane), and a filter paper 5 are arranged in sequence. The components on the base plate 1 are connected end to end in sequence, and the functions of each component are as follows: The sample pad is used to receive the sample to be tested, such as saliva and urine.

[0045] The conjugate pad is used to store quantum dot-labeled antibodies. During detection, the quantum dot-labeled antibody solution is sprayed onto the conjugate pad and then dried at 37°C. In addition, the conjugate pad is treated with a conjugate pad treatment solution to prevent non-specific adsorption. The conjugate pad treatment solution typically contains 0.5% BSA (Bovine Serum Albumin) and 2% sucrose.

[0046] The NC membrane is used to set the detection line (T line) and control line (C line), with the C line close to the conjugate pad and the T line away from the conjugate pad. Specifically, in this embodiment, the T line and C line are set by spraying a solution, where the T line is obtained by spraying with a coated antigen-BSA conjugate solution and the C line is obtained by spraying with a goat anti-mouse IgG antibody solution. The concentration of the coated antigen-BSA conjugate solution can be 0.2~3.0 mg / mL, and the concentration of the goat anti-mouse IgG antibody solution can be 0.2~3.0 mg / mL. As an example, in this embodiment, the concentration of the coated antigen-BSA conjugate solution is 1 mg / mL, and the concentration of the goat anti-mouse IgG antibody solution is 0.5 mg / mL.

[0047] The filter paper can drive the chromatographic flow, allowing the sample at the sample pad to move towards the filter paper.

[0048] The detection principle of the fluorescent immunochromatographic test strip in this application is as follows: Drug molecules in the sample bind to monoclonal antibodies in quantum dot-labeled antibodies, competitively inhibiting the binding of monoclonal antibodies to the T-line antigen. Therefore, the T-line signal intensity is negatively correlated with drug concentration. Quantitative analysis is performed using a fluorescence reader, or the fluorescence intensity is observed visually. The C-line can verify the detection effectiveness.

[0049] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0050] Example 1 This embodiment provides a quantum dot-labeled antibody, which includes quantum dot particles emitting a wavelength of 525±5nm and an antimorphine antibody. The antimorphine antibody is coupled to the surface of the quantum dot particles via a copolymer of maleic anhydride and styrene activated by EDC / NHS solution. The mass ratio of quantum dot particles to monoclonal antibody is 10:1.

[0051] Example 2 The main difference between this embodiment and Embodiment 1 is that the anti-methamphetamine antibody is used instead of the anti-morphine antibody, and the mass ratio of quantum dot particles to monoclonal antibody is 50:1.

[0052] Example 3 The main difference between this embodiment and Embodiment 1 is that anticocaine antibody is used instead of antimorphine antibody, and the mass ratio of quantum dot particles to monoclonal antibody is 30:1.

[0053] Example 4 This embodiment provides a quantum dot-labeled antibody, comprising quantum dot particles with emission wavelengths of 525±5nm, 570±5nm, and 615±5nm, and a monoclonal antibody. The monoclonal antibody is coupled to the surface of the quantum dot particles via a copolymer of maleic anhydride and styrene activated by EDC / NHS solution. The monoclonal antibody includes anti-morphine antibody, anti-metamphenicol antibody, and anti-cocaine antibody, which are grafted onto the surface of the quantum dot particles with emission wavelengths of 525±5nm, 570±5nm, and 615±5nm, respectively. The mass ratio of the quantum dot particles to their corresponding monoclonal antibodies is 10:1.

[0054] Application examples The quantum dot-labeled antibody from the examples was used in a fluorescent immunochromatographic test strip, the structure of which is shown above. Detection was then performed on the strip, and the results showed that: Taking Example 1 as an example, its detection limit reaches 0.05 ng / mL, and its linear range is 0.1-100 ng / mL, which is 50 times more sensitive than existing colloidal gold test strips. Moreover, the quantum dot-labeled antibody in Example 1 has high stability, and its fluorescence intensity can still be maintained at over 95% after being stored at 4°C for 6 months.

[0055] Taking Example 4 as an example, it was found that the quantum dot-labeled antibody in this application can effectively label antibodies against different drugs, enabling simultaneous detection of morphine, methamphetamine, and cocaine, with a cross-reactivity rate of <5%. This indicates that the quantum dot-labeled antibody in this application supports multi-target detection and is suitable for rapid on-site screening scenarios such as public security and customs.

[0056] The above are merely embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A quantum dot-labeled antibody, characterized in that, It includes quantum dot particles and monoclonal antibodies, wherein the monoclonal antibodies are grafted onto the surface of the quantum dot particles via grafting groups; the grafting groups include modifying groups activated by an activator, and the modifying groups include at least one of carboxyl groups or amino groups.

2. The quantum dot-labeled antibody according to claim 1, characterized in that, The quantum dot-labeled antibody emits light at a wavelength of 500-800 nm.

3. The quantum dot-labeled antibody according to claim 1 or 2, characterized in that, The quantum dot particles have a particle size of 100nm~300nm.

4. The quantum dot-labeled antibody according to claim 1, characterized in that, The monoclonal antibody includes at least one of antimorphine antibody, anti-metabolic antibody, anti-cocaine antibody, anti-etomilate antibody, or anti-methamphetamine antibody.

5. The quantum dot-labeled antibody according to claim 1, characterized in that, The activator includes at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, or sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide.

6. The quantum dot-labeled antibody according to claim 1, characterized in that, The mass ratio of the quantum dot particles to the monoclonal antibody is 10:1 to 50:

1.

7. The method for preparing quantum dot-labeled antibodies according to any one of claims 1 to 6, characterized in that, It includes the following steps: Quantum dot particles are treated with a surface modifier to create a modified group on the surface of the quantum dot particles. The modified group is then activated with an activator, and a monoclonal antibody is subsequently coupled to the activated modified group.

8. A fluorescent immunochromatographic test strip, characterized in that, It includes a detection line, a control line, and a quantum dot-labeled antibody as described in any one of claims 1 to 6.

9. The fluorescent immunochromatographic test strip according to claim 8, characterized in that, The detection line is obtained by spraying an antigen-BSA conjugate solution, and the control line is obtained by spraying a goat anti-mouse IgG antibody solution.

10. The fluorescent immunochromatographic test strip according to claim 9, characterized in that, The concentration of the coated antigen-BSA conjugate solution is 0.2~3.0 mg / mL, and the concentration of the goat anti-mouse IgG antibody solution is 0.2~3.0 mg / mL.

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