CN113264535B

CN113264535B - Silicon dioxide nano particle with bent spikes and preparation method thereof

Info

Publication number: CN113264535B
Application number: CN202110717436.2A
Authority: CN
Inventors: 徐天兆
Original assignee: Shanghai Qiansu Biotechnology Co ltd
Current assignee: Shanghai Qiansu Biotechnology Co ltd
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2021-12-31
Anticipated expiration: 2041-06-28
Also published as: CN113264535A

Abstract

The invention belongs to the field of biological medicine chemistry, and particularly relates to a spike bending virus-like silicon dioxide nano particle and a preparation method thereof. The specific technical scheme is as follows: the silica nanoparticle comprises an inner core and spikes, wherein the diameter of the inner core is 5-100 nm, and a plurality of nanometer spikes with controllable quantity and shape grow in situ on the surface of the inner core. The invention provides the small-size virus-like silica nano-particles which can accurately control the diameter of the inner core, the number of the spikes and the shapes of the spikes for the first time; and further synthesizing into functional virus-shaped silicon dioxide nano particles which can be rapidly degraded; and further, the transfection kit is prepared by using the recombinant plasmid, so that the transfection efficiency is high, and the cell survival rate is high.

Description

Silicon dioxide nano particle with bent spikes and preparation method thereof

Technical Field

The invention belongs to the field of biomedical chemistry, and particularly relates to a silicon dioxide nanoparticle with a bent spike and a preparation method thereof.

Background

Compared with the traditional medicine, the nano-technology-based medicine delivery, targeting property of diagnosis and treatment system, bioavailability and the like have obvious advantages. In various complex applications, it is often desirable that nanocarriers adhere rapidly across biological membranes like viruses and concentrate in large numbers at the site of a lesion. Currently, research on viroid nanoparticles is limited, and if surface chemistry modification is carried out on the viroid nanoparticles, cytotoxicity may be increased; if a rough surface structure is built on the surface of the material, the known building process is very complicated and the controllability of the building process is poor. Therefore, growing some nanostructures in situ on the surface of the material to form an anti-virus structure is one of the most potential ways.

Although some silica nanoparticle materials with an anti-virus structure can be prepared, the prepared nano materials are generally large in size and difficult to control within 100nm or even 80nm, and the materials with smaller sizes can penetrate biological membranes/cross biological barriers more easily. Meanwhile, in the existing research, only two options of growing spikes in situ and not growing spikes are provided, and the number and the shape of the spikes cannot be controlled, so that the application scene of the nano material is limited. In addition, the known silicon dioxide nano material has slow biodegradation and single function, and is difficult to meet increasingly complex biological application scenes.

In addition, the spikes of the silica nanoparticles prepared in the prior art are all long and straight, and cannot be prepared into other shapes. Whereas curved spikes are more easily associated with cells or penetrate biological membranes/cross biological barriers than long straight spikes.

In summary, there is a need for a biodegradable silica nanoparticle with a curved surface spike in the fields of scientific research and medical application.

Disclosure of Invention

The invention aims to provide a silicon dioxide nano particle with a bent spike and a preparation method thereof.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: the silica nanoparticle comprises an inner core and spikes, wherein the diameter of the inner core is 5-100 nm, and a plurality of nano spikes with curved shapes in quantity grow on the surface of the inner core in situ.

Preferably, the inner core is a hollow sphere or a concentric sphere structure.

Preferably, the number of the nano-spikes is more than or equal to 10.

Preferably, the spikes are cylindrical or oblate cylindrical in shape.

Correspondingly, the preparation method of the silicon dioxide nano particles comprises the steps of stirring a silicon source, a catalyst, a surfactant and water at 70-80 ℃ to form a microemulsion system, and continuously stirring for reaction to obtain the silicon dioxide nano particles.

Preferably, the silicon source comprises tetraethoxysilane; and/or; the catalyst is NaOH or tetrapropylammonium hydroxide; and/or; the surfactant is cetyl trimethyl ammonium bromide.

Preferably, the silicon source is ethyl orthosilicate, the catalyst is NaOH, and the surfactant is cetyl trimethyl ammonium bromide, wherein the mass ratio of the cetyl trimethyl ammonium bromide: NaOH: ethyl orthosilicate: cyclohexane: h₂O＝2：0.05：7：23.4：100。

Preferably, the silicon source is ethyl orthosilicate, the catalyst is tetrapropylammonium hydroxide, the surfactant is cetyl trimethyl ammonium bromide, and the mass ratio of the cetyl trimethyl ammonium bromide: tetrapropylammonium hydroxide: ethyl orthosilicate: cyclohexane: h₂O＝2：0.28：7：23.4：100。

Preferably, the reaction system also comprises rare earth elements.

Correspondingly, the reagent, the test paper, the detection card and the kit are prepared by utilizing the silicon dioxide nano particles.

The invention has the following beneficial effects:

the invention discovers for the first time that: under the condition of jointly controlling the dosage and the temperature of each reactant, a stable micro-emulsion system is formed, so that the silicon dioxide nano-particles with bent spikes can be obtained. In addition, when tetrapropylammonium hydroxide is used, silica nanoparticles having bent spikes can also be obtained. Bent spikes are more likely to bind to cells or penetrate biological membranes, crossing biological barriers. The invention enriches the types of the silicon dioxide nano particles and increases the application direction of the particles

In order to further improve the applicability, the invention also synthesizes the functional virus-shaped silicon dioxide nano particles which can be quickly degraded by a method of introducing a tetrasulfide bond silicon source or introducing a rare earth element. And further, the transfection kit is prepared by using the recombinant plasmid, so that the transfection efficiency is high, and the cell survival rate is high.

Drawings

FIG. 1 is an electron microscope scanning image of silica nanoparticles with 10-15 nano-columns;

FIG. 2 is an electron microscope scanning image of silica nanoparticles with 30-35 nano-columns;

FIG. 3 is an electron microscope scanning image of silica nanoparticles with a number of nanopillars of 50-60;

FIG. 4 is an electron microscope scan of silica nanoparticles with a core size of 30 nm;

FIG. 5 is an electron microscope scan of silica nanoparticles with a core size of 45 nm;

FIG. 6 is an electron microscope scan of silica nanoparticles with an inner core size of 65 nm;

FIG. 7 is an electron microscope scan of silica nanoparticles with a core size of 50 nm;

FIG. 8 is a scanning image of a projection electron microscope of silica nanoparticles having an inner core size of 10 nm;

FIG. 9 is a scanning electron microscope scan of silica nanoparticles with a core size of 10 nm;

FIG. 10 is an electron microscope scan of silica nanoparticles with a core size of 18 nm;

FIG. 11 is an electron microscope scan of silica nanoparticles with a core size of 30 nm;

FIG. 12 is a scanning electron microscope projection image of silica nanoparticles having an overall size of 30 nm;

FIG. 13 is a scanning electron microscope scan of silica nanoparticles having an overall size of 30 nm;

FIG. 14 is an electron microscope scanning image of silicon dioxide nanoparticles with spike bending obtained by regulating temperature;

FIG. 15 is an electron microscope scan of a spike bend silica nanoparticle obtained with a modified catalyst;

FIG. 16 is a transmission electron micrograph of silica nanoparticles in a GSH solution for 0 h;

FIG. 17 is a transmission electron micrograph of silica nanoparticles in a GSH solution for 1 h;

fig. 18 is a projection electron micrograph of silica nanoparticles in GSH solution for 4 h.

Detailed Description

The invention provides a small-size inner core virus-imitating silicon dioxide nano particle. The silicon dioxide nano particles consist of an inner core and spikes, wherein the particle size of the inner core is 5-100 nm, preferably 5-80 nm. The inner core is a porous spherical structure or a concentric spherical structure; the concentric spherical structure means that the whole inner core is spherical, a through hole is arranged in the middle of the spherical structure, and the cross section of the concentric spherical structure is concentric. And growing a plurality of nano spikes with controllable quantity and shape on the surface of the inner core in situ, wherein the length of the spikes is 6-70 nm, the cross section width of the spikes is 6-10 nm, and the quantity of the spikes can be 15, 35, 60 or other. The spikes are cylindrical or flat-column shaped, and the spikes are long, straight or curved.

The more preferable scheme is as follows: the silica nanoparticles can be rapidly biodegradable.

The invention also provides a preparation process of the silicon dioxide nano particles, which comprises the following steps:

1. preparing mixed aqueous solution of CTAB (cetyl trimethyl ammonium bromide) and NaOH, wherein the preparation method comprises the following steps: in terms of mass ratio, CTAB：NaOH：H₂O ═ 0.1 to 2: 0.05: 100, adding CTAB and NaOH into water, heating to 60 ℃, and stirring to dissolve.

2. According to the mass ratio, CTAB: NaOH: TEOS: cyclohexane: h₂O ═ 0.1 to 2: 0.05: 7: (2-23): 100, TEOS (tetraethyl orthosilicate) and cyclohexane were added. And (3) rapidly stirring at 60 ℃ (400-1500 rpm) to form a stable microemulsion system. And continuously stirring and reacting for 36-72 hours to obtain the required silicon dioxide nano particles.

Wherein, the diameter of the inner core is gradually increased along with the increase of CTAB concentration, and is reduced along with the increase of stirring speed. When only CTAB is used, the inner core is spherical; when CTAB and gemini surfactant are used in combination (the gemini surfactant is N, N' -bi (tetradecyl dimethyl) -1, 2-dibromide-ethanediaminium salt, the gemini surfactant and CTAB are equal in mass), the inner core is in a concentric sphere shape.

In one embodiment, the remaining conditions are unchanged, and when the reaction temperature is increased from 60 ℃ to 70-80 ℃, the spikes are bent from a long straight shape. The other implementation mode is as follows: and changing NaOH into tetrapropylammonium hydroxide with the same molar weight under the condition that other conditions are not changed, wherein the spikes are bent from long straight shape. In some cases, curved spikes are more likely to bind to cells, or penetrate biological membranes/cross biological barriers, than long straight spikes.

In one embodiment, the remaining conditions are unchanged, the stirring speed is set at 600rpm, the reaction temperature is increased from 60 ℃ to 70 ℃, and CTAB is replaced by gemini surfactant (N, N' -ditetradecyldimethyl-1, 2-dibromide-ethylenediammonium salt, abbreviated as C_14-2-14) The spike shape is changed from cylindrical to flat cylindrical. Compared with cylindrical spikes, the flat-column spikes with the nanometer scale can be matched with the shapes of special biological membrane surface proteins, and certain special biological effects are generated, such as easier targeting, influence on surface protein mechanical force factors, virus inhibition and the like. Therefore, the shape of the surface nano structure is accurately controlled, and the method has important biomedical research and application values.

The preferable scheme is as follows: when silicon is introduced, other ions are simultaneously doped, so that functional silica nanoparticles are obtained. The silane containing the tetrasulfide bond and/or the rare earth element are doped, so that the silicon dioxide nano particles can obtain the biodegradable performance. For example, incorporation of Gd can impart both biodegradation and nuclear magnetic imaging functionality to the silica nanoparticles; for another example, by doping Mn/Ce, the obtained silica nanoparticles can be not only biodegradable, but also used for preparing inorganic nanoenzymes.

In one embodiment, the incorporation of tetrasulfide bond-containing silanes can effectively increase the biodegradability of the silica nanoparticles. The specific implementation mode is as follows: using TEOS and bis- [3- (triethoxysilyl) propyl group]-tetrasulfide silane (Bis [3- (triethoxysilyl) propyl)]tetrasulfide, abbreviated as bis-sulfurr silane) is a mixed silicon source, replaces TEOS in the step 2, adjusts the dosage proportion of each substance, and has the same steps as the rest steps, thereby preparing the silicon dioxide nano particles capable of being rapidly biodegraded. In the whole system, according to the mass ratio, CTAB: NaOH: TEOS: bis-sulfurs silane: cyclohexane: h₂O ═ 0.1 to 2: 0.05: 6: (0.1-2): (2-23): 100. the greater the amount of bis-sulfurs silane used, the faster the degradation rate.

3. After the reaction, the aqueous phase was centrifuged, and the centrifuged product was washed with deionized water and ethanol three times each. The washed product was dispersed in a hydrochloric acid-ethanol solution (hydrochloric acid: ethanol ═ 1: 100, v/v). Extracting surfactant (CTAB) under heating and refluxing at 80 deg.C, and repeatedly extracting for 3 times, each for 12 hr. And (4) performing centrifugal separation again, washing the centrifugate three times by using deionized water and ethanol, and performing vacuum drying at room temperature to obtain a final product.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The first embodiment is as follows: preparation of silica nanoparticles with different numbers of Nano-spikes

Adding CTAB into 20mL of water, stirring at the speed of 500rpm at the temperature of 60 ℃, stirring until the CTAB is fully dissolved, then adding 0.01g of NaOH, continuing to stir for 30 minutes, then adding a mixed solution of cyclohexane and TEOS (4.68g of cyclohexane and 1.4g of TEOS), and continuing to stir at 60 ℃ for reaction for 48 hours to obtain the silica nano material with the virus-like structure. In the specific reaction process, when the consumption of CTAB is respectively 0.15g, 0.2g and 0.3g (when the concentration of CTAB is respectively 0.75 wt%, 1.0 wt% and 1.5 wt%), the quantity of the obtained silica nanoparticle surface nano-columns with the virus-like structure is respectively as follows: 10-15 (as shown in figure 1), 30-35 (as shown in figure 2) and 50-60 (as shown in figure 3).

Example two: preparation of silica nanoparticles with different core diameters

1. Obtaining small-size (30-80 nm) silicon dioxide nanoparticles: according to the mass ratio, CTAB: NaOH: h₂O ═ 0.1 to 2: 0.05: preparing mixed aqueous solution of CTAB and NaOH at 100 and 60 ℃. And adding TEOS and cyclohexane, wherein the mass ratio of CTAB: NaOH: TEOS: cyclohexane: h₂O ═ 0.1 to 2: 0.05: 7: 23: 100, and rapidly stirring (400-1500 rpm) at 60 ℃ to form a stable microemulsion system. And continuously stirring and reacting for 48 hours to obtain the silica nano material with the virus-like structure.

By adjusting the stirring speed, and simultaneously adjusting the initial concentration of CTAB, the size of the viroid silica nanoparticles can be accurately adjusted. The control parameters, corresponding kernel sizes and corresponding graphs are shown in table 1.

TABLE 1 comparative table for silica nanoparticle size control

It was found that the inner core of the resulting nanoparticles was reduced by merely increasing the stirring speed, but this also affected the formation of surface nanocolumns in many cases, and a good pseudoviral structure could not be obtained. Further, we have found that if the stirring speed is increased while the surfactant concentration is adjusted (decreased), it is possible to ensure the growth and formation of surface nanocolumns, resulting in a viroid-like structure. For example, when the concentration of CTAB is 1.5 wt% and the stirring speed is 600rpm, the virus-like silica nano-particles with 65nm inner cores are obtained; however, if the stirring speed is simply increased to 1000rpm, viral silica spheres cannot be obtained, and only ordinary silica spheres without a nanopillar on the surface can be obtained. If we simultaneously increase the stirring rate to 1000rpm and simultaneously decrease the surfactant concentration to 0.25 wt%, viroid-like silica nanoparticles having an inner core of 30nm can be obtained. Therefore, we believe that, in general, 2 parameters need to be simultaneously regulated so as not to affect the formation of surface nano-pillars when the core size of the virus-like silica nanoparticles is regulated.

2. Obtaining the silicon dioxide nano particles with ultra-small core size (the core is 10-30 nm):

meanwhile, the stirring speed and the concentration of CTAB are adjusted, and the virus-like silica nano particles with ultra-small inner core size cannot be obtained. We further explore and find that adjusting stirring speed, CTAB concentration and TEOS initial concentration simultaneously (but ensuring the total amount to be unchanged, so we can ensure the change of the initial concentration and the total amount of TEOS to be unchanged by adding TEOS in multiple times, specifically, adding TEOS in 2-3 parts in batches, adding TEOS every 1 time, adding TEOS every 12 h), and adding TEOS in multiple times to obtain virus-like nanoparticles with ultra-small cores. In the present embodiment, CTAB: NaOH: TEOS: cyclohexane: h₂O ═ 0.5: 0.05: 7: 23: 100. the stirring speed was 800rpm and the TEOS was added in equal portions (for example, in group 2, in 2 portions, 0.7g each), as specified in the following table, with a 12 hour time interval between two consecutive TEOS additions and a 60 ℃ temperature. Further, virus-like silica nanoparticles having 10nm, 18nm and 30nm inner cores were obtained.

TABLE 2 comparative table for silica nanoparticle size control

3. Obtaining ultra-small sized viroid nanoparticles: 0.08g of CTAB was added to 20mL of water, the stirring speed was 800rpm, the temperature was 60 ℃, the mixture was stirred until the CTAB was sufficiently dissolved, 0.01g of NaOH was added, the stirring was continued for 30 minutes, a mixed solution of cyclohexane and TEOS (3g of cyclohexane and 1.4g of TEOS) was added, and the reaction was continued at 70 ℃ and 800rpm for 48 hours, whereby viroid-like nanoparticles having an overall size of about 30nm were obtained (FIGS. 12 and 13).

The research finds that: meanwhile, the consumption of cyclohexane and CTAB is reduced, the rotating speed is improved, and the virus-imitating nano particles with ultra-small sizes can be obtained; and the step of adding TEOS in batches is the key for obtaining the virus-like nanoparticles with ultra-small core size.

Example three: preparation of silica nanoparticles with spikes of specific shape

1. The spike shape was controlled by varying the reaction temperature.

Adding 0.4g of CTAB into 20mL of water, stirring at 600rpm and 70 ℃ until the CTAB is fully dissolved, then adding 0.01g of NaOH, continuing to stir for 30 minutes, then adding a mixed solution of cyclohexane and TEOS (4.68g of cyclohexane and 1.4g of TEOS), and continuing to stir and react for 48 hours at 70 ℃ to obtain the silica nano material with the virus-like structure, wherein the spikes of the silica nano material are obviously bent. The scanning electron microscope is shown in FIG. 14.

2. The spike shape is controlled by changing the catalyst.

Adding 0.4g of CTAB into 20mL of water, stirring at 600rpm and 60 ℃ until the CTAB is fully dissolved, then adding 0.056g of tetrapropylammonium hydroxide, continuing to stir for 30 minutes, then adding a mixed solution of cyclohexane and TEOS (4.68g of cyclohexane and 1.4g of TEOS), and continuing to stir and react for 48 hours at 70 ℃ to obtain the silica nano-material with the virus-like structure, wherein the spikes of the silica nano-material are obviously bent. The scanning electron microscope is shown in FIG. 15.

Example four: preparation of rapidly biodegradable silica nanoparticles

1. 20mL of water, 0.4g of CTAB and 0.01g of NaOH were added, heated to 60 ℃ and dissolved with stirring. Then adding a mixed solution of TEOS, bis-sulfury silane and cyclohexane (4.68g of cyclohexane +1.2g of TEOS +0.3g of bis-sulfury silane), wherein the mass ratio of various substances in the whole system is CTAB: NaOH: TEOS: bis-sulfurs silane: cyclohexane: h₂O is 1: 0.05: 6: 1.5: 23: 100, and rapidly stirring at the temperature of 60 ℃ to form a stable microemulsion system. And continuously stirring and reacting for 48 hours to obtain the rapidly degradable silica nano particles with the virus-like structure.

2. The synthesized silica nanoparticles were dispersed in a 10mM GSH (reduced glutathione) solution, and the dissolution of the silica nanoparticles was observed at 37 ℃. The degradation of the viromimetic silica nanoparticles started about 1 hour and was complete about 4 hours. Fig. 16, 17, 18 are tem images at 0h, 1h and 4h in GSH solution, respectively.

Example five: kit for preparing silica nanoparticles by utilizing rapid biodegradation

1. The plasmids, siRNA, microRNA mics and microRNA inhibitors used in this example were diluted in serum-free DMEM medium or MEM medium from GIBICO.

Transfection kits were prepared using the silica nanoparticles prepared in example four. The kit is specifically shown in table 3. Of course, kits and the like may also be prepared by those skilled in the art according to methods well known in the art. It should be noted that: table 3 shows the proposed system for different plates and nucleic acid formats.

Table 3 display table for each system

2. The desired cells (suspension cells or adherent cells) are transfected using the kit. According to the conventional technology in the field. After transfection and culture, the mRNA and protein levels were measured. Cell inoculation: cells were seeded the day before transfection and the next day the cell density was around 80%. Preparation of transfection mixture: the transfection reagent (silica nanoparticles) and nucleic acid (siRNA) were diluted separately using serum-free medium, and after 5min of incubation, the two were mixed and incubated at room temperature for about 20 min. Transfection: adding the mixture into the culture hole, slightly shaking the culture plate, and mixing uniformly. Culturing in an incubator at 37 ℃ for 18-72 h, and detecting the gene inhibition effect. Generally, the fluorescent protein can be observed to express in 24 hours, mRNA level is detected in 24-72 hours, and protein level is detected in 36-72 hours.

This example tested 8 different cells: miapaca-2, MKN-74, NCI-1299, MDA-MB-231, cho, A549, HEK293, RBL-2H3 cells. Transfection efficiency and survival rate were tested separately.

And (3) testing efficiency: when the cells were grown in 24-well plates until the cell fusion rate reached 70%, the silica nanoparticle transfection kit and Lip2000 transfection reagent (cat 11668019, Thermo Fisher) prepared in example four of the present invention were used to transfect GFP (cat VT1110, elite) and 0.5 μ g GFP per well cell and 20pmol of fluorescently labeled siRNA negative control NC per well, respectively. The green fluorescence was observed 48h after transfection using a fluorescence microscope and photographed, and the results are shown in Table 4 (no fluorescence plot is provided due to the black and white plot limitation). All data are expressed as (mean ± sd) and processed using SPSS 22.0 statistical software. Comparisons between groups were analyzed using one-way anova. P < 0.01 indicates significant statistical differences.

TABLE 4 comparison of cell transfection efficiencies

The kit provided by the invention has high transfection efficiency on several cells to be tested. Wherein, the transfection efficiency of the A549 cells, the NCI-1299 cells and the RBL-2H3 cells is obviously higher than that of the cationic liposome Lip2000 transfection reagent.

And (3) survival rate testing: when 293 cells grow in a 24-well plate until the cell fusion rate reaches 70%, a transfection reagent prepared by the silica nanoparticle transfection kit and a Lip2000 transfection reagent are respectively added for incubation for different times, and then the cell survival rate is determined. After the co-incubation for 6h, 12h, 24h and 48h, 10 μ L of CCK8 solution (product number CK04, DOJINDO) was added to each well, incubation was performed for 1-4 h at 37 ℃, absorbance at 450nm was measured by an enzyme reader, and the cell survival rate was calculated, with the results shown in Table 5.

TABLE 5 comparison of cell viability

Treatment group	6h	12h	24h	48h
					The invention	99.078±0.2691	97.0478±0.2168	94.6512±0.0294	91.8467±0.2517
lipo2000	99.032±0.1482	96.9634±0.0247	94.2191±0.2034	88.6425±0.3412

When the co-incubation time is not less than 6h, the cell survival rate of the kit provided by the invention is obviously higher than that of the Lip2000 transfection reagent. The kit provided by the invention has good biocompatibility, low immunogenicity and low toxicity, and has better safety and high cell survival rate when used for gene transfection compared with the common Lip2000 transfection reagent.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes, modifications, alterations, and substitutions which may be made by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A silica nanoparticle characterized by: the silica nano particles comprise an inner core and spikes, the diameter of the inner core is 5-100 nm, and a plurality of nano spikes with controllable quantity and shape grow in situ on the surface of the inner core; the spikes are curved in shape.

2. Silica nanoparticles according to claim 1, characterized in that: the inner core is a hollow sphere or a concentric spherical structure.

3. Silica nanoparticles according to claim 1, characterized in that: the number of the nano spikes is more than or equal to 10.

4. Silica nanoparticles according to claim 1, characterized in that: the spikes are cylindrical or flat-column shaped.

5. A method for preparing silica nanoparticles according to any one of claims 1 to 4, characterized in that: the reaction system comprises a silicon source, a catalyst, a surfactant and water, all substances in the reaction system are stirred at 70-80 ℃ to form a microemulsion system, and the materials are continuously stirred for reaction to obtain the silicon dioxide nano particles.

6. The method for preparing silica nanoparticles according to claim 5, wherein: the silicon source comprises tetraethoxysilane; the catalyst is NaOH or tetrapropylammonium hydroxide; the surfactant is cetyl trimethyl ammonium bromide.

7. The method of preparing silica nanoparticles according to claim 6, wherein: the silicon source is ethyl orthosilicate, the catalyst is NaOH, the surfactant is cetyl trimethyl ammonium bromide, and the mass ratio of the cetyl trimethyl ammonium bromide to the sodium hydroxide is as follows: NaOH: ethyl orthosilicate: cyclohexane: h₂O＝2：0.05：7：23.4：100。

8. The method of preparing silica nanoparticles according to claim 6, wherein: the silicon source is ethyl orthosilicate, the catalyst is tetrapropylammonium hydroxide, the surfactant is hexadecyl trimethyl ammonium bromide, and the mass ratio of the hexadecyl trimethyl ammonium bromide is as follows: tetrapropylammonium hydroxide: ethyl orthosilicate: cyclohexane: h₂O＝2：0.28：7：23.4：100。

9. The method for preparing silica nanoparticles according to claim 5, wherein: the reaction system also comprises rare earth elements.

10. A reagent, a test paper, a detection card and a kit prepared by using the silica nanoparticles according to any one of claims 1 to 4.