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

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

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CN107598159A
CN107598159A CN201710754491.2A CN201710754491A CN107598159A CN 107598159 A CN107598159 A CN 107598159A CN 201710754491 A CN201710754491 A CN 201710754491A CN 107598159 A CN107598159 A CN 107598159A
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noble metal
gnrs
nanometer material
metal nanometer
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CN107598159B (en
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李海芸
吴晓春
颜姣
王黎明
陈春英
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National Center for Nanosccience and Technology China
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National Center for Nanosccience and Technology China
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Abstract

The invention provides a kind of noble metal nanometer material with nitric oxide synthase activity and its application, including：GNRs@Au, GNRs@Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@SiO2In any one or at least two combination.The noble metal nanometer material of the present invention, can be catalyzed L arginine and NADPH reacts and generates NO, have the function that to significantly improve the horizontal 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 field, it is related to the quasi-enzyme catalytic property of noble metal nanometer material, and in particular to one Noble metal nanometer material and its application of the kind with nitric oxide synthase activity.
Background technology
Nitric oxide (NO) is a kind of intracellular important messenger molecule, its physiological function by extensive concern with In-depth study.1998, three scientists in the U.S. obtained promise because finding that NO is endothelium derived relaxing factor (EDRF) Bel's physiology and medicine is encouraged.NO not only plays an important role in cardiovascular system, and in immunological regulation, cancer Morbidity and treatment, and exercise important function in the physiology course such as tissue damage reparation.Therefore, the disease treatment based on NO More and more paid close attention to by people.
At present, the level for improving NO mainly passes through two ways：One kind is to utilize catalyst NO donor S-nitrosoglutathiones Sulfydryl adduction species (RSNO), such as S-nitrosoglutathione albumin (AlbSNO), S-nitrosoglutathione cysteine (CysNO) and S-nitrosoglutathione Glutathione (GSNO) etc., discharge NO；Another kind is the expression or regulation and control nitricoxide synthase by influenceing nitricoxide synthase Activity so as to adjusting internal NO concentration.
The RSNO releases NO using gold nano grain in blood serum induced such as Jia, it was demonstrated that gold nano grain has to 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 increasing NO donors, have also appeared Under the influence of near infrared light, upper conversion nano particle catalysis NO donor controlled releases 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 typically catalyzed L-arginine oxidation by nitricoxide synthase and produced, and reaction is divided into two steps：The first step That L-arginine is oxidized into N ω-liydroxy-L-arginine under the catalytic action of nitricoxide synthase, second step be N ω- Liydroxy-L-arginine continues to be oxidized generation Cit and produces NO, and two-step reaction process is required for consuming oxygen and reduction Type DPN I (NADPH).1992, researcher pointed out that horseradish peroxidase can be catalyzed N ω-hydroxyl-L- essence ammonia for the first time It is that nitricoxide synthase catalysis L-arginine changes into L- melons that acid, which produces Cit and NO, wherein N ω-liydroxy-L-arginine, Propylhomoserin and NO intermediate product (Boucher J L, Genet A, Vadon S, et al.Formation of Nitrogen 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 find horseradish peroxidase can be catalyzed hydroxycarbamide produce 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, there is presently no find to have the similar of identical function with nitricoxide synthase Thing, it can directly be catalyzed L-arginine and produce Cit and NO.
In recent years, nano enzyme field emerges rapidly, and the nano enzyme of different-shape, yardstick and material emerges in an endless stream.Compared to Native enzyme, nano enzyme is more economical, more stable, is also easier to large-scale production and prepares, it has also become the study hotspot in each field.Your gold Metal nano-particle catalyst occupies an important position in catalytic field, is shown 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 on the nano enzyme with nitric oxide synthase activity is found no.
The content 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 should With the noble metal nanometer material can be catalyzed L-arginine and NADPH reacts and generates Cit and NO, and thin in people source Intracellular shows nitric oxide synthase activity, significantly improves intracellular NO level.
In a first aspect, the invention provides a kind of noble metal nanometer material with nitric oxide synthase activity, including： 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, obtain GNRs@Au, GNRs@Ag, GNRs@AuAg, GNRs@ Pd, GNRs@Pt or GNRs@SiO2, there is nitric oxide synthase activity, catalysis L-arginine reacts with NADPH and generates L- Citrulling and NO, significantly improve intracellular NO level.
Second aspect, the invention provides a kind of noble metal nanometer material catalysis L-arginine production as described in relation to the first aspect Raw NO method, comprises the following steps：
(1) L-arginine solution is added into buffer solution；
(2) noble metal nanometer material is added；
(3) NADPH solution is added, produces NO.
Preferably, step (1) described buffer solution is included in Tris-HCl buffer solutions, PBS or phosphate buffer Any one or at least two combination, preferably Tris-HCl buffer solutions.
Preferably, the concentration of step (1) described buffer solution is 10-100mM, for example, can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, preferably 50mM.
Preferably, the pH value of step (1) described buffer solution is 7.0-8.0, for example, can be 7.0,7.1,7.2,7.3, 7.4th, 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 is 1-1000mM, for example, 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, more preferably 50mM。
Preferably, the volume ratio of step (1) the L-arginine solution and the buffer solution 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, be preferably (0.3-30):100, more preferably 3:100。
Preferably, also include to step (1) buffer solution and L-arginine before step (2) adds noble metal nanometer material The step of CTAB solution is added in the mixed liquor of solution.
Preferably, the concentration of the CTAB solution is 1-100mM, for example, 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 solution 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 is 1.0-10nM, for example, 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 solution is (1-50): 1000, such as 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 is 10-1000mM, for example, 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, more preferably 50mM.
Preferably, the volume ratio of step (3) the NADPH solution and step (1) described buffer solution 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, be preferably (0.7-7):10, more 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 divide 3 additions.
As optimal technical scheme, a kind of noble metal nanometer material catalysis L-arginine generation NO method, 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 solution that pH value is 7.0-8.0 Acid solution, the volume ratio of the L-arginine solution and the buffer solution is (0.1-200):100；
(2) CTAB that concentration is 1-100mM is added into the mixed liquor of step (1) buffer solution and L-arginine solution Solution, the volume ratio of the CTAB solution and the buffer solution 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 solution are added Product ratio is (1-50):1000；
(4) the NADPH solution that concentration is 10-1000mM is added, the volume ratio of the NADPH solution and the buffer solution 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 invention provides the side that a kind of noble metal nanometer material is catalyzed L-arginine generation NO in the cell Method, comprise 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 is included 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 individual, such as can be 1 × 105It is individual, 2 × 105Individual, 3×105It is individual, 4 × 105It is individual or 5 × 105It is individual.
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 closing GNRs@ SiO2-NH2Or cysteine closing 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 is 1-20h, for example, 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, more preferably 2- 12h。
Fourth aspect, the invention provides one kind detection noble metal nanometer material to be catalyzed caused by L-arginine in the cell NO method, comprises the following steps：
1 ') culture medium containing NO fluorescence probes is added into cell；
2 ') in CO2Incubation step 1 ' in incubator) cell；
3 ') the intracellular NO of flow cytometry analysis fluorescence intensity is used.
Preferably, step 1 ') the NO fluorescence probes include 4- amino -5- methylamino -2,7- difluoro fluorescein diethyls Acid esters and/or 4,5- diaminourea fluorescein(e) diacetate, preferably 4- amino -5- methylaminos -2,7- difluoro fluorescein diethyl Acid esters.
Preferably, step 1 ') concentration of the NO fluorescence probes 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 probes are loaded in cell, realize the real-time detection of NO in cell.
Compared with prior art, the present invention has the advantages that：
(1) noble metal nanometer material of the invention has nitric oxide synthase activity, can be catalyzed L-arginine and NADPH React and produce NO；
(2) as GNRs@Au-CTAB final concentration of 6pM, NO growing amount is maximum；
(3) the GNRs@AuAg-CTAB of smooth surface have optimal catalytic performance, NO growing amount highest；
(4) it is intracellular in THP1, as GNRs@SiO2-NH2Concentration be 60 μ g/mL, incubation time is when being 12h, NO life It is maximum into amount；
(5) it is intracellular in HUVEC, as GNRs@SiO2-NH2Concentration be 60 μ g/mL, incubation time is when being 6h, NO life It is maximum into amount.
Brief description of the drawings
Fig. 1 (a) be NO growing amount and noble metal nanometer material concentration relationship curve, Fig. 1 (b) be NO growing amount with The relation block diagram of the species of noble metal nanometer material；
The growing amount and GNRs@SiO that Fig. 2 (a) is THP1 cells NO2-NH2Concentration and incubation time relation block diagram, The growing amount and GNRs@SiO that Fig. 2 (b) is THP1 cells NO2-NH2, glutathione closing GNRs@SiO2-NH2And cysteine Close GNRs@SiO2-NH2Concentration relation block diagram；
The growing amount and GNRs@SiO that Fig. 3 (a) is HUVEC cells NO2-NH2Concentration and incubation time relation column Figure, the growing amount and GNRs@SiO that Fig. 3 (b) is HUVEC cells NO2-NH2, glutathione closing GNRs@SiO2-NH2With half Guang Propylhomoserin closing GNRs@SiO2-NH2Concentration relation block diagram.
Embodiment
For the technological means and its effect that the present invention is taken is expanded on further, with reference to embodiments with accompanying drawing to this hair It is bright to be further described.It is understood that embodiment described herein is used only for explaining the present invention, rather than Limitation of the invention.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art, Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from The conventional products of acquisition.
Influence of the concentration of the noble metal nanometer material of embodiment 1 to NO growing amounts
30 μ L L-arginines (50mM) are taken then to add 50 μ in 1mL Tris-HCl buffer solutions (50mM, pH=7.4) L CTAB solution (10mM), the GNRs@Au-CTAB that 1.7 μ L, 10 μ L, 14 μ L and 27 μ L concentration are 1nM are separately added into after mixing； NO detecting electrodes are placed in reaction solution, submergence 2-3mm, after electrode is stable, into reaction solution according to 100 μ L-100 μ L-500 μ L mode adds NADPH solution (50mM), per minor tick 1min；The electricity that detection third time is added after NADPH solution Changing value (Δ I) is flowed, makees noble metal nano structure concentration-Δ I curves.
Influence of the species of the noble metal nanometer material of embodiment 2 to NO growing amounts
30 μ L L-arginines (50mM) are taken then to add 5 μ L in 1mL Tris-HCl buffer solutions (50mM, pH=7.4) CTAB solution (0.1M), GNRs@Au-CTAB, GNRs@Ag- that 2 μ L concentration are 5nM (final concentration of 6pM) are separately added into after mixing CTAB, GNRs@AuAg-CTAB (coarse), GNRs@AuAg-CTAB (smooth), GNRs@Pd-CTAB and GNRs@Pt-CTAB；Will NO detecting electrodes are placed in reaction solution, submergence 2-3mm, after electrode is stable, into reaction solution according to 100 μ L-100 μ L-500 μ L mode adds NADPH solution (50mM), per minor tick 1min；The electric current that detection third time is added after NADPH solution Changing value (Δ I).
Embodiment 3
3 μ L L-arginines (500mM) are taken then to add 50 μ in 1mL Tris-HCl buffer solutions (50mM, pH=7.4) L CTAB solution (10mM), adds the GNRs@Au-CTAB that 14 μ L concentration are 1nM (final concentration of 8pM) after mixing, piping and druming mixes； NO detecting electrodes are placed in reaction solution, submergence 2-3mm, after electrode is stable, into reaction solution according to 350 μ L-350 μ L mode adds NADPH solution (50mM), per minor tick 1min；Second of curent change added after NADPH solution of detection It is worth (Δ I).
Embodiment 4
300 μ L L-arginines (5mM) are taken then to add 5 μ L CTAB in 1mL PBSs (50mM, pH=7.4) Solution (100mM), the GNRs@Au-CTAB that 1 μ L concentration is 10nM (final concentration of 16pM) are added after mixing；By NO detecting electrodes It is placed in reaction solution, submergence 2-3mm, after electrode is stable, 70 μ L NADPH solution is added into reaction solution (500mM), detection current variation value (Δ I).
Embodiment 5
1 μ L L-arginines (1M) are taken then to add 500 μ L in 1mL Tris-HCl buffer solutions (10mM, pH=7.0) CTAB solution (1mM), the GNRs@Au-CTAB that 17 μ L concentration are 0.1nM (final concentration of 1pM) are added after mixing；NO is detected into electricity Pole is placed in reaction solution, submergence 2-3mm, and after electrode is stable, 1mL-1mL-1mL-1mL-1mL is added into reaction solution NADPH solution (10mM), detection current variation value (Δ I).
Embodiment 6
2mL L-arginines (1mM) are taken to add 50 μ L after mixing in 1mL phosphate buffers (100mM, pH=8.0) Concentration is 1nM (final concentration of 20pM) GNRs@Au, and piping and druming mixes；NO detecting electrodes are placed in reaction solution, submergence is 2-3mm, after electrode is stable, 10 μ L NADPH solution (1M), detection current variation value (Δ I) are added into reaction solution.
Comparative example 1
Compared with Example 1, the final concentration of 0.05pM of noble metal nanometer material GNRs@Au-CTAB, other conditions and reality It is identical to apply example 1.
Comparative example 2
Compared with Example 1, the final concentration of 25pM of noble metal nanometer material GNRs@Au-CTAB, other conditions are with implementing Example 1 is identical.
Experimental result is as shown in table 1.
The in vitro study of the class nitric oxide enzymatic activity of the noble metal nanometer material of table 1
From embodiment 1 and Fig. 1 (a) as can be seen that NO growing amount and GNRs@Au-CTAB concentration within the specific limits It is proportionate：With the increase of GNRs@Au-CTAB concentration, NO growing amount increases, final concentration of as GNRs@Au-CTAB During 6pM, NO growing amount is maximum；With the further increase of GNRs@Au-CTAB concentration, NO growing amount is gradually reduced, can Can be due to that excessive GNRs@Au-CTAB assemble in the solution, specific surface area diminishes, and catalytic performance weakens.
From embodiment 2 and Fig. 1 (b) as can be seen that NO growing amount is relevant with the species of noble metal nanometer material：Put down on surface Sliding GNRs@AuAg-CTAB have optimal catalytic performance, NO growing amount highest.
From the point of view of embodiment 1-6 contrasts, concentration is that the GNRs@AuAg-CTAB of 6pM smooth surface have optimal catalysis Performance.
Compared with Example 1, the GNRs@Au-CTAB of comparative example 1 concentration is relatively low, can not effectively be catalyzed L-arginine and NADPH reaction generations NO；The GNRs Au-CTAB of comparative example 2 concentration is higher, and aggregation in the solution turns into larger micro- of particle diameter Rice structure, catalytic performance significantly reduce.
Catalytic performance of the noble metal nanometer material of embodiment 7 in people's Acute Monocytic Leukemia Cell Line
(1) to 4 × 106Personal acute monocytic leukemia (THP1) cell adds 5 μM of 4- amino -5- methyl of 1mL Amino -2,7- difluoro fluorescein(e) diacetate (DAF-FM DA) NO fluorescence probe solution, after being resuspended in centrifuge tube, 37 DEG C, 5%CO220min is incubated in incubator, is centrifuged off the NO probes of unloaded；
(2) cell is spread to 12 orifice plates, addition and contains GNRs@SiO2-NH2Mass concentration be respectively 0 μ g/mL, 10 μ g/ ML, 30 μ g/mL and 60 μ g/mL 1640 culture mediums, in 37 DEG C, 5%CO22h, 6h and 12h are incubated in incubator respectively；
(3) after being centrifuged off unnecessary nano particle, cell is resuspended using the DMEM culture mediums without phenol red serum-free, with stream Formula cell instrument detects intracellular NO fluorescence intensity.
Embodiment 8
Compared with Example 7, noble metal nanometer material uses glutathione closing GNRs@SiO2-NH2, other conditions and reality It is identical to apply example 7.
Embodiment 9
Compared with Example 7, noble metal nanometer material uses cysteine closing GNRs@SiO2-NH2, other conditions and reality It is identical to apply example 7.
Catalytic performance of the noble metal nanometer material of embodiment 10 in human umbilical vein endothelial cell
(1) human umbilical vein endothelial cell (HUVEC) is inoculated into 6 orifice plates, per hole 2 × 105Individual cell, was incubated Night；
(2) former culture medium is removed and with PBS 1-2 times, addition 200 μ L, 5 μM of DAF-FM DANO fluorescence probes per hole Solution, in 37 DEG C, 5%CO220min is incubated in incubator, removes the NO probes of unloaded and with PBS 2 times；
(3) add and contain GNRs@SiO2-NH2Mass concentration be respectively 0 μ g/mL, 10 μ g/mL, 30 μ g/mL and 60 μ g/ ML 1640 culture mediums, in 37 DEG C, 5%CO22h, 6h and 12h are incubated in incubator respectively；
(4) remove culture medium and with after PBS, cell dissociation is got off and with the DMEM culture mediums without phenol red serum-free Cell is resuspended, with the intracellular NO of flow cytomery fluorescence intensity.
Embodiment 11
Compared with Example 10, noble metal nanometer material uses glutathione closing GNRs@SiO2-NH2, other conditions with Embodiment 10 is identical.
Embodiment 12
Compared with Example 10, noble metal nanometer material uses cysteine closing GNRs@SiO2-NH2, other conditions with Embodiment 10 is identical.
Embodiment 13
(1) to 1 × 106It is glimmering that personal acute monocytic leukemia (THP1) cell adds 1 μM of DAF-FM DA NO of 1mL Light probe solution, after being resuspended in centrifuge tube, in 37 DEG C, 5%CO25min is incubated in incubator, is centrifuged off the NO of unloaded Probe；
(2) cell is spread to 12 orifice plates, addition and contains GNRs@SiO2Mass concentration be 100 μ g/mL 1640 culture Base, in 37 DEG C, 5%CO21h is incubated in incubator；
(3) after being centrifuged off unnecessary nano particle, cell is resuspended using the DMEM culture mediums without phenol red serum-free, with stream Formula cell instrument detects intracellular NO fluorescence intensity.
Embodiment 14
(1) human umbilical vein endothelial cell (HUVEC) is inoculated into 6 orifice plates, per hole 5 × 105Individual cell, was incubated Night；
(2) former culture medium is removed and with PBS 1-2 times, addition 200 μ L, 10 μM of 4,5- diaminourea fluoresceins two per hole Acetic acid esters (DAF-2Diacetate) NO fluorescence probe solution, in 37 DEG C, 5%CO260min is incubated in incubator, remove without NO probes of load and with PBS 2 times；
(3) add and contain GNRs@SiO2-NH2Mass concentration be 100 μ g/mL 1640 culture mediums, in 37 DEG C, 5%CO2 20h is incubated in incubator；
(4) remove culture medium and with after PBS, cell dissociation is got off and with the DMEM culture mediums without phenol red serum-free Cell is resuspended, with the intracellular NO of flow cytomery fluorescence intensity.
As shown in Fig. 2 (a), in embodiment 7, the fluorescence intensity of THP1 cells is with GNRs@SiO2-NH2Mass concentration and The increase of incubation time and increase, as GNRs@SiO2-NH2Concentration be 60 μ g/mL, incubation time is when being 12h, THP1 cells Fluorescence intensity is most strong；As shown in Fig. 2 (b), embodiment 8-9 uses glutathione or cysteine closing GNRs@SiO2-NH2Work Behind property site, GNRs@SiO2-NH2Class nitric oxide enzymatic activity be substantially suppressed.
As shown in Fig. 3 (a), in embodiment 10, the fluorescence intensity of HUVEC cells is with GNRs@SiO2-NH2Mass concentration Increase with incubation time and increase, as GNRs@SiO2-NH2Concentration be 60 μ g/mL, incubation time is when being 6h, HUVEC cells Fluorescence intensity it is most strong；As shown in Fig. 3 (b), embodiment 11-12 uses glutathione or cysteine closing GNRs@SiO2-NH2 Avtive spot after, GNRs@SiO2-NH2Class nitric oxide enzymatic activity be substantially suppressed.
Compared with Example 7, the GNRs@SiO of embodiment 132The unmodified NH in surface2, surface is in negative electrical charge, into cell GNRs@SiO2Reduce, NO growing amount is reduced；Embodiment 14 uses DAF-2Diacetate NO fluorescence probes, is formed with NO Fluorescence-causing substance stability is lower slightly, and obtained fluorescence intensity is relatively low.
In summary, noble metal nanometer material of the invention has nitric oxide synthase activity, can be catalyzed L-arginine Reacted with NADPH and produce NO；When GNRs@Au-CTAB concentration is 6pM, NO growing amount is maximum；Smooth surface GNRs@AuAg-CTAB have optimal catalytic performance, NO growing amount highest；It is intracellular in THP1, as GNRs@SiO2-NH2's When concentration is 60 μ g/mL, incubation time is 12h, NO growing amount is maximum；It is intracellular in HUVEC, as GNRs@SiO2-NH2It is dense Spend for 60 μ g/mL, incubation time when being 6h, NO growing amount is maximum.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art 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., within the scope of all falling within protection scope of the present invention and disclosing.

Claims (10)

  1. A kind of 1. noble metal nanometer material with nitric oxide synthase activity, it is characterised in that including：GNRs@Au、GNRs@ Ag, GNRs@AuAg, GNRs@Pd, GNRs@Pt or GNRs@SiO2In any one or at least two combination.
  2. 2. a kind of method that noble metal nanometer material catalysis L-arginine as claimed in claim 1 produces NO, it is characterised in that Comprise the following steps：
    (1) L-arginine solution is added into buffer solution；
    (2) noble metal nanometer material is added；
    (3) NADPH solution is added, produces NO.
  3. 3. according to the method for claim 2, it is characterised in that step (1) described buffer solution include Tris-HCl buffer solutions, In PBS or phosphate buffer any one or at least two combination, preferably Tris-HCl buffer solutions；
    Preferably, the concentration of step (1) described buffer solution is 10-100mM, preferably 50mM；
    Preferably, the pH value of step (1) described buffer solution is 7.0-8.0, preferably 7.4；
    Preferably, the concentration of step (1) the L-arginine solution is 1-1000mM, preferably 5-500mM, is more preferably 50mM；
    Preferably, the volume ratio of step (1) the L-arginine solution and the buffer solution is (0.1-200):100, be preferably (0.3-30):100, more preferably 3:100.
  4. 4. according to the method in claim 2 or 3, it is characterised in that before step (2) adds noble metal nanometer material also The step of including adding CTAB solution into the mixed liquor of step (1) buffer solution and L-arginine solution；
    Preferably, the concentration of the CTAB solution is 1-100mM, preferably 10mM；
    Preferably, the volume ratio of the CTAB solution and step (1) described buffer solution is (1-100):200, preferably 1:20；
    Preferably, the concentration of step (2) described noble metal nanometer material is 0.1-10nM, preferably 1-5nM；
    Preferably, the volume ratio of step (2) noble metal nanometer material and step (1) described buffer solution is (1-50):1000；
    Preferably, step (2) the noble metal nanometer material surface modification has CTAB.
  5. 5. according to the method described in claim any one of 2-4, it is characterised in that the concentration of step (3) the NADPH solution is 10-1000mM, preferably 50-500mM, more preferably 50mM；
    Preferably, the volume ratio of step (3) the NADPH solution and step (1) described buffer solution is (0.1-50):10, preferably For (0.7-7):10, more preferably 7:10；
    Preferably, step (3) the NADPH solution divides 1-5 addition, preferably divides 3 additions.
  6. 6. according to the method described in claim any one of 2-5, it is characterised in that comprise the following steps：
    (1) it is 10-100mM to concentration, it is molten to add the L-arginine that concentration is 1-1000mM in the buffer solution that pH value is 7.0-8.0 Liquid, the volume ratio of the L-arginine solution and the buffer solution 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 solution and L-arginine solution Liquid, the volume ratio of the CTAB solution and the buffer solution 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 solution are added Product ratio is (1-50):1000；
    (4) the NADPH solution that concentration is 10-1000mM is added, the volume ratio of the NADPH solution and the buffer solution is (0.1-50):10；
    (5) under the catalytic action of noble metal nanometer material, L-arginine reacts with NADPH, generates NO.
  7. 7. a kind of noble metal nanometer material as claimed in claim 1 is catalyzed the method that L-arginine produces NO in the cell, its It is characterised by, comprises the following steps：
    1) culture medium containing noble metal nanometer material is added into cell；
    2) in CO2Incubation step 1 in incubator) cell.
  8. 8. according to the method for claim 7, it is characterised in that the step 1) cell includes the white blood of people's Acute monocytic Sick cell and/or human umbilical vein endothelial cell；
    Preferably, the quantity of the step 1) cell is (1-5) × 105It is individual；
    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 closing GNRs@SiO2-NH2 Or cysteine closing 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, preferably 10-60 μ g/mL；
    Preferably, the time of the step 2) incubation is 1-20h, preferably 2-12h.
  9. A kind of 9. method for detecting the NO as caused by the methods described of claim 7 or 8, it is characterised in that comprise the following steps：
    1 ') culture medium containing NO fluorescence probes is added into cell；
    2 ') in CO2Incubation step 1 ' in incubator) cell；
    3 ') the intracellular NO of flow cytometry analysis fluorescence intensity is used.
  10. 10. the NO fluorescence probes include 4- amino -5- according to the method for claim 9, it is characterised in that step 1 ') Methylamino -2,7- difluoro fluorescein(e) diacetate and/or 4,5- diaminourea fluorescein(e) diacetate, preferably 4- amino -5- Methylamino -2,7- difluoro fluorescein(e) diacetates；
    Preferably, step 1 ') concentration of the NO fluorescence probes is 1-10 μM, preferably 5 μM；
    Preferably, step 2 ') time of the incubation is 5-60min, preferably 20min.
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