Preparation method of high-fluorescence carbon quantum dot, high-fluorescence carbon quantum dot and application of high-fluorescence carbon quantum dot in FK506 detection

Abstract

The invention discloses a preparation method of a high-fluorescence carbon quantum dot, the high-fluorescence carbon quantum dot and application of the high-fluorescence carbon quantum dot in FK506 detection. According to the method, a double-ligand synergistic modification strategy is adopted, 2-aminoterephthalic acid and L-cysteine are used as surface modification ligands, the high-fluorescence carbon quantum dots are synthesized in a citric acid / urea hydrothermal system, the quantum yield of the high-fluorescence carbon quantum dots is up to 85%, the high-fluorescence carbon quantum dots are further combined with a corresponding biological material to construct a targeted FK506 detection kit, and the detection kit is used for detecting the FK506. And the detection limit on the FK506 is as low as 0.16 ng / mL. According to the present invention, the sensitivity bottleneck of the traditional immunochromatography technology is broken through, the advantages of low cost, high stability, simple operation, short detection time and the like are provided, the bedside rapid detection and the home monitoring of the post-transplantation patient can be cooperatively achieved, and the important clinical significance is provided for guiding the tacrolimus medication adjustment and effectively preventing the acute rejection reaction.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of nanomaterial preparation and in vitro diagnostic technology. Specifically, it relates to a method for preparing highly fluorescent carbon quantum dots, the highly fluorescent carbon quantum dots and their application in FK506 detection. Background Technology

[0002] FK506 (tacrolimus) is a macrolide immunosuppressant that inhibits organ rejection by blocking T cell activation and the generation of cytotoxic T cells. It is one of the core drugs currently used for anti-immune rejection therapy after organ transplantation, and its blood concentration is directly related to the immune status of the transplant recipient and the drug toxicity.

[0003] Currently, the main methods for detecting FK506 blood drug concentrations include HPLC-MS, chemiluminescence immunoassay, and immunochromatography. Among these: HPLC-MS has the advantages of high sensitivity, strong specificity, and strict quality control standards, making it the "gold standard" for clinical testing. However, due to the high cost of related instruments and the need for professional technicians to operate and maintain them, as well as the relatively complex and time-consuming operation process, the adoption rate of HPLC-MS in clinical practice is low, especially in some primary healthcare institutions where its application is limited. Chemiluminescence immunoassay has advantages such as high sensitivity, good specificity, wide detection range and high degree of automation. However, it has high operational requirements and requires strict control of experimental conditions (such as temperature and pH value) to ensure the stability of detection results. The sample processing process also needs to strictly follow the operating procedures, otherwise it may affect the accuracy of the detection results. In addition, in some cases, there may be cross-reactions between antibodies and other drugs or metabolites, which may interfere with the accuracy of the detection results. Compared to the aforementioned methods, which suffer from drawbacks such as high cost, complex operation, strict requirements for testing environment, and inability to be integrated with point-of-care testing, immunochromatography is relatively simple to operate, does not require complex instruments and equipment, can produce test results in a short time, is portable, and has low cost, making it more suitable for routine clinical monitoring and large-scale applications. However, immunochromatography is affected by many factors, especially its traditional colloidal gold labeling technology, which has a significant shortcoming in sensitivity (detection limit >2ng / mL), and cannot meet the needs of precise clinical medication.

[0004] In conclusion, researchers in this field believe that the technical approach of developing novel detection materials to address the current problem of insufficient sensitivity in immunochromatographic detection is feasible and has application value, and is a goal that the industry urgently needs to achieve. Summary of the Invention

[0005] To overcome the limitations of existing immunochromatographic techniques in terms of insufficient sensitivity for FK506 detection, this invention provides a novel detection material for FK506 detection by immunochromatography. This material is synthesized using ligand-engineered modified carbon quantum dots, which improves the fluorescence quantum yield of carbon quantum dots and their detection sensitivity for FK506, providing real-time data support for personalized medication for organ transplant patients.

[0006] The improved technical solution of this invention is as follows: A method for preparing highly fluorescent carbon quantum dots includes the following steps: A1. Add citric acid, urea and 2-aminoterephthalic acid to ultrapure water, heat and stir until completely dissolved to obtain a precursor solution; A2. Adjust the pH of the precursor solution to 3.0~4.0, add L-cysteine, heat and stir until completely dissolved, transfer the entire solution system to a high-pressure reactor, purge with nitrogen, and carry out a hydrothermal reaction by programmed temperature control. After the reaction is completed, allow it to cool naturally to room temperature. A3. Collect the cooled product from the high-pressure reactor, and after dialysis purification and freeze-drying, obtain the target product.

[0007] Preferably, the ratio of the total mass of citric acid, urea, and 2-aminoterephthalic acid to the volume of ultrapure water is 4~5:30, with the mass unit being grams and the volume unit being milliliters; for example, 4g:30mL, 4.5g:30mL, 5g:30mL.

[0008] Preferably, in step A2, the mass ratio of L-cysteine ​​to the volume of ultrapure water is 0.1~0.2:30, with the mass unit being grams and the volume unit being milliliters; for example, 0.1g:30mL, 0.15g:30mL, 0.2g:30mL.

[0009] Preferably, in steps A1 and A2, the dissolution conditions are: heating temperature of 45~60℃, stirring time of 0.5~4h, and stirring speed of 300~800rpm.

[0010] Preferably, in step A2, the hydrothermal reaction by programmed temperature control includes two stages, wherein the reaction temperature of the first stage is 180°C and the reaction time is 2-3 hours, and the reaction temperature of the second stage is 210°C and the reaction time is 6-8 hours.

[0011] Preferably, in step A3, the cooled product needs to be pre-filtered through a 0.2~0.3 μm microporous membrane before dialysis purification, and the molecular weight cutoff during purification is 1 kDa.

[0012] A high-fluorescence carbon quantum dot includes a carbon quantum dot matrix and a ligand material modified on the surface of the carbon quantum dot matrix, wherein the ligand material includes 2-aminoterephthalic acid and L-cysteine.

[0013] Preferably, the mass ratio of the carbon quantum dot matrix to 2-aminoterephthalic acid and L-cysteine ​​is 39:3~6:1~2; for example, 39:3:1, 39:4:1, 39:5:2, 39:6:1 or 39:6:2.

[0014] Preferably, the mass ratio of 2-aminoterephthalic acid to L-cysteine ​​is 4.5:1.

[0015] According to an embodiment of the present invention, the spectral range of the highly fluorescent carbon quantum dots is 520~600nm. Preferably, the spectral position of the highly fluorescent carbon quantum dots is 540nm.

[0016] According to an embodiment of the present invention, the particle size of the highly fluorescent carbon quantum dots is 2-5 nm. Preferably, the particle size of the highly fluorescent carbon quantum dots is 3.5 nm.

[0017] According to an embodiment of the present invention, the luminescence efficiency of the high-fluorescence carbon quantum dots is 70-85%. Preferably, the luminescence efficiency of the high-fluorescence carbon quantum dots is 85%.

[0018] An application of highly fluorescent carbon quantum dots in the detection of FK506 involves dissolving highly fluorescent carbon quantum dots in ultrapure water and mixing them with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and sodium N-hydroxysuccinimide sulfonate (sulfo-NHS). After activation at room temperature, a detection antibody targeting FK506 is added. After mixing thoroughly, the mixture is ultrafiltered and centrifuged, and the supernatant is collected to obtain a highly fluorescent carbon quantum dot probe for detecting FK506.

[0019] It should be noted that during ultrafiltration centrifugation, if the target molecules are significantly smaller than the filter membrane pore size, ultrafiltration centrifuge tubes can be used to remove pyrogens, clarify, and separate large molecular pollutants. In this case, the target molecules will pass through the filter membrane and remain in the centrifuge tube. Conversely, if the target molecules are much larger than the filter membrane pore size, small molecular pollutants will be filtered out. In this case, the target molecules cannot pass through the filter membrane and are concentrated in the filtration device. In this invention, the molecular weight of the highly fluorescent carbon quantum dot probe is obviously larger than that of the antibody and pure carbon quantum dots, and therefore it is retained in the upper layer.

[0020] Preferably, the molar ratio of the high-fluorescence carbon quantum dots to EDC is 1:2~5, and the molar ratio of EDC to sulfo-NHS is 2~5:1; more preferably, the molar ratio of the high-fluorescence carbon quantum dots to EDC and sulfo-NHS is 2:4:1~2.

[0021] Preferably, the molar ratio of the highly fluorescent carbon quantum dots to the detection antibody targeting FK506 is 1 to 5:1.

[0022] Preferably, the prepared high-fluorescence carbon quantum dot probe is refrigerated at a temperature of 1-5°C.

[0023] Preferably, the room temperature activation time is 15–60 min during the preparation of the highly fluorescent carbon quantum dot probe.

[0024] An FK506 detection kit includes a detection solution, an immunochromatographic test strip, and an instruction manual. The detection solution contains a highly fluorescent carbon quantum dot probe prepared according to the above application. The immunochromatographic test strip contains a capture antibody targeting FK506, and the capture antibody does not cross-react with the detection antibody targeting FK506.

[0025] The method of using the FK506 detection kit is as follows: First, mix the sample and the test solution evenly, then drop it onto the immunochromatographic test strip and react for 15 minutes. Finally, use a portable fluorescence detector to detect whether fluorescence appears on the C and T lines of the immunochromatographic test strip and collect fluorescence intensity data.

[0026] Based on the principle of antibody-antigen specific recognition, if the sample contains human FK506-related antigen, the T line will show green fluorescence. Conversely, if the sample does not contain human FK506-related antigen, the T line will show almost no fluorescence. The higher the fluorescence intensity, the higher the concentration of human FK506-related antigen in the sample. Conversely, the lower the fluorescence intensity, the lower the concentration of human FK506-related antigen. The C line is the quality control line. When the C line shows no fluorescence, it indicates that the test strip has deteriorated.

[0027] According to an embodiment of the present invention, the detection time of the FK506 detection kit is 10-20 minutes, preferably 15 minutes.

[0028] According to an embodiment of the present invention, the detection linear range of the FK506 detection kit is 0.5~30 ng / mL, and the detection limit can be as low as 0.16 ng / mL.

[0029] The technical solution of the present invention has at least the following beneficial effects: 1. This invention employs a dual-ligand synergistic modification strategy, introducing 2-aminoterephthalic acid and L-cysteine ​​sequentially into a citric acid / urea hydrothermal system to successfully construct highly fluorescent carbon quantum dots with amino / carboxyl dual active surfaces, achieving a fluorescence quantum yield of up to 85%.

[0030] 2. The highly fluorescent carbon quantum dots prepared in this invention can be used as fluorescent tags for rapid detection of FK506. The kits using these as detection materials have the advantages of simple and rapid operation, short detection time, and easy result interpretation. They are of great clinical significance for enabling home monitoring of transplant patients, guiding the adjustment of tacrolimus medication, and effectively preventing acute rejection.

[0031] 3. This invention overcomes the sensitivity limitations of traditional methods for FK506 detection, with a detection limit (LOD) as low as 0.16 ng / mL, while enabling rapid point-of-care testing within 15 minutes, providing real-time data support for personalized medication for organ transplant patients. Attached Figure Description

[0032] Figure 1 This is a fluorescence image of the high-fluorescence carbon quantum dots in Example 1; Figure 2 This is a transmission electron microscope image of the high-fluorescence carbon quantum dots in Example 1; Figure 3 The quantum yield diagram of ordinary carbon quantum dots in Comparative Example 1; Figure 4 The quantum yield diagram of the high-fluorescence carbon quantum dots in Example 1; Figure 5 This is a comparison of the PL spectra of the high-fluorescence carbon quantum dots in Example 1 and the ordinary carbon quantum dots in Comparative Example 1, where the blue line represents the high-fluorescence carbon quantum dots and the yellow line represents the ordinary carbon quantum dots. Figure 6 This is a comparison curve of the FK506 concentration standard conversion between the high-fluorescence carbon quantum dot probe in Example 2 and the ordinary carbon quantum dot probe in Comparative Example 2. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0034] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0035] Comparative Example 1 The preparation steps for ordinary carbon quantum dots are as follows: A1. Weigh 2.1 g of citric acid and 1.8 g of urea and dissolve them in 30 mL of ultrapure water. Stir magnetically for 30 min at 50℃ and 600 rpm until the mixture is completely dissolved. A2. Transfer the entire solution system to a polytetrafluoroethylene high-pressure reactor, purge with nitrogen to remove oxygen, and carry out a hydrothermal reaction by programmed temperature control. The first stage is carried out at 180℃ for 2 hours, and the second stage is carried out at 210℃ for 6 hours. After the reaction is completed, allow it to cool naturally to room temperature. A3. Collect the coolant in the high-pressure reactor, first filter it through a 0.22 μm microporous membrane, then dialyze it (molecular weight cutoff 1 kDa) for 48 h, replace the dialysis solution every 6 h, and finally freeze dry it to obtain the target product.

[0036] Comparative Example 2 The process of using ordinary carbon quantum dots for FK506 detection is as follows: B1. Dissolve the ordinary carbon quantum dot solution prepared in Comparative Example 1 in ultrapure water (1 mg / mL), add EDC and sulfo-NHS, wherein the molar ratio of ordinary carbon quantum dots to EDC is 1:2 and the molar ratio of EDC to sulfo-NHS is 4:1, vortex and oscillate until uniform, and activate at room temperature for 30 min. B2. Add the detection antibody targeting FK506 to the activated solution, and react in a shaker at 37°C for 30 minutes. B3. After the reaction is complete, use an ultrafiltration centrifuge tube with a molecular weight cutoff of 50 kDa to collect the upper layer of residue, store it at 4°C for later use, and obtain a common carbon quantum dot probe for detecting FK506. B4. Stack the sample pad (glass fiber), absorbent pad, NC membrane (detection / reference line), and absorbent pad in sequence. Place the capture antibody targeting FK506 on the detection line of the NC membrane to assemble a traditional immunochromatographic test strip. B5. Dilute the ordinary carbon quantum dot probe to 1 mg / mL as the detection solution, add it to the sample to be tested and mix well. Then, take 100 μL of the mixture and drop it onto the traditional immunochromatographic test strip and react for 15 min. B6. The fluorescence intensity value of the T line was collected using a portable fluorescence detector to obtain the FK506 concentration standard conversion curve.

[0037] Example 1 The preparation steps of fluorescent carbon quantum dots provided by this invention are as follows: A1. Weigh 2.1 g of citric acid, 1.8 g of urea, and 0.6 g of 2-aminoterephthalic acid and dissolve them in 30 mL of ultrapure water. Stir magnetically for 30 min at 50 °C and 600 rpm to completely dissolve the mixture and obtain the precursor solution. A2. Adjust the pH of the precursor solution to 3.0~4.0, add 0.2g L-cysteine, and stir to dissolve under the same conditions as in A1. Transfer the entire solution system to a polytetrafluoroethylene high-pressure reactor, purge with nitrogen to remove oxygen, and carry out a hydrothermal reaction by programmed temperature control. The first stage is carried out at 180℃ for 2 h, and the second stage is carried out at 210℃ for 6 h. After the reaction is completed, allow it to cool naturally to room temperature. A3. Collect the coolant in the high-pressure reactor, first filter it through a 0.22 μm microporous membrane, then dialyze it (with a molecular weight cutoff of 1 kDa) for 48 h, changing the dialysis solution every 6 h, and finally freeze-dry it to obtain the target product.

[0038] The carbon quantum dots prepared in this embodiment and Comparative Example 1 were tested respectively, and the results were obtained. Figures 1-4 .in, Figure 1 The image shows the fluorescence of highly fluorescent carbon quantum dots. Figure 2 This is a transmission electron microscope image of highly fluorescent carbon quantum dots, further measured... Figure 2 The particle size of medium- to high-fluorescence carbon quantum dots is approximately 3.5 nm; Figure 3 The quantum yield map of the high-fluorescence carbon quantum dots was obtained using an FLS980 instrument, and its quantum yield value is approximately 85%. Figure 4 The quantum yield of ordinary carbon quantum dots, measured by an FLS980 instrument, is approximately 44%. This demonstrates that the high-fluorescence carbon quantum dots provided by this invention have a significant advantage in luminescence efficiency and are suitable for high-sensitivity detection.

[0039] The carbon quantum dots prepared in this embodiment and Comparative Example 1 were subjected to luminescence tests, and the results are shown in [reference]. Figure 5 Within the wavelength range of 500~670nm, the fluorescence intensity of the high-fluorescence carbon quantum dots provided by this invention is significantly higher than that of ordinary carbon quantum dots. In particular, the difference is most obvious when the emission peak position is 540nm, which is the emission wavelength of the fluorescence detector most suitable for the scheme of this invention.

[0040] Example 2 The process of using highly fluorescent carbon quantum dots for FK506 detection is as follows: B1. Dissolve the high-fluorescence carbon quantum dot solution prepared in Example 1 in ultrapure water (1 mg / mL), add EDC and sulfo-NHS, wherein the molar ratio of high-fluorescence carbon quantum dots to EDC is 1:2, and the molar ratio of EDC to sulfo-NHS is 4:1. Vortex and oscillate until uniform, and activate at room temperature for 30 min. B2. Add the detection antibody targeting FK506 to the activated solution, and react in a shaker at 37°C for 30 minutes. B3. After the reaction is complete, use an ultrafiltration centrifuge tube with a molecular weight cutoff of 50 kDa to collect the upper layer of residue, store it at 4°C for later use, and obtain a high-fluorescence carbon quantum dot probe for detecting FK506. B4. The sample pad (glass fiber), absorbent pad, NC membrane (detection / reference line), and absorbent pad are stacked in sequence. A capture antibody targeting FK506 is placed on the detection line of the NC membrane to assemble the immunochromatographic test strip of the present invention. B5. Dilute the high-fluorescence carbon quantum dot probe to 1 mg / mL as the detection solution, add it to the sample to be tested and mix well. Then, take 100 μL of the mixture and drop it onto the immunochromatographic test strip of the present invention and react for 15 min. B6. The fluorescence intensity value of the T line was collected using a portable fluorescence detector to obtain the FK506 concentration standard conversion curve.

[0041] In this embodiment and Comparative Example 2, the detection antibody was FKBP11 Recombinant antibody PBSOnly (Detector), purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number 84478-3-PBS; the capture antibody was FKBP11 Recombinant antibody, purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number 84478-4-PBS.

[0042] In this embodiment and Comparative Example 2, the standard curve used to convert the FK506 concentration is as follows: Figure 6 As shown, the linear range of the high-fluorescence carbon quantum dot probe provided by the present invention is 0.5~30 ng / mL, and the detection limit is as low as 0.16 ng / mL, while the detection limit of ordinary carbon quantum dot probes for FK506 concentration can only reach 5 ng / mL, which is a huge difference. The modified carbon quantum dot probe of the present invention improves the sensitivity of FK506 detection by an order of magnitude.

[0043] The embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing highly fluorescent carbon quantum dots, characterized in that, Includes the following steps: A1. Add citric acid, urea and 2-aminoterephthalic acid to ultrapure water, heat and stir until completely dissolved to obtain a precursor solution; A2. Adjust the pH of the precursor solution to 3.0~4.0, add L-cysteine, heat and stir until completely dissolved, transfer the entire solution system to a high-pressure reactor, purge with nitrogen, and carry out a hydrothermal reaction by programmed temperature control. After the reaction is completed, allow it to cool naturally to room temperature. A3. Collect the cooled product from the high-pressure reactor, and after dialysis purification and freeze-drying, obtain the target product.

2. The method for preparing high-fluorescence carbon quantum dots according to claim 1, characterized in that, The ratio of the total mass of citric acid, urea, and 2-aminoterephthalic acid to the volume of ultrapure water is 4~5:30, and the ratio of the mass of L-cysteine ​​to the volume of ultrapure water is 0.1~0.2:

30. The unit of mass measurement is grams, and the unit of volume measurement is milliliters.

3. The method for preparing high-fluorescence carbon quantum dots according to claim 1, characterized in that, In steps A1 and A2, the dissolution conditions are: heating temperature of 45~60℃, stirring time of 0.5~4h, and stirring speed of 300~800rpm.

4. The method for preparing high-fluorescence carbon quantum dots according to claim 1, characterized in that, In step A2, the hydrothermal reaction by programmed temperature control includes two stages, wherein the reaction temperature of the first stage is 180°C and the reaction time is 2-3 hours, and the reaction temperature of the second stage is 210°C and the reaction time is 6-8 hours.

5. The method for preparing high-fluorescence carbon quantum dots according to claim 1, characterized in that, In step A3, the cooled product needs to be pre-filtered through a 0.2~0.3 μm microporous membrane before dialysis purification, and the molecular weight cutoff during purification is 1 kDa.

6. The highly fluorescent carbon quantum dots prepared by the method according to any one of claims 1 to 5, characterized in that, It includes a carbon quantum dot matrix and ligand materials modified on the surface of the carbon quantum dot matrix, wherein the ligand materials include 2-aminoterephthalic acid and L-cysteine.

7. The high-fluorescence carbon quantum dot according to claim 6, characterized in that, The mass ratio of the carbon quantum dot matrix to 2-aminoterephthalic acid and L-cysteine ​​is 39:3~6:1~2.

8. The application of the high-fluorescence carbon quantum dots as described in claim 7 in FK506 detection, characterized in that, Highly fluorescent carbon quantum dots were dissolved in ultrapure water and mixed with EDC and sulfo-NHS. After activation at room temperature, a detection antibody targeting FK506 was added. After mixing evenly, the mixture was ultrafiltered and centrifuged. The supernatant was collected to obtain a highly fluorescent carbon quantum dot probe for detecting FK506.

9. The application of the high-fluorescence carbon quantum dots according to claim 8 in FK506 detection, characterized in that, The molar ratio of the highly fluorescent carbon quantum dots to EDC is 1:2~5, the molar ratio of EDC to sulfo-NHS is 2~5:1, and the molar concentration ratio of the highly fluorescent carbon quantum dots to the detection antibody targeting FK506 is 1~5:

1.

10. An FK506 detection kit, characterized in that, The test solution includes a detection solution, an immunochromatographic test strip, and an instruction manual. The detection solution contains a highly fluorescent carbon quantum dot probe prepared according to claim 8 or 9. The immunochromatographic test strip contains a capture antibody targeting FK506, and the capture antibody does not cross-react with the detection antibody targeting FK506.

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