A photoluminescence-resonance light scattering dual-mode biological detection method
Through the photoluminescence-resonant light scattering dual-mode detection method, combined with materials such as quantum dots and gold nanoparticles, the problem of fluorescence detection is solved, and high sensitivity and low cost biomarker detection is achieved.
Patent Information
- Application Number
- CN202211576966.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-09
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2042-12-09
AI Technical Summary
The existing fluorescence detection methods are susceptible to interference and surrounding environment when detecting biomarkers, and the multimode detection methods require a variety of instruments and complex material preparation processes, resulting in high cost and poor stability.
The photoluminescence-resonance light scattering dual-mode detection method is used to combine quantum dots, gold nanoparticles, gold nanostars or gold nanorods with aptamer molecules, and photoluminescence-resonance light scattering detection is performed after co-incubation and mixing, which simplifies the detection process and improves stability and sensitivity.
It realizes that without increasing equipment costs, the sensitivity and reliability of biomarker detection are improved, the detection process is simplified, and the detection cost is reduced.
Smart Images
Figure 221208164357
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomarker analysis, and in particular to a photoluminescence-resonance light scattering dual-mode biological detection method. Background Art
[0002] Liquid biopsies for cancer-related biomarkers hold promise for advancing early cancer screening. However, the concentrations of biomarkers such as proteins and miRNA molecules in body fluids are typically low, making the development of highly sensitive trace detection technologies a critical yet challenging task. Fluorescence detection has become a leading method in laboratory and clinical testing due to its high sensitivity and ease of use. However, its results are susceptible to interferences and environmental factors. Improving the reliability of this method remains a critical issue that needs to be addressed.
[0003] Dual-mode or even multimodal detection strategies that combine fluorescence detection with surface-enhanced Raman scattering, colorimetry, or electrochemistry have made significant progress in improving the reliability of test results. However, the bioprobes used in these methods are often prepared by combining multiple functional materials. The cumbersome composite material preparation process may affect the uniformity and stability of the probe signal. In addition, most of these methods require detection on different test instruments, and the relatively high detection cost efficiency limits the clinical promotion of these methods. Developing a cost-effective and stable dual-mode detection method is a significant task.
[0004] Resonance light scattering is a common method for detecting biomolecules, and its spectral characteristics are related to the size, structure, and concentration of the particles. Numerous studies have explored the use of the resonance light scattering properties of nanoparticles to detect biomolecules, often demonstrating good sensitivity and accuracy. Resonance light scattering spectra can be acquired using a fluorescence spectrometer. Therefore, combining resonance light scattering with fluorescence detection eliminates the need for additional detection equipment and sample processing, potentially improving the cost-efficiency and stability of detection while maintaining sensitivity and ease of use. Based on these ideas, the design of a dual-mode photoluminescence-resonance light scattering bioassay method has significant research significance and practical application value. Summary of the Invention
[0005] The purpose of the present invention is to provide a photoluminescence-resonance light scattering dual-mode biological detection method.
[0006] A photoluminescence-resonance light scattering dual-mode detection kit comprising: quantum dots, gold nanoparticles, gold nanostars or gold nanorods, an amino-modified aptamer molecule solution, and a thiol-modified aptamer molecule solution;
[0007] The aptamer is a human immunoglobulin aptamer, a carcinoembryonic antigen aptamer, an alpha-fetoprotein aptamer and / or a microRNA molecule aptamer;
[0008] The microRNA molecule is miRNA-155, miRNA-141 or miRNA-21;
[0009] The amino-modified miRNA-141 molecular aptamer has a molecular sequence of: 5'-(NH2-C6)-TTTTTTGCCATCTTTACCA-3';
[0010] The molecular sequence of the thiol-modified miRNA-141 molecular aptamer is: 5'-GACAGTGTTATTTTTT-(SH-C3)-3';
[0011] The quantum dots are quantum dot dispersions; the gold nanoparticles, gold nanostars or gold nanorods are gold nanoparticles, gold nanostars or gold nanorod dispersions;
[0012] The quantum dots are ZnSe, CdTe or CdSe;
[0013] Photoluminescence-resonance light scattering dual-mode detection system, which includes:
[0014] 1) incubating the quantum dots with the amino-modified aptamer molecule solution;
[0015] 2) co-incubating the gold nanoparticles, gold nanostars or gold nanorods with a solution of thiol-modified aptamer molecules;
[0016] 3) After incubation, the mixture was mixed and detected using photoluminescence-resonance light scattering dual mode;
[0017] The co-incubation conditions are as follows: incubation at room temperature in the dark for 0.5 to 2.5 hours;
[0018] The photoluminescence signal is 480-620 nm; the excitation wavelength of the resonance light scattering is 365 nm;
[0019] Photoluminescence-resonance light scattering dual-mode biological detection method, which includes:
[0020] 1) Disperse the quantum dots in a buffer solution, add a coupling agent, and magnetically stir at room temperature in the dark for 0.4-1 hour. After magnetic stirring, centrifuge. Collect the precipitate, wash the product with water, and disperse it in a buffer solution. Add a solution of amino-modified aptamer molecules corresponding to the analyte and incubate at room temperature in the dark for 0.5-2.5 hours.
[0021] 2) Disperse the gold nanoparticles, gold nanostars, or gold nanorods in a buffer solution, add the solution of the thiol-modified aptamer corresponding to the analyte, and incubate in the dark at room temperature for 0.5–2.5 h.
[0022] 3) mixing the solutions obtained in step 1) and step 2), and performing photoluminescence-resonance light scattering dual-mode detection;
[0023] The analyte is a human immunoglobulin G HIgG, carcinoembryonic antigen CEA, alpha-fetoprotein AFP, miRNA-155, miRNA-141 or miRNA-21 type microRNA molecule.
[0024] The quantum dot dispersion is prepared by the following method:
[0025] 1) Dissolve zinc acetate dihydrate or cadmium chloride pentahydrate powder in ultrapure water, add surface modifier, stir magnetically at room temperature, and add 1 mol / L sodium hydroxide solution dropwise to adjust the pH to 10.0-12.8;
[0026] 2) Dispersing selenium powder or tellurium powder in ultrapure water, adding sodium borohydride powder, and reacting in a nitrogen atmosphere to obtain a sodium selenide or sodium telluride precursor solution;
[0027] 3) adding the sodium selenide or sodium telluride precursor solution obtained in step 2) to the zinc or cadmium solution prepared in step 1), and reacting the mixed solution at 95-105° C. in a nitrogen atmosphere for 2-6 hours; after the reaction is completed, cooling to room temperature, adding isopropanol, centrifuging, washing with water, collecting the precipitate to obtain quantum dots, and then adding the quantum dots to a buffer solution;
[0028] The molar mass ratio of the zinc acetate dihydrate or cadmium chloride pentahydrate, surfactant, selenium powder or tellurium powder and sodium borohydride is 1:0.3:0.5:2; the surfactant is mercaptopropionic acid, thioglycolic acid or L-cysteine;
[0029] The gold nanoparticle dispersion is prepared by the following method: heating a chloroauric acid solution to boiling, adding a sodium citrate dihydrate aqueous solution, keeping the solution boiling for 15 to 25 minutes, and then cooling it to room temperature to obtain a gold nanoparticle dispersion;
[0030] The gold nanostar dispersion is prepared by taking 2 mL of a 0.25 mM chloroauric acid solution, adjusting the pH to 11.5 with a sodium hydroxide solution, adding 16 μL of a 40 mM hydroxylamine hydrochloride solution and 160 μL of the gold nanoparticle dispersion, and magnetically stirring at room temperature for 1 minute until the solution turns blue; collecting the precipitate after centrifugation, and dispersing the precipitate in a phosphate buffer solution;
[0031] The gold nanorod dispersion is prepared by the following method: taking 0.7294 g of hexadecyltrimethylammonium bromide powder and placing it in 20 mL of ultrapure water; taking 0.0176 g of ascorbic acid powder and placing it in 1 mL of ultrapure water; taking 0.0114 g of sodium borohydride powder and dissolving it in 3 mL of ultrapure water to obtain a solution with a molar concentration of 0.1 M, and then diluting it to 0.01 M; adding 100 μL of chloroauric acid aqueous solution to 2 mL of hexadecyltrimethylammonium bromide solution at room temperature, magnetically stirring for 1 minute, and then adding 10 μL of silver nitrate aqueous solution, and magnetically stirring for 1 minute; adding 100 μL of hydrochloric acid and 16 μL of ascorbic acid solution to the above solution; adding 4.8 μL of gold seed solution to the above solution, magnetically stirring to mix, and then standing at room temperature in the dark overnight; the obtained sample is centrifuged to collect the precipitate, washed with ultrapure water, and finally dispersed in 2 mL of ultrapure water;
[0032] The preparation process of the gold seed comprises the following steps: adding 12.5 μL of chloroauric acid solution to 500 μL of hexadecyltrimethylammonium bromide solution, mixing by magnetic stirring, adding 30 μL of 0.01 M sodium borohydride aqueous solution, and standing in the dark for 30 minutes.
[0033] The present invention provides a photoluminescence-resonance light scattering dual-mode detection kit, which includes: quantum dots, gold nanoparticles, gold nanostars or gold nanorods, amino-modified aptamer molecule solution, and thiol-modified aptamer molecule solution; a photoluminescence-resonance light scattering dual-mode detection system, which includes: 1) co-incubating the quantum dots with the amino-modified aptamer molecule solution; 2) co-incubating the gold nanoparticles, gold nanostars or gold nanorods with the thiol-modified aptamer molecule solution; 3) mixing after incubation, and adopting photoluminescence-resonance light scattering dual-mode detection; a photoluminescence-resonance light scattering dual-mode biological detection method, which includes: 1) dispersing the quantum dots in a buffer solution; The method comprises the following steps: 1) adding a coupling agent to a solution, and magnetically stirring the solution in the dark at room temperature for 0.4 to 1 hour; after the magnetic stirring is completed, centrifuging the solution; collecting the precipitate, washing the product with water, and dispersing the precipitate in a buffer solution; adding an amino-modified aptamer molecule solution corresponding to the analyte, and incubating the solution in the dark at room temperature for 0.5 to 2.5 hours; 2) taking the gold nanoparticles, gold nanostars or gold nanorods, dispersing them in a buffer solution, adding the thiol-modified aptamer molecule solution corresponding to the analyte, and incubating the solution in the dark at room temperature for 0.5 to 2.5 hours; 3) mixing the solutions obtained in step 1) and step 2), and performing photoluminescence-resonance light scattering dual-mode detection; the results show that the detection system has good sensitivity and reliability for the detection of biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 Spectrum of miRNA-141 detected by photoluminescence and resonance light scattering. DETAILED DESCRIPTION
[0035] Example 1 Preparation of CdTe quantum dots and gold nanoparticles dual-mode detection system and its detection of miRNA-141 molecules
[0036] 1. Prepare CdTe quantum dots using the reflux method, the steps are:
[0037] 1) Add 10 mmol of sodium borohydride powder and 2.5 mmol of tellurium powder to a three-necked flask, add 20 mL of ultrapure water, and react with magnetic stirring for 2 h to obtain a sodium telluride precursor solution.
[0038] 2) Dissolve 1 mmol of cadmium chloride hemipentahydrate powder in 100 mL of ultrapure water, add 156 μL of 3-mercaptopropionic acid and 0.118 g of L-cysteine; stir magnetically until the powder is completely dissolved, then add 1 mol / L sodium hydroxide solution dropwise to adjust the pH to 11.0 to obtain a cadmium precursor solution;
[0039] 3) Take 2 mL of sodium telluride precursor solution and quickly inject it into 100 mL of cadmium precursor solution. Condensation reflux is carried out at 100°C for 4 hours. After the reaction is completed, it is naturally cooled to room temperature. 200 mL of isopropanol is added to the resulting quantum dot solution. After centrifugation, the quantum dots are dispersed in water to obtain a CdTe quantum dot dispersion.
[0040] 4) Take 1 mL of CdTe quantum dot dispersion, centrifuge (8000 rpm, 10 min), collect the precipitate, and obtain CdTe quantum dots.
[0041] 2. Surface DNA functionalization of CdTe quantum dots: 1 mL of the CdTe quantum dot dispersion obtained in step 1 was centrifuged (8000 rpm, 10 min), the precipitate was collected, and then dispersed in a morpholineethanesulfonic acid buffer solution with a pH of 5.5. 1 mg of EDC and 1 mg of NHS powder were then added, and the reaction was carried out in the dark for 10 min. 1 mL of a 10 μM amino-modified single-stranded DNA (NH2-ssDNA) solution was added to the above solution, the reaction was carried out in the dark for 30 min, and then the solution was centrifuged (5000 rpm, 10 min), washed three times, and finally the precipitate was dispersed in 1 mL of phosphate buffer.
[0042] 3. The sodium citrate reduction method was used to prepare a gold nanoparticle dispersion. The steps were as follows: 0.04 mL of chloroauric acid solution was dissolved in 20 mL of ultrapure water and heated to boiling under magnetic stirring; 0.08 mL of 1% sodium citrate aqueous solution was injected into the boiling solution and reacted for 20 minutes to obtain a wine-red gold nanoparticle dispersion.
[0043] 4. Functionalization of the gold nanoparticle surface with DNA molecules: Take 1 mL of the gold nanoparticle dispersion obtained in step 2, add 1 mL of a 10 μM thiol-modified single-stranded DNA (SH-ssDNA) solution, oscillate in the dark for 1 hour, and then centrifuge (8000 rpm, 16 min) and wash three times. Finally, disperse the precipitate in 1 mL of phosphate buffer solution.
[0044] 5. Construction of a photoluminescence-resonance light scattering dual-mode detection system and detection of miRNA-141, the steps are as follows: mix the quantum dot solution obtained in step 2 and the gold nanoparticle dispersion obtained in step 4, and use a fluorescence spectrophotometer to measure the initial photoluminescence signal intensity and resonance light scattering signal intensity of the system; add 1 mL of the analyte miRNA-141 solution at different concentrations (0, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM and 10 nM, respectively) to the above mixture, incubate at room temperature in the dark for 1 h, and then measure the photoluminescence and resonance light scattering signal intensities.
[0045] The sequence of the miRNA-141 molecule used in Example 1 is: 5'-UAACACUGUCUGGUAAAGAUGG-3';
[0046] The amino-modified single-stranded DNA (NH2-ssDNA) molecule sequence used in Example 1 is: 5'-(NH2-C6)-TTTTTTGCCATCTTTACCA-3';
[0047] The sequence of the sulfhydryl-modified single-stranded DNA (SH-ssDNA) molecule used in Example 1 is: 5'-GACAGTGTTATTTTTT-(SH-C3)-3';
[0048] The photoluminescence signal peak of the CdTe quantum dots is located at 540 nm, and the wavelength of the excitation light used is 365 nm.
[0049] like Figure 1 As shown, the dual-mode detection system of CdTe quantum dots and gold nanoparticles has both photoluminescence and resonance light scattering properties. The results show that the photoluminescence signal intensity of the dual-mode detection system of CdTe quantum dots and gold nanoparticles gradually decreases with the increase of miRNA-141 molecular concentration, while the resonance light scattering signal intensity gradually increases.
[0050] Example 2 Preparation of CdSe quantum dots and gold nanostar dual-mode detection system and its detection of miRNA-141 molecules
[0051] 1. Prepare CdSe quantum dots using the reflux method. The steps are:
[0052] 1) Place 10 mmol of sodium borohydride powder and 2.5 mmol of selenium powder in a three-necked flask, add 20 mL of ultrapure water, and react under magnetic stirring for 1 hour to obtain a sodium selenide precursor solution.
[0053] 2) Dissolve 0.5 mmol of cadmium chloride hemipentahydrate powder in 50 mL of ultrapure water and add 1.0 mmol of 3-mercaptopropionic acid. Stir magnetically until the powder is completely dissolved, then add 1 mol / L sodium hydroxide solution to adjust the solution pH to 10.0 to obtain a cadmium precursor solution.
[0054] 3) Take 1.6 mL of sodium hydride selenide precursor solution and inject it into 50 mL of cadmium precursor solution. Condensation reflux is carried out at 100°C for 4 hours. After the reaction is completed, it is naturally cooled to room temperature. Isopropanol is added to the resulting quantum dot solution to precipitate the quantum dots. After centrifugation and washing, the quantum dots are dispersed in water for later use to obtain a CdSe quantum dot dispersion.
[0055] 2. Surface DNA molecule functionalization of CdSe quantum dots: 1 mL of the CdSe quantum dot dispersion obtained in step 1 was centrifuged, the precipitate was collected, and then dispersed in a morpholineethanesulfonic acid buffer solution with a pH of 5.5. 1 mg of EDC and 1 mg of NHS powder were then added, and the mixture was reacted in the dark for 10 min. 1 mL of a 10 μM amino-modified single-stranded DNA (NH2-ssDNA) solution was added to the solution obtained in the above step, the mixture was reacted in the dark for 30 min, centrifuged, and washed three times. Finally, the precipitate was dispersed in 1 mL of phosphate buffer.
[0056] 3. Gold nanostars were prepared by the in situ growth method. The steps were as follows: 0.04 mL of 0.25 mM chloroauric acid solution was dissolved in 20 mL of ultrapure water and heated to boiling under magnetic stirring; 0.08 mL of 1% sodium citrate aqueous solution was injected into the boiling solution and reacted for 20 min to obtain a wine-red gold nanoparticle dispersion; 2 mL of 0.25 mM chloroauric acid solution was taken, the pH was adjusted to 11.5 with sodium hydroxide solution, 16 μL of 40 mM hydroxylamine hydrochloride solution and 160 μL of the above gold nanoparticle dispersion were added, and the solution turned blue after magnetic stirring at room temperature for 1 min; after centrifugation, the precipitate was dispersed in phosphate buffer solution.
[0057] 4. Functionalization of the gold nanostar surface with DNA molecules: Take 1 mL of the gold nanostar dispersion obtained in step 2, add 1 mL of 10 μM thiol-modified single-stranded DNA (SH-ssDNA) solution, shake in the dark for 1 hour, and then centrifuge and wash three times. Finally, disperse the precipitate in 1 mL of phosphate buffer solution.
[0058] 5. Construction of a photoluminescence-resonance light scattering dual-mode detection system and detection of miRNA-141, the steps are as follows: mix the CdSe quantum dot dispersion obtained in step 2 and the gold nanostar dispersion obtained in step 4, and use a fluorescence spectrophotometer to measure the initial photoluminescence signal intensity and resonance light scattering signal intensity of the system; add 1 mL of the analyte miRNA-141 solution at different concentrations (0, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM and 50 nM) to the above mixture, incubate at room temperature in the dark for 1 h, and then measure the photoluminescence and resonance light scattering signal intensities.
[0059] The sequence of the miRNA-141 molecule used in Example 2 is: 5'-UAACACUGUCUGGUAAAGAUGG-3';
[0060] The amino-modified single-stranded DNA (NH2-ssDNA) molecule sequence used in Example 2 is: 5'-(NH2-C6)-TTTTTTGCCATCTTTACCA-3';
[0061] The sequence of the sulfhydryl-modified single-stranded DNA (SH-ssDNA) molecule used in Example 2 is: 5'-GACAGTGTTATTTTTT-(SH-C3)-3';
[0062] The photoluminescence signal spectrum of the CdSe quantum dots is located at 620 nm, and the wavelength of the excitation light used is 365 nm.
[0063] The results showed that the photoluminescence signal intensity of the obtained CdSe quantum dots and gold nanostars dual-mode detection system gradually decreased with the increase of miRNA-141 molecular concentration; the resonance light scattering signal intensity gradually increased.
[0064] Example 3 Preparation of ZnSe quantum dots and gold nanomaterials dual-mode detection system and its detection of miRNA-21 molecules
[0065] 1. Preparation of ZnSe quantum dots: Dissolve 1 mmol of zinc acetate dihydrate powder in ultrapure water. Magnetic stirring is used until the powder is completely dissolved. Add 0.2 mmol of mercaptopropionic acid and adjust the pH of the solution to 12.8 with 1 mol / L sodium hydroxide solution. Transfer the zinc precursor solution to a 100 mL three-necked flask. Disperse selenium powder in 10 mL of ultrapure water and transfer it to a 50 mL three-necked flask. Add 2 mmol of sodium borohydride powder to the three-necked flask and react for 30 minutes to obtain a sodium selenium hydride precursor solution. Add the resulting sodium selenium hydride precursor solution to the zinc precursor solution. Raise the temperature to 100°C and allow the reaction to continue for 6 hours. After the reaction, collect the particles by centrifugation, wash with water, and finally disperse them in water.
[0066] 2. Biofunctionalization of the ZnSe quantum dots surface, the steps are as follows: replacing the CdTe quantum dots in Example 1 with the obtained ZnSe nanoparticles, and coupling amino-modified single-stranded DNA molecules complementary to the miRNA-21 molecular sequence on the surface of the ZnSe quantum dots using the EDC / NHS condensation method.
[0067] 3. Prepare gold nanoparticles. The steps are the same as the gold nanoparticle preparation method in Example 1.
[0068] 4. Biofunctionalization of the gold nanoparticle surface: the steps are the same as those for the preparation of gold nanoparticles in Example 1.
[0069] 5. Photoluminescence-resonance light scattering dual-mode detection process, the steps are as follows: the solutions obtained in steps 2 and 4 are mixed to form an initial dual-mode detection system, the photoluminescence spectrum of the mixed solution is collected under the excitation of 365 nm laser, and the photoluminescence spectrum is measured at λ ex =λ em The resonance light scattering spectrum of the detection system was scanned under the following conditions; miRNA-21 solutions of different concentrations were added to the mixed system, and after incubation in the dark at room temperature for 2 h, the photoluminescence spectrum and resonance light scattering spectrum were collected under the same instrument parameters to complete the dual-mode detection process.
[0070] The photoluminescence peak of the ZnSe quantum dots is located at 480 nm, and the wavelength of the excitation light used is 365 nm.
[0071] Results: The photoluminescence signal intensity of the obtained ZnSe quantum dots and gold nanoparticles dual-mode detection system gradually decreased with the increase of miRNA-21 molecular concentration; the resonance light scattering signal intensity gradually increased.
[0072] Example 4 Preparation of CdSe quantum dots and gold nanorods dual-mode detection system and its detection of miRNA-141 molecules
[0073] The gold nanoparticles in Example 2 were replaced with gold nanorods, and the remaining steps were the same.
[0074] The preparation process of the gold nanorods comprises the following steps: dissolving 0.7294 g of hexadecyltrimethylammonium bromide powder in 20 mL of ultrapure water; dissolving 0.0176 g of ascorbic acid powder in 1 mL of ultrapure water; dissolving 0.0114 g of sodium borohydride powder in 3 mL of ultrapure water to obtain a solution with a molar concentration of 0.1 M, which is then diluted to 0.01 M; adding 100 μL of chloroauric acid aqueous solution to 2 mL of hexadecyltrimethylammonium bromide solution at room temperature, magnetically stirring for 1 minute, and then adding 10 μL of silver nitrate aqueous solution, magnetically stirring for 1 minute; adding 100 μL of hydrochloric acid and 16 μL of ascorbic acid solution to the above solution; adding 4.8 μL of gold seed solution to the above solution, magnetically stirring to mix, and then standing at room temperature in the dark overnight; the obtained sample is centrifuged to collect the precipitate, washed with ultrapure water, and finally dispersed in 2 mL of ultrapure water;
[0075] The preparation process of the gold seed comprises the following steps: adding 12.5 μL of chloroauric acid solution to 500 μL of hexadecyltrimethylammonium bromide solution, mixing by magnetic stirring, adding 30 μL of 0.01 M sodium borohydride aqueous solution, and standing in the dark for 30 minutes.
[0076] The photoluminescence signal spectrum of the CdSe dual-mode nanoprobe is located at 620 nm, and the excitation light wavelength used is 365 nm.
[0077] Results: The photoluminescence signal intensity of the obtained CdSe quantum dots and gold nanorods dual-mode detection system gradually decreased with the increase of miRNA-141 molecular concentration; while the resonance light scattering signal intensity gradually increased.
[0078] Example 5 Preparation of a Quantum Dot and Gold Nanoparticle Dual-Mode Detection System and Its Detection of Marker Molecules
[0079] The CdTe quantum dots prepared in Example 1 were replaced with commercially available CdTe quantum dots, and the remaining steps were the same.
[0080] Example 6 Preparation of Quantum Dot and Gold Nanostar Dual-Mode Detection System and Detection of Marker Molecules
[0081] The CdSe quantum dots prepared in Example 2 were replaced with commercially available CdSe quantum dots, and the remaining steps were the same.
[0082] Example 7 Preparation of Quantum Dot and Gold Nanoparticle Dual-Mode Detection System and Detection of Marker Molecules
[0083] The ZnSe quantum dots prepared in Example 3 were replaced with commercially available ZnSe quantum dots, and the remaining steps were the same.
[0084] Example 8 Preparation of Quantum Dot and Gold Nanoparticle Dual-Mode Detection System and Detection of Marker Molecules
[0085] The gold nanoparticles prepared in Example 1 were replaced with commercially available gold nanoparticles, and the remaining steps were the same.
[0086] Example 9 Preparation of Quantum Dot and Gold Nanostar Dual-Mode Detection System and Detection of Marker Molecules
[0087] The gold nanostars prepared in Example 2 were replaced with commercially available gold nanostars, and the remaining steps were the same.
[0088] Example 10 Preparation of a Quantum Dot and Gold Nanoparticle Dual-Mode Detection System and Its Detection of Marker Molecules
[0089] The gold nanoparticles prepared in Example 3 were replaced with commercially available gold nanoparticles, and the remaining steps were the same.
Claims
1. A photoluminescence-resonance light scattering dual-mode detection kit comprising: Quantum dots, gold nanoparticles, gold nanostars or gold nanorods, amino-modified aptamer molecule solutions, and thiol-modified aptamer molecule solutions; The aptamer is a human immunoglobulin aptamer, a carcinoembryonic antigen aptamer, an alpha-fetoprotein aptamer or a microRNA molecule aptamer; The microRNA molecule is miRNA-155, miRNA-141 or miRNA-21; The quantum dots are quantum dot dispersions; the gold nanoparticles, gold nanostars or gold nanorods are gold nanoparticles, gold nanostars or gold nanorod dispersions; The quantum dots are ZnSe, CdTe or CdSe.
2. A detection method based on a photoluminescence-resonance light scattering dual-mode detection kit, characterized in that: The photoluminescence-resonance light scattering dual-mode detection kit according to claim 1 comprises: 1) co-incubating the quantum dots with the amino-modified aptamer molecules; 2) co-incubating the gold nanoparticles, gold nanostars or gold nanorods with the thiol-modified aptamer molecules; 3) After incubation, the mixture is mixed and human immunoglobulin G (HIgG), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), miRNA-155, miRNA-141, or miRNA-21 microRNA molecules are detected using photoluminescence-resonance light scattering dual mode.
3. The detection method based on the photoluminescence-resonance light scattering dual-mode detection kit according to claim 2, characterized in that: The co-incubation conditions are as follows: incubation at room temperature in the dark for 0.5 to 2.5 hours.
4. The detection method based on the photoluminescence-resonance light scattering dual-mode detection kit according to claim 3, characterized in that: The photoluminescence signal is 480-620 nm; the excitation light wavelength of the resonance light scattering is 365 nm.
5. A detection method based on a photoluminescence-resonance light scattering dual-mode detection kit, using the photoluminescence-resonance light scattering dual-mode detection kit according to claim 1, comprising: 1) Disperse the quantum dots in a buffer solution, add a coupling agent, and stir magnetically in the dark at room temperature for 0.4-1 h; After magnetic stirring, centrifuge; Collect the precipitate, wash the product with water, and disperse it in a buffer solution; add the amino-modified aptamer molecule solution corresponding to the analyte and incubate at room temperature in the dark for 0.5-2.5 h; 2) Disperse the gold nanoparticles, gold nanostars, or gold nanorods in a buffer solution, add the solution of the thiol-modified aptamer corresponding to the analyte, and incubate in the dark at room temperature for 0.5–2.5 h. 3) The solutions obtained in step 1) and step 2) are mixed, and human immunoglobulin G HIgG, carcinoembryonic antigen CEA, alpha-fetoprotein AFP, miRNA-155, miRNA-141 or miRNA-21 type microRNA molecules are detected using photoluminescence-resonance light scattering dual mode.