Preparation method of fluorescence immune probe of quantum dot labeled antibody
By preparing CdSe/ZnS quantum dot fluorescent immunoprobes, the problem of limited detection effect of traditional fluorescent dyes was solved, and highly selective and sensitive fluorescent immunoassay was achieved. The photostability and biocompatibility of quantum dots improved the detection effect.
Patent Information
- Application Number
- CN202511340110.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-18
- Publication Date
- 2025-12-30
AI Technical Summary
In existing fluorescence immunoassay techniques, traditional fluorescent dyes have poor photobleaching ability, narrow excitation spectrum, and sensitivity to temperature or pH, which limits the detection effect. Furthermore, research on quantum dot-labeled antibody fluorescent immunoprobes has not been widely reported, making it difficult to achieve detection with high selectivity and high sensitivity.
CdSe/ZnS quantum dots were activated with coupling agents 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then bound to antibodies via a coupling reaction to prepare CdSe/ZnS quantum dot fluorescent immunoprobes. The reaction conditions, including temperature, time, and centrifugation parameters, were optimized.
Quantum dots improve the selectivity and sensitivity of fluorescent immunoassay probes, possessing strong photostability and good biocompatibility. Their flexible surface modification enables highly efficient fluorescence detection.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of fluorescent immunological probe preparation, and particularly relates to a fluorescent immunological probe preparation method of quantum dot labeled antibodies. BACKGROUND
[0002] The fluorescent immunological analysis technology is an analysis technology based on the specific and reversible binding reaction of antigens and antibodies, and has the advantages of high sensitivity, simple operation and low cost, and is widely used. However, the traditional fluorescent dyes have the disadvantages of poor anti-photobleaching ability, narrow excitation spectrum and sensitivity to temperature or pH, which seriously limits the development of the fluorescent analysis method.
[0003] In recent years, with the continuous development of nanotechnology, carbon quantum dots (CDs), semiconductor quantum dots (QDs) and noble metal nanoclusters with fluorescent emission have been developed for the design of fluorescent probes, and have shown good development prospects and application value. Among them, the semiconductor quantum dots have the advantages of strong light stability, good biocompatibility and flexible surface modification.
[0004]
[0005] In order to improve the detection effect of the fluorescent immunological analysis method, quantum dot labeled antibodies with high selectivity and high sensitivity need to be synthesized. Although there is no extensive report on the fluorescent immunological probe of quantum dot labeled antibodies at present, the design and synthesis of effective fluorescent immunological probes are still a technical difficulty in the immunological analysis technology. SUMMARY
[0006] The purpose of the present application is to provide a preparation method of a fluorescent immunological probe of quantum dot labeled antibodies. The quantum dot fluorescent probe provided by the present application has high sensitivity and good selectivity.
[0007] The purpose of the present application can be achieved by the following measures:
[0008] A preparation method of a quantum dot fluorescent probe, characterized in that it is prepared by the following steps:
[0009] a. adding CdSe / ZnS quantum dots into a coupling agent for activation to obtain an activated quantum dot solution; wherein the coupling agent is 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide and N-hydroxysuccinimide;
[0010] b. mixing the activated quantum dot solution and an antibody buffer solution for a coupling reaction to obtain the CdSe / ZnS quantum dot fluorescent immunological probe;
[0011] c. centrifuging the coupling product and then storing the obtained precipitate dispersed in a buffer solution.
[0012]
[0013] The preparation method of the CdSe / ZnS quantum dot fluorescent immunological probe is characterized in that the activation temperature in step a is room temperature, and the reaction time is 0.5-1.5 h.
[0014] The preparation method of the CdSe / ZnS quantum dot fluorescent immunological probe is characterized in that in step b, the molar ratio of the quantum dots, the antibody, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide is preferably 1:10-30:50-150:100-300; the buffer solution is 2-morpholinoethanesulfonic acid, the pH of the buffer solution is 5-7; the reaction temperature is room temperature, and the reaction time is preferably 2-5 h.
[0015] The preparation method of the CdSe / ZnS quantum dot fluorescent immunological probe is characterized in that in step c, the centrifugation temperature is 4°C, the rotation speed is 8000-12000 rpm, and the time is preferably 10-30 min; the buffer solution is a PBS buffer solution.
[0016] Further optimization mode:
[0017] The CdSe / ZnS quantum dots are added into a reactor, 2-morpholinoethanesulfonic acid buffer solution is supplemented, the pH is preferably 5-7, and the final concentration is 1 mg / ml, and the mixture is uniformly stirred. The CdSe / ZnS quantum dots and the coupling agent described in the above scheme are mixed for activation to obtain an activated quantum dot solution; the activated quantum dot solution and the 2-morpholinoethanesulfonic acid buffer solution are mixed for a coupling reaction to obtain the CdSe / ZnS quantum dot fluorescent immunological probe. The CdSe / ZnS quantum dots and the coupling agent described in the above scheme are mixed for activation to obtain an activated quantum dot solution in the present application. In the present application, the coupling agent is preferably 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) and N-hydroxysuccinimide (NHS), the activation temperature is preferably room temperature, and the activation time is preferably 0.5-1.5 h. After obtaining the activated quantum dot solution, the activated quantum dot solution and the 2-morpholinoethanesulfonic acid buffer solution are mixed for a coupling reaction to obtain the CdSe / ZnS quantum dot fluorescent immunological probe.
[0018] In the present application, when preparing the CdSe / ZnS quantum dot fluorescent immune probe, the molar ratio of the quantum dots, the antibody, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is preferably 1:10-30:50-150:100-300; the concentration of the 2-morpholinoethanesulfonic acid buffer is preferably 0.05 mol / L, and the pH is preferably 5-7, more preferably 6.5; the activated quantum dot solution is preferably added dropwise into the 2-morpholinoethanesulfonic acid buffer solution, the temperature of the coupling reaction is preferably room temperature, and the time of the coupling reaction is preferably 2-5 h.
[0019] After the coupling reaction is completed, the obtained reaction solution is preferably centrifuged, the centrifugation is preferably 15-30 min, the centrifugation speed is 8000-12000 r / min, more preferably 20 min and 10000 r / min, to obtain the CdSe / ZnS quantum dot fluorescent immune probe solution after coupling, and the collected precipitate is dispersed in the PBS buffer solution to obtain the CdSe / ZnS quantum dot fluorescent immune probe solution.
[0020] Advantages:
[0021] Compared with the prior art, the present application has the following advantages:
[0022] The present application obtains the CdSe / ZnS quantum dot fluorescent immune probe by activating ester reaction and coupling with the antibody, replaces the traditional organic dye with the quantum dots to improve the selectivity and sensitivity of the fluorescent immune probe, and the quantum dots have the advantages of strong light stability, good biocompatibility and flexible surface modification compared with the traditional organic dye. DETAILED DESCRIPTION
[0023] The present application is further illustrated by the following examples, but the present application is not limited in the scope of the examples.
[0024]
[0025] CdSe / ZnS quantum dots 12.5 μl are added into a reactor, and 2-morpholinoethanesulfonic acid buffer solution (pH=6.5) is supplemented to a final concentration of 1 mg / ml, and stirred at room temperature for 5 min to make it uniform. 3.54 μl of EDC solution and 0.575 μl of N-hydroxysuccinimide solution are added dropwise into the reaction, and reacted for 30 min. 30 μl of antibody (0.65 mg / ml) is slowly added dropwise into the CdSe / ZnS quantum dot solution. Then, continuous stirring is carried out at room temperature for 3 h. After the coupling reaction is completed, the reaction product is separated by centrifugation at 10000 rpm for 20 min at 4 ℃, and the precipitate is dispersed in PBS buffer solution (pH=7.4) to a concentration of 0.5 mg / ml for standby use.
[0026] Although the invention has been described in detail with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art should be able to make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the invention should be considered as defined by the appended claims.
Claims
1. A method for preparing a CdSe / ZnS quantum dot fluorescent immuno-probe, characterized in that, It is prepared by the following steps: a. adding CdSe / ZnS quantum dots to a coupling agent for activation to obtain an activated quantum dot solution; wherein the coupling agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide; b. mixing the activated quantum dot solution and a buffer solution of an antibody for a coupling reaction to obtain the CdSe / ZnS quantum dot fluorescent immunological probe; c. centrifuging the coupling product, and then dispersing the obtained precipitate in a buffer solution for storage.
2. The method for preparing a CdSe / ZnS quantum dot fluorescent immunoprobe according to claim 1, characterized in that, The activation temperature in step a is room temperature, and the reaction time is 0.5-1.5 h.
3. The method for preparing a CdSe / ZnS quantum dot fluorescent immunoprobe according to claim 1, characterized in that, In step b, the molar ratio of the quantum dots, the antibody, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is preferably 1:10-30:50-150:100-300; the buffer solution is 2-morpholinoethanesulfonic acid, the pH of the buffer solution is 5-7, the reaction temperature is room temperature, and the reaction time is preferably 2-5 h.
4. The method for preparing a CdSe / ZnS quantum dot fluorescent immunoprobe according to claim 1, characterized in that, In step c, the centrifugation temperature is 4°C, the rotation speed is 8000-12000 rpm, and the time is preferably 10-30 min; the buffer solution is a PBS buffer solution.