Nanoparticles and their use in the preparation of surface-enhanced raman scattering test kits

Abstract

This disclosure provides a method for preparing nanoparticles, the method comprising: mixing amino-functionalized polyethylene nanoparticles with nano-metal particles to obtain polyethylene nanoparticles with the nano-metal particles bound to their surface; mixing the polyethylene nanoparticles with the nano-metal particles bound to their surface with triethanolamine, hexadecyltrimethylammonium bromide, and tetraethoxysilane to obtain polyethylene nanoparticles coated with a mesoporous silica shell; and calcining the polyethylene nanoparticles coated with the mesoporous silica shell to obtain calcined particles. This disclosure also provides the nanoparticles prepared by this method and their use in the preparation of surface-enhanced Raman scattering (SERS) detection kits. The nanoparticles of this disclosure further mitigate the "protein crown" effect of SERS-labeled nanoprobes, improving the stability and accuracy of the Raman signal.

Description

Technical Field

[0001] This disclosure relates to the field of measurement, and more specifically, to a method for preparing nanoparticles, the nanoparticles prepared by the method, and the use of the nanoparticles in the preparation of a surface-enhanced Raman scattering detection kit. Background Technology

[0002] Once nanomaterials are introduced into a biological environment, proteins and other biomolecules from the surrounding environment will quickly adsorb onto the surface of the nanomaterials, thereby generating a biomolecular layer composed of proteins. This "protein crown" effect directly affects the generation of Raman signals of the analyte molecules.

[0003] CN111474162A discloses a SERS-labeled nanoprobe with a gold-hexamethylenetetramine-mesoporous silica structure. It employs a one-pot two-step method to encapsulate gold nanoparticles with mesoporous silica. First, activated gold nanoparticles are mixed with hexadecyltrimethylammonium bromide (CTAB) and tetraethyl orthosilicate (TEOS) to form a dense silica layer on the surface of the gold nanoparticles, serving as a reduction template. Then, ammonia is used for reduction, creating loose pores on the template surface and forming a thin layer of mesoporous silica on the surface of the gold nanoparticles. Excess CTAB is then washed away to obtain the mesoporous silica-encapsulated gold nanoparticles, i.e., the SERS-labeled nanoprobe with the gold-hexamethylenetetramine-mesoporous silica structure. The mesoporous silica coating on the probe surface forms a molecular sieve-like structure, avoiding the influence of intracellular biomolecules such as proteins on Raman signal molecules, further improving the stability and accuracy of the Raman signal.

[0004] However, experimental studies have found that existing surface-enhanced Raman scattering (SERS) labeled nanoprobes still have the problem of residual adsorbed proteins forming a "protein crown" effect, and the stability and accuracy of Raman signals still need to be improved. Summary of the Invention

[0005] The purpose of this disclosure is to further mitigate the “protein crown” effect of SERS-labeled nanoprobes and improve the stability and accuracy of Raman signals.

[0006] To achieve the above objectives, this disclosure provides a method for preparing nanoparticles, the method comprising: mixing amino-functionalized polyethylene nanoparticles with nano-metal particles to obtain polyethylene nanoparticles with the nano-metal particles bound to the particle surface; mixing the polyethylene nanoparticles with the nano-metal particles bound to the particle surface with triethanolamine, hexadecyltrimethylammonium bromide and tetraethoxysilane to obtain polyethylene nanoparticles coated with a mesoporous silica shell; and calcining the polyethylene nanoparticles coated with the mesoporous silica shell to obtain calcined particles.

[0007] Optionally, the nano-metal particles have a particle size of 15-20 nm; the hollow mesoporous silica microspheres have a particle size of 300-700 nm and a wall thickness of 50-200 nm.

[0008] Optionally, the polyethylene nanoparticles have a particle size of 100-650 nm.

[0009] Optionally, the method further includes: dispersing polyethylene nanoparticles in ethanol, then adding 3-aminopropyltriethoxysilane and stirring at 10-30°C for 10-30 hours to obtain amino-functionalized polyethylene nanoparticles. The amount of ethanol used is 10-30 mL and the amount of 3-aminopropyltriethoxysilane is 50-400 μL relative to 100 mg of the polyethylene nanoparticles.

[0010] Optionally, wherein the nano-metal particles are gold nanoparticles, the method further includes preparing gold nanoparticles by heating an aqueous solution of chloroauric acid to boiling, and then adding an aqueous solution of trisodium citrate to turn the liquid purple-red, thereby obtaining gold nanoparticles.

[0011] Optionally, the concentration of the chloroauric acid aqueous solution is 0.1-10 mM, the concentration of the trisodium citrate aqueous solution is 0.5-2 wt%, and the amount of trisodium citrate used is 1-2 g relative to 1 mmol of chloroauric acid.

[0012] Optionally, the amount of gold nanoparticles used is 0.01-0.05 mmol, the amount of triethanolamine is 20-160 mg, the amount of hexadecyltrimethylammonium bromide is 25-200 mg, and the amount of tetraethoxysilane is 50-600 μL, relative to 100 mg of the polyethylene nanoparticles.

[0013] Optionally, the amount of 3-mercaptophenylboronic acid used is 0.1-0.5 mmol, the amount of 4-mercaptobenzoic acid used is 0.1-0.5 mmol, the amount of ethanol used is 10-30 mL, and the amount of liposomes used is 10-100 mg, relative to 100 mg of the hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall.

[0014] Optionally, the calcination is carried out in air at a temperature of 450-550°C for 2-8 hours.

[0015] This disclosure also provides nanoparticles prepared by the method described above.

[0016] This disclosure also provides the use of the nanoparticles in the preparation of a detection kit for surface-enhanced Raman scattering.

[0017] Through the above technical solution, the mesoporous silica shell in the nanoparticles of this disclosure forms an ideal hollow mesoporous structure after calcination. This structure can adsorb more hydroxide ions in aqueous solution, resulting in lower electronegativity. Consequently, it is more difficult for the nanoparticles to bind with negatively charged protein molecules in aqueous solution to form a "protein crown" effect. Furthermore, the nano-metal particles in the nanoparticles of this disclosure exhibit better Raman scattering surface enhancement capabilities after calcination. Therefore, this disclosure further improves the stability and accuracy of the Raman signal.

[0018] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0020] Figure 1 This is a schematic diagram of the structure of the calcined nanoparticles in Example 1.

[0021] Figure 2 This is a schematic diagram of the structure of the uncalcined nanoparticles in Comparative Example 1. Specific implementation methods

[0022] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0023] This disclosure provides a method for preparing nanoparticles, the method comprising: mixing amino-functionalized polyethylene nanoparticles with nano-metal particles to obtain polyethylene nanoparticles with the nano-metal particles bound to the particle surface; mixing the polyethylene nanoparticles with the nano-metal particles bound to the particle surface with triethanolamine, hexadecyltrimethylammonium bromide and tetraethoxysilane to obtain polyethylene nanoparticles coated with a mesoporous silica shell; and calcining the polyethylene nanoparticles coated with the mesoporous silica shell to obtain calcined particles.

[0024] Optionally, the nano-metal particles have a particle size of 15-20 nm; the hollow mesoporous silica microspheres have a particle size of 300-700 nm and a wall thickness of 50-200 nm.

[0025] Optionally, the polyethylene nanoparticles have a particle size of 100-650 nm.

[0026] Optionally, the method further includes: dispersing polyethylene nanoparticles in ethanol, then adding 3-aminopropyltriethoxysilane and stirring at 10-30°C for 10-30 hours to obtain amino-functionalized polyethylene nanoparticles. The amount of ethanol used is 10-30 mL and the amount of 3-aminopropyltriethoxysilane is 50-400 μL relative to 100 mg of the polyethylene nanoparticles.

[0027] Optionally, wherein the nano-metal particles are gold nanoparticles, the method further includes preparing gold nanoparticles by heating an aqueous solution of chloroauric acid to boiling, and then adding an aqueous solution of trisodium citrate to turn the liquid purple-red, thereby obtaining gold nanoparticles.

[0028] Optionally, the concentration of the chloroauric acid aqueous solution is 0.1-10 mM, the concentration of the trisodium citrate aqueous solution is 0.5-2 wt%, and the amount of trisodium citrate used is 1-2 g relative to 1 mmol of chloroauric acid.

[0029] Optionally, the amount of gold nanoparticles used is 0.01-0.05 mmol, the amount of triethanolamine is 20-160 mg, the amount of hexadecyltrimethylammonium bromide is 25-200 mg, and the amount of tetraethoxysilane is 50-600 μL, relative to 100 mg of the polyethylene nanoparticles.

[0030] Optionally, the amount of 3-mercaptophenylboronic acid used is 0.1-0.5 mmol, the amount of 4-mercaptobenzoic acid used is 0.1-0.5 mmol, the amount of ethanol used is 10-30 mL, and the amount of liposomes used is 10-100 mg, relative to 100 mg of the hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall.

[0031] Optionally, the calcination is carried out in air at a temperature of 450-550°C for 2-8 hours.

[0032] This disclosure also provides nanoparticles prepared by the method described above.

[0033] This disclosure also provides the use of the nanoparticles in the preparation of a detection kit for surface-enhanced Raman scattering.

[0034] The present invention will be further described in detail below through examples.

[0035] Example 1

[0036] 150 μL of HAuCl4 (100 mM) was added to 30 mL of ultrapure water, and the mixture was heated to 100 °C to boil. A solution of trisodium citrate dihydrate (1.5 mL) was slowly added to the above solution using a syringe. The color of the mixture changed from brownish-yellow to purplish-red, yielding gold particles with a diameter of approximately 15-20 nm.

[0037] 50 mg of polyethylene (PS) particles (150 nm in diameter) were uniformly dispersed in 10 mL of ethanol solution. 75 μL of LAPTES was added, and the mixture was stirred at room temperature for 12 h to obtain amino-functionalized PS particles (PS-NH2). The product was washed 3-5 times by centrifugation with ethanol. The obtained PS-NH2 nanoparticles were added to the synthesized gold particles and stirred at room temperature for 24 h. The nanoparticles obtained by centrifugation were washed 3 times with ethanol and dispersed in 20 mL of water to obtain polyethylene nanoparticles with the aforementioned metal nanoparticles bound to their surface.

[0038] 30 mg of triethanolamine (TEOA) and 50 mg of cetyltrimethylammonium bromide (CTAB) were added to polyethylene nanoparticles with the aforementioned metal nanoparticles attached to their surface. The mixed solution was heated to 80 °C in an oil bath. 100 μL of tetraethoxysilane (TEOS) was slowly added dropwise. After reacting for 2 hours, a layer of mesoporous silica grew on the particle surface, resulting in polyethylene nanoparticles encapsulated in a mesoporous silica shell. After the reaction was complete, the nanoparticles were washed five times with N,N-dimethylformamide (DMF) to dissolve them, yielding hollow mesoporous silica microspheres with metal nanoparticles loaded on their inner walls.

[0039] Hollow mesoporous silica microspheres with inner walls loaded with nano-metal particles were calcined in air at 500°C for 4 hours, and then naturally cooled to obtain the nanoparticles of this embodiment. A schematic diagram of the structure of the nanoparticles is shown below. Figure 1 As shown, the Zeta potential is -18.8mV.

[0040] Comparative Example 1

[0041] 150 μL of HAuCl4 (100 mM) was added to 30 mL of ultrapure water, and the mixture was heated to 100 °C to boil. A solution of trisodium citrate dihydrate (1.5 mL) was slowly added to the above solution using a syringe. The color of the mixture changed from brownish-yellow to purplish-red, yielding gold particles with a diameter of approximately 15-20 nm.

[0042] 50 mg of polyethylene (PS) particles (150 nm in diameter) were uniformly dispersed in 10 mL of ethanol solution. 75 μL of LAPTES was added, and the mixture was stirred at room temperature for 12 h to obtain amino-functionalized PS particles (PS-NH2). The product was washed 3-5 times by centrifugation with ethanol. The obtained PS-NH2 nanoparticles were added to the synthesized gold particles and stirred at room temperature for 24 h. The nanoparticles obtained by centrifugation were washed 3 times with ethanol and dispersed in 20 mL of water to obtain polyethylene nanoparticles with the aforementioned metal nanoparticles bound to their surface.

[0043] 30 mg of triethanolamine (TEOA) and 50 mg of cetyltrimethylammonium bromide (CTAB) were added to polyethylene nanoparticles with the aforementioned metal nanoparticles attached to their surface. The mixed solution was heated to 80°C in an oil bath. 100 μL of tetraethoxysilane (TEOS) was slowly added dropwise. After reacting for 2 hours, a layer of mesoporous silica grew on the particle surface, resulting in polyethylene nanoparticles encapsulated in a mesoporous silica shell. After the reaction was complete, the nanoparticles were washed five times with N,N-dimethylformamide (DMF) to dissolve them, yielding hollow mesoporous silica microspheres with metal nanoparticles loaded on their inner walls, which are the nanoparticles of this comparative example. A schematic diagram of the structure of this nanoparticle is shown below. Figure 2 As shown, the Zeta potential is -7.42mV.

[0044] Test Example 1

[0045] The protein crown effect of nanoparticles in Example 1 and Comparative Example 1 was determined.

[0046] 10 mg of nanoparticles from Example 1 and Comparative Example 1 were respectively suspended in a solution containing CV(10) -5 mol / L), R6G(10 -5 The mixture was incubated and stirred for 15 min in a combined solution of 1 mol / L BSA and 35 mg / mL BSA. 15 μL of the solution was then added to the silicon wafer surface and air-dried before Raman spectroscopy was performed.

[0047] The detection conditions for Raman spectroscopy included: excitation light of 633 nm, and recording of each Raman spectrum at 400-1600 cm⁻¹. -1 The signal value within the range, for a time of 20 seconds, using 520cm -1 The signal value of the peak at that point is used for correction.

[0048] Table 1

[0049]

[0050] As can be seen from the data in Table 1, under BSA interference of 35 mg / mL, the relative intensity values ​​of the Raman spectra of the nanoparticles of Example 1 for small molecules CV and R6G are significantly higher than those of the uncalcined nanoparticles of Comparative Example 1. This indicates that the nanoparticles of this disclosure further reduce the "protein crown" effect of SERS-labeled nanoprobes and improve the stability and accuracy of the Raman signal.

[0051] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0052] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0053] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A method for preparing nanoparticles, characterized in that, The method includes: Amino-functionalized polyethylene nanoparticles are mixed with nano-metal particles to obtain polyethylene nanoparticles with the nano-metal particles attached to the particle surface. The polyethylene nanoparticles with the nano-metal particles bonded to the particle surface are mixed with triethanolamine, hexadecyltrimethylammonium bromide and tetraethoxysilane to obtain polyethylene nanoparticles with a mesoporous silica shell. Polyethylene nanoparticles encapsulated with a mesoporous silica shell were calcined to obtain calcined hollow mesoporous silica microspheres.

2. The method according to claim 1, wherein, The nano-metal particles have a particle size of 15-20 nm; the hollow mesoporous silica microspheres have a particle size of 300-700 nm and a wall thickness of 50-200 nm.

3. The method according to claim 1, wherein, The polyethylene nanoparticles have a particle size of 100-650 nm; The method further includes: dispersing polyethylene nanoparticles in ethanol, then adding 3-aminopropyltriethoxysilane and stirring at 10-30°C for 10-30 hours to obtain amino-functionalized polyethylene nanoparticles. The amount of ethanol used is 10-30 mL and the amount of 3-aminopropyltriethoxysilane is 50-400 μL relative to 100 mg of the polyethylene nanoparticles.

4. The method according to claim 1 or 3, wherein, The nano-metal particles are gold nanoparticles, and the method further includes preparing gold nanoparticles by the following steps: Chloroauric acid aqueous solution was heated to boiling, and then trisodium citrate aqueous solution was added to turn the liquid purple-red, thus obtaining nano-gold.

5. The method according to claim 4, wherein, The concentration of the chloroauric acid aqueous solution is 0.1-10 mM, the concentration of the trisodium citrate aqueous solution is 0.5-2 wt%, and the amount of trisodium citrate used is 1-2 g relative to 1 mmol of chloroauric acid.

6. The method according to claim 1, wherein, Relative to 100 mg of the polyethylene nanoparticles, the amount of gold nanoparticles used is 0.01-0.05 mmol, the amount of triethanolamine is 20-160 mg, the amount of hexadecyltrimethylammonium bromide is 25-200 mg, and the amount of tetraethoxysilane is 50-600 μL.

7. The method according to claim 1, wherein, Compared to 100 mg of hollow mesoporous silica microspheres with nano-metal particles loaded on the inner wall, the amount of 3-mercaptophenylboronic acid used is 0.1-0.5 mmol, the amount of 4-mercaptobenzoic acid used is 0.1-0.5 mmol, the amount of ethanol used is 10-30 mL, and the amount of liposomes used is 10-100 mg.

8. The method according to claim 1, wherein, The calcination is carried out in air at a temperature of 450-550°C for 2-8 hours.

9. Nanoparticles prepared by the method according to any one of claims 1-8.

10. Use of the nanoparticles of claim 9 in the preparation of a surface-enhanced Raman scattering detection kit.

Images