CN111334499B - Urease-nano gold composite material with adjustable urease activity and preparation method and application thereof - Google Patents
Urease-nano gold composite material with adjustable urease activity and preparation method and application thereof Download PDFInfo
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- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/05—Metallic powder characterised by the size or surface area of the particles
- B22F1/054—Nanosized particles
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- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/07—Metallic powder characterised by particles having a nanoscale microstructure
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- B22F9/00—Making metallic powder or suspensions thereof
- B22F9/16—Making metallic powder or suspensions thereof using chemical processes
- B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
- B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/58—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving urea or urease
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01005—Urease (3.5.1.5)
Abstract
The invention discloses a urease-nano gold composite material with adjustable urease activity, a preparation method and application thereof. The composite material is formed by combining urease and nano gold, wherein the mass ratio of the urease to the nano gold is (0.5-1.5). The urease modified nano gold synthesized by the invention can effectively control the catalytic activity of urease through photo-thermal effect. The mixture of urea and phenol is used as a substrate, and under the condition of laser irradiation, the decomposition degree of the urease modified nano gold on the substrate is greatly improved within the same reaction time. By changing the irradiation power of laser, the decomposition degree of the urease modified nano gold on the substrate is changed, and the catalytic activity of the urease can be adjusted by changing the laser power.
Description
Technical Field
The invention belongs to the technical field of nano materials and biological detection, and particularly relates to a urease-nano gold composite material with adjustable urease activity, and a preparation method and application thereof.
Background
Urease is nickel-containing oligoenzyme, has absolute specificity, can specifically catalyze urea to decompose and release ammonia and carbon dioxide, and is commonly used for measuring urea in urine and blood. In general, the activity of urease is strongest at about 60℃and the detection efficiency is low due to the low activity at room temperature. At present, the detection speed is mainly realized by heating, but the heating mode often needs a water bath or an oven, so that the detection method is huge and complex. In recent years, the nano material with photo-thermal effect has been widely focused, and the nano particles are equivalent to the biological molecules in size, so that the functionalized nano gold has the optimal biological recognition capability and the sensing, marking and energy conversion properties of the nano particles after the nano particles are combined with the biological molecules. The porous hollow nanospheres have excellent photo-thermal properties and can release heat under the irradiation of 808nm near infrared light. Therefore, after the urease is modified to the porous hollow gold nano-gold surface, the photo-thermal property of the nano-gold can be utilized to adjust the activity of the urease and control the enzymatic decomposition of urea.
Disclosure of Invention
The invention provides a urease-nano gold composite material with adjustable urease activity, a preparation method and application thereof.
The urease-nano gold composite material with adjustable urease activity is formed by combining urease and nano gold, wherein the mass ratio of the urease to the nano gold is (0.5-1.5) to (0.5-1.5).
The nano gold is porous nano gold, and urease can be combined to the surface of the nano gold through sulfhydryl or electrostatic adsorption.
The nano gold is spherical, and the average diameter of the nano gold is 100-150 nm.
The preparation method of the urease-nano gold composite material with adjustable urease activity comprises the following steps:
1) Preparation of spherical porous nano gold: adding PVP solution into hydroquinone solvent, then adding AgNO 3 Adding chloroauric acid solution dropwise into the above solution in dark place, oscillating at the same time, continuing oscillating after dripping, standing, adding concentrated ammonia water, reacting on a shaking table, and separating after reactionThe core is used for obtaining porous hollow spherical nano gold; dispersing the gold nanoparticles in water to obtain a gold nanoparticle solution;
2) Preparation of urease-nano gold composite material: adding the nano gold solution obtained in the step (1) into a urease solution, mixing, vibrating, reacting and centrifuging to obtain the urease-nano gold composite material.
In the step 1), the concentration of PVP solution is 80-100 mM, the concentration of hydroquinone is 25-30 mM, and AgNO 3 The concentration of the solution is 50-70 mM, and the concentration of the chloroauric acid solution is 20-30 mM; PVP solution, benzenediol and AgNO 3 The volume ratio of the solution to the chloroauric acid solution is (1700-1900) (60-70) (3-5) (60-70); continuously oscillating for 4-6 min, standing for 50-70 min, wherein the concentration of the concentrated ammonia water is 12-14 mM, and the volume ratio of the concentrated ammonia water to the chloroauric acid solution is (1-3) (6-7); the reaction temperature of the shaking table is 35-40 ℃, and the reaction time of the shaking table is 1-3 h; the concentration of the nano gold solution is 1-2 mg/mL.
In the step 2), the concentration of the urease solution is 0.5-1.5 mg/mL, and the volume ratio of the urease solution to the nano-gold solution is (3-4): 2-3; the oscillation reaction time is 6-12 h.
The invention relates to application of the urease-nano gold composite material in urea detection.
The principle of the invention is as follows: because the sulfhydryl and gold can form stable gold sulfide bond, the urease structure contains sulfhydryl, so that the urease can be combined on the surface of nano gold through sulfhydryl, and because the nano gold is porous spherical nano gold, the urease can be adsorbed on the surface of nano gold or in a pore canal through static electricity, but the urease is mainly distributed on the surface of nano gold.
The invention has the beneficial effects that:
the urease modified nano gold synthesized by the invention can effectively control the catalytic activity of urease through photo-thermal effect. The mixture of urea and phenol is used as a substrate, and under the condition of laser irradiation, the decomposition degree of the urease modified nano gold on the substrate is greatly improved within the same reaction time. By changing the irradiation power of laser, the decomposition degree of the urease modified nano gold on the substrate is changed, and the catalytic activity of the urease can be adjusted by changing the laser power.
Drawings
FIG. 1 is a schematic diagram of the synthesis of urease-modified nanogold;
FIG. 2 shows laser irradiation of urease-modified nanogold (a) and urease (b) for 10min (power 1.5W/cm 2 ) A subsequent absorption spectrum;
FIG. 3 is a graph showing absorption spectra of urease-modified nanogold after irradiation for 10min at different laser powers.
Detailed Description
To facilitate understanding by those skilled in the art, the present invention provides a specific embodiment of urease-modified nanogold.
Example 1
(1) Preparation of hollow porous nano gold
To 18mL of 90mM PVP solution was added 640. Mu.L of 28mM hydroquinone, 40. Mu.L, 60mM AgNO 3 After the solution is protected from light, 640 mu L of 25mM chloroauric acid is added dropwise, shaking is carried out while adding, standing is carried out for 60min after 5min of intense shaking, 200 mu L of concentrated ammonia water is added, and after 2h of reaction is carried out on the solution under a shaking table at 37 ℃, the solution is dispersed into 2mL of water after centrifugation, thus obtaining 1.5mg/mL of porous hollow nano gold solution.
(2) Synthesis of urease-modified nanogold
Adding 240 mu L of the nano gold solution obtained in the step (1) into 360 mu L of urease, mixing, vibrating overnight, and centrifuging to obtain the urease functionalized nano gold.
(3) Photothermal control of urease activity
Using a power of 1.5W/cm 2 Near infrared light with the wavelength of 808nm irradiates the urease modified nano gold spheres and the urease solution alone for 10min respectively, the results are shown in fig. 2, and the results show that: the urease activity on the urease functionalized nano gold spheres is greatly improved.
The urease activity is controlled by adjusting the irradiation power, the porous nano gold has a photo-thermal effect, and when the nano gold solution is irradiated by 808nm laser, the laser light energy can be converted into heat energy, so that the temperature of the solution is increased. The higher the laser irradiation power, the more remarkable the rise in solution temperature is, so that when the laser irradiation power is from 1.3W/cm 2 Increase to 1.5W/cm 2 When the urease activity on the urease functionalized nano gold spheres is gradually enhanced. However, as the laser irradiation power continues to increase, the urease activity on the urease functionalized gold nanospheres gradually decreases due to the heat inactivation of the enzyme caused by the excessive temperature. The results are shown in fig. 3, and the results indicate that: the laser irradiation power was 1.5W/cm 2 When the urease functionalized nano gold spheres have the maximum urease activity.
Example 2
(1) Preparation of hollow porous nano gold
To 17mL of 100mM PVP solution was added 600. Mu.L of 30mM hydroquinone, 50. Mu.L of 50mM AgNO 3 After the solution is protected from light, 600 mu L of 30mM chloroauric acid is added dropwise, shaking is carried out while adding, standing is carried out for 70min after shaking vigorously for 6min, 300 mu L of 12mM ammonia water is added, and after 3h of reaction under a shaking table at 35 ℃, the solution is centrifugally dispersed into 1.5mL of water to obtain 2mg/mL of porous hollow nano gold solution.
(2) Synthesis of urease-modified nanogold
And (3) adding 300 mu L of the nano gold solution obtained in the step (1) into 400 mu L of urease with the concentration of 1.5mg/mL, mixing, vibrating overnight, and centrifuging to obtain the urease functionalized nano gold.
Example 3
(1) Preparation of hollow porous nano gold
To 19mL of 80mM PVP solution was added 700. Mu.L of 25mM hydroquinone, 30. Mu.L of 70mM AgNO 3 After the solution is protected from light, 700 mu L of 20mM chloroauric acid is added dropwise, shaking is carried out while adding, standing is carried out for 50min after 4min of intense shaking, 100 mu L of 14mM ammonia water is added, and after reaction is carried out for 1h at a temperature of 40 ℃ in a shaking table, the solution is centrifugally dispersed into 3mL of water, and thus 1.0mg/mL of porous hollow nano gold solution is obtained.
(2) Synthesis of urease-modified nanogold
Adding 200 mu L of the nano gold solution obtained in the step (1) into 300 mu L of urease with concentration of 0.5mg/mL, mixing, shaking overnight, and centrifuging to obtain the urease functionalized nano gold.
Claims (4)
1. The urease-nano gold composite material is formed by combining urease and nano gold, wherein the mass ratio of the urease to the nano gold is (0.5-1.5); the nano gold is porous nano gold, and urease is fixed on the surface of the nano gold through sulfhydryl and electrostatic adsorption; the nano gold is spherical, and the average diameter of the nano gold is 100-150 nm; the preparation method comprises the following steps:
1) Preparation of spherical porous nano gold: adding AgNO into PVP solution and hydroquinone solvent 3 Adding chloroauric acid solution dropwise in dark place while shaking, continuing shaking after the dropwise addition, standing, adding concentrated ammonia water, reacting under a shaking table, and centrifuging after the reaction is finished to obtain spherical porous nano gold; dispersing the gold nanoparticles in water to obtain a gold nanoparticle solution;
2) Preparation of urease-nano gold composite material: adding the nano-gold solution obtained in the step 1) into a urease solution, mixing, vibrating and reacting, and centrifuging to obtain a urease-nano-gold composite material;
in the step 1), the concentration of hydroquinone is 25-30 mM;
in the step 2), the concentration of the urease solution is 0.5-1.5 mg/mL, and the volume ratio of the urease solution to the nano-gold solution is (3-4): 2-3; the oscillation reaction time is 6-12 h.
2. The method for preparing urease-nanogold composite material with adjustable urease activity according to claim 1, wherein in the step 1), PVP solution has a concentration of 80-100 mM, agNO 3 The concentration of the solution is 50-70 mM, and the concentration of the chloroauric acid solution is 20-30 mM; PVP solution, benzenediol and AgNO 3 The volume ratio of the solution to the chloroauric acid solution is (1700-1900): (60-70): (3-5): (60-70); continuing to shake for 4-6 min, standing for 50-70 min, wherein the concentration of the concentrated ammonia water is 12-14 mM, and the volume ratio of the concentrated ammonia water to the chloroauric acid solution is (1-3) (6-7).
3. The method for preparing the urease-nanogold composite material with adjustable urease activity according to claim 1, wherein the reaction temperature of a shaking table is 35-40 ℃ and the reaction time of the shaking table is 1-3 h; the concentration of the nano gold solution is 1-2 mg/mL.
4. Use of a urease-nanogold composite material prepared according to the preparation method of any one of claims 1 to 3 in the preparation of a reagent for detecting urea.
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CN108949145A (en) * | 2018-09-12 | 2018-12-07 | 福建医科大学 | Urase-fluorescent au nanocluster material and preparation method thereof |
CN109738417A (en) * | 2019-01-25 | 2019-05-10 | 中南大学 | A method of tumour cell is detected using porous gold nanosphere |
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CN108949145A (en) * | 2018-09-12 | 2018-12-07 | 福建医科大学 | Urase-fluorescent au nanocluster material and preparation method thereof |
CN109738417A (en) * | 2019-01-25 | 2019-05-10 | 中南大学 | A method of tumour cell is detected using porous gold nanosphere |
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Jing Xu等.Gold nanoparticles bound on microgel particles and their application as an enzyme support.《Nanotechnology》.2007,第18卷(第18期),265704. * |
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