Gold nanoparticles encapsulating reporter molecules, methods of making and using the same

Abstract

The application provides a gold nanoparticle wrapped with a reporter molecule, a preparation method and application thereof. The method is based on 50nm gold nanospheres, and the reporter molecule 4-MBA is wrapped. A prepared base liquid can maintain stable SERS enhancement performance, and a surface concave-convex structure is beneficial to enhancing a local electromagnetic field effect, so that the enhancement efficiency on the reporter molecule is improved, and application advantages in SERS detection are embodied.

Description

Technical Field

[0001] This invention relates to the field of chemical reagents, and in particular to gold nanoparticles encapsulating reporter molecules, their preparation methods, and applications. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) is a highly sensitive analytical method widely used in chemistry, life sciences, environmental monitoring, and other fields. SERS significantly enhances weak Raman scattering signals through the localized field enhancement effect of metal nanoparticles on light, thereby achieving highly sensitive detection of low-concentration molecules. Specifically, SERS utilizes the localized electromagnetic field enhancement effect generated on the surface of metal nanoparticles (such as gold and silver), which significantly enhances the Raman signal when molecules interact with the nanoparticles. This effect originates from the surface plasmon resonance (SPR) phenomenon; the plasma oscillations excited by the nanoparticles enhance the Raman scattering signal of the molecules. This allows SERS to detect extremely low concentrations of molecules, overcoming the limitations of traditional Raman spectroscopy in analyzing low-concentration samples.

[0003] Due to its ultra-high sensitivity, SERS technology has been widely used in chemical analysis, life sciences, and environmental monitoring. In chemistry, SERS can accurately detect trace amounts of chemical substances; in life sciences, it can be used to detect pathological biomarkers and drug molecules; and in environmental monitoring, SERS can monitor pollutant concentrations in real time. With its rapid, non-invasive, and efficient characteristics, SERS technology has demonstrated enormous potential and significant value in various scientific research and practical applications.

[0004] In the application of gold nanoparticles, improving the reporter molecule encapsulation efficiency and enhancing the SERS signal is a key issue. Existing technologies mainly enhance the SERS signal by improving the morphology and surface modification of gold nanoparticles. However, encapsulating reporter molecules with a single gold nanoparticle, especially when multiple different reporter molecules are encapsulated in its core-shell structure, often faces problems such as uneven encapsulation, unsatisfactory signal enhancement, and core-shell structure instability. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide gold nanoparticles encapsulating reporter molecules, their preparation method and application, to solve the problems of uneven encapsulation and insufficient signal enhancement in the prior art.

[0006] One aspect of the present invention provides a method for preparing gold nanoparticles encapsulating reporter molecules, the method comprising the following steps: (1) Under ice-water bath conditions, a solution of 50 nm gold nanospheres was ultrasonically mixed with an alcoholic solution of the reporter molecule; (2) Centrifuge, remove the supernatant, disperse the precipitate in buffer solution, and obtain the primary solution of reporter molecule modified gold nanospheres. (3) Mix the obtained reporter molecule-modified gold nanospheres primary solution with HAuCl4·3H2O solution; (4) After stirring, centrifuge multiple times to remove excess reactants and byproducts, disperse in buffer solution to obtain a pure gold nanoparticle solution. (5) Add the reporter molecule solution and mix by sonication in ice water; (6) Centrifugation was performed multiple times to obtain reporter molecule-modified gold nanospheres as a precipitate.

[0007] 4-MBA, 4-mercaptobenzoic acid, CAS No. 1074-36-8.

[0008] Further, the preparation method of the 50nm gold nanospheres includes the following: mixing a 0.0985g / L chloroauric acid aqueous solution with a 3.78g / L sodium borohydride aqueous solution while the solution is boiling, stirring until the solution cools naturally to room temperature, centrifuging to remove the supernatant, resuspending in deionized water and ultrasonically dispersing, repeating centrifugation and washing to remove residual reactants, and obtaining 50nm gold nanospheres.

[0009] Furthermore, the concentration of the gold nanosphere solution is 1:0.8-1.2 to the concentration of the reporter molecule.

[0010] Furthermore, the concentration of the gold nanosphere solution is 0.25 mM, the concentration of the reporter molecule is 0.25 mM, and the volume ratio of the gold nanosphere solution to the alcoholic solution of the 4-MBA reporter molecule is 1000:1.

[0011] Furthermore, the reporting molecule is selected from one of TFMBA, 4-MBA, 4-NBT, and DTNB.

[0012] Furthermore, in step (1), the solvent for the centrifuged gold nanosphere solution is BSPP, and the solvent for the alcohol solution of the reporter molecule is ethanol.

[0013] Furthermore, the ultrasound time in step (1) is 5-10 minutes.

[0014] Furthermore, the centrifugation conditions in step (2) are 2000-6000 rpm for 5-15 minutes; the buffer solution is HEPES solution with pH = 5.5-6.5.

[0015] Further, in step (3), the concentration of the HAuCl4·3H2O solution is 0.1mM-100mM. Further, it can also be 0.1mM-3mM, 3mM-10mM, or 10mM-100mM, and the volume ratio of the primary solution of gold nanospheres to HAuCl4·3H2O is 1:0.2-0.4.

[0016] Furthermore, the stirring time in step (4) is 20-40 min at 500-900 rpm, and the centrifugation conditions are 3000-5000 rpm for 5-10 min.

[0017] Furthermore, in step (5), the concentration of the reporting molecule is 1 mM, and the volume ratio of the reporting molecule to the pure gold nanoparticle solution is 1-8:1000.

[0018] Furthermore, the conditions for step (6) are 2000-5000 rpm and 5-10 min.

[0019] Another aspect of the present invention provides gold nanoparticles encapsulating reporter molecules prepared by the above method.

[0020] Another aspect of the present invention provides an enhancing agent for surface-enhanced Raman scattering, the agent containing the aforementioned gold nanoparticles encapsulating the reporter molecule.

[0021] Another aspect of the present invention provides the application of the aforementioned gold nanoparticles encapsulating reporter molecules in surface-enhanced Raman scattering.

[0022] As described above, the gold nanoparticles encapsulating reporter molecules of the present invention have the following beneficial effects: The method in this application can effectively modulate the enhancement effect of gold nanoparticles in SERS by controlling the structural characteristics of gold nanoparticles, providing a more efficient, stable and tunable nanoprobe platform, and offering new technical support for fields such as bioanalysis and environmental monitoring. Attached Figure Description

[0023] Figure 1 This is a flowchart of a method for preparing gold nanoparticles with an uneven core-shell structure in which both the core and shell encapsulate reporter molecules, as provided in an embodiment of the present invention. Figure 2 This is a TEM image of the concave-convex core-shell gold nanoparticles prepared in Example 1 of the present invention (both the core and the shell are coated with 4-MBA reporter molecules). Figure 3 This is a TEM image of the concave-convex core-shell gold nanoparticles prepared in Example 2 of the present invention; Figure 4This is a TEM image of the concave-convex core-shell gold nanoparticles prepared in Example 3 of the present invention; Figure 5 This is a TEM image of the concave-convex core-shell gold nanoparticles prepared in Example 4 of the present invention; Figure 6 This is a TEM image of the concave-convex core-shell gold nanoparticles prepared in Example 5 of the present invention; Figure 7 Examples 1–4 show different amounts of tetrachloroauric acid added (1×10⁻⁶). -7 mol, 3×10 -6 mol, 2×10 -5 mol and 2×10 -4 SERS spectra of concave-convex core-shell gold nanoparticles prepared under the condition of mol) were obtained. The amount of 4-MBA used in each example was the same. The test was conducted using 785 nm excitation light with an excitation power of 15 mW and an integration time of 5 s. Figure 8 (a) Comparison of SERS spectra of 10 μL and 0.1 mM 4-MBA reporter molecules encapsulated in gold nanospheres and convex-concave core-shell gold nanoparticles, respectively, in Example 5, wherein the amount of HAuCl4·3H2O added was 2 × 10⁻⁶. -5 mol; Figures (b) and (c) are magnified views, corresponding to the SERS spectra of the concave-convex core-shell gold nanoparticles and gold nanospheres, respectively; Figure 9 (a) Comparison of SERS spectra of 20 μL and 0.1 mM 4-MBA reporter molecules encapsulated in gold nanospheres and convex-core-shell gold nanoparticles, respectively, in Example 6, wherein the amount of HAuCl4·3H2O added was 2 × 10⁻⁶. -5 mol; Figures (b) and (c) are magnified views, corresponding to the SERS spectra of the concave-convex core-shell gold nanoparticles and gold nanospheres, respectively; Figure 10 (a) Comparison of SERS spectra of 40 μL and 0.1 mM 4-MBA reporter molecules encapsulated in gold nanospheres and concave-convex core-shell gold nanoparticles, respectively, in Example 7, wherein the amount of HAuCl4·3H2O added was 2×10 -5 mol; Figures (b) and (c) are magnified views, corresponding to the SERS spectra of the concave-convex core-shell gold nanoparticles and gold nanospheres, respectively; Detailed Implementation

[0024] We achieved signal enhancement in SERS by precisely controlling the gold core size, shell thickness, and morphology of the micro / nanostructures. Specifically, we used 45 nm gold nanoparticles as the core and formed a shell with multi-spherical shapes and uneven micro / nanostructures on its surface. This design significantly enhanced the Raman signal while maintaining the chemical and thermal stability of the particles.

[0025] Through innovative encapsulation technology, this invention not only improves the encapsulation efficiency of reporter molecules but also significantly enhances SERS signal intensity, with an enhancement factor of over 10-fold. Simultaneously, the optimized gold nanoparticle structure enhances its application potential in bioassays, particularly in low-concentration molecular detection and multiplex labeling analysis.

[0026] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0027] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0028] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

[0029] Example 1: Preparation of gold nanoparticles encapsulating reporter molecules 1. Pretreatment of glassware: Prepare a 2% aqueous solution of laboratory-grade surfactant cleaning solution, place it in an ultrasonic cleaner and ultrasonically clean for 15 minutes; then rinse thoroughly with deionized water 3 times, dry in an oven at 80°C, and then blow dry with nitrogen gas for later use.

[0030] 2. Accurately weigh 9.85 mg of tetrachloroauric acid (HAuCl4) and transfer it to a 100 mL volumetric flask. Then, dilute to the mark with deionized water. Gently invert the flask to mix, and you will obtain a 0.25 mM HAuCl4 solution.

[0031] 3. Place 100 mL of 0.25 mM HAuCl4 working solution into a 250 mL three-necked flask, assemble a reflux condenser, and insert a temperature probe and magnetic stir bar. Stir at 400 rpm and heat to boiling.

[0032] 4. Accurately weigh 0.882 g of trisodium citrate dihydrate. Dissolve in approximately 80 mL of deionized water. After complete dissolution, bring the volume to 100 mL with deionized water and mix thoroughly to obtain a 30 mM trisodium citrate solution.

[0033] 5. Once the HAuCl4 solution boils, quickly add 1 mL of 30 mM trisodium citrate in one continuous stream. Maintain boiling and continue stirring for 10 minutes.

[0034] 6. Turn off the heating device and continue stirring until the solution cools to room temperature naturally.

[0035] 7. Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Avoid disturbing the precipitate. Resuspend in an equal volume of deionized water and sonicate for 30 s. Repeat centrifugation and washing three times to remove residual reactants.

[0036] 8. Suspend the washed gold nanoparticles in 100 mL of pH 7.4, 1 mM HEPES buffer and mix thoroughly.

[0037] 9. The 0.25mM 50 nm gold nanospheres obtained above were left to stand at room temperature for 24 hours.

[0038] 10. Transfer 5 mL of 0.25 mM 50 nm gold nanospheres to a centrifuge tube and centrifuge at 2000-6000 rpm for 5-15 minutes. Discard the supernatant and retain the precipitate.

[0039] 11. Weigh approximately 3 mg of bis(p-sulfonated)phenylphosphine dihydrate dipotassium salt (BSPP) and dissolve it in 5 mL of deionized water. Stir until completely dissolved to prepare an approximately 1 mM BSPP solution.

[0040] 12. Disperse the precipitate in 5 mL of 1 mM BSPP to ensure uniform particle dispersion.

[0041] 13. Dissolve approximately 7.7 mg of 4-MBA reporter molecule in 500 mL of ethanol to prepare an approximately 0.1 mM 4-MBA solution. 14. Under ice-water bath conditions, sonicate for 5 minutes using an ultrasonic processor to thoroughly mix the gold nanoparticle solution with the reporter molecule in 5 μL of 0.1 mM ethanol solution. Slowly add the reporter molecule solution to the gold nanoparticle solution, ensuring uniform dispersion.

[0042] 15. Centrifuge at 4000 rpm for 10 minutes to remove the supernatant. Redisperse the precipitate in 5 mL of HEPES buffer (50 mM, pH 5.5-6.5) to obtain “reporter molecule modified gold nanospheres”.

[0043] 16. Mix 5 ml of "Raman reporter molecule modified gold nanospheres" with 1 ml of 0.1 mM (1×10⁻⁶) gold nanospheres. -7 mol) HAuCl4·3H2O mixture.

[0044] 17. Stir the mixture at 50 rpm for 40 minutes at room temperature.

[0045] 18. Centrifuge at 3000 rpm for 10 minutes, repeating the centrifugation process multiple times to remove excess reactants and byproducts. Redisperse the particles in 5 mL of 1 mM BSPP buffer to ensure a pure gold nanoparticle solution.

[0046] 19. Add 5 μL of a 1 mM reporter molecule to the solution and sonicate in ice water for 5-20 minutes.

[0047] 20. Centrifuge three times at 2000 rpm for 10 minutes each time, and disperse in 5 mL of 1 mM BSPP buffer.

[0048] 21. The prepared concave-convex shell-shaped nanoprobes are stored at 4°C for later use.

[0049] Example 2 1. Pretreatment of glassware: Prepare a 2% aqueous solution of laboratory-grade surfactant cleaning solution, place it in an ultrasonic cleaner and ultrasonically clean for 15 minutes; then rinse thoroughly with deionized water 3 times, dry in an oven at 80°C, and then blow dry with nitrogen gas for later use.

[0050] 2. Accurately weigh 9.85 mg of tetrachloroauric acid (HAuCl4) and transfer it to a 100 mL volumetric flask. Then, dilute to the mark with deionized water. Gently invert the flask to mix, and you will obtain a 0.25 mM HAuCl4 solution.

[0051] 3. Place 100 mL of 0.25 mM HAuCl4 working solution into a 250 mL three-necked flask, assemble a reflux condenser, and insert a temperature probe and magnetic stir bar. Stir at 400 rpm and heat to boiling.

[0052] 4. Accurately weigh 0.882 g of trisodium citrate dihydrate. Dissolve in approximately 80 mL of deionized water. After complete dissolution, bring the volume to 100 mL with deionized water and mix thoroughly to obtain a 30 mM trisodium citrate solution.

[0053] 5. Once the HAuCl4 solution boils, quickly add 1 mL of 30 mM trisodium citrate in one continuous stream. Maintain boiling and continue stirring for 10 minutes.

[0054] 6. Turn off the heating device and continue stirring until the solution cools to room temperature naturally.

[0055] 7. Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Avoid disturbing the precipitate. Resuspend in an equal volume of deionized water and sonicate for 30 s. Repeat centrifugation and washing three times to remove residual reactants.

[0056] 8. Suspend the washed gold nanoparticles in 100 mL of pH 7.4, 1 mM HEPES buffer and mix thoroughly.

[0057] 9. The 0.25mM 50nm gold nanospheres obtained above were left to stand at room temperature for 24 hours.

[0058] 10. Transfer 5 mL of 0.25 mM 50 nm gold nanospheres to a centrifuge tube and centrifuge at 2000-6000 rpm for 5-15 minutes. Discard the supernatant and retain the precipitate.

[0059] 11. Weigh approximately 3 mg of bis(p-sulfonated)phenylphosphine dihydrate dipotassium salt (BSPP) and dissolve it in 5 mL of deionized water. Stir until completely dissolved to prepare an approximately 1 mM BSPP solution.

[0060] 12. Disperse the precipitate in 5 mL of 1 mM BSPP to ensure uniform particle dispersion.

[0061] 13. Dissolve approximately 7.7 mg of 4-MBA reporter molecule in 500 mL of ethanol to prepare an approximately 0.1 mM 4-MBA solution. 14. Under ice-water bath conditions, sonicate for 5 minutes using an ultrasonic processor to thoroughly mix the gold nanoparticle solution with the reporter molecule in 5 μL of 0.1 mM ethanol solution. Slowly add the reporter molecule solution to the gold nanoparticle solution, ensuring uniform dispersion.

[0062] 15. Centrifuge at 2000 rpm for 15 minutes to remove the supernatant. Redisperse the precipitate in 5 mL of HEPES buffer (50 mM, pH 5.5-6.5) to obtain “reporter molecule modified gold nanospheres”.

[0063] 16. Mix 5 ml of "Raman reporter molecule modified gold nanospheres" with 1 ml of 3 mM (3 × 10⁻⁶) solution. -6 mol) HAuCl4·3H2O mixture 17. At room temperature, stir the mixture at 500 rpm for 40 minutes. 18. Centrifuge at 5000 rpm for 5 minutes, repeating the centrifugation process multiple times to remove excess reactants and byproducts. Redisperse the particles in 5 mL of 1 mM BSPP buffer to ensure a pure gold nanoparticle solution.

[0064] 19. Add 5 μL of a 1 mM reporter molecule to the solution and sonicate in ice water for 5-20 minutes.

[0065] 20. Centrifuge at 5000 rpm for 5 minutes four times, and disperse in 5 mL of 1 mM BSPP buffer.

[0066] 21. The prepared concave-convex shell-shaped nanoprobes are stored at 4°C for later use.

[0067] Example 3 1. Pretreatment of glassware: Prepare a 2% aqueous solution of laboratory-grade surfactant cleaning solution, place it in an ultrasonic cleaner and ultrasonically clean for 15 minutes; then rinse thoroughly with deionized water 3 times, dry in an oven at 80°C, and then blow dry with nitrogen gas for later use.

[0068] 2. Accurately weigh 9.85 mg of tetrachloroauric acid (HAuCl4) and transfer it to a 100 mL volumetric flask. Then, dilute to the mark with deionized water. Gently invert the flask to mix, and you will obtain a 0.25 mM HAuCl4 solution.

[0069] 3. Place 100 mL of 0.25 mM HAuCl4 working solution into a 250 mL three-necked flask, assemble a reflux condenser, and insert a temperature probe and magnetic stir bar. Stir at 400 rpm and heat to boiling.

[0070] 4. Accurately weigh 0.882 g of trisodium citrate dihydrate. Dissolve in approximately 80 mL of deionized water. After complete dissolution, bring the volume to 100 mL with deionized water and mix thoroughly to obtain a 30 mM trisodium citrate solution.

[0071] 5. Once the HAuCl4 solution boils, quickly add 1 mL of 30 mM trisodium citrate in one continuous stream. Maintain boiling and continue stirring for 10 minutes.

[0072] 6. Turn off the heating device and continue stirring until the solution cools to room temperature naturally.

[0073] 7. Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Avoid disturbing the precipitate. Resuspend in an equal volume of deionized water and sonicate for 30 s. Repeat centrifugation and washing three times to remove residual reactants.

[0074] 8. Suspend the washed gold nanoparticles in 100 mL of pH 7.4, 1 mM HEPES buffer and mix thoroughly.

[0075] 9. The 0.25mM 50nm gold nanospheres obtained above were left to stand at room temperature for 24 hours.

[0076] 10. Transfer 5 mL of 0.25 mM 50 nm gold nanospheres to a centrifuge tube and centrifuge at 2000-6000 rpm for 5-15 minutes. Discard the supernatant and retain the precipitate.

[0077] 11. Weigh approximately 3 mg of bis(p-sulfonated)phenylphosphine dihydrate dipotassium salt (BSPP) and dissolve it in 5 mL of deionized water. Stir until completely dissolved to prepare an approximately 1 mM BSPP solution.

[0078] 12. Disperse the precipitate in 5 mL of 1 mM BSPP to ensure uniform particle dispersion.

[0079] 13. Dissolve approximately 7.7 mg of 4-MBA reporter molecules in 500 mL of ethanol to prepare an approximately 0.1 mM 4-MBA solution.

[0080] 14. Under ice-water bath conditions, sonicate for 5 minutes using an ultrasonic processor to thoroughly mix the gold nanoparticle solution with the reporter molecule in 5 μL of 0.1 mM ethanol solution. Slowly add the reporter molecule solution to the gold nanoparticle solution, ensuring uniform dispersion.

[0081] 15. Centrifuge at 6000 rpm for 5 minutes to remove the supernatant. Redisperse the precipitate in 5 mL of HEPES buffer (50 mM, pH 5.5-6.5) to obtain "reporter molecule modified gold nanospheres".

[0082] 16. Mix 5 mL of "Raman reporter molecule modified gold nanospheres" with 2 mL of 10 mM (2 × 10⁻⁶) solution. -5 mol) HAuCl4·3H2O mixture.

[0083] 17. Stir the mixture at 900 rpm for 20 minutes at room temperature.

[0084] 18. Centrifuge at 5000 rpm for 5 minutes, repeating the centrifugation process multiple times to remove excess reactants and byproducts. Redisperse the particles in 5 mL of 1 mM BSPP buffer to ensure a pure gold nanoparticle solution.

[0085] 19. Add 5 μL of a 1 mM reporter molecule to the solution and sonicate in ice water for 5-20 minutes.

[0086] 20. Centrifuge multiple times at 3500 rpm for 8 minutes each time, and disperse in 5 mL of 1 mM BSPP buffer.

[0087] 21. The prepared concave-convex shell-shaped nanoprobes are stored at 4°C for later use.

[0088] Example 4 1. Pretreatment of glassware: Prepare a 2% aqueous solution of laboratory-grade surfactant cleaning solution, place it in an ultrasonic cleaner and ultrasonically clean for 15 minutes; then rinse thoroughly with deionized water 3 times, dry in an oven at 80°C, and then blow dry with nitrogen gas for later use.

[0089] 2. Accurately weigh 9.85 mg of tetrachloroauric acid (HAuCl4) and transfer it to a 100 mL volumetric flask. Then, dilute to the mark with deionized water. Gently invert the flask to mix, and you will obtain a 0.25 mM HAuCl4 solution.

[0090] 3. Place 100 mL of 0.25 mM HAuCl4 working solution into a 250 mL three-necked flask, assemble a reflux condenser, and insert a temperature probe and magnetic stir bar. Stir at 400 rpm and heat to boiling.

[0091] 4. Accurately weigh 0.882 g of trisodium citrate dihydrate. Dissolve in approximately 80 mL of deionized water. After complete dissolution, bring the volume to 100 mL with deionized water and mix thoroughly to obtain a 30 mM trisodium citrate solution.

[0092] 5. Once the HAuCl4 solution boils, quickly add 1 mL of 30 mM trisodium citrate in one continuous stream. Maintain boiling and continue stirring for 10 minutes.

[0093] 6. Turn off the heating device and continue stirring until the solution cools to room temperature naturally.

[0094] 7. Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Avoid disturbing the precipitate. Resuspend in an equal volume of deionized water and sonicate for 30 s. Repeat centrifugation and washing three times to remove residual reactants.

[0095] 8. Suspend the washed gold nanoparticles in 100 mL of pH 7.4, 1 mM HEPES buffer and mix thoroughly.

[0096] 9. The 0.25mM 50nm gold nanospheres obtained above were left to stand at room temperature for 24 hours.

[0097] 10. Transfer 5 mL of 0.25 mM 50 nm gold nanospheres to a centrifuge tube and centrifuge at 2000-6000 rpm for 5-15 minutes. Discard the supernatant and retain the precipitate.

[0098] 11. Weigh approximately 3 mg of bis(p-sulfonated)phenylphosphine dihydrate dipotassium salt (BSPP) and dissolve it in 5 mL of deionized water. Stir until completely dissolved to prepare an approximately 1 mM BSPP solution.

[0099] 12. Disperse the precipitate in 5 mL of 1 mM BSPP to ensure uniform particle dispersion.

[0100] 13. Dissolve approximately 7.7 mg of 4-MBA reporter molecules in 500 mL of ethanol to prepare an approximately 0.1 mM 4-MBA solution.

[0101] 14. Under ice-water bath conditions, sonicate for 5 minutes using an ultrasonic processor to thoroughly mix the gold nanoparticle solution with the reporter molecule in 5 μL of 0.1 mM ethanol solution. Slowly add the reporter molecule solution to the gold nanoparticle solution, ensuring uniform dispersion.

[0102] 15. Centrifuge at 3000 rpm for 12 minutes to remove the supernatant. Redisperse the precipitate in 5 mL of HEPES buffer (50 mM, pH 5.5-6.5) to obtain “reporter molecule modified gold nanospheres”.

[0103] 16. Mix 5 mL of "Raman reporter molecule modified gold nanospheres" with 2 mL of 100 mM (2 × 10⁻⁶) solution. -4 mol) HAuCl4·3H2O mixture.

[0104] 17. At room temperature, stir the mixture at 500-900 rpm for 20-40 minutes.

[0105] 18. Centrifuge at 4000 rpm for 6 minutes, repeating the centrifugation process multiple times to remove excess reactants and byproducts. Redisperse the particles in 5 mL of 1 mM BSPP buffer to ensure a pure gold nanoparticle solution.

[0106] 19. Add 5 μL of a 1 mM reporter molecule to the solution and sonicate in ice water for 5-20 minutes.

[0107] 20. Centrifuge 5 times at 4000 rpm for 6 minutes each time, and disperse in 5 mL of 1 mM BSPP buffer.

[0108] 21. The prepared concave-convex shell-shaped nanoprobes are stored at 4°C for later use.

[0109] Example 5 1. Pretreatment of glassware: Prepare a 2% aqueous solution of laboratory-grade surfactant cleaning solution, place it in an ultrasonic cleaner and ultrasonically clean for 15 minutes; then rinse thoroughly with deionized water 3 times, dry in an oven at 80°C, and then blow dry with nitrogen gas for later use.

[0110] 2. Accurately weigh 9.85 mg of tetrachloroauric acid (HAuCl4) and transfer it to a 100 mL volumetric flask. Then, dilute to the mark with deionized water. Gently invert the flask to mix, and you will obtain a 0.25 mM HAuCl4 solution.

[0111] 3. Place 100 mL of 0.25 mM HAuCl4 working solution into a 250 mL three-necked flask, assemble a reflux condenser, and insert a temperature probe and magnetic stir bar. Stir at 400 rpm and heat to boiling.

[0112] 4. Accurately weigh 0.882 g of trisodium citrate dihydrate. Dissolve in approximately 80 mL of deionized water. After complete dissolution, bring the volume to 100 mL with deionized water and mix thoroughly to obtain a 30 mM trisodium citrate solution.

[0113] 5. Once the HAuCl4 solution boils, quickly add 1 mL of 30 mM trisodium citrate in one continuous stream. Maintain boiling and continue stirring for 10 minutes.

[0114] 6. Turn off the heating device and continue stirring until the solution cools to room temperature naturally.

[0115] 7. Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Avoid disturbing the precipitate. Resuspend in an equal volume of deionized water and sonicate for 30 s. Repeat centrifugation and washing three times to remove residual reactants.

[0116] 8. Suspend the washed gold nanoparticles in 100 mL of pH 7.4, 1 mM HEPES buffer and mix thoroughly.

[0117] 9. The 0.25mM 50nm gold nanospheres obtained above were left to stand at room temperature for 24 hours.

[0118] 10. Transfer 5 mL of 0.25 mM 50 nm gold nanospheres to a centrifuge tube and centrifuge at 2000-6000 rpm for 5-15 minutes. Discard the supernatant and retain the precipitate.

[0119] 11. Weigh approximately 3 mg of bis(p-sulfonated)phenylphosphine dihydrate dipotassium salt (BSPP) and dissolve it in 5 mL of deionized water. Stir until completely dissolved to prepare an approximately 1 mM BSPP solution.

[0120] 12. Disperse the precipitate in 5 mL of 1 mM BSPP to ensure uniform particle dispersion.

[0121] 13. Dissolve approximately 7.7 mg of 4-MBA reporter molecules in 500 mL of ethanol to prepare an approximately 0.1 mM 4-MBA solution.

[0122] 14. Under ice-water bath conditions, sonicate for 5 minutes using an ultrasonic processor to thoroughly mix the gold nanoparticle solution with the reporter molecule in 10 μL of 0.1 mM ethanol solution. Slowly add the reporter molecule solution to the gold nanoparticle solution to ensure uniform dispersion.

[0123] 15. Centrifuge at 4000 rpm for 8 minutes to remove the supernatant. Redisperse the precipitate in 5 mL of HEPES buffer (50 mM, pH 5.5-6.5) to obtain "reporter molecule modified gold nanospheres".

[0124] 16. Mix 5 mL of "Raman reporter molecule modified gold nanospheres" with 2 mL of 10 mM (2 × 10⁻⁶) solution. -5 mol) HAuCl4·3H2O mixture.

[0125] 17. Stir the mixture at 800 rpm for 35 minutes at room temperature.

[0126] 18. Centrifuge at 4000 rpm for 6 minutes, repeating the centrifugation process multiple times to remove excess reactants and byproducts. Redisperse the particles in 5 mL of 1 mM BSPP buffer to ensure a pure gold nanoparticle solution.

[0127] 19. Add 10 μL of a 1 mM reporter molecule to the solution and sonicate in ice water for 5-20 minutes.

[0128] 20. Centrifuge three times at 3000 rpm for 8 minutes each time, and disperse in 5 mL of 1 mM BSPP buffer.

[0129] 21. The prepared concave-convex shell-shaped nanoprobes are stored at 4°C for later use.

[0130] Example 6 1. Pretreatment of glassware: Prepare a 2% aqueous solution of laboratory-grade surfactant cleaning solution, place it in an ultrasonic cleaner and ultrasonically clean for 15 minutes; then rinse thoroughly with deionized water 3 times, dry in an oven at 80°C, and then blow dry with nitrogen gas for later use.

[0131] 2. Accurately weigh 9.85 mg of tetrachloroauric acid (HAuCl4) and transfer it to a 100 mL volumetric flask. Then, dilute to the mark with deionized water. Gently invert the flask to mix, and you will obtain a 0.25 mM HAuCl4 solution.

[0132] 3. Place 100 mL of 0.25 mM HAuCl4 working solution into a 250 mL three-necked flask, assemble a reflux condenser, and insert a temperature probe and magnetic stir bar. Stir at 400 rpm and heat to boiling.

[0133] 4. Accurately weigh 0.882 g of trisodium citrate dihydrate. Dissolve in approximately 80 mL of deionized water. After complete dissolution, bring the volume to 100 mL with deionized water and mix thoroughly to obtain a 30 mM trisodium citrate solution.

[0134] 5. Once the HAuCl4 solution boils, quickly add 1 mL of 30 mM trisodium citrate in one continuous stream. Maintain boiling and continue stirring for 10 minutes.

[0135] 6. Turn off the heating device and continue stirring until the solution cools to room temperature naturally.

[0136] 7. Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Avoid disturbing the precipitate. Resuspend in an equal volume of deionized water and sonicate for 30 s. Repeat centrifugation and washing three times to remove residual reactants.

[0137] 8. Suspend the washed gold nanoparticles in 100 mL of pH 7.4, 1 mM HEPES buffer and mix thoroughly.

[0138] 9. The 0.25mM 50nm gold nanospheres obtained above were left to stand at room temperature for 24 hours.

[0139] 10. Transfer 5 mL of 0.25 mM 50 nm gold nanospheres to a centrifuge tube and centrifuge at 2000-6000 rpm for 5-15 minutes. Discard the supernatant and retain the precipitate.

[0140] 11. Weigh approximately 3 mg of bis(p-sulfonated)phenylphosphine dihydrate dipotassium salt (BSPP) and dissolve it in 5 mL of deionized water. Stir until completely dissolved to prepare an approximately 1 mM BSPP solution.

[0141] 12. Disperse the precipitate in 5 mL of 1 mM BSPP to ensure uniform particle dispersion.

[0142] 13. Dissolve approximately 7.7 mg of 4-MBA reporter molecules in 500 mL of ethanol to prepare an approximately 0.1 mM 4-MBA solution.

[0143] 14. Under ice-water bath conditions, sonicate for 5 minutes using an ultrasonic processor to thoroughly mix the gold nanoparticle solution with the reporter molecule in 20 μL of 0.1 mM ethanol solution. Slowly add the reporter molecule solution to the gold nanoparticle solution, ensuring uniform dispersion.

[0144] 15. Centrifuge at 5000 rpm for 6 minutes to remove the supernatant. Redisperse the precipitate in 5 mL of HEPES buffer (50 mM, pH 5.5-6.5) to obtain “reporter molecule modified gold nanospheres”.

[0145] 16. Mix 5 mL of "Raman reporter molecule modified gold nanospheres" with 2 mL of 10 mM (2 × 10⁻⁶) solution. -5 mol) HAuCl4·3H2O mixture.

[0146] 17. Stir the mixture at 700 rpm for 30 minutes at room temperature.

[0147] 18. Centrifuge at 5000 rpm for 5 minutes, repeating the centrifugation process multiple times to remove excess reactants and byproducts. Redisperse the particles in 5 mL of 1 mM BSPP buffer to ensure a pure gold nanoparticle solution.

[0148] 19. Add 20 μL of a 1 mM reporter molecule to the solution and sonicate in ice water for 5-20 minutes.

[0149] 20. Centrifuge 4 times at 2000 rpm for 10 minutes each time, and disperse in 5 mL of 1 mM BSPP buffer.

[0150] 21. The prepared concave-convex shell-shaped nanoprobes are stored at 4°C for later use.

[0151] Example 7 1. Pretreatment of glassware: Prepare a 2% aqueous solution of laboratory-grade surfactant cleaning solution, place it in an ultrasonic cleaner and ultrasonically clean for 15 minutes; then rinse thoroughly with deionized water 3 times, dry in an oven at 80°C, and then blow dry with nitrogen gas for later use.

[0152] 2. Accurately weigh 9.85 mg of tetrachloroauric acid (HAuCl4) and transfer it to a 100 mL volumetric flask. Then, dilute to the mark with deionized water. Gently invert the flask to mix, and you will obtain a 0.25 mM HAuCl4 solution.

[0153] 3. Place 100 mL of 0.25 mM HAuCl4 working solution into a 250 mL three-necked flask, assemble a reflux condenser, and insert a temperature probe and magnetic stir bar. Stir at 400 rpm and heat to boiling.

[0154] 4. Accurately weigh 0.882 g of trisodium citrate dihydrate. Dissolve in approximately 80 mL of deionized water. After complete dissolution, bring the volume to 100 mL with deionized water and mix thoroughly to obtain a 30 mM trisodium citrate solution.

[0155] 5. Once the HAuCl4 solution boils, quickly add 1 mL of 30 mM trisodium citrate in one continuous stream. Maintain boiling and continue stirring for 10 minutes.

[0156] 6. Turn off the heating device and continue stirring until the solution cools to room temperature naturally.

[0157] 7. Centrifuge at 5000 rpm for 10 min at room temperature and discard the supernatant. Avoid disturbing the precipitate. Resuspend in an equal volume of deionized water and sonicate for 30 s. Repeat centrifugation and washing three times to remove residual reactants.

[0158] 8. Suspend the washed gold nanoparticles in 100 mL of pH 7.4, 1 mM HEPES buffer and mix thoroughly.

[0159] 9. The 0.25mM 50nm gold nanospheres obtained above were left to stand at room temperature for 24 hours.

[0160] 10. Transfer 5 mL of 0.25 mM 50 nm gold nanospheres to a centrifuge tube and centrifuge at 2000-6000 rpm for 5-15 minutes. Discard the supernatant and retain the precipitate.

[0161] 11. Weigh approximately 3 mg of bis(p-sulfonated)phenylphosphine dihydrate dipotassium salt (BSPP) and dissolve it in 5 mL of deionized water. Stir until completely dissolved to prepare an approximately 1 mM BSPP solution.

[0162] 12. Disperse the precipitate in 5 mL of 1 mM BSPP to ensure uniform particle dispersion.

[0163] 13. Dissolve approximately 7.7 mg of 4-MBA reporter molecules in 500 mL of ethanol to prepare an approximately 0.1 mM 4-MBA solution.

[0164] 14. Under ice-water bath conditions, sonicate for 5 minutes using an ultrasonic processor to thoroughly mix the gold nanoparticle solution with the reporter molecule in 40 μL of 0.1 mM ethanol solution. Slowly add the reporter molecule solution to the gold nanoparticle solution, ensuring uniform dispersion.

[0165] 15. Centrifuge at 4000 rpm for 10 minutes to remove the supernatant. Redisperse the precipitate in 5 mL of HEPES buffer (50 mM, pH 5.5-6.5) to obtain “reporter molecule modified gold nanospheres”.

[0166] 16. Mix 5 mL of "Raman reporter molecule modified gold nanospheres" with 2 mL of 10 mM (2 × 10⁻⁶) solution. -5 mol) HAuCl4·3H2O mixture.

[0167] 17. Stir the mixture at 600 rpm for 30 minutes at room temperature.

[0168] 18. Centrifuge at 3000 rpm for 10 minutes, repeating the centrifugation process multiple times to remove excess reactants and byproducts. Redisperse the particles in 5 mL of 1 mM BSPP buffer to ensure a pure gold nanoparticle solution.

[0169] 19. Add 40 μL of a 1 mM reporter molecule to the solution and sonicate in ice water for 5-20 minutes.

[0170] 20. Centrifuge three times at 5000 rpm for 5 minutes each time, and disperse in 5 mL of 1 mM BSPP buffer.

[0171] 21. The prepared concave-convex shell-shaped nanoprobes are stored at 4°C for later use.

[0172] Example 8: Transmission electron microscope image Figure 2-6 Transmission electron microscopy (TEM) images of the convex-concave core-shell gold nanoparticles prepared in Examples 1-5 are shown. As can be seen from the figures, the nanoparticles obtained in each example all have a clear and complete core-shell structure, and the core and shell surfaces of the particles are uniformly coated with 4-MBA reporter molecules.

[0173] The above results indicate that the concave-convex core-shell gold nanoparticles prepared by the method of the present invention have good structural consistency and dispersibility, and the 4-MBA reporter molecules encapsulated therein are stably distributed on the surface of the core and shell of the particles, which is beneficial to achieving a stable and reproducible SERS signal enhancement effect.

[0174] Example 9: Effect of tetrachloroauric acid content on SERS enhancement of concave-convex core-shell gold nanoparticles Figure 7 The SERS spectra of the irregular core-shell gold nanoparticles obtained in Examples 1-4 under different tetrachloroauric acid addition amounts are shown. The tetrachloroauric acid addition amounts are 1×10⁻⁶. -7 mol, 3×10 -6 mol, 2×10 -5 mol and 2×10 -4 The amount of 4-MBA reporter molecule encapsulated in each example was kept consistent. All SERS measurements were performed under 785 nm excitation light with an excitation power of 15 mW and an integration time of 5 s.

[0175] Under different tetrachloroauric acid addition conditions, 4-MBA at 1078 cm⁻¹ -1 The intensity of the SERS characteristic peak at the location showed a clear dependence. When the amount of tetrachloroauric acid was 1×10⁻⁶, the intensity of the characteristic peak at that location showed a significant dependence. -7The SERS signal was lowest at mol; as the amount of tetrachloroauric acid increased to 3×10 -6 mol, 1078 cm -1 The intensity of the characteristic peak at that point began to increase significantly; when the dosage was further increased to 2×10 -5 At mol, the SERS signal continued to rise, indicating that the concave-convex core-shell structure gradually formed and tended to be perfected, which is conducive to the construction of electromagnetic hotspots.

[0176] However, when the amount of tetrachloroauric acid continued to increase to 2×10 -4 At mol, the SERS signal of 4-MBA showed a decreasing trend. This may be due to the excessive growth of the outer gold shell, which leads to passivation of the uneven structure, a decrease in hotspot density, and an increase in the effective enhancement distance between the reporting molecules and the metal surface, thereby weakening the local electromagnetic field enhancement effect.

[0177] The above results indicate that the amount of tetrachloroauric acid added is a key parameter for regulating the SERS performance of core-shell gold nanoparticles, and there exists an optimal range for its addition. Under suitable tetrachloroauric acid dosage conditions, a SERS substrate with a reasonable structure and significant enhancement effect can be obtained, while excessively low or high dosages are detrimental to the enhancement of the SERS signal.

[0178] Example 10: SERS comparison of concave-convex core-shell gold nanoparticles and spherical gold nanoparticles encapsulating 4-MBA reporter molecules Compare Figure 8 In Example 5, the amount of tetrachloroauric acid (HAuCl4·3H2O) added was 2×10 -5 SERS spectra of gold nanospheres and concave-shell gold nanoparticles were compared under the condition of mol; Figures (b) and (c) are magnified views, corresponding to the SERS spectra of concave-shell gold nanoparticles and gold nanospheres, respectively.

[0179] Figure 9 The above are SERS spectra comparisons obtained by encapsulating 20 μL and 0.1 mM 4-MBA reporter molecules in gold nanospheres and concave-shell gold nanoparticles, respectively, under the same tetrachloroauric acid addition conditions in Example 6; Figure (b) and Figure (c) are magnified SERS spectra of concave-shell gold nanoparticles and gold nanospheres, respectively.

[0180] Figure 10 The above are comparison SERS spectra obtained by encapsulating 40 μL and 0.1 mM of 4-MBA reporter molecules in gold nanospheres and concave-shell gold nanoparticles, respectively, under the same tetrachloroauric acid addition conditions in Example 7; Figure (b) and Figure (c) are magnified SERS spectra of concave-shell gold nanoparticles and gold nanospheres, respectively.

[0181] Combination Figure 8 -10 indicates that, with the same amount of tetrachloroauric acid added (2×10), -5 Under the condition of mol), as the amount of 4-MBA reporter molecule encapsulation increased from 10 μL to 20 μL and 40 μL, the SERS signal intensity obtained by the concave-convex core-shell gold nanoparticles under each corresponding condition was higher than that of gold nanospheres. Under different reporter molecule encapsulation conditions, the concave-convex core-shell gold nanoparticles were able to obtain relatively clear characteristic peaks and the spectrum signal-to-noise ratio was good.

[0182] The above results show that, compared with gold nanospheres, the concave-convex core-shell gold nanoparticles of the present invention can maintain stable SERS enhancement performance under certain reporter molecule encapsulation conditions. Their concave-convex surface structure is beneficial to enhancing the local electromagnetic field effect, thereby improving the enhancement efficiency of reporter molecules, demonstrating their application advantages in SERS detection.

Claims

1. A method for preparing gold nanoparticles encapsulating reporter molecules, characterized in that, The method includes at least: (1) Under ice-water bath conditions, a solution of 50 nm gold nanospheres was ultrasonically mixed with an alcoholic solution of the reporter molecule; (2) Centrifuge, remove the supernatant, disperse the precipitate in buffer solution, and obtain a primary solution of reporter molecule modified gold nanospheres. (3) Mix the obtained reporter molecule-modified gold nanospheres primary solution with HAuCl4·3H2O solution; (4) After stirring, centrifuge multiple times to remove excess reactants and byproducts, disperse in buffer solution to obtain a pure gold nanoparticle solution. (5) Add the reporter molecule solution and mix by sonication in ice water; (6) Centrifugation was performed multiple times to obtain reporter molecule-modified gold nanospheres as a precipitate.

2. The method according to claim 1, characterized in that: The preparation method of the 50nm gold nanospheres includes the following steps: mixing a 0.0985g / L chloroauric acid aqueous solution with a 3.78g / L sodium borohydride aqueous solution while the solution is boiling, stirring until the solution cools naturally to room temperature, centrifuging to remove the supernatant, resuspending in deionized water and ultrasonically dispersing, and repeating centrifugation and washing to remove residual reactants to obtain 50nm gold nanospheres.

3. The method according to claim 1, characterized in that: The concentration of the gold nanosphere solution is 0.25 mM, the concentration of the reporter molecule is 0.25 mM, and the volume ratio of the gold nanosphere solution to the alcohol solution of the reporter molecule is 1000:

1.

4. The method according to claim 1, characterized in that: In step (1), the solvent for the centrifuged gold nanosphere solution is BSPP, and the solvent for the alcohol solution of the reporter molecule is ethanol; the sonication time is 5-10 min, and the reporter molecule is selected from TFMBA, 4-MBA, 4-NBT, and DTNB.

5. The method according to claim 1, characterized in that: The centrifugation conditions in step (2) are 2000-6000 rpm for 5-15 minutes; the buffer solution is HEPES solution with pH=5.5-6.

5.

6. The method according to claim 1, characterized in that: In step (3), the concentration of HAuCl4·3H2O solution is 0.1mM-100mM, and the volume ratio of the primary solution of gold nanospheres to HAuCl4·3H2O is 1:0.2-0.

4.

7. The method according to claim 1, characterized in that: In step (4), the stirring time is 20-40 min at 500-900 rpm, and the centrifugation conditions are 3000-5000 rpm for 5-10 min; in step (5), the concentration of the reporting molecule is 1 mM, and the volume ratio of the reporting molecule to the pure gold nanoparticle solution is 1-8:1000; in step (6), the conditions are 2000-5000 rpm for 5-10 min.

8. Gold nanoparticles encapsulating reporter molecules prepared by the method of any one of claims 1-7.

9. An enhancing agent for surface-enhanced Raman scattering, characterized in that, The reagent contains gold nanoparticles that encapsulate the reporter molecule as described in claim 8.

10. The application of gold nanoparticles encapsulating reporter molecules as described in claim 8 in surface-enhanced Raman scattering.

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