CN107598159A - A kind of noble metal nanometer material and its application with nitric oxide synthase activity - Google Patents

Abstract

The invention provides a noble metal nanomaterial with nitric oxide synthase activity and its application, including any of GNRs@Au, GNRs@Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@ _SiO2 One or a combination of at least two. The noble metal nanomaterial of the present invention can catalyze the reaction between L-arginine and NADPH to generate NO, and has the effect of significantly increasing the level of intracellular NO.

Description

A noble metal nanomaterial with nitric oxide synthase activity and its application

technical field

The invention belongs to the field of artificial simulated enzymes, relates to the enzyme-like catalytic properties of noble metal nanomaterials, in particular to a noble metal nanomaterial with nitric oxide synthase activity and its application.

Background technique

Nitric oxide (NO) is an important messenger molecule in cells, and its physiological function has received extensive attention and in-depth research. In 1998, three American scientists won the Nobel Prize in Physiology and Medicine for discovering that NO is Endothelial Cell Relaxation Factor (EDRF). NO not only plays an important role in the cardiovascular system, but also plays an important role in physiological processes such as immune regulation, cancer pathogenesis and treatment, and tissue damage repair. Therefore, NO-based disease treatment has received more and more attention.

At present, there are two main ways to increase the level of NO: one is to use catalysts to catalyze the NO donor S-nitrososulfhydryl adducts (RSNO), such as S-nitrosoalbumin (AlbSNO), S-nitrosoalbumin Nitrocysteine (CysNO) and S-nitrosoglutathione (GSNO), etc., release NO; the other is by affecting the expression of nitric oxide synthase or regulating the activity of nitric oxide synthase. Regulates the concentration of NO in the body.

Jia et al. used gold nanoparticles to induce RSNO in serum to release NO, and proved that gold nanoparticles can induce RSNO (Jia H Y, Liu Y, Zhang X J, et al. Potential Oxidative Stress of Gold Nanoparticles by Induced-NO Releasing in Serum. Journal of the American Chemical Society, 2008, 131(1):40-41.). With the emergence of more and more NO donors, there have also been some studies on the controlled release of NO from NO donors catalyzed by upconversion nanoparticles under the influence of near-infrared light (Zhang X, Tian G, Yin W, et al. al. Controllable Generation of Nitric Oxide by Near‐Infrared‐Sensitized Upconversion Nanoparticles for Tumor Therapy. Advanced Functional Materials, 2015, 25(20): 3049-3056.).

Endogenous NO is generally produced by the oxidation of L-arginine catalyzed by nitric oxide synthase. The reaction is divided into two steps: the first step is that L-arginine is oxidized to Nω- Hydroxy-L-arginine, the second step is that Nω-hydroxy-L-arginine continues to be oxidized to L-citrulline and produces NO. The two-step reaction process requires the consumption of oxygen and reduced coenzyme II (NADPH) . In 1992, researchers first pointed out that horseradish peroxidase can catalyze Nω-hydroxy-L-arginine to produce L-citrulline and NO, in which Nω-hydroxy-L-arginine is the synthesis of nitric oxide Enzyme-catalyzed conversion of L-arginine into L-citrulline and NO intermediates (Boucher J L, Genet A, Vadon S, et al.Formation of NitrogenOxides and Citrulline upon Oxidation of Nω-hydroxy-L-arginine by Hemeproteins.Biochemical and Biophysical Research Communications, 1992, 184(3): 1158-1164.). In 2002, a new study found that horseradish peroxidase can catalyze hydroxyurea to produce NO (Huang J, Sommers E M, Kim-Shapiro D B, et al. Horseradish peroxidase catalyzed nitricoxide formation from hydroxyurea. Journal of the American Chemical Society, 2002 , 124(13):3473-3480.). However, no analogs with the same function as nitric oxide synthase have been found so far, which can directly catalyze L-arginine to produce L-citrulline and NO.

In recent years, the field of nanozymes has risen rapidly, and nanozymes with different shapes, sizes and materials emerge in an endless stream. Compared with natural enzymes, nanozymes are more economical, more stable, and easier to produce on a large scale, and have become a research hotspot in various fields. Noble metal nanoparticle catalysts play an important role in the field of catalysis. Solid catalysts doped with noble metal nanoparticles show good catalytic activity, such as platinum nanoparticles with peroxidase, catalase, superoxide dismutase and oxidase activity of four enzymes. So far, no research reports on nanozymes with nitric oxide synthase activity have been found.

Contents of the invention

In view of the above problems, the present invention provides a noble metal nanomaterial with nitric oxide synthase activity and its application. The noble metal nanomaterial can catalyze the reaction between L-arginine and NADPH to generate L-citrulline and NO, and in Human cells exhibit nitric oxide synthase activity, significantly increasing the level of intracellular NO.

In a first aspect, the present invention provides a noble metal nanomaterial having nitric oxide synthase activity, comprising: GNRs@Au, GNRs@Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@SiO ₂ Any one or a combination of at least two.

The noble metal nanomaterials provided by the present invention take gold nanorods (gold nanorods, GNRs) as the core, and respectively coat Au, Ag, Au/Ag alloy, Pd, Pt or SiO ₂ outer layers on the surface to obtain GNRs@Au, GNRs @Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@SiO ₂ , have nitric oxide synthase activity, catalyze the reaction between L-arginine and NADPH to generate L-citrulline and NO, and significantly improve the Inner NO levels.

In a second aspect, the present invention provides a method for the noble metal nanomaterial catalyzing L-arginine to produce NO as described in the first aspect, comprising the following steps:

(1) adding L-arginine solution to the buffer;

(2) adding precious metal nanomaterials;

(3) Add NADPH solution to produce NO.

Preferably, the buffer in step (1) includes any one or a combination of at least two of Tris-HCl buffer, PBS buffer or phosphate buffer, preferably Tris-HCl buffer.

Preferably, the concentration of the buffer in step (1) is 10-100 mM, such as 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, preferably 50 mM.

Preferably, the buffer solution in step (1) has a pH value of 7.0-8.0, such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, preferably 7.4.

Preferably, the concentration of the L-arginine solution in step (1) is 1-1000mM, such as 1mM, 5mM, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM , 550mM, 600mM, 650mM, 700mM, 750mM, 800mM, 850mM, 900mM, 950mM or 1000mM, preferably 5-500mM, more preferably 50mM.

Preferably, the volume ratio of the L-arginine solution to the buffer in step (1) is (0.1-200):100, for example, it can be 0.1:100, 0.15:100, 0.3:100, 0.6:100 , 0.9:100, 1.5:100, 3:100, 6:100, 9:100, 15:100, 30:100, 60:100, 90:100, 150:100 or 200:100, preferably (0.3- 30):100, more preferably 3:100.

Preferably, before adding the noble metal nanomaterial in step (2), a step of adding CTAB solution to the mixture of buffer solution and L-arginine solution in step (1) is also included.

Preferably, the concentration of the CTAB solution is 1-100mM, such as 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM , 85mM, 90mM, 95mM or 100mM, preferably 10mM.

Preferably, the volume ratio of the CTAB solution to the buffer in step (1) is (1-100):200, such as 1:200, 5:200, 1:20, 2:20, 3:20 , 4:20, 5:20, 6:20, 7:20, 8:20, 9:20 or 1:2, preferably 1:20.

Preferably, the concentration of the noble metal nanomaterial in step (2) is 1.0-10nM, such as 1.0nM, 2.0nM, 3.0nM, 4.0nM, 5.0nM, 6.0nM, 7.0nM, 8.0nM, 9.0nM or 10nM , preferably 1-5 nM.

Preferably, the volume ratio of the noble metal nanomaterial in step (2) to the buffer in step (1) is (1-50):1000, such as 1:1000, 5:1000, 10:1000, 15: 1000, 20:1000, 25:1000, 30:1000, 35:1000, 40:1000, 45:1000 or 50:1000.

Preferably, the surface of the noble metal nanomaterial in step (2) is modified with CTAB.

Preferably, the concentration of the NADPH solution in step (3) is 10-1000mM, such as 10mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM , 750mM, 800mM, 850mM, 900mM, 950mM or 1000mM, preferably 50-500mM, more preferably 50mM.

Preferably, the volume ratio of the NADPH solution described in step (3) to the buffer solution described in step (1) is (0.1-50):10, such as 0.1:50, 0.35:10, 0.5:10, 0.7:10 , 3.5:10, 5:10, 7:10, 35:10 or 50:10, preferably (0.7-7):10, more preferably 7:10.

Preferably, the NADPH solution in step (3) is added in 1-5 times, for example, 1 time, 2 times, 3 times, 4 times or 5 times, preferably in 3 times.

As a preferred technical solution, a method for producing NO from L-arginine catalyzed by noble metal nanomaterials comprises the following steps:

(1) Adding a concentration of 1-1000mM L-arginine solution to a buffer solution with a pH value of 7.0-8.0 at a concentration of 10-100mM, the volume ratio of the L-arginine solution to the buffer solution For (0.1-200): 100;

(2) adding the CTAB solution that concentration is 1-100mM in the mixed solution of buffer solution and L-arginine solution described in step (1), the volume ratio of described CTAB solution and described buffer solution is (1-100 ):200;

(3) adding a noble metal nanomaterial with a concentration of 1-5nM, the volume ratio of the noble metal nanomaterial to the buffer is (1-50):1000;

(4) adding a NADPH solution with a concentration of 10-1000mM, the volume ratio of the NADPH solution to the buffer is (0.1-50):10;

(5) Under the catalysis of noble metal nanomaterials, L-arginine reacts with NADPH to generate NO.

In a third aspect, the present invention provides a method for the noble metal nanomaterials to catalyze L-arginine to produce NO in cells, comprising the following steps:

1) adding a culture medium containing noble metal nanomaterials to the cells;

2) Incubate the cells described in step 1) in a CO ₂ incubator.

Preferably, the cells in step 1) include human acute monocytic leukemia cells and/or human umbilical vein endothelial cells.

Preferably, the number of cells in step 1) is (1-5)×10 ⁵ , for example, 1×10 ⁵ , 2×10 ⁵ , 3×10 ⁵ , 4×10 ⁵ or 5 × 10 ⁵ pcs.

Preferably, the surface of the noble metal nanomaterial in step 1) is modified with NH ₂ .

Preferably, the noble metal nanomaterial in step 1) includes any one of GNRs@SiO ₂ -NH ₂ , glutathione-blocked GNRs@SiO ₂ -NH ₂ or cysteine-blocked GNRs@SiO ₂ -NH ₂ or a combination of at least two.

Preferably, the mass concentration of the noble metal nanomaterial in step 1) is 1-100 μg/mL, such as 1 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL mL, 35μg/mL, 40μg/mL, 45μg/mL, 50μg/mL, 55μg/mL, 60μg/mL, 65μg/mL, 70μg/mL, 75μg/mL, 80μg/mL, 85μg/mL, 90μg/mL, 95 μg/mL or 100 μg/mL, preferably 10-60 μg/mL.

Preferably, the incubation time in step 2) is 1-20h, such as 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h , 17h, 18h, 19h or 20h, preferably 1-15h, more preferably 2-12h.

In a fourth aspect, the present invention provides a method for detecting NO produced by noble metal nanomaterials in cells catalyzing L-arginine, comprising the following steps:

1') adding the medium containing the NO fluorescent probe to the cells;

2') incubating the cells described in step 1') in a CO ₂ incubator;

3') The fluorescence intensity of intracellular NO was analyzed by flow cytometry.

Preferably, the NO fluorescent probe described in step 1') includes 4-amino-5-methylamino-2,7-difluorofluorescein diacetate and/or 4,5-diaminofluorescein diacetic acid ester, preferably 4-amino-5-methylamino-2,7-difluorofluorescein diacetate.

Preferably, the concentration of the NO fluorescent probe in step 1') is 1-10 μM, such as 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or 10 μM, preferably 5 μM.

Preferably, the incubation time in step 2') is 5-60min, such as 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or 60min, preferably 20min.

In the present invention, the NO fluorescent probe is loaded in the cells to realize the real-time detection of NO in the cells.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The noble metal nanomaterial of the present invention has nitric oxide synthase activity, which can catalyze the reaction between L-arginine and NADPH to produce NO;

(2) When the final concentration of GNRs@Au-CTAB was 6pM, the NO generation was the largest;

(3) GNRs@AuAg-CTAB with a smooth surface has the best catalytic performance and the highest NO generation;

(4) In THP1 cells, when the concentration of GNRs@SiO ₂ -NH ₂ was 60μg/mL and the incubation time was 12h, the production of NO was the largest;

(5) In HUVEC cells, when the concentration of GNRs@SiO ₂ -NH ₂ was 60μg/mL and the incubation time was 6h, the production of NO was the largest.

Description of drawings

Figure 1(a) is the curve of the relationship between the amount of NO generation and the concentration of the noble metal nanomaterial, and Figure 1(b) is a histogram of the relationship between the amount of NO generation and the type of the noble metal nanomaterial;

Figure 2(a) is a histogram of the relationship between NO production in THP1 cells and the concentration and incubation time of GNRs@SiO ₂ -NH ₂ , and Figure 2(b) is a histogram of the relationship between NO production in THP1 cells and GNRs@SiO ₂ -NH ₂ , histogram of the relationship between the concentration of glutathione-blocked GNRs@SiO ₂ -NH ₂ and cysteine-blocked GNRs@SiO ₂ -NH ₂ ;

Figure 3(a) is a histogram of the relationship between NO production in HUVEC cells and the concentration and incubation time of GNRs@SiO ₂ -NH ₂ , and Figure 3(b) is a histogram showing the relationship between NO production in HUVEC cells and GNRs@SiO ₂ -NH ₂ , Histogram of the relationship between the concentration of glutathione-blocked GNRs@SiO ₂ -NH ₂ and cysteine-blocked GNRs@SiO ₂ -NH ₂ .

detailed description

In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through regular channels.

The influence of the concentration of the noble metal nanomaterial on the amount of NO generation of embodiment 1

Take 30 μL of L-arginine (50 mM) in 1 mL of Tris-HCl buffer (50 mM, pH=7.4), then add 50 μL of CTAB solution (10 mM), mix well and add 1.7 μL, 10 μL, 14 μL and 27 μL of 1nM of GNRs@Au-CTAB; put the NO detection electrode in the reaction solution, the immersion depth is 2-3mm, after the electrode is stable, add NADPH solution (50mM) to the reaction solution in the manner of 100μL-100μL-500μL, every The time interval is 1 min; the current change value (ΔI) after the third addition of NADPH solution is detected, and the concentration-ΔI curve of the noble metal nanostructure is drawn.

Embodiment 2 The impact of the type of noble metal nanomaterials on the amount of NO generation

Take 30μL of L-arginine (50mM) in 1mL of Tris-HCl buffer (50mM, pH=7.4), then add 5μL of L-arginine solution (0.1M), mix well and add 2μL of it at a concentration of 5nM (final concentration is 6pM) The GNRs@Au-CTAB, GNRs@Ag-CTAB, GNRs@AuAg-CTAB (rough), GNRs@AuAg-CTAB (smooth), GNRs@Pd-CTAB and GNRs@Pt-CTAB; the NO detection electrodes were placed in the reaction In the liquid, the immersion depth is 2-3mm. After the electrode is stable, add NADPH solution (50mM) into the reaction solution in the form of 100μL-100μL-500μL, with an interval of 1min each time; detect the current change after adding the NADPH solution for the third time value (ΔI).

Example 3

Take 3 μL of L-arginine (500 mM) in 1 mL of Tris-HCl buffer (50 mM, pH=7.4), then add 50 μL of CTAB solution (10 mM), mix well and add 14 μL of 1 nM (final concentration of 8 pM) GNRs@Au-CTAB, pipette and mix well; put the NO detection electrode in the reaction solution, the immersion depth is 2-3mm, after the electrode is stable, add NADPH solution (50mM) into the reaction solution in the form of 350μL-350μL, every The time interval is 1 min; the current change value (ΔI) after adding the NADPH solution for the second time is detected.

Example 4

Take 300μL L-arginine (5mM) in 1mL PBS buffer (50mM, pH=7.4), then add 5μL CTAB solution (100mM), mix well and add 1μL GNRs@ with a concentration of 10nM (final concentration 16pM) Au-CTAB; place the NO detection electrode in the reaction solution with an immersion depth of 2-3 mm. After the electrode is stable, add 70 μL of NADPH solution (500 mM) to the reaction solution to detect the current change value (ΔI).

Example 5

Take 1 μL of L-arginine (1M) in 1 mL of Tris-HCl buffer (10 mM, pH=7.0), then add 500 μL of L-arginine solution (1 mM), mix well and add 17 μL of 0.1 nM (final concentration of 1 pM) GNRs@Au-CTAB; put the NO detection electrode in the reaction solution, and the immersion depth is 2-3mm. After the electrode is stable, add 1mL-1mL-1mL-1mL-1mL-1mLNADPH solution (10mM) to the reaction solution to detect the current change value (ΔI).

Example 6

Take 2mL of L-arginine (1mM) in 1mL of phosphate buffer (100mM, pH=8.0), mix well, add 50μL of GNRs@Au with a concentration of 1nM (final concentration of 20pM), pipette and mix well; The detection electrode is placed in the reaction solution with an immersion depth of 2-3 mm. After the electrode is stabilized, 10 μL of NADPH solution (1M) is added to the reaction solution to detect the current change value (ΔI).

Comparative example 1

Compared with Example 1, the final concentration of noble metal nanomaterials is GNRs@Au-CTAB of 0.05pM, and other conditions are the same as Example 1.

Comparative example 2

Compared with Example 1, the final concentration of noble metal nanomaterials is GNRs@Au-CTAB of 25pM, and other conditions are the same as Example 1.

The experimental results are shown in Table 1.

Table 1 In vitro studies of the nitric oxide-like enzyme activity of noble metal nanomaterials

From Example 1 and Figure 1(a), it can be seen that the amount of NO produced is positively correlated with the concentration of GNRs@Au-CTAB within a certain range: with the increase of the concentration of GNRs@Au-CTAB, the amount of NO produced increases Large, when the final concentration of GNRs@Au-CTAB is 6pM, the NO production is the largest; with the further increase of the GNRs@Au-CTAB concentration, the NO production gradually decreases, which may be due to the excessive GNRs@ Au-CTAB aggregated in the solution, the specific surface area became smaller, and the catalytic performance was weakened.

From Example 2 and Figure 1(b), it can be seen that the amount of NO generated is related to the type of noble metal nanomaterials: GNRs@AuAg-CTAB with a smooth surface has the best catalytic performance, and the amount of NO generated is the highest.

From the comparison of Examples 1-6, GNRs@AuAg-CTAB with a smooth surface at a concentration of 6 pM has the best catalytic performance.

Compared with Example 1, the concentration of GNRs@Au-CTAB in Comparative Example 1 was lower, which could not effectively catalyze the reaction of L-arginine and NADPH to generate NO; the concentration of GNRs@Au-CTAB in Comparative Example 2 was higher, and the Agglomerates in the solution to form a micron structure with a larger particle size, and the catalytic performance is significantly reduced.

Catalytic performance of embodiment 7 noble metal nanomaterials in human acute monocytic leukemia cells

(1) Add 1 mL of 5 μM 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM DA) NO to 4×10 ⁶ human acute monocytic leukemia (THP1) cells Fluorescent probe solution, resuspended in a centrifuge tube, incubated at 37°C, 5% CO ₂ incubator for 20min, centrifuged to remove unloaded NO probe;

(2) Spread the cells into a 12-well plate, add 1640 medium containing GNRs@SiO ₂ -NH ₂ at a mass concentration of 0 μg/mL, 10 μg/mL, 30 μg/mL and 60 μg/mL, at 37 °C, 5 Incubate for 2h, 6h and 12h respectively in %CO ₂ incubator;

(3) After centrifuging to remove excess nanoparticles, the cells were resuspended in DMEM medium without phenol red and serum, and the fluorescence intensity of intracellular NO was detected by flow cytometry.

Example 8

Compared with Example 7, glutathione is used to block GNRs@SiO ₂ -NH ₂ for noble metal nanomaterials, and other conditions are the same as Example 7.

Example 9

Compared with Example 7, the noble metal nanomaterial uses cysteine to block GNRs@SiO ₂ -NH ₂ , and other conditions are the same as Example 7.

Catalytic performance of embodiment 10 noble metal nanomaterials in human umbilical vein endothelial cells

(1) Inoculate human umbilical vein endothelial cells (HUVEC) into 6-well plates, ² ×105 cells per well, and incubate overnight;

(2) Remove the original medium and wash with PBS 1-2 times, add 200 μL of 5 μM DAF-FM DANO fluorescent probe solution to each well, incubate at 37 °C, 5% CO ₂ incubator for 20 min, remove unloaded NO probe And wash 2 times with PBS;

(3) Add 1640 culture medium containing GNRs@SiO ₂ -NH ₂ with mass concentrations of 0 μg/mL, 10 μg/mL, 30 μg/mL and 60 μg/mL, and incubate at 37°C and 5% CO ₂ incubator 2h, 6h and 12h;

(4) After removing the medium and washing with PBS, the cells were digested and resuspended in DMEM medium without phenol red and serum, and the fluorescence intensity of intracellular NO was detected by flow cytometry.

Example 11

Compared with Example 10, the noble metal nanomaterial uses glutathione to block GNRs@SiO ₂ -NH ₂ , and other conditions are the same as Example 10.

Example 12

Compared with Example 10, the noble metal nanomaterial uses cysteine to block GNRs@SiO ₂ -NH ₂ , and other conditions are the same as Example 10.

Example 13

(1) Add 1mL of 1μDAF-FM DANO fluorescent probe solution to ¹ ×106 human acute monocytic leukemia (THP1) cells, resuspend in a centrifuge tube, and incubate in a 37°C, 5% _CO2 incubator 5min, centrifuge to remove unloaded NO probe;

(2) Spread the cells into a 12-well plate, add 1640 medium containing GNRs@SiO ₂ with a mass concentration of 100 μg/mL, and incubate for 1 h at 37°C in a 5% CO ₂ incubator;

(3) After centrifuging to remove excess nanoparticles, the cells were resuspended in DMEM medium without phenol red and serum, and the fluorescence intensity of intracellular NO was detected by flow cytometry.

Example 14

(1) Inoculate human umbilical vein vascular endothelial cells (HUVEC) into 6-well plates, ⁵ ×105 cells per well, and incubate overnight;

(2) Remove the original medium and wash with PBS 1-2 times, add 200 μL of 10 μM 4,5-diaminofluorescein diacetate (DAF-2Diacetate) NO fluorescent probe solution to each well, at 37 ° C, 5% CO ₂ Incubate in the incubator for 60 minutes, remove the unloaded NO probe and wash it twice with PBS;

(3) Add 1640 medium containing GNRs@SiO ₂ -NH ₂ with a mass concentration of 100 μg/mL, and incubate at 37° C. in a 5% CO ₂ incubator for 20 h;

(4) After removing the medium and washing with PBS, the cells were digested and resuspended in DMEM medium without phenol red and serum, and the fluorescence intensity of intracellular NO was detected by flow cytometry.

As shown in Figure 2(a), in Example 7, the fluorescence intensity of THP1 cells increases with the increase of the mass concentration of GNRs@SiO ₂ -NH ₂ and the incubation time, when the concentration of GNRs@SiO ₂ -NH ₂ When the concentration is 60 μg/mL and the incubation time is 12h, the fluorescence intensity of THP1 cells is the strongest; as shown in Figure 2(b), Examples 8-9 use glutathione or cysteine to block GNRs@SiO ₂ -NH ₂ , the nitric oxide-like activity of GNRs@SiO ₂ -NH ₂ was significantly inhibited.

As shown in Figure 3(a), in Example 10, the fluorescence intensity of HUVEC cells increased with the increase of the mass concentration of GNRs@SiO ₂ -NH ₂ and the incubation time, when the concentration of GNRs@SiO ₂ -NH ₂ When the concentration was 60 μg/mL and the incubation time was 6 h, the fluorescence intensity of HUVEC cells was the strongest; as shown in Figure 3(b), Examples 11-12 used glutathione or cysteine to block GNRs@SiO ₂ -NH ₂ , the nitric oxide-like activity of GNRs@SiO ₂ -NH ₂ was significantly inhibited.

Compared with Example 7, the surface of GNRs@SiO ₂ in Example 13 is not modified with NH ₂ , the surface is negatively charged, the GNRs@SiO ₂ entering the cell is reduced, and the production of NO is reduced; Example 14 uses DAF-2Diacetate NO fluorescence The stability of the fluorescent product formed by the probe and NO is slightly lower, and the obtained fluorescence intensity is lower.

In summary, the noble metal nanomaterials of the present invention have nitric oxide synthase activity, which can catalyze the reaction between L-arginine and NADPH to produce NO; when the concentration of GNRs@Au-CTAB is 6pM, the amount of NO produced is the largest ; GNRs@AuAg-CTAB with a smooth surface has the best catalytic performance, and the production of NO is the highest; in THP1 cells, when the concentration of GNRs@SiO ₂ -NH ₂ is 60μg/mL and the incubation time is 12h, the NO production The production amount was the largest; in HUVEC cells, when the concentration of GNRs@SiO ₂ -NH ₂ was 60μg/mL and the incubation time was 6h, the production amount of NO was the largest.

The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. A noble metal nanomaterial with nitric oxide synthase activity, characterized in that it includes any one of GNRs@Au, GNRs@Ag, GNRs@ _AuAg , GNRs@Pd, GNRs@Pt or GNRs@SiO one or a combination of at least two. 2. a kind of method that noble metal nanomaterial catalyzes L-arginine as claimed in claim 1 produces NO, is characterized in that, comprises the steps: (1) adding L-arginine solution to the buffer; (2) adding precious metal nanomaterials; (3) Add NADPH solution to produce NO. 3. method according to claim 2, is characterized in that, step (1) described buffer comprises any one or the combination of at least two in Tris-HCl buffer, PBS buffer or phosphate buffer, Preferably Tris-HCl buffer; Preferably, the concentration of the buffer in step (1) is 10-100mM, preferably 50mM; Preferably, the pH value of the buffer in step (1) is 7.0-8.0, preferably 7.4; Preferably, the concentration of the L-arginine solution in step (1) is 1-1000mM, preferably 5-500mM, more preferably 50mM; Preferably, the volume ratio of the L-arginine solution to the buffer in step (1) is (0.1-200):100, preferably (0.3-30):100, more preferably 3:100. 4. according to the described method of claim 2 or 3, it is characterized in that, before step (2) adds noble metal nanomaterial, also comprises adding CTAB solution in the mixed liquor of step (1) damping fluid and L-arginine solution A step of; Preferably, the concentration of the CTAB solution is 1-100mM, preferably 10mM; Preferably, the volume ratio of the CTAB solution to the buffer in step (1) is (1-100):200, preferably 1:20; Preferably, the concentration of the noble metal nanomaterial in step (2) is 0.1-10nM, preferably 1-5nM; Preferably, the volume ratio of the noble metal nanomaterial in step (2) to the buffer in step (1) is (1-50):1000; Preferably, the surface of the noble metal nanomaterial in step (2) is modified with CTAB. 5. The method according to any one of claims 2-4, wherein the concentration of the NADPH solution in step (3) is 10-1000mM, preferably 50-500mM, more preferably 50mM; Preferably, the volume ratio of the NADPH solution in step (3) to the buffer in step (1) is (0.1-50):10, preferably (0.7-7):10, more preferably 7:10; Preferably, the NADPH solution in step (3) is added in 1-5 times, preferably in 3 times. 6. The method according to any one of claims 2-5, characterized in that, comprising the steps of: (1) Adding a concentration of 1-1000mM L-arginine solution to a buffer solution with a pH value of 7.0-8.0 at a concentration of 10-100mM, the volume ratio of the L-arginine solution to the buffer solution For (0.1-200): 100; (2) adding the CTAB solution that concentration is 1-100mM in the mixed solution of buffer solution and L-arginine solution described in step (1), the volume ratio of described CTAB solution and described buffer solution is (1-100 ):200; (3) adding noble metal nanomaterials with a concentration of 0.1-10nM, the volume ratio of the noble metal nanomaterials to the buffer is (1-50):1000; (4) adding a NADPH solution with a concentration of 10-1000mM, the volume ratio of the NADPH solution to the buffer is (0.1-50):10; (5) Under the catalysis of noble metal nanomaterials, L-arginine reacts with NADPH to generate NO. 7. a kind of noble metal nanomaterial as claimed in claim 1 catalyzes the method that L-arginine produces NO in the cell, is characterized in that, comprises the steps: 1) adding a culture medium containing noble metal nanomaterials to the cells; 2) Incubate the cells described in step 1) in a CO ₂ incubator. 8. The method according to claim 7, wherein the cells in step 1) comprise human acute monocytic leukemia cells and/or human umbilical vein endothelial cells; Preferably, the number of cells in step 1) is (1-5)×10 ⁵ ; Preferably, the surface of the noble metal nanomaterial in step 1) is modified with NH ₂ ; Preferably, the noble metal nanomaterial in step 1) includes any one of GNRs@SiO ₂ -NH ₂ , glutathione-blocked GNRs@SiO ₂ -NH ₂ or cysteine-blocked GNRs@SiO ₂ -NH ₂ or a combination of at least two; Preferably, the mass concentration of the noble metal nanomaterial in step 1) is 1-100 μg/mL, preferably 10-60 μg/mL; Preferably, the incubation time in step 2) is 1-20 h, preferably 2-12 h. 9. A method for detecting the NO produced by the method according to claim 7 or 8, comprising the steps of: 1') adding the medium containing the NO fluorescent probe to the cells; 2') incubating the cells described in step 1') in a CO ₂ incubator; 3') The fluorescence intensity of intracellular NO was analyzed by flow cytometry. 10. The method according to claim 9, characterized in that, step 1') said NO fluorescent probe comprises 4-amino-5-methylamino-2,7-difluorofluorescein diacetate and/ or 4,5-diaminofluorescein diacetate, preferably 4-amino-5-methylamino-2,7-difluorofluorescein diacetate; Preferably, the concentration of the NO fluorescent probe described in step 1') is 1-10 μM, preferably 5 μM; Preferably, the incubation time of step 2') is 5-60min, preferably 20min.