CN107598159B

CN107598159B - A kind of noble metal nanometer material and its application with nitric oxide synthase activity

Info

Publication number: CN107598159B
Application number: CN201710754491.2A
Authority: CN
Inventors: 李海芸; 吴晓春; 颜姣; 王黎明; 陈春英
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2017-08-29
Filing date: 2017-08-29
Publication date: 2019-08-09
Anticipated expiration: 2037-08-29
Also published as: CN107598159A

Abstract

The present invention provides a kind of noble metal nanometer material with nitric oxide synthase activity and its applications, comprising: GNRs@Au, GNRs@Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@SiO₂In any one or at least two combination.Noble metal nanometer material of the invention, can be catalyzed L-arginine and NADPH reacts and generates NO, have the function of significantly improving the level of intracellular NO.

Description

A kind of noble metal nanometer material and its application with nitric oxide synthase activity

Technical field

The invention belongs to artificial mimic enzyme fields, are related to the quasi-enzyme catalytic property of noble metal nanometer material, and in particular to one Kind has noble metal nanometer material and its application of nitric oxide synthase activity.

Background technique

Nitric oxide (NO) is the important messenger molecule of intracellular one kind, physiological function by extensive concern with In-depth study.1998, three scientists in the U.S. obtained promise due to finding that NO is endothelium derived relaxing factor (EDRF) The prize of Bell's physiology and medicine.NO not only plays an important role in cardiovascular system, but also in immunological regulation, cancer Important function is exercised in the physiology courses such as morbidity and treatment and tissue damage reparation.Therefore, based on the disease treatment of NO It is more and more paid close attention to by people.

Currently, the level for improving NO mainly passes through two ways: one is utilize catalyst NO donor S-nitrosoglutathione Sulfydryl adduction species (RSNO), such as S-nitrosoglutathione albumin (AlbSNO), S-nitrosoglutathione cysteine (CysNO) and S-nitrosoglutathione Glutathione (GSNO) etc. discharges NO；Another kind is the expression or regulation nitricoxide synthase by influencing nitricoxide synthase Activity to adjust the concentration of internal NO.

Jia etc. using gold nano grain it is blood serum induced in RSNO discharge NO, it was demonstrated that gold nano grain has RSNO Inducing action (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 appearance of more and more NO donors, have also appeared Under the influence of near infrared light, it is upper conversion nano particle catalysis NO donor controlled release NO research (Zhang X, Tian G, Yin W,et 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 catalyzed L-arginine oxidation by nitricoxide synthase and generates, and reaction is divided into two steps: the first step It is that L-arginine is oxidized into N ω-liydroxy-L-arginine under the catalytic action of nitricoxide synthase, second step is N ω- Liydroxy-L-arginine, which continues to be oxidized, to be generated L-citrulline and generates NO, and two-step reaction process requires consumption oxygen and reduction Type Coenzyme I I (NADPH).1992, researcher pointed out that horseradish peroxidase can be catalyzed N ω-hydroxyl-L- essence ammonia for the first time Acid generates L-citrulline and NO, and wherein N ω-liydroxy-L-arginine is that nitricoxide synthase catalysis L-arginine is converted to L- melon Intermediate product (Boucher J L, Genet A, Vadon S, the et al.Formation of Nitrogen of propylhomoserin and NO Oxides and Citrulline upon Oxidation of Nω-hydroxy-L-arginine by Hemeproteins.Biochemical and Biophysical Research Communications,1992,184(3): 1158-1164.).2002, new research discovery horseradish peroxidase can be catalyzed hydroxycarbamide generate NO (Huang J, Sommers E M,Kim-Shapiro D B,et al.Horseradish peroxidase catalyzed nitric oxide formation from hydroxyurea.Journal of the American Chemical Society, 2002,124(13):3473-3480.).However, with the same function similar with nitricoxide synthase there is presently no finding Object can directly be catalyzed L-arginine and generate L-citrulline and NO.

In recent years, nano enzyme field emerges rapidly, and the nano enzyme of different-shape, scale and material emerges one after another.Compared to Native enzyme, nano enzyme is more economical, more stable, is also easier to large-scale production preparation, it has also become the research hotspot in each field.Your gold Metal nano-particle catalyst occupies an important position in catalytic field, shows mixed with the solid catalyst of noble metal nano particles Good catalytic activity, as Pt nanoparticle has peroxidase, catalase, superoxide dismutase and oxidizing ferment four The activity of kind enzyme.Up to the present, the research report about the nano enzyme with nitric oxide synthase activity is found no.

Summary of the invention

In view of the above-mentioned problems, the present invention provides a kind of noble metal nanometer material with nitric oxide synthase activity and its answers With the noble metal nanometer material can be catalyzed L-arginine and NADPH reacts and generates L-citrulline and NO, and thin in source of people Performance nitric oxide synthase activity intracellular, significantly improves the level of intracellular NO.

In a first aspect, the present invention provides a kind of noble metal nanometer materials with nitric oxide synthase activity, comprising: GNRs@Au, GNRs@Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@SiO₂In any one or at least two Combination.

Noble metal nanometer material provided by the invention with gold nanorods (gold nanorods, GNRs) for kernel, and in table Face is coated with Au, Ag, Au/Ag alloy, Pd, Pt or SiO respectively₂Outer layer obtains GNRs@Au, GNRs@Ag, GNRs@AuAg, GNRs@ Pd, GNRs@Pt or GNRs@SiO₂, there is nitric oxide synthase activity, catalysis L-arginine and NADPH, which react, generates L- Citrulling and NO significantly improve the level of intracellular NO.

Second aspect, the present invention provides a kind of noble metal nanometer material catalysis L-arginines as described in relation to the first aspect to produce The method of raw NO, includes the following steps:

(1) L-arginine solution is added into buffer；

(2) noble metal nanometer material is added；

(3) NADPH solution is added, generates NO.

Preferably, step (1) described buffer includes in Tris-HCl buffer, PBS buffer solution or phosphate buffer Any one or at least two combination, preferably Tris-HCl buffer.

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

Preferably, the pH value of step (1) described buffer is 7.0-8.0, for example, can be 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 step (1) the L-arginine solution be 1-1000mM, such as can be 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, further preferably 50mM。

Preferably, the volume ratio of step (1) the L-arginine solution and the buffer is (0.1-200): 100, example Such as 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, further preferably 3:100。

It preferably, further include to step (1) buffer and L-arginine before noble metal nanometer material is added in step (2) The step of CTAB solution is added in the mixed liquor of solution.

Preferably, the concentration of the CTAB solution be 1-100mM, such as can be 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 and step (1) described buffer is (1-100): 200, such as can be with It is 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 step (2) described noble metal nanometer material be 1.0-10nM, such as can be 1.0nM, 2.0nM, 3.0nM, 4.0nM, 5.0nM, 6.0nM, 7.0nM, 8.0nM, 9.0nM or 10nM, preferably 1-5nM.

Preferably, the volume ratio of step (2) noble metal nanometer material and step (1) described buffer is (1-50): 1000, for example, can be 1:1000,5:1000,10:1000,15:1000,20:1000,25:1000,30:1000,35:1000, 40:1000,45:1000 or 50:1000.

Preferably, step (2) the noble metal nanometer material surface modification has CTAB.

Preferably, the concentration of step (3) the NADPH solution be 10-1000mM, such as can be 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, further preferably 50mM.

Preferably, the volume ratio of step (3) the NADPH solution and step (1) described buffer is (0.1-50): 10, Such as can be 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, further preferably 7:10.

Preferably, step (3) the NADPH solution divides 1-5 addition, such as can be 1 time, 2 times, 3 times, 4 times or 5 It is secondary, preferably it is added three times.

As optimal technical scheme, a kind of method that noble metal nanometer material catalysis L-arginine generates NO, including it is as follows Step:

(1) it is 10-100mM to concentration, the L- essence ammonia that concentration is 1-1000mM is added in the buffer that pH value is 7.0-8.0 The volume ratio of acid solution, the L-arginine solution and the buffer is (0.1-200): 100；

(2) CTAB that concentration is 1-100mM is added into the mixed liquor of step (1) buffer and L-arginine solution The volume ratio of solution, the CTAB solution and the buffer is (1-100): 200；

(3) noble metal nanometer material that concentration is 1-5nM, the body of the noble metal nanometer material and the buffer is added Product is than being (1-50): 1000；

(4) the NADPH solution that concentration is 10-1000mM is added, the volume ratio of the NADPH solution and the buffer is (0.1-50):10；

(5) under the catalytic action of noble metal nanometer material, L-arginine reacts with NADPH, generates NO.

The third aspect, the present invention provides a kind of noble metal nanometer materials to be catalyzed the side that L-arginine generates NO in the cell Method includes the following steps:

1) culture medium containing noble metal nanometer material is added into cell；

2) in CO₂Incubation step 1 in incubator) cell.

Preferably, the step 1) cell includes in people's Acute Monocytic Leukemia Cell Line and/or human umbilical vein Chrotoplast.

Preferably, the quantity of the step 1) cell is (1-5) × 10⁵It is a, such as can be 1 × 10⁵It is a, 2 × 10⁵A, 3×10⁵It is a, 4 × 10⁵It is a or 5 × 10⁵It is a.

Preferably, step 1) the noble metal nanometer material surface modification has NH₂。

Preferably, the step 1) noble metal nanometer material includes GNRs@SiO₂-NH₂, glutathione close GNRs@ SiO₂-NH₂Or cysteine closes GNRs@SiO₂-NH₂In any one or at least two combination.

Preferably, the mass concentration of the step 1) noble metal nanometer material is 1-100 μ g/mL, such as can be 1 μ g/ mL、5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL、35μg/mL、40μg/mL、45μg/mL、50μ g/mL、55μg/mL、60μg/mL、65μg/mL、70μg/mL、75μg/mL、80μg/mL、85μg/mL、90μg/mL、95μg/mL Or 100 μ g/mL, preferably 10-60 μ g/mL.

Preferably, the time of the step 2) incubation be 1-20h, such as can be 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h or 20h, preferably 1-15h, further preferably 2- 12h。

Fourth aspect, the present invention provides a kind of detection noble metal nanometer materials to be catalyzed L-arginine generation in the cell The method of NO, includes the following steps:

1 ') culture medium containing NO fluorescence probe is added into cell；

2 ') in CO₂Incubation step 1 ' in incubator) cell；

3 ') fluorescence intensity of the intracellular NO of flow cytometry analysis is used.

Preferably, step 1 ') the NO fluorescence probe includes 4- amino -5- methylamino -2,7- difluoro fluorescein diethyl Acid esters and/or 4,5- diamino fluorescein(e) diacetate, preferably 4- amino -5- methylamino -2,7- difluoro fluorescein diethyl Acid esters.

Preferably, step 1 ') concentration of the NO fluorescence probe is 1-10 μM, such as can be 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, step 2 ') time of the incubation is 5-60min, such as can be 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min or 60min, preferably 20min.

In the present invention, NO fluorescence probe is loaded in cell, realizes the real-time detection of NO in cell.

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

(1) noble metal nanometer material of the invention has nitric oxide synthase activity, can be catalyzed L-arginine and NADPH It reacts and generates NO；

(2) as the final concentration of 6pM of GNRs@Au-CTAB, the production quantity of NO is maximum；

(3) the GNRs@AuAg-CTAB of smooth surface has optimal catalytic performance, the production quantity highest of NO；

(4) intracellular in THP1, as GNRs@SiO₂-NH₂Concentration be 60 μ g/mL, when incubation time is 12h, the life of NO It is maximum at amount；

(5) intracellular in HUVEC, as GNRs@SiO₂-NH₂Concentration be 60 μ g/mL, when incubation time is 6h, the life of NO It is maximum at amount.

Detailed description of the invention

Fig. 1 (a) be NO production quantity and noble metal nanometer material concentration relationship curve, Fig. 1 (b) be NO production quantity with The relationship histogram of the type of noble metal nanometer material；

The production quantity and GNRs@SiO that Fig. 2 (a) is THP1 cell NO₂-NH₂Concentration and incubation time relationship histogram, The production quantity and GNRs@SiO that Fig. 2 (b) is THP1 cell NO₂-NH₂, glutathione close GNRs@SiO₂-NH₂And cysteine Close GNRs@SiO₂-NH₂Concentration relationship histogram；

The production quantity and GNRs@SiO that Fig. 3 (a) is HUVEC cell NO₂-NH₂Concentration and incubation time relationship column Figure, the production quantity and GNRs@SiO that Fig. 3 (b) is HUVEC cell NO₂-NH₂, glutathione close GNRs@SiO₂-NH₂With half Guang Propylhomoserin closes GNRs@SiO₂-NH₂Concentration relationship histogram.

Specific embodiment

The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than Limitation of the invention.

In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art, Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from The conventional products of acquisition.

Influence of the concentration of 1 noble metal nanometer material of embodiment to NO production quantity

It takes 30 μ L L-arginines (50mM) in 1mL Tris-HCl buffer (50mM, pH=7.4), 50 μ is then added L CTAB solution (10mM) is separately added into the GNRs@Au-CTAB that 1.7 μ L, 10 μ L, 14 μ L and 27 μ L concentration are 1nM after mixing； NO detecting electrode is placed in reaction solution, submergence 2-3mm, after electrode is stablized, into reaction solution according to 100 μ L-100 The mode of μ L-500 μ L is added NADPH solution (50mM), every minor tick 1min；The electricity after NADPH solution is added in detection third time It flows changing value (Δ I), makees noble metal nano structure concentration-Δ I curve.

Influence of the type of 2 noble metal nanometer material of embodiment to NO production quantity

It takes 30 μ L L-arginines (50mM) in 1mL Tris-HCl buffer (50mM, pH=7.4), 5 μ L is then added CTAB solution (0.1M) is separately added into GNRs@Au-CTAB, GNRs@Ag- that 2 μ L concentration are 5nM (final concentration of 6pM) after mixing CTAB, GNRs@AuAg-CTAB (coarse), GNRs@AuAg-CTAB (smooth), GNRs@Pd-CTAB and GNRs@Pt-CTAB；It will NO detecting electrode is placed in reaction solution, submergence 2-3mm, after electrode is stablized, into reaction solution according to 100 μ L-100 μ The mode of L-500 μ L is added NADPH solution (50mM), every minor tick 1min；The electric current after NADPH solution is added in detection third time Changing value (Δ I).

Embodiment 3

It takes 3 μ L L-arginines (500mM) in 1mL Tris-HCl buffer (50mM, pH=7.4), 50 μ is then added L CTAB solution (10mM), is added the GNRs@Au-CTAB that 14 μ L concentration are 1nM (final concentration of 8pM) after mixing, piping and druming mixes； NO detecting electrode is placed in reaction solution, submergence 2-3mm, after electrode is stablized, into reaction solution according to 350 μ L-350 The mode of μ L is added NADPH solution (50mM), every minor tick 1min；Second of curent change being added after NADPH solution of detection It is worth (Δ I).

Embodiment 4

It takes 300 μ L L-arginines (5mM) in 1mL PBS buffer solution (50mM, pH=7.4), 5 μ L CTAB is then added The GNRs@Au-CTAB that 1 μ L concentration is 10nM (final concentration of 16pM) is added in solution (100mM) after mixing；By NO detecting electrode It is placed in reaction solution, submergence 2-3mm, after electrode is stablized, 70 μ L NADPH solution is added into reaction solution (500mM) is detected current variation value (Δ I).

Embodiment 5

It takes 1 μ L L-arginine (1M) in 1mL Tris-HCl buffer (10mM, pH=7.0), 500 μ L is then added The GNRs@Au-CTAB that 17 μ L concentration are 0.1nM (final concentration of 1pM) is added in CTAB solution (1mM) after mixing；NO is detected into electricity Pole is placed in reaction solution, submergence 2-3mm, and after electrode is stablized, 1mL-1mL-1mL-1mL-1mL is added into reaction solution NADPH solution (10mM) detects current variation value (Δ I).

Embodiment 6

It takes 2mL L-arginine (1mM) in 1mL phosphate buffer (100mM, pH=8.0), 50 μ L is added after mixing Concentration is the GNRs@Au of 1nM (final concentration of 20pM), and piping and druming mixes；NO detecting electrode is placed in reaction solution, submergence is 10 μ L NADPH solution (1M) are added into reaction solution after electrode is stablized by 2-3mm, detect current variation value (Δ I).

Comparative example 1

Compared with Example 1, the GNRs@Au-CTAB of the final concentration of 0.05pM of noble metal nanometer material, other conditions and reality It is identical to apply example 1.

Comparative example 2

Compared with Example 1, the GNRs@Au-CTAB of the final concentration of 25pM of noble metal nanometer material, other conditions and implementation Example 1 is identical.

Experimental result is as shown in table 1.

The active in vitro study of class Nitric oxide syntheses of 1 noble metal nanometer material of table

From embodiment 1 and Fig. 1 (a) as can be seen that the production quantity of NO and the concentration of GNRs@Au-CTAB in a certain range Be positively correlated: with the increase of the concentration of GNRs@Au-CTAB, the production quantity of NO increases, final concentration of as GNRs@Au-CTAB When 6pM, the production quantity of NO is maximum；As the concentration of GNRs@Au-CTAB further increases, the production quantity of NO is gradually reduced, can It can be since excessive GNRs@Au-CTAB assembles in the solution, specific surface area becomes smaller, and catalytic performance weakens.

From embodiment 2 and Fig. 1 (b) as can be seen that the production quantity of NO is related with the type of noble metal nanometer material: surface is flat Sliding GNRs@AuAg-CTAB has optimal catalytic performance, the production quantity highest of NO.

From the point of view of embodiment 1-6 comparison, concentration is that the GNRs@AuAg-CTAB of the smooth surface of 6pM has optimal catalysis Performance.

Compared with Example 1, the concentration of the GNRs@Au-CTAB of comparative example 1 is lower, can not effectively be catalyzed L-arginine and NADPH reaction generates NO；The concentration of the GNRs Au-CTAB of comparative example 2 is higher, and it is biggish micro- to become partial size for aggregation in the solution Rice structure, catalytic performance significantly reduce.

Catalytic performance of 7 noble metal nanometer material of embodiment in people's Acute Monocytic Leukemia Cell Line

(1) to 4 × 10⁶5 μM of 4- amino -5- methyl of 1mL are added in personal acute monocytic leukemia (THP1) cell Amino -2,7- difluoro fluorescein(e) diacetate (DAF-FM DA) NO fluorescence probe solution, after being resuspended in centrifuge tube, 37 DEG C, 5%CO₂It is incubated for 20min in incubator, is centrifuged off the NO probe of unloaded；

(2) cell is spread to 12 orifice plates, is added and contains GNRs@SiO₂-NH₂Mass concentration be respectively 0 μ g/mL, 10 μ g/ 1640 culture mediums of mL, 30 μ g/mL and 60 μ g/mL, in 37 DEG C, 5%CO₂2h, 6h and 12h are incubated in incubator respectively；

(3) after being centrifuged off extra nano particle, cell is resuspended using the DMEM culture medium without phenol red serum-free, with stream Formula cell instrument detects the fluorescence intensity of intracellular NO.

Embodiment 8

Compared with Example 7, noble metal nanometer material closes GNRs@SiO using glutathione₂-NH₂, other conditions and reality It is identical to apply example 7.

Embodiment 9

Compared with Example 7, noble metal nanometer material closes GNRs@SiO using cysteine₂-NH₂, other conditions and reality It is identical to apply example 7.

Catalytic performance of 10 noble metal nanometer material of embodiment in human umbilical vein endothelial cell

(1) human umbilical vein endothelial cell (HUVEC) is inoculated into 6 orifice plates, every hole 2 × 10⁵A cell, was incubated for Night；

(2) it removes former culture medium and is cleaned 1-2 times with PBS, 200 μ L, 5 μM of DAF-FM DANO fluorescence probes are added in every hole Solution, in 37 DEG C, 5%CO₂It is incubated for 20min in incubator, remove the NO probe of unloaded and is cleaned 2 times with PBS；

(3) it is added and contains GNRs@SiO₂-NH₂Mass concentration be respectively 0 μ g/mL, 10 μ g/mL, 30 μ g/mL and 60 μ g/ 1640 culture mediums of mL, in 37 DEG C, 5%CO₂2h, 6h and 12h are incubated in incubator respectively；

(4) after removing culture medium and being cleaned with PBS, the DMEM culture medium without phenol red serum-free will be used together under cell dissociation Cell is resuspended, with the fluorescence intensity of the intracellular NO of flow cytomery.

Embodiment 11

Compared with Example 10, noble metal nanometer material closes GNRs@SiO using glutathione₂-NH₂, other conditions with Embodiment 10 is identical.

Embodiment 12

Compared with Example 10, noble metal nanometer material closes GNRs@SiO using cysteine₂-NH₂, other conditions with Embodiment 10 is identical.

Embodiment 13

(1) to 1 × 10⁶It is glimmering that 1 μM of DAF-FM DA NO of 1mL is added in personal acute monocytic leukemia (THP1) cell Light probe solution, after being resuspended in centrifuge tube, in 37 DEG C, 5%CO₂It is incubated for 5min in incubator, is centrifuged off the NO of unloaded Probe；

(2) cell is spread to 12 orifice plates, is added and contains GNRs@SiO₂Mass concentration be 100 μ g/mL 1640 culture Base, in 37 DEG C, 5%CO₂1h is incubated in incubator；

Embodiment 14

(1) human umbilical vein endothelial cell (HUVEC) is inoculated into 6 orifice plates, every hole 5 × 10⁵A cell, was incubated for Night；

(2) it removes former culture medium and is cleaned 1-2 times with PBS, 200 μ L, 10 μM of 4,5- diamino fluoresceins two are added in every hole Acetic acid esters (DAF-2Diacetate) NO fluorescence probe solution, in 37 DEG C, 5%CO₂Be incubated for 60min in incubator, remove without The NO probe of load is simultaneously cleaned 2 times with PBS；

(3) it is added and contains GNRs@SiO₂-NH₂Mass concentration be 100 μ g/mL 1640 culture mediums, in 37 DEG C, 5%CO₂ 20h is incubated in incubator；

As shown in Fig. 2 (a), in embodiment 7, the fluorescence intensity of THP1 cell is with GNRs@SiO₂-NH₂Mass concentration and The increase of incubation time and increase, as GNRs@SiO₂-NH₂Concentration be 60 μ g/mL, when incubation time is 12h, THP1 cell Fluorescence intensity is most strong；As shown in Fig. 2 (b), embodiment 8-9 closes GNRs@SiO using glutathione or cysteine₂-NH₂Work Behind property site, GNRs@SiO₂-NH₂Class nitric oxide enzymatic activity be obviously suppressed.

As shown in Fig. 3 (a), in embodiment 10, the fluorescence intensity of HUVEC cell is with GNRs@SiO₂-NH₂Mass concentration Increase with incubation time and increase, as GNRs@SiO₂-NH₂Concentration be 60 μ g/mL, when incubation time is 6h, HUVEC cell Fluorescence intensity it is most strong；As shown in Fig. 3 (b), embodiment 11-12 closes GNRs@SiO using glutathione or cysteine₂-NH₂ Active site after, GNRs@SiO₂-NH₂Class nitric oxide enzymatic activity be obviously suppressed.

Compared with Example 7, the GNRs@SiO of embodiment 13₂The unmodified NH in surface₂, surface is in negative electrical charge, into cell GNRs@SiO₂It reduces, the production quantity of NO is reduced；Embodiment 14 uses DAF-2Diacetate NO fluorescence probe, is formed with NO Fluorescence-causing substance stability is slightly lower, and obtained fluorescence intensity is lower.

In conclusion noble metal nanometer material of the invention has nitric oxide synthase activity, L-arginine can be catalyzed It reacts with NADPH and generates NO；When the concentration of GNRs@Au-CTAB is 6pM, the production quantity of NO is maximum；Smooth surface GNRs@AuAg-CTAB has optimal catalytic performance, the production quantity highest of NO；It is intracellular in THP1, as GNRs@SiO₂-NH₂'s When concentration is 60 μ g/mL, incubation time is 12h, the production quantity of NO is maximum；It is intracellular in HUVEC, as GNRs@SiO₂-NH₂It is dense When degree is 60 μ g/mL, incubation time is 6h, the production quantity of NO is maximum.

The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.

Claims

1. a kind of noble metal nanometer material with nitric oxide synthase activity characterized by comprising GNRs@Au, GNRs@ Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@SiO₂In any one or at least two combination.

2. a kind of method that noble metal nanometer material catalysis L-arginine as described in claim 1 generates NO, which is characterized in that Include the following steps:

(1) L-arginine solution is added into buffer；

(2) noble metal nanometer material is added；

(3) NADPH solution is added, generates NO.

3. according to the method described in claim 2, it is characterized in that, step (1) described buffer include Tris-HCl buffer, In PBS buffer solution or phosphate buffer any one or at least two combination.

4. according to the method described in claim 3, it is characterized in that, step (1) buffer is Tris-HCl buffer.

5. according to the method described in claim 2, it is characterized in that, the concentration of step (1) described buffer is 10-100mM.

6. according to the method described in claim 5, it is characterized in that, the concentration of step (1) described buffer is 50mM.

7. according to the method described in claim 2, it is characterized in that, the pH value of step (1) described buffer is 7.0-8.0.

8. the method according to the description of claim 7 is characterized in that the pH value of step (1) described buffer is 7.4.

9. according to the method described in claim 2, it is characterized in that, the concentration of step (1) the L-arginine solution is 1- 1000mM。

10. according to the method described in claim 9, it is characterized in that, the concentration of step (1) the L-arginine solution is 5- 500mM。

11. according to the method described in claim 10, it is characterized in that, the concentration of step (1) the L-arginine solution is 50mM。

12. according to the method described in claim 2, it is characterized in that, step (1) the L-arginine solution and the buffer Volume ratio be (0.1-200): 100.

13. according to the method for claim 12, which is characterized in that step (1) the L-arginine solution and the buffering The volume ratio of liquid is (0.3-30): 100.

14. according to the method for claim 13, which is characterized in that step (1) the L-arginine solution and the buffering The volume ratio of liquid is 3:100.

15. according to the method described in claim 2, it is characterized in that, also being wrapped before noble metal nanometer material is added in step (2) Include the step of CTAB solution is added into the mixed liquor of step (1) buffer and L-arginine solution.

16. according to the method for claim 15, which is characterized in that the concentration of the CTAB solution is 1-100mM.

17. according to the method for claim 16, which is characterized in that the concentration of the CTAB solution is 10mM.

18. according to the method for claim 15, which is characterized in that the CTAB solution and step (1) described buffer Volume ratio is (1-100): 200.

19. according to the method for claim 18, which is characterized in that the CTAB solution and step (1) described buffer Volume ratio is 1:20.

20. according to the method described in claim 2, it is characterized in that, the concentration of step (2) described noble metal nanometer material is 0.1-10nM。

21. according to the method for claim 20, which is characterized in that the concentration of step (2) described noble metal nanometer material is 1-5nM。

22. according to the method described in claim 2, it is characterized in that, step (2) noble metal nanometer material and step (1) The volume ratio of the buffer is (1-50): 1000.

23. according to the method described in claim 2, it is characterized in that, step (2) the noble metal nanometer material surface modification has CTAB。

24. according to the method described in claim 2, it is characterized in that, the concentration of step (3) the NADPH solution is 10- 1000mM。

25. according to the method for claim 24, which is characterized in that the concentration of step (3) the NADPH solution is 50- 500mM。

26. according to the method for claim 25, which is characterized in that the concentration of step (3) the NADPH solution is 50mM.

27. according to the method described in claim 2, it is characterized in that, step (3) the NADPH solution and step (1) are described slow The volume ratio of fliud flushing is (0.1-50): 10.

28. according to the method for claim 27, which is characterized in that step (3) the NADPH solution and step (1) are described The volume ratio of buffer is (0.7-7): 10.

29. according to the method for claim 28, which is characterized in that step (3) the NADPH solution and step (1) are described The volume ratio of buffer is 7:10.

30. according to the method described in claim 2, it is characterized in that, step (3) the NADPH solution divides 1-5 addition.

31. according to the method for claim 30, which is characterized in that step (3) the NADPH solution is added three times.

32. according to the method described in claim 2, it is characterized by comprising the following steps:

It (1) is 10-100mM to concentration, it is molten that the L-arginine that concentration is 1-1000mM is added in the buffer that pH value is 7.0-8.0 The volume ratio of liquid, the L-arginine solution and the buffer is (0.1-200): 100；

(2) it is molten that the CTAB that concentration is 1-100mM is added into the mixed liquor of step (1) buffer and L-arginine solution The volume ratio of liquid, the CTAB solution and the buffer is (1-100): 200；

(3) noble metal nanometer material that concentration is 0.1-10nM, the body of the noble metal nanometer material and the buffer is added Product is than being (1-50): 1000；

33. a kind of noble metal nanometer material as described in claim 1 is catalyzed the method that L-arginine generates NO in the cell, It is characterized in that, includes the following steps:

2) in CO₂Incubation step 1 in incubator) cell.

34. according to the method for claim 33, which is characterized in that the step 1) cell includes that people's Acute monocytic is white Blood disease cell and/or human umbilical vein endothelial cell.

35. according to the method for claim 33, which is characterized in that the quantity of the step 1) cell is (1-5) × 10⁵It is a.

36. according to the method for claim 33, which is characterized in that step 1) the noble metal nanometer material surface modification has NH₂。

37. according to the method for claim 33, which is characterized in that the step 1) noble metal nanometer material includes GNRs@ SiO₂-NH₂, glutathione close GNRs@SiO₂-NH₂Or cysteine closes GNRs@SiO₂-NH₂In any one or at least Two kinds of combination.

38. according to the method for claim 33, which is characterized in that the mass concentration of the step 1) noble metal nanometer material For 1-100 μ g/mL.

39. according to the method for claim 38, which is characterized in that the mass concentration of the step 1) noble metal nanometer material For 10-60 μ g/mL.

40. according to the method for claim 33, which is characterized in that the time of the step 2) incubation is 1-20h.

41. according to the method for claim 40, which is characterized in that the time of the step 2) incubation is 2-12h.

42. the method for the NO that a kind of detection such as any one of claim 33-41 the method generates, which is characterized in that including Following steps:

1 ') culture medium containing NO fluorescence probe is added into cell；

2 ') in CO₂Incubation step 1 ' in incubator) cell；

43. according to the method for claim 42, which is characterized in that step 1 ') the NO fluorescence probe includes 4- amino -5- Methylamino -2,7- difluoro fluorescein(e) diacetate and/or 4,5- diamino fluorescein(e) diacetate.

44. according to the method for claim 43, which is characterized in that step 1 ') the NO fluorescence probe is 4- amino -5- first Base amino -2,7- difluoro fluorescein(e) diacetate.

45. according to the method for claim 42, which is characterized in that step 1 ') concentration of the NO fluorescence probe is 1-10 μ M。

46. according to the method for claim 45, which is characterized in that step 1 ') concentration of the NO fluorescence probe is 5 μM.

47. according to the method for claim 42, which is characterized in that step 2 ') time of the incubation is 5-60min.

48. according to the method for claim 47, which is characterized in that step 2 ') time of the incubation is 20min.