CN110102344B - Application of gold nanorods modified by cysteine as peroxidase-like enzyme - Google Patents
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 51
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- 235000018417 cysteine Nutrition 0.000 title claims abstract description 36
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 27
- 238000011534 incubation Methods 0.000 claims description 15
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- WHOHXJZQBJXAKL-DFWYDOINSA-N Mecysteine hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CS WHOHXJZQBJXAKL-DFWYDOINSA-N 0.000 description 2
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- 229910052737 gold Inorganic materials 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
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- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 1
- 229940025294 hemin Drugs 0.000 description 1
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/0234—Nitrogen-, phosphorus-, arsenic- or antimony-containing compounds
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- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/0234—Nitrogen-, phosphorus-, arsenic- or antimony-containing compounds
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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Abstract
The invention provides application of gold nanorods modified by cysteine as peroxidase-like enzyme; the application provided by the invention develops a new application of gold nanorods modified by cysteine molecules, and the gold nanorods (AuNR) are covalently modified by cysteine, so that the affinity of the gold nanorods with a substrate can be remarkably enhanced, and the peroxidase-like activity of the gold nanorods is further remarkably improved; the catalytic oxidation substrate activity is improved by 20 times under the acidic condition, the peroxidase-like activity and the controllable oxidation to the substrate in time and space are further enhanced by the unique local photothermal effect of the gold nanorods, a new thought is provided for comprehensively balancing catalytic sites and substrate affinity to achieve the optimization of the catalytic activity, and the method has important value for practical application.
Description
Technical Field
The invention belongs to the technical field of nano biology, and relates to application of gold nanorods modified by cysteine as peroxidase-like enzyme.
Background
The natural enzyme is protein or RNA which is produced by organisms and has substrate selectivity and high catalytic activity, is a high-efficiency biocatalyst and plays an important role in life activities. However, due to the high production cost and the inability to withstand extreme conditions such as high temperature, strong acid, strong base, etc., the industrial application of natural enzymes is greatly limited. Therefore, people aim at the enzyme mimics, which are substitutes for natural enzymes. Nanoenzymes are a new class of developed mimic enzymes, and are nanomaterials with enzyme-like activity (chem.soc.rev.,2019,48, 1004-1076). From the chemical composition, the nano-enzyme is mainly divided into nano-enzyme composed of oxide, metal and carbon-based nano-materials. From the catalytic reaction type, the currently discovered nanoenzymes are mainly oxidoreductases, such as oxidase-like enzyme, peroxidase-like enzyme, catalase-like enzyme and superoxide dismutase-like enzyme. Because of its low cost, high stability, and long-term availability, nanoenzymes have been used in research in fields such as biological detection, environmental management, disease diagnosis (chem. rev.,2019), (Nat Nanotechnol,2007,2, 577-.
The selectivity of the nanoenzyme to the substrate is often lower due to the lack of natural selection process of the natural enzyme compared with the natural enzyme, so how to improve the substrate selectivity of the nanoenzyme is a challenging problem in the research of the nanoenzyme. There are currently few advances that are relevant. Some scholars design the nano enzyme from the perspective of improving the selectivity of the substrate, and the catalytic activity of the nano enzyme is obviously improved. Ferroferric oxide magnetic nanoparticles (Fe)3O4MNPs) have peroxidase-like activity and can catalyze H2O2And (3) oxidizing a chromogenic substrate such as 3,3',5,5' -Tetramethylbenzidine (TMB). But it is paired with H2O2The affinity of the enzyme is much lower than that of Horse Radish Peroxidase (HRP) (Fe)3O4The Km value of (B) is about 40 times larger than that of HRP). The Yan project group (chem. Commun.2016,53,424-427) used for the reference of specific binding H in HRP2O2A functional group histidine at the site, modified to Fe3O4The surface of the nanoparticles. The experimental result shows that the histidine modified Fe3O4Nanoparticle pair H2O2The apparent affinity of (A) is improved by more than 10 times. Theoretical simulation shows that Fe3O4Histidine on the surface of the nanoparticles can react with H2O2Hydrogen bond formation, H enhancement2O2Adsorption on the surface of the particles. The Liu project group (J.Am.chem.Soc.,2017,139,5412-5419) significantly increases the substrate affinity of the nanoenzyme by using the molecular imprinting technology. They dispersed the chromogenic substrate in a polymer gel and coated with Fe3O4The surface of the nanoenzyme is washed, the chromogenic substrate is then eluted, the imprinting of the molecular template of the chromogenic substrate is left in the gel, and a 'pocket' for increasing the substrate affinity is constructed "To obtain Fe3O4As a center, polymer as a shell, Fe3O4And the polymer interface contains a nanogel with a characteristic hole of a chromogenic substrate. In addition, polymer monomers with charges are introduced into the nanogel, so that the charges of the nanogel are opposite to those of a chromogenic substrate, and the catalytic specificity (compared with the original Fe) to the substrates such as TMB, 2' -biazonitrogen-bis-3-ethylbenzthiazoline-6-sulfonic Acid (ABTS) and the like is further improved3O4Nanoparticles in contrast to k of negatively charged nanogels with TMB-characterized voidscatThe Km value is improved by about 15 times). They also demonstrated that the process also increased CeO2Enzyme-like activity of nanoparticles, gold nanoparticles. Itamar Willner et al (J.am. chem.Soc.,2016,138,164-72) modifies a catalytically active G-quadruplex with hemin and binds to a nucleic acid aptamer to give an aptamer/nucleic acid mimic enzyme. By selecting an appropriate aptamer, its affinity for the reaction substrate can be increased, thereby increasing the peroxidase-like activity of the aptamer/nucleic acid mimic enzyme. At the same time, they demonstrated that the conformation change of the aptamer/nucleic acid mimic enzyme also affects its catalytic activity.
CN105749907A discloses a photocatalytic material and a preparation method and application thereof, wherein the photocatalytic material is a core-shell structure formed by wrapping gold nanorods with nano titanium oxide, and the nano titanium oxide is wrapped on the surfaces of the gold nanorods through chemical connection with surface modification molecules of the gold nanorods. The photocatalytic material is prepared by modifying the whole surface of a gold nanorod by adopting organic molecules containing sulfydryl and carboxyl, and then the nano titanium oxide is prepared, and the nano titanium oxide wraps the gold nanorod through the interaction of a surface modification molecular group of the gold nanorod and a surface group of the nano titanium oxide.
According to the existing reports, the methods for improving the substrate affinity of the nanoenzyme are few, and the application of the micromolecule modified gold nanorod in the catalytic oxidation reaction is not researched. Therefore, the development of a new application of the gold nanorod has important significance for the application in the field of catalysis.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the application of gold nanorods modified by cysteine as peroxidase-like enzyme so as to solve the problems of low substrate affinity and low catalytic activity of the existing catalyst.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides the use of cysteine modified gold nanorods as a peroxidase-like enzyme.
The application provided by the invention develops a new application of gold nanorods modified by cysteine molecules, and the gold nanorods (AuNR) are covalently modified by cysteine, so that the affinity of the gold nanorods with a substrate can be remarkably enhanced, and the peroxidase-like activity of the gold nanorods is further remarkably improved.
Preferably, the cysteine is L-cysteine and/or D-cysteine.
Preferably, the modification is a covalent modification.
The covalent modification described herein refers to the gold-sulfur covalent bond formed between the gold atom and cysteine.
Compared with the prior art, the cysteine and the nano-particles are in covalent bond interaction, the cysteine is prevented from falling off the surfaces of the particles, the chromogenic substrate and the cysteine are combined through electrostatic (hydrogen bond) interaction to bring the substrate to the surfaces of the particles, the substrate affinity is enhanced, and the catalytic activity is obviously improved. Although covalent modification occupies part of the catalytically active site, in the actual reaction process, the promotion of catalytic activity is more critical as the substrate is brought around the catalytically active site. Therefore, the invention also provides a new idea of comprehensively balancing catalytic sites and substrate affinity to achieve optimization of catalytic activity.
Preferably, the covalent modification method is: and mixing the gold nanorods with CTAB for the first time, adding cysteine for incubation, and then adding CTAB for the second time for mixing to obtain cysteine modified gold nanorods.
Preferably, the concentration ratio of the gold nanorods to the cysteine is 1 (5 x 10)5～2×107) For example, it may be 1: 5X 105、1:1×106、1:4×106、1:8×106、1:1×107Or 1: 2X 107And so on.
In the invention, with the increase of the concentration of cysteine, the affinity of the gold nanorod substrate is also improved, and the peroxidase-like activity of the gold nanorod substrate is also improved, but when the concentration of cysteine is too high, the area of the gold nanorod modified by cysteine is too large, the active site of the gold nanorod is less exposed, the catalytic activity is influenced, and the peroxidase-like activity of the gold nanorod is not obviously increased.
Preferably, the concentration ratio of CTAB to cysteine in the first mixing is (8-12): 1; for example, 8:1, 9:1, 10:1, 11:1, 12:1, etc., preferably 10: 1.
In the invention, the consumption of CTAB is more during the first mixing, so as to wrap more gold nanorods and prevent excessive cysteine from modifying the surfaces of the gold nanorods. If CTAB is used in a smaller amount, the cysteine-modified gold nanorods may agglomerate.
Preferably, CTAB is used in an amount of 0.6-1 mM during the second mixing.
For subsequent experiments, after the second mixing, the solution needs to be diluted and other reagents are added to perform catalytic reaction. At this point, too high a CTAB concentration slows down the catalytic reaction rate, so we want to reduce the CTAB addition. If the concentration is too high, an additional centrifugation step is required, resulting in loss of AuNR-Cys.
Preferably, the incubation temperature is 25-45 deg.C, such as 25 deg.C, 27 deg.C, 28 deg.C, 30 deg.C, 31 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 38 deg.C, 39 deg.C, 40 deg.C, 42 deg.C, 43 deg.C or 45 deg.C.
Preferably, the incubation time is 0.5-1 h, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h or 1 h.
Preferably, the cysteine-modified gold nanorods act as a peroxidase-like enzyme in an oxidation reaction.
Preferably, the oxidizing agent of the oxidation reaction is hydrogen peroxide.
In the invention, the oxidation reaction realizes 20 times of activity improvement of catalytic oxidation substrate under acidic condition. In addition, the unique local photothermal effect of the gold nanorods further enhances the peroxidase-like activity and the time and space controllable oxidation of the substrate.
Preferably, the concentration of the hydrogen peroxide is 10 to 50mM, and may be, for example, 10mM, 12mM, 14mM, 15mM, 20mM, 23mM, 25mM, 30mM, 38mM, 40mM, 42mM, 45mM, or 50 mM.
Preferably, the oxidation reaction is carried out at a pH of 4.0 to 7.0, for example, the pH may be 4.0, 4.1, 4.2, 4.5, 4.6, 4.8, 5.0, 5.3, 5.5, 5.9, 6.0, 6.1, 6.3, 6.5, 6.8, 7.0, or the like. It is generally preferred that the pH is around 4.6.
Preferably, the temperature of the oxidation reaction is 25 to 60 ℃, and may be, for example, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃. Preferably 45 to 50 ℃.
In the present invention, the temperature of the oxidation reaction has an influence on the reaction rate. Generally, the higher the reaction temperature, the higher the reaction rate. However, when the reaction temperature is raised to a certain degree, for example, 60 ℃, the reaction rate tends to decrease.
Preferably, the oxidation reaction is performed under irradiation of a light source.
In the invention, because the gold nanorods have unique photo-thermal effect, the activity of peroxidase-like enzymes can be improved by irradiating the gold nanorods with a certain light source in the process of carrying out oxidation reaction, and the catalysis efficiency is improved.
The wavelength of the light source is matched with the plasmon absorption band of the specially used nano rod, and is generally 630-1500 nm, for example, 630nm, 700nm, 800nm, 900nm, 1000nm, 1100nm, 1200nm, 1300nm, 1400nm or 1500 nm.
Preferably, the substrate of the oxidation reaction is 3,3',5,5' -Tetramethylbenzidine (TMB).
The method provided by the invention can specifically improve the reaction rate with TMB as a substrate. Compared with the method using other substances as substrates, the method has the advantage that the reaction rate is improved more remarkably.
Preferably, the substrate for the oxidation reaction has a concentration of 0.1 to 1mM, and may be, for example, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, or 1 mM.
Compared with the prior art, the invention has the following beneficial effects:
the application provided by the invention develops a new application of the gold nanorod modified by the cysteine molecule, and the gold nanorod covalently modified by the cysteine can obviously enhance the affinity with a substrate, so that the peroxidase-like activity of the gold nanorod is obviously improved. Compared with the existing method, the surface modification molecule cysteine selected by the invention is in covalent bond interaction with the nano-particles, so that the cysteine is prevented from falling off from the particle surface, and the chromogenic substrate and the cysteine are combined through electrostatic (hydrogen bond) interaction to bring the substrate to the particle surface, thereby enhancing the substrate affinity and remarkably improving the catalytic activity. Although covalent modification occupies part of the catalytically active site, in the actual reaction process, the promotion of catalytic activity is more critical as the substrate is brought around the catalytically active site. Therefore, the invention also provides a new idea of comprehensively balancing catalytic sites and substrate affinity to achieve optimization of catalytic activity.
The oxidation reaction of the invention realizes that the activity of the catalytic oxidation substrate is improved by 20 times under the acidic condition, and the peroxidase-like activity and the controllable oxidation of the substrate in time and space are further enhanced by the unique local photothermal effect of the gold nanorods, thereby having important value for practical application.
Drawings
FIG. 1A is an extinction spectrum of peroxidase-like activity of AuNR-Cys80 in a water bath at 25 ℃ in example 1 to oxidize TMB to an oxidized product.
FIG. 1B is an extinction spectrum of the peroxidase-like activity of AuNR in a 25 ℃ water bath in example 1 to oxidize TMB to an oxidized product.
FIG. 2 is a transmission electron micrograph (scale 100nm) of AuNR in example 1.
FIG. 3 is a bar graph of the rate of oxidation of TMB by the peroxidase-like activity in a water bath at 25 ℃ to oxidation products for AuNR-CysX of example 2 at various ratios.
FIG. 4 is a line graph of the rate of peroxidase-like activity of AuNR-Cys80 prepared in example 2 at various incubation times in a 25 ℃ water bath to oxidize TMB to oxidized products.
FIG. 5A is a bar graph of the rates at which peroxidase-like activities in water baths of 25 deg.C, 35 deg.C, 45 deg.C and 60 deg.C in AuNR and AuNR-Cys80, respectively, oxidize TMB to oxidation products in example 3.
FIG. 5B is a plot of the rate scatter and the rate ratio of the peroxidase-like activity in water baths of AuNR and AuNR-Cys80 at 25 deg.C, 35 deg.C, 45 deg.C, and 60 deg.C, respectively, to oxidize TMB to oxidized products in example 3.
FIG. 6A is a bar graph of the rates at which peroxidase-like activities of AuNR and AuNR-Cys80 in a 25 ℃ water bath oxidize different substrates to oxidation products, respectively, in example 4.
FIG. 6B is a scatterplot of the rate and rate ratio of peroxidase-like activity of AuNR and AuNR-Cys80 in a 25 ℃ water bath to oxidize different substrates to oxidation products, respectively, in example 4.
FIG. 7 is a bar graph of the rates at which various surface-modified AuNRs of example 5 oxidize TMB to oxidation products, respectively, by peroxidase-like activity in a water bath at 25 ℃.
FIG. 8 is a bar graph of the rate of peroxidase-like activity of oxidizing TMB to oxidation products in example 6 with AuNR-Cys80 in a dark state in a 45 ℃ water bath or with 3W 1064nm laser irradiation.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The kit and the instruments used in the examples of the present invention are as follows: cetyl trimethylammonium bromide (CTAB), 3',5,5' -tetramethylbenzidine hydrochloride (tmb.2hcl), Glutathione (GSH) were purchased from Amresco; concentrated hydrochloric acid was purchased from Beijing chemical plant; 30% aqueous hydrogen peroxide, o-phenylenediamine (OPD) from Aladdin; l-cysteine, D-cysteine, N-acetyl-L-cysteine, sodium ascorbate available from Sigma Aldrich trade, Inc.; l-cysteine methyl ester hydrochloride (Cys-OMe & HCl) was purchased from Chiloeia (Shanghai) chemical industry development Co., Ltd. Incubation of AuNR and L-cysteine solution was carried out in a digital display constant temperature water bath (Katsuka Ke-Cai instruments Ltd.); the UV-VISIBLE-NIR extinction spectra were measured with a UV-VISIBLE spectrophotometer (Agilent Cary 60) at 25-60 ℃.
TMBox is a product of oxidation of 3,3',5,5' -Tetramethylbenzidine (TMB) by hydrogen peroxide, and has a characteristic absorption peak of 655nm in ultraviolet-visible spectrum (epsilon: 39000M)-1·cm-1) Here, the amount of TMBox produced was evaluated by the change in the intensity of the absorption peak at 655 nm. The invention firstly researches the effect of gold nanorods (AuNR) and gold nanorods (AuNR-Cys) coated with L-Cys on TMB oxidation, and utilizes AuNR and AuNR-Cys coated with CTAB with the same concentration (the coating means that CTAB and gold nanorods are connected through electrostatic interaction) and with the same particle concentration to react with TMB hydrochloride solution and H-Cys at the same temperature and pH value2O2The solutions were mixed and the formation of the TMBox was characterized.
Example 1
This example provides methods for L-Cys modification of AuNR and methods for determining the ability of AuNR-Cys and AuNR peroxidase-like activity to oxidize TMB
Peroxidase-like activity test of AuNR-Cys: 1mL of the 0.48nM gold nanorod (AuNR) solution after centrifugation was centrifuged once, the supernatant was removed, 500. mu.L of 10mM CTAB solution was added back, and the mixture was shaken up. mu.L of the solution was taken out, 12. mu.L of 10mM L-Cys solution was added thereto, diluted to 1.5mL, incubated in a water bath at 30 ℃ for 30min, centrifuged, the supernatant was removed, 75. mu.L of 1mM CTAB solution was added thereto, and shaken to give CTAB-coated AuNR-Cys. To the above solution were added 20. mu.L of 2mM dilute hydrochloric acid, 20. mu.L of 1M H in that order2O2And 20. mu.L of 32mM TMB in water, diluted to 1.5mL and mixed quicklyAnd (4) homogenizing. The change of extinction spectrum was recorded every 1min with an ultraviolet-visible spectrophotometer under a water bath condition of 25 ℃. As shown in particular in fig. 1A.
Peroxidase-like activity test of AuNR: 1mL of the gold nanorods after centrifugation was centrifuged once at 0.48nM, the supernatant was removed, and 500. mu.L of 10mM CTAB solution was added back and shaken up. mu.L of the solution was taken out from the above solution, diluted to 1.5mL, incubated in a water bath at 30 ℃ for 30min, centrifuged, the supernatant was removed, and 75. mu.L of 1mM CTAB solution was added thereto, and shaken to give CTAB-coated AuNR. To the above solution was added 20. mu.L of 2mM dilute hydrochloric acid, 20. mu.L of 1M H2O2And 20. mu.L of 32mM TMB in water, diluted to 1.5mL and mixed quickly. The change of extinction spectrum was recorded every 2min with an ultraviolet-visible spectrophotometer under a water bath condition of 25 ℃. As shown in particular in fig. 1B.
As can be seen from the comparison between fig. 1A and fig. 1B, under the same conditions, the peroxidase-like activity of AuNR-Cys is higher than that of AuNR, i.e., covalent modification of L-Cys enhances the peroxidase-like activity of AuNR.
Example 2
This example tests the effect of L-Cys concentration and incubation time on the Activity of AuNR-Cys peroxidase
In example 1, L-Cys solutions were added in amounts of 6, 12, 60, 120 and 240. mu.L during incubation of L-Cys and AuNR, respectively, and the products obtained after incubation centrifugation were designated AuNR-Cys40, AuNR-Cys80, AuNR-Cys400, AuNR-Cys800 and AuNR-Cys 1600. To AuNR-CysX (X: 0-1600. mu.M, X represents the concentration of Cys in the incubation solution), 75. mu.L of 1mM CTAB solution was added and shaken to give CTAB-coated AuNR-CysX. To the above solution was added 20. mu.L of 2mM dilute hydrochloric acid, 20. mu.L of 1M H2O2The solution, 20. mu.L of 32mM TMB solution, was diluted to 1.5mL and mixed quickly. The change of extinction spectrum was recorded every 1min with an ultraviolet-visible spectrophotometer under a water bath condition of 25 ℃. The specific results are shown in FIG. 3.
As can be seen in FIG. 3, as the L-Cys concentration increases, the oxidation rate of TMB increases; however, the rate of AuNR-Cys1600 has not risen significantly.
In example 1, Cys and AuNR incubationIn the process, the incubation time is set to 0.5h, 3h and 15h, and 3 AuNR-Cys are obtained. To each AuNR-Cys was added 75. mu.L of 1mM CTAB solution, and shaken to give CTAB-coated AuNR-Cys. To the above solution was added 20. mu.L of 2mM dilute hydrochloric acid, 20. mu.L of 1M H2O2And 20. mu.L of 32mM TMB solution, diluted to 1.5mL and mixed rapidly. The change of extinction spectrum was recorded every 1min with an ultraviolet-visible spectrophotometer under a water bath condition of 25 ℃. The specific results are shown in FIG. 4.
As can be seen from FIG. 4, the rate of oxidation of TMB increased with increasing incubation time during the preparation process.
Example 3
This example measures the peroxidase-like Activity of AuNR-Cys at different temperatures for TMB oxidizing ability
AuNR-Cys80 was prepared as in example 1. To the above solution was added 20. mu.L of 2mM dilute hydrochloric acid, 20. mu.L of 1M H2O2And 20. mu.L of 32mM TMB in water, diluted to 1.5mL and mixed quickly. The change of extinction spectrum was recorded every 1min with an ultraviolet-visible spectrophotometer under the conditions of 25 deg.C, 35 deg.C, 45 deg.C or 60 deg.C water bath. The specific results are shown in fig. 5A and 5B.
From the combined results of fig. 5A and fig. 5B, it can be seen that the activity of AuNR-Cys and AuNR increases after the temperature rises, and the increasing trend of AuNR-Cys rate becomes slower when the temperature rises to 60 ℃. The optimal reaction temperature is about 45-50 ℃ in comprehensive view.
Example 4
This example measures the peroxidase-like Activity of AuNR-Cys for the ability to oxidize different substrates
AuNR-Cys80 was prepared as in example 1. To the above solution was added 20. mu.L of 1M H2O2The solution, 30. mu.L of 100mM OPD solution, was diluted to 1.5mL and mixed rapidly. The change of extinction spectrum was recorded every 2min with an ultraviolet-visible spectrophotometer under the conditions of 25 deg.C, 35 deg.C, 45 deg.C or 60 deg.C water bath.
AuNR-Cys80 was prepared as in example 1. To the above solution was added 20. mu.L of 1M H2O2The solution was diluted to 1 μ L with 15 μ L of 10mM sodium ascorbate (NaA)5mL and mix quickly. The change of extinction spectrum was recorded every 2min with an ultraviolet-visible spectrophotometer under the conditions of 25 deg.C, 35 deg.C, 45 deg.C or 60 deg.C water bath. Specific results are shown in fig. 6A and 6B.
The results of fig. 6A and 6B show that the activity of AuNR-Cys peroxidase-like enzyme is more potent in oxidizing TMB, indicating that it has the specificity of TMB substrate.
Example 5
This example measures the ability of various surface-modified AuNR-type peroxidase activities to oxidize TMB
AuNR-D-Cys80, AuNR-LNAC80, AuNR-Cys-OMe80 or AuNR-GSH80 were prepared by changing the Cys solution in the incubation solution of L-Cys and AuNR in example 1 to 12. mu.L of 10mM D-Cystein, N-acetyl-L-Cysteine (LNAC), L-Cysteine methyl ester hydrochloride (Cys-OMe. HCl) or Glutathione (GSH) solution. To each of the above solutions was added 20. mu.L of 2mM dilute hydrochloric acid, and 20. mu.L of 1M H in that order2O2And 20. mu.L of 32mM TMB in water, diluted to 1.5mL and mixed quickly. The change of extinction spectrum was recorded every 1min with an ultraviolet-visible spectrophotometer under a water bath condition of 25 ℃. The specific results are shown in FIG. 7.
As is clear from the results shown in FIG. 7, the activities of L-Cys, D-Cystein and LNAC-modified AuNR peroxidases were stronger. Most preferred is L-Cys or D-Cysteine.
Example 6
This example measures the peroxidase-like activity of AuNR-Cys in the dark state or under laser irradiation for TMB oxidation.
AuNR-Cys was prepared as in example 1. To the above solution were added 20. mu.L of 2mM dilute hydrochloric acid, 20. mu.L of 1M H in that order2O2And 20. mu.L of 32mM TMB in water, diluted to 1.5mL and mixed quickly. The solution was irradiated with a 1064nm laser at a laser power of 3W in a water bath at 25 ℃ and a temperature of about 45 ℃. The change in extinction spectrum was recorded every 1min with an ultraviolet-visible spectrophotometer.
AuNR-Cys was prepared as in example 1. To the above solution were added 20. mu.L of 2mM dilute hydrochloric acid, 20. mu.L of 1M H in that order2O2And 20. mu.L of 32mM TMB in water, diluted to 1.5mL and mixed quickly. The change of extinction spectrum was recorded every 1min with an ultraviolet-visible spectrophotometer under a water bath condition of 45 ℃. The specific results are shown in FIG. 8.
From the results of fig. 8, it can be seen that the activity of AuNR-Cys is significantly higher than that of the dark state and the oxidation rate of TMB is higher under the same solution temperature of laser irradiation.
The applicants state that the present invention illustrates the application of cysteine modified gold nanorods of the present invention as a peroxidase-like enzyme by the above examples, but the present invention is not limited to the above process steps, i.e., it does not mean that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (15)
1. The application of gold nanorods modified by cysteine as peroxidase-like enzyme is characterized in that the modification is covalent modification;
the covalent modification method comprises the following steps: mixing gold nanorods and CTAB for the first time, adding cysteine for incubation, centrifuging, and then adding CTAB for the second time to obtain cysteine modified gold nanorods;
the concentration ratio of the gold nanorods to the cysteine is 1 (5 multiplied by 10)5～2×107)；
The concentration ratio of CTAB to cysteine is (8-12) to 1 during the first mixing;
the amount of CTAB used in the second mixing is 0.6-1 mM.
2. Use according to claim 1, wherein the cysteine is L-cysteine and/or D-cysteine.
3. The use according to claim 1, wherein the concentration ratio of CTAB to cysteine at the first mixing is 10: 1.
4. Use according to claim 1, wherein the incubation temperature is 25-45 ℃.
5. The use according to claim 1, wherein the incubation time is 0.5 to 1 h.
6. The use according to claim 1, wherein the cysteine-modified gold nanorods act as a peroxidase-like enzyme in an oxidation reaction.
7. Use according to claim 6, wherein the oxidizing agent of the oxidation reaction is hydrogen peroxide.
8. The use according to claim 7, wherein the concentration of hydrogen peroxide is 10 to 50 mM.
9. The use according to claim 6, wherein the oxidation is carried out at a pH of 4.0 to 7.0.
10. The use according to claim 6, wherein the temperature of the oxidation reaction is 25 to 60 ℃.
11. The use according to claim 10, wherein the temperature of the oxidation reaction is 45 to 50 ℃.
12. The use according to claim 6, wherein the oxidation reaction is carried out under irradiation with a light source.
13. The use according to claim 12, wherein the light source has a wavelength of 630 to 1500 nm.
14. Use according to claim 6, wherein the substrate of the oxidation reaction is 3,3',5,5' -tetramethylbenzidine.
15. The use according to claim 14, wherein the substrate for the oxidation reaction is present in a concentration of 0.1 to 1 mM.
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