Quantum dot-colloidal gold composite probe as well as preparation method and application thereof

Abstract

The invention discloses a quantum dot-colloidal gold composite probe. The composite probe is composed of a quantum dot core layer, a colloidal gold shell layer and a biological coupling layer, wherein the quantum dot core layer is composed of one of sulfur quantum dots, carbon quantum dots, CdSe / ZnS quantum dots, InP / ZnS quantum dots or perovskite quantum dots, the colloidal gold shell layer wraps the surfaces of the quantum dots, and the biological coupling layer is connected with targeting molecules through a difunctional coupling agent. The invention also discloses a preparation method of the quantum dot-colloidal gold composite probe. The invention also discloses a multi-mode detection method based on the quantum dot-colloidal gold composite probe. In addition, the invention also discloses a tumor diagnosis kit. The quantum dot-colloidal gold composite probe provided by the invention utilizes high fluorescence intensity and light stability of the quantum dots, combines the advantages of the quantum dots and colloidal gold, and provides an efficient and accurate method for tumor detection through specific binding and high-sensitivity detection.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection, and in particular to a quantum dot-colloidal gold composite probe and a preparation method and application thereof. Background Art

[0002] Quantum dots and colloidal gold, two important nanomaterials, are currently widely used in fields such as biomedicine and analytical testing. Specifically, colloidal gold refers to a colloidal solution composed of gold nanoparticles dispersed in a liquid medium; these gold nanoparticles typically have diameters between 1 and 100 nanometers and possess unique optical, electronic, and catalytic properties. Quantum dots (QDs) are a type of semiconductor nanocrystals with sizes between 1 and 10 nanometers. Because their size is close to or smaller than the coherence length of electrons, quantum dots often exhibit significant quantum confinement effects, where the movement of electrons and holes is restricted, resulting in unique optical, electrical, and physical properties.

[0003] Quantum dots are widely used in biolabeling, imaging, and sensing due to their unique optical properties, such as tunable fluorescence emission wavelength, high quantum yield, and good photostability. Colloidal gold, on the other hand, has shown great potential in biomedical diagnostics, drug delivery, and catalysis due to its good biocompatibility, stable chemical properties, and ease of preparation into different shapes and sizes. However, the use of quantum dots or colloidal gold alone often fails to meet the needs of complex applications. Therefore, in order to meet the requirements of higher detection sensitivity, stability, and specificity, it is very necessary to improve the performance of colloidal gold and quantum dots. Therefore, the development of a composite material that combines the advantages of quantum dots and colloidal gold has broad application prospects. Summary of the Invention

[0004] The purpose of the present invention is to provide a quantum dot-colloidal gold composite probe and its preparation method and application.

[0005] In order to achieve the above object, the solution of the present invention is: A quantum dot-colloidal gold composite probe, comprising a quantum dot core layer, a colloidal gold shell layer, and a biocoupling layer; wherein the quantum dot core layer is composed of one of sulfur quantum dots, carbon quantum dots, CdSe / ZnS quantum dots, InP / ZnS quantum dots, or perovskite quantum dots; the colloidal gold shell layer is wrapped around the surface of the quantum dots; and the biocoupling layer is connected to a targeting molecule via a bifunctional coupling agent.

[0006] Preferably, the particle size of the quantum dots is in the range of 2-10 nm; the thickness of the colloidal gold shell is 5-20 nm, and the surface roughness is ≤3 nm; and the coupling agent is an organic compound containing thiol and carboxyl functional groups.

[0007] Preferably, a 1-3 nm silicon dioxide isolation layer is provided between the quantum dot core layer and the colloidal gold shell layer, and the quantum dot fluorescence quantum yield is ≥80%, and the colloidal gold extinction coefficient is ≥3×10 8 M -1 cm -1 .

[0008] Preferably, the targeting molecule is selected from at least one of anti-EGFR monoclonal antibodies, PD-1, HER2 aptamers or EpCAM nucleic acid aptamers, and the coupling density is 10-50 molecules / particle.

[0009] A method for preparing a quantum dot-colloidal gold composite probe, the specific steps are as follows: Step I: Under nitrogen protection, the quantum dots were dispersed in an ethanol solution containing 3-aminopropyltriethoxysilane and reacted at 60°C for 2 h to form an amino surface; Step II: Mixing chloroauric acid solution and quantum dots in a molar ratio of 1:5-1:20, adding a reducing agent for in situ reduction, and controlling the pH to 5.0-6.5; Step III: Add the bifunctional coupling agent Sulfo-SMCC to activate the carboxyl group at 4°C; Step IV: Coupling with the target molecule in pH 7.4 phosphate buffer, ultrafiltration purification and storage at 4°C.

[0010] Preferably, a gradient temperature increase method is used in step II, and the specific operation is as follows: The first stage: maintain at 25℃ for 10min to form crystal nuclei; The second stage: the temperature was raised to 100°C at 0.5°C / min to complete the shell growth; The third stage: sudden cooling to 4°C to terminate the reaction.

[0011] Preferably, the temperature for activating the carboxyl groups in step III is 4°C.

[0012] The multimodal detection method of the aforementioned quantum dot-colloidal gold composite probe comprises: The sample to be tested was incubated with the composite probe at a volume ratio of 1:50-1:200 for 30 minutes; Synchronous: a) Fluorescence detection: excitation wavelength 400-550nm, detection of emission spectrum; b) Surface-enhanced Raman spectroscopy: 785 nm laser excitation, collecting 1500-1700 cm -1 characteristic peaks; c) Colorimetric detection: Measure the change in absorbance at 520 nm.

[0013] A tumor diagnostic kit comprising: The aforementioned quantum dot-colloidal gold composite probe 0.1-1 mg / mL; Multimodal test strips, including: - Fluorescence readout zone (coated with quencher-modified capture probes) - Colorimetric control card (including standard colorimetric gradient).

[0014] Preferably, it further comprises a stabilization buffer, which consists of: Tris-HCl 25 mM, trehalose 5% (w / v), BSA 1% (w / v), pH 7.4±0.2.

[0015] The principles of the quantum dot-colloidal gold composite probe and its preparation method and application provided by the present invention are as follows: The quantum dot-colloidal gold composite probe provided by the present invention is mainly based on the characteristics of quantum dots and colloidal gold and their ability to bind to biomolecules. Specifically, the quantum dots in the composite probe have unique optical properties, such as a wide excitation spectrum, a narrow emission spectrum, good monochromaticity and color tunability, high fluorescence intensity, and good photochemical stability; these properties make quantum dots highly sensitive and specific in biomolecule detection. The colloidal gold in the composite probe has good optical properties and biocompatibility. The method provided by the present invention combines quantum dots and colloidal gold together through a chemical reaction method to form a composite probe, and then combines quantum dot nanoparticles and biomolecule marker antibodies through coupling. This composite probe combines the high sensitivity of quantum dots with the stability and easy detectability of colloidal gold.

[0016] The advantages of the quantum dot-colloidal gold composite probe provided by the present invention are as follows: (1) The quantum dot-colloidal gold composite probe provided by the present invention utilizes the high fluorescence intensity and photostability of quantum dots to significantly improve the detection sensitivity.

[0017] (2) The quantum dot-colloidal gold composite probe provided by the present invention can be applied to tumor detection, and the accuracy of the detection results can be ensured by combining specific antibodies with tumor markers.

[0018] (3) The quantum dot-colloidal gold composite probe provided by the present invention can realize the simultaneous detection of multiple tumor markers on the same detection platform.

[0019] (4) The quantum dot-colloidal gold composite probe and its preparation method provided by the present invention also have the advantages of simple operation and suitability for on-site rapid detection.

[0020] (5) The quantum dot-colloidal gold composite probe provided by the present invention combines the advantages of quantum dots and colloidal gold, and provides an efficient and accurate method for tumor detection through specific binding and high-sensitivity detection. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the examples. It should also be understood that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of the present invention. The specific mass, reaction time, temperature, process parameters, etc. in the examples are only examples within the appropriate ranges, and any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the scope of the present invention.

[0022] Unless otherwise specified, all reagents used were commercial reagents and were not further purified before use.

[0023] In the examples used in the invention, 1.4 g of sublimed sulfur, 4.0 g of sodium hydroxide, and 50 ml of deionized water were added to a 150 ml round-bottom flask. The resulting mixture was then transferred to an ultrasonic reactor and subjected to ultrasonic oscillation (80 kHz, 50 W) at 70°C for 1 hour. The resulting mixture was then placed in a microwave reactor and subjected to microwave reaction at 80°C for 1.5 hours at a microwave power of 300 W. After the reaction was complete, the mixture was cooled to room temperature and the solution was placed in a centrifuge tube and subjected to a 1500 rpm rotation. -1 The solution was centrifuged at a speed of 0.5 h, and then the obtained solution was passed through a dialysis membrane with a molecular cutoff of 3500 Da to obtain a yellow transparent sulfur quantum dot solution; finally, the sulfur quantum dot solution obtained after dialysis was freeze-dried to obtain a light yellow solid, which is sulfur quantum dots, recorded as SDs.

[0024] The steps for preparing the carbon quantum dots used in the examples of the present invention are as follows: 2.5 g of ginkgo leaf powder and 150 ml of deionized water were added to a 250 ml round-bottom flask and stirred at room temperature for 0.5 h. The round-bottom flask was sealed and moved into an ultrasonic reactor for ultrasonic oscillation reaction at 85 ° C (80 kHz, 100 w) for 1 h. After the reaction was completed, it was naturally cooled to room temperature and allowed to stand for 45 min. The above solution was taken into a centrifuge tube and centrifuged at a speed of 1500 r / min for 0.5 h. The obtained solution was then passed through a dialysis membrane with a molecular cutoff of 3500 Da to obtain a yellow transparent carbon quantum dot solution. Finally, the carbon quantum dot solution obtained after dialysis was freeze-dried to obtain a light yellow solid, which is carbon quantum dots, recorded as CDs.

[0025] The serum sample used in the Examples of the present invention is an artificial serum sample to which PD-L1-specific antibodies are added, wherein the concentration of the PD-L1-specific antibodies is 100 ng / ml.

[0026] Example 1 The preparation method of quantum dot-colloidal gold composite probe has the following specific steps: Step I: Under nitrogen protection, 20 mg of sulfur quantum dots were dispersed in 20 ml of an ethanol solution containing 24 mg of 3-aminopropyltriethoxysilane. The resulting mixture was reacted at 60°C for 2 h and then cooled to room temperature to obtain a surface amino-modified sulfur quantum dot solution. Step II: Add 10 mL of 0.01% chloroauric acid solution to the surface amino-modified sulfur quantum dot solution prepared in step I and mix evenly. Then quickly add 10 mL of 1% sodium citrate solution, and heat the resulting mixed solution in a gradient heating process. The specific heating program is as follows: The first stage: maintain at 25℃ for 10min to form crystal nuclei; The second stage: the temperature was raised to 100°C at 0.5°C / min to complete the shell growth; The third stage: sudden cooling to 4°C to terminate the reaction; After heating, a sulfur quantum dot-colloidal gold composite probe precursor solution is obtained; Step III: Add 30 mg of the bifunctional coupling agent Sulfo-SMCC to the sulfur quantum dot-colloidal gold precursor solution prepared in step II, react the resulting mixture at 4°C for 1 hour, centrifuge the resulting reaction mixture at 6500 r / min, and collect the resulting solid particles to obtain sulfur quantum dot-colloidal gold composite probe precursor particles with surface modification of the coupling agent; Step IV: Add the sulfur quantum dot-colloidal gold composite probe precursor particles of the surface-modified coupling agent prepared in Step III to 10 mL of phosphate buffer and disperse evenly. Then, add 0.1 mg of PD-1 targeting antibody molecules and incubate for 1 hour at pH 7.4. Then, the resulting mixture is purified by ultrafiltration and stored at 4°C. The resulting quantum dot-colloidal gold composite probe is recorded as PD-1-SDs-CG.

[0027] Example 2 The preparation method of quantum dot-colloidal gold composite probe refers to Example 1, except that the sulfur quantum dots in step I are replaced with carbon quantum dots, and the obtained quantum dot-colloidal gold composite probe is recorded as PD-1-CDs-CG.

[0028] Example 3 The preparation method of quantum dot-colloidal gold composite probe refers to Example 1, except that the sulfur quantum dots in step I are replaced with CdSe / ZnS quantum dots, and the obtained quantum dot-colloidal gold composite probe is recorded as PD-1-CdSe / ZnS-CG.

[0029] Example 4 The preparation method of quantum dot-colloidal gold composite probe refers to Example 1, except that the sulfur quantum dots in step I are replaced with carbon quantum dots, and the targeting antibody molecule PD-1 in step IV is replaced with EGFR. The obtained quantum dot-colloidal gold composite probe is recorded as EGFR-CDs-CG.

[0030] Example 5 The preparation method of quantum dot-colloidal gold composite probe refers to Example 1, except that the sulfur quantum dots in step I are replaced with carbon quantum dots, and the targeting antibody molecule PD-1 in step IV is replaced with HER2. The obtained quantum dot-colloidal gold composite probe is recorded as HER2-CDs-CG.

[0031] Example 6 The preparation of quantum dot-colloidal gold composite probe test paper was carried out according to the reference (Wu Yaoyao, Sun Chongsi, Hu Qiushi, et al. Quantitative detection of multiple drugs in saliva based on quantum dot immunochromatography technology [J]. Journal of Qingdao University of Science and Technology (Natural Science Edition), 2025, 46 (2): 23-28.), and the specific steps are as follows: The test strips are based on a nitrocellulose membrane (NC membrane) and feature a laminated design. The primary components include a PVC base, a sample pad, a conjugate pad, an absorbent pad, and the NC membrane. The NC membrane, the key carrier for the antigen-antibody reaction, is located on the PVC base and pre-sprayed with a test line (T line) and a control line (C line). The T line immobilizes PD-L1-specific antibodies, while the C line immobilizes the targeting antibody molecule PD-1. The conjugate pad is used to load the quantum dot-colloidal gold composite probe PD-1-SDs-CG prepared in Example 1. The sample pad ensures uniform sample distribution and prevents nonspecific interference. It is also soaked in Tris-HCl buffer (pH 7.4) to maintain the activity of the targeting antibody molecule PD-1. The absorbent pad is placed at the end to quickly absorb excess reaction solution and prevent backflow interference. All components are assembled sequentially on a laminator and cut into 5 mm wide strips. The strips are sealed and stored in a dry room. The prepared test strip will be used for subsequent quantum dot fluorescence signal detection, and the fluorescence intensity of its T line and C line can be used to determine the content of PD-L1-specific antibodies and the sensitivity and specificity of the detection.

[0032] The steps for sample testing are as follows: A 200 μL artificial serum sample was diluted in a 1:10 Tris-HCl buffer (25 mM Tris-HCl, 5% (w / v) trehalose, 1% (w / v) BSA, pH 7.4) and then dripped onto the sample pad of the test strip. Capillary action allowed the serum sample to flow along the nitrocellulose membrane, gradually passing through the conjugate pad and detection zone. The quantum dot-colloidal gold composite probe, PD-1-SDs-CG, pre-loaded on the conjugate pad, bound to the target PD-L1-specific antibody in the sample. The complex migrated to the T- and C-line regions. After reaction, the quantum dot fluorescence signal was observed and quantified using a fluorescence microscope and a microplate reader. The excitation wavelength was 520 nm. Results showed that the fluorescence signal intensity of the quantum dot-colloidal gold composite probe, PD-1-SDs-CG, for PD-L1 detection was 1680 ± 130.

[0033] Example 7 The preparation of quantum dot-colloidal gold composite probe test paper was carried out in accordance with Example 6, except that the quantum dot-colloidal gold composite probe PD-1-SDs-CG was replaced with the quantum dot-colloidal gold composite probe PD-1-CDs-CG. The sample detection steps were also carried out in accordance with Example 6. The results showed that when the quantum dot-colloidal gold composite probe PD-1-CDs-CG was used for PD-L1 detection, the fluorescence signal intensity was 1920 ± 150.

[0034] Example 8 The preparation of quantum dot-colloidal gold composite probe test paper was carried out in accordance with Example 6, except that the quantum dot-colloidal gold composite probe PD-1-SDs-CG was replaced with the quantum dot-colloidal gold composite probe PD-1-CdSe / ZnS-CG. The sample detection steps were also carried out in accordance with Example 6. The results showed that when the quantum dot-colloidal gold composite probe PD-1-CdSe / ZnS-CG was used for PD-L1 detection, the fluorescence signal intensity was 1750 ± 140.

[0035] Example 9 The present invention also provides a tumor diagnosis kit comprising: Quantum dot-colloidal gold composite probe 0.1-1 mg / mL; Multimodal test strips, including: - Fluorescence readout zone (coated with quencher-modified capture probes) - Colorimetric control card (including standard colorimetric gradient).

[0036] Also contains a stabilization buffer consisting of: Tris-HCl 25 mM, trehalose 5% (w / v), BSA 1% (w / v), pH 7.4±0.2.

[0037] Wherein, the quantum dot-colloidal gold composite probe is one of PD-1-SDs-CG, PD-1-CDs-CG or PD-1-CdSe / ZnS-CG.

Claims

1. A quantum dot-colloidal gold composite probe, characterized in that: The composite probe consists of a quantum dot core layer, a colloidal gold shell layer and a biocoupling layer; wherein the quantum dot core layer is composed of one of sulfur quantum dots, carbon quantum dots, CdSe / ZnS quantum dots, InP / ZnS quantum dots or perovskite quantum dots, the colloidal gold shell layer is wrapped around the surface of the quantum dots, and the biocoupling layer is connected to the targeting molecule through a bifunctional coupling agent.

2. The quantum dot-colloidal gold composite probe according to claim 1, characterized in that: The particle size of the quantum dots is in the range of 2-10 nm; the thickness of the colloidal gold shell is 5-20 nm, and the surface roughness is ≤3 nm; and the coupling agent is an organic compound containing thiol and carboxyl functional groups.

3. The quantum dot-colloidal gold composite probe according to claim 1, characterized in that: A 1-3 nm silicon dioxide isolation layer is provided between the quantum dot core layer and the colloidal gold shell layer, and the quantum dot fluorescence quantum yield is ≥80%, and the colloidal gold extinction coefficient is ≥3×10 8 M -1 cm -1 .

4. The quantum dot-colloidal gold composite probe according to claim 1, characterized in that: The targeting molecule is selected from at least one of anti-EGFR monoclonal antibodies, PD-1, HER2 aptamers or EpCAM nucleic acid aptamers, and the coupling density is 10-50 molecules / particle.

5. A method for preparing a quantum dot-colloidal gold composite probe, characterized in that: The specific steps are as follows: Step I: Under nitrogen protection, the quantum dots were dispersed in an ethanol solution containing 3-aminopropyltriethoxysilane and reacted at 60°C for 2 h to form an amino surface; Step II: Mixing chloroauric acid solution and quantum dots in a molar ratio of 1:5-1:20, adding a reducing agent for in situ reduction, and controlling the pH to 5.0-6.5; Step III: Add the bifunctional coupling agent Sulfo-SMCC and activate the carboxyl group; Step IV: Add the targeting molecule and couple it in pH 7.4 phosphate buffer. Purify it by ultrafiltration and store it at 4°C.

6. The method for preparing the quantum dot-colloidal gold composite probe according to claim 5, characterized in that: In step II, a gradient temperature increase method is used, and the specific operation is as follows: The first stage: maintain at 25℃ for 10min to form crystal nuclei; The second stage: the temperature was raised to 100°C at 0.5°C / min to complete the shell growth; The third stage: sudden cooling to 4°C to terminate the reaction.

7. The method for preparing the quantum dot-colloidal gold composite probe according to claim 5, characterized in that: The temperature for activating the carboxyl groups in step III is 4°C.

8. A multimodal detection method based on the composite probe according to any one of claims 1 to 4, characterized in that: include: The sample to be tested was incubated with the composite probe at a volume ratio of 1:50-1:200 for 30 minutes; Synchronous: a) Fluorescence detection: excitation wavelength 400-550nm, detection of emission spectrum; b) Surface-enhanced Raman spectroscopy: 785 nm laser excitation, collecting 1500-1700 cm -1 characteristic peaks; c) Colorimetric detection: Measure the change in absorbance at 520 nm.

9. A tumor diagnostic kit, characterized in that: Include: 0.1-1 mg / mL of the composite probe according to any one of claims 1-4; Multimodal test strips, including: - Fluorescence readout zone (coated with quencher-modified capture probes) - Colorimetric control card (including standard colorimetric gradient).

10. The kit according to claim 9, characterized in that Also contains a stabilization buffer consisting of: Tris-HCl 25 mM, trehalose 5% (w / v), BSA 1% (w / v), pH 7.4±0.2.