CN107824800B - Preparation method of sea urchin-shaped gold nanoparticles and method for labeling protein - Google Patents
Preparation method of sea urchin-shaped gold nanoparticles and method for labeling protein Download PDFInfo
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- B22—CASTING; POWDER METALLURGY
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
A preparation method of sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 10-25 minutes to fully finish the reaction, then adding citric acid or potassium carbonate to adjust the pH value, and adjusting the pH value to 5.5-8.5. Compared with the prior art, the method has the advantages of rapidness, no need of green synthesis of gold seeds and the like.
Description
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a preparation method of sea urchin-shaped gold nanoparticles and a method for labeling proteins by the gold nanoparticles.
Background
The nano particles have unique properties of light, electricity, magnetism, catalysis and the like, and have wide application prospects in the fields of catalysis, electronics, optics, biomedicine and the like, so that the nano particles are widely concerned. Nanoparticles having an uneven and spiny structure are an important component of three-dimensional nanomaterials, and have various names according to their different classifications, such as star-shaped, multi-pin-shaped, and uneven gold particles. Due to the rough surface and the high specific surface area, the plasmon resonance in a near infrared region can be enhanced, and the electromagnetic field enhancement effect is concentrated on the tip of the nanoparticle, so that a plurality of hot spots are formed on the surface of the same nanoparticle. Therefore, nanoparticles with non-smooth surfaces are effectively applied to Surface Enhanced Raman Scattering (SERS), electrochemical applications, nuclear magnetic imaging, chemiluminescence sensing, cellular imaging, and photothermal spectroscopy analysis therapies.
The existing synthesis methods of the unsmooth nano particles mainly comprise a seed method, an in-situ growth method and a bionic synthesis method. Some methods require seed synthesis in advance, so that the synthesis steps are complicated and time-consuming, some methods require toxic surfactants or macromolecular polymers, and the methods are not easy to obtain and have poor biocompatibility, which is contrary to the concept of green chemistry.
The echinoid gold nanoparticles can generate abundant hot points due to the special surface energy, and are widely applied as an ideal SERS substrate, but the traditional echinoid gold nanoparticles are poor in stability after being synthesized and are easy to aggregate and flocculate; in biomedical application, protein, nucleic acid and other biomolecules cannot be directly coupled and labeled like colloidal gold.
In addition, echinoid gold nanoparticles are greatly different from common colloidal gold, particularly the surface charge property has certain obstacle in marking biomolecules such as protein, the echinoid gold nanoparticles are easy to agglomerate in biological buffer solution, and the echinoid gold nanoparticles cannot be redissolved in the buffer solution to form agglomerate dead gold once being centrifuged after marking, so that further application is limited.
Therefore, the sea urchin-shaped nano gold particles which have simple preparation method and accurate marking result and do not generate agglomeration and flocculation, the preparation method thereof and the method for marking protein by the nano gold particles are provided.
Disclosure of Invention
The invention discloses a preparation method of sea urchin-shaped gold nanoparticles, which is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 10-25 minutes to fully finish the reaction, then adding citric acid or potassium carbonate to adjust the pH value, and adjusting the pH value to 5.5-8.5.
Adding 1-5 parts by weight of the N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, wherein the concentration is 0.06-0.1mol/L, and the PH is 7.2-7.5; the added chloroauric acid solution is 0.5-1.5 weight parts, and the concentration is 0.01-0.03 mol/L.
In actual use: the pH value is determined according to the principle that the pH value after adding citric acid or potassium carbonate is equal to the isoelectric point value +1 of the labeled protein, for example: the isoelectric point of the marked staphylococcus aureus protein A is 5.5, and the pH value of the prepared sea urchin-shaped nanogold particles is 6.5 according to the isoelectric point of the marked protein and the isoelectric point + 1.
A protein labeling method using sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring and standing for 10-25 minutes to fully complete the reaction, then adding citric acid or potassium carbonate to adjust the pH value to the isoelectric point +1 of the marked protein, adjusting the pH value to the isoelectric point of the marked staphylococcus aureus protein A to be 5.5, adjusting the pH value of the prepared sea urchin-shaped nano gold particles to be 6.5 according to the isoelectric point of the marked protein to be +1, carrying out ultrasonic treatment for 8-15 minutes when adding citric acid or potassium carbonate, stirring at 200-300 rpm, adding the marked protein while stirring, reducing the stirring speed to 80-120 rpm after the addition is completed, reacting for 2-6 hours, then adding hydrolyzed casein sodium fragments and sodium polystyrene sulfonate, and continuing the ultrasonic stirring reaction for 20-40 minutes, and centrifugally collecting to obtain the protein functional sea urchin-shaped nano gold particles.
Adding hydrolyzed casein sodium fragment with final concentration of 0.5% -1.5%, namely adding 0.5g hydrolyzed casein sodium fragment into 100ml of the above solution, and dissolving to obtain solution with concentration of 0.5%; the final concentration of the sodium polystyrene sulfonate is 0.1-0.5%, namely the concentration of 0.2% after 0.2g of sodium polystyrene sulfonate is added into 100ml of the solution and dissolved, the molecule of the sodium polystyrene sulfonate is 20000-40000, and the concentration ratio of the hydrolyzed casein sodium fragment to the sodium polystyrene sulfonate is controlled to be 3: 7-5: 5, namely the mass percent of the hydrolyzed casein sodium fragment is 30-50%, and the rest is sodium polystyrene sulfonate.
The conventional casein used at present has low solubility in water, is not easy to dissolve and is difficult to prepare, so the hydrolyzed casein fragment used is sodium salt of casein after treatment, and is dissolved when preparing a solution.
In actual use: the method comprises the following steps:
(1) rapidly adding 200 microliters of chloroauric acid solution with the concentration of 0.025mol/L into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution with certain concentration and pH value at one time, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue to indicate that echinoid nano gold particles are formed, stopping stirring, and standing for 15 minutes to fully complete the reaction;
(2) adding a proper amount of citric acid or potassium hydroxide into the prepared sea urchin-shaped nano gold particles according to the isoelectric point of the marked protein and the isoelectric point +1, stirring uniformly by ultrasonic at 200rpm, adding the marked protein with a certain concentration, reacting for 2-6 hours,
(3) adding the mixed solution of the hydrolyzed casein sodium fragment and the sodium polystyrene sulfonate, continuing to perform ultrasonic stirring reaction for 1 hour, and finally performing centrifugal collection to obtain the sea urchin-shaped gold nanoparticles with the protein marker coupling functionality.
The scheme can be further optimized, limited and improved as follows:
the concentration of the N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution in the step (1) is controlled to be 0.06-0.1 mol/L; controlling the pH value to be 7.2-7.5; the final concentration of the added chloroauric acid is controlled to be 0.005-1%.
And (2) adding the chloroauric acid in the step (1) while stirring, and fully and uniformly mixing the added chloroauric acid for reaction.
All reagents of the sea urchin-shaped gold nanoparticles regulated in the step (2) need to be citric acid and potassium hydroxide, and the optimal concentration of the citric acid is 0.5mol/L citric acid; the optimal concentration of potassium hydroxide is as follows: 0.01mol/L of the mixture,
step (2) the pH of the echinoid gold nanoparticles prepared according to the step (1) is 7.0, the pH is adjusted according to the isoelectric point +1 of the coupled labeled protein, for example, when the isoelectric point of the coupled labeled protein is 5.5, the pH value of the echinoid gold nanoparticles is adjusted to 6.5, 0.5mol/L citric acid is added for adjustment, for example, when the isoelectric point of the coupled labeled protein is 7.5, the pH value of the echinoid gold nanoparticles is adjusted to 8.5, 0.01mol/L potassium hydroxide is added for adjustment.
Adding a mixed solution of hydrolyzed sodium caseinate fragments and sodium polystyrene sulfonate in the step (3), wherein the final concentration of the hydrolyzed sodium caseinate fragments in the mixed solution is 0.5-1.5%; the final concentration of the sodium polystyrene sulfonate is 0.1 to 0.5 percent; the molecule of the sodium polystyrene sulfonate is 20000-40000, the two components are mixed and used together, and either component can cause failure of protein labeling or influence subsequent use.
The method comprises the following specific steps: adding 10ml of 0.08mol of N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution with pH7.5 into a clean 100ml three-neck flask, stirring at 20O rpm, quickly adding 10ml of chloroauric acid solution with the concentration of 0.025mol/L at one time, continuously stirring and uniformly mixing, reducing the rotating speed to 100rpm, slowly stirring until the color is changed from colorless transparency to dark blue to indicate that echinoid nano gold particles are formed, stopping stirring, standing for 15 minutes and fully completing the reaction; and (3) measuring the particle size of the prepared sea urchin-shaped gold nanoparticles: good dispersibility, uniform particle size distribution, and particle size of about 254 nm.
Marking staphylococcus aureus protein A by 5ml of echinoid gold nanoparticles, wherein the isoelectric point of the marked staphylococcus aureus protein A is 5.5, the pH value of the prepared echinoid gold nanoparticles to be regulated is 6.5 according to the isoelectric point of the marked protein and the isoelectric point of the marked protein is +1, the pH value of the prepared echinoid gold nanoparticles is about 7.3, the prepared echinoid gold nanoparticles are regulated to 6.5 by 0.5mol/L citric acid, then the echinoid gold nanoparticles are added with ultrasonic 200rpm and stirred uniformly, the staphylococcus aureus protein A is added, mixed solution with 0.7 percent of hydrolyzed casein sodium fragment and 0.3 percent of polystyrene sodium sulfonate is added after reaction for 6 hours, the reaction is continued for 1 hour, and finally, the product is obtained by centrifugal collection, the echinoid gold nanoparticles marked and coupled by the staphylococcus aureus protein A can be used as an immune probe to detect antibodies in serum, and the images are the negative, the positive, the negative and the negative, the negative and the positive and, Positive test results.
Compared with the prior art, the invention has the advantages of good seed extraction effect, simple structure and the like.
Therefore, the echinoid gold nanoparticles are prepared by a rapid and green synthesis method without gold seeds, weak reducing agents are added at one time under the condition of neutral alkali bias to synthesize the echinoid gold nanoparticles, and parameters such as particle size, morphology and the like of the echinoid gold nanoparticles are adjusted by the amount of the weak reducing agents. More importantly, echinoid gold nanoparticles are greatly different from common colloidal gold, particularly the surface charge property has certain obstacle in marking biomolecules such as protein, the echinoid gold nanoparticles are easy to agglomerate in biological buffer solution, and the echinoid gold nanoparticles cannot be redissolved in the buffer solution to form agglomerate dead gold once being centrifuged after marking, so that further application is limited. The patent designs a preparation method specially aiming at sea urchin-shaped gold nanoparticles and a marking method using the gold nanoparticles, so that protein coupling marking of the sea urchin-shaped gold nanoparticles is realized, the activity of marked compound protein cannot be changed, and precipitates are loose after centrifugation and easy to redissolve, so that the gold nanoparticles can be used in immunochromatography test strips and immune percolation.
Drawings
FIG. 1 is a graph showing the results of particle size measurement of sea urchin-like gold nanoparticles prepared.
FIG. 2 shows the result of detecting the negative and positive serum antibodies of tuberculosis patients (left: negative; right: positive) using the sea urchin-like gold nanoparticles with the coupling function of staphylococcus aureus protein A marker as a probe.
Detailed Description
Example 1: a preparation method of sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully and uniformly stirring, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 10 minutes to fully finish the reaction, and then adding citric acid or potassium carbonate to adjust the pH value to 6.5.
Adding 2 parts by weight of the N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, wherein the concentration is 0.1mol/L, and the PH is 7.4; the chloroauric acid solution was added in an amount of 1.0 part by weight and at a concentration of 0.03 mol/L.
In actual use: the pH value is determined according to the principle that the pH value after adding citric acid or potassium carbonate is equal to the isoelectric point value +1 of the labeled protein, for example: the isoelectric point of the marked staphylococcus aureus protein A is 5.5, and the pH value of the prepared sea urchin-shaped nanogold particles is 6.5 according to the isoelectric point of the marked protein and the isoelectric point + 1.
Example 2: a preparation method of sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully and uniformly stirring, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 25 minutes to fully finish the reaction, and then adding citric acid or potassium carbonate to adjust the pH value to 8.5.
Adding 1 part by weight of the N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, wherein the concentration is 0.06mol/L, and the PH is 7.2; the chloroauric acid solution was added in an amount of 0.5 parts by weight and at a concentration of 0.02 mol/L.
In actual use: the pH value is determined according to the principle that the pH value after adding citric acid or potassium carbonate is equal to the isoelectric point value +1 of the labeled protein, for example: the isoelectric point of the marked protein is 7.5, and the pH value of the prepared sea urchin-shaped nanogold particles is 8.5 according to the isoelectric point of the marked protein and the isoelectric point + 1.
Example 3: a preparation method of sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully and uniformly stirring, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 15 minutes to fully finish the reaction, and then adding citric acid or potassium carbonate to adjust the pH value to 5.5.
5 parts by weight of the N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution is added, the concentration is 0.08mol/L, and the PH is 7.5; the chloroauric acid solution was added in an amount of 0.7 parts by weight and at a concentration of 0.01 mol/L.
In actual use: the pH value is determined according to the principle that the pH value after adding citric acid or potassium carbonate is equal to the isoelectric point value +1 of the labeled protein, for example: the isoelectric point of the marked protein is 6.5, and the pH value of the prepared sea urchin-shaped nanogold particles is 7.5 according to the isoelectric point of the marked protein and the isoelectric point of the prepared sea urchin-shaped nanogold particles.
Example 4: a preparation method of sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully and uniformly stirring, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 20 minutes to fully finish the reaction, and then adding citric acid or potassium carbonate to adjust the pH value to 7.5.
Adding 3 parts by weight of the N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, wherein the concentration is 0.09mol/L, and the PH is 7.3; the chloroauric acid solution was added in an amount of 1.5 parts by weight and at a concentration of 0.01 mol/L.
Example 5: a preparation method of sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully and uniformly stirring, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 22 minutes to fully finish the reaction, and then adding citric acid or potassium carbonate to adjust the pH value to 7.0.
4 parts by weight of the N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution is added, the concentration is 0.07mol/L, and the PH is 7.5; the chloroauric acid solution was added in an amount of 1.3 parts by weight and at a concentration of 0.01 mol/L.
Example 6: a protein labeling method using sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring and standing for 11 minutes to fully finish the reaction, then adding citric acid or potassium carbonate to adjust the pH value to the isoelectric point +1 of the marked protein, adjusting the pH value to the isoelectric point of the marked staphylococcus aureus protein A to be 5.5, adjusting the pH value of the prepared sea urchin-shaped nano gold particles to be 6.5 according to the isoelectric point +1 of the marked protein, carrying out ultrasonic treatment for 8 minutes when adding citric acid or potassium carbonate, stirring at 250rpm, adding the marked protein while stirring, reducing the stirring speed to 110rpm after the addition is finished, reacting for 2 hours, then adding hydrolyzed casein sodium fragment and polystyrene sodium sulfonate, continuing the ultrasonic stirring reaction for 35 minutes, and centrifuging and collecting the protein functional sea urchin-shaped nano gold particles.
Adding hydrolyzed casein sodium fragment with final concentration of 0.75%, namely adding 0.5g of hydrolyzed casein sodium fragment into 100ml of the above solution to dissolve to obtain a solution with concentration of 0.5%; the final concentration of the sodium polystyrene sulfonate is 0.5 percent, namely the concentration of the dissolved sodium polystyrene sulfonate is 0.2 percent after 0.2g of sodium polystyrene sulfonate is added into 100ml of the solution, the molecule of the sodium polystyrene sulfonate is 20000, the concentration ratio of the hydrolyzed casein sodium fragment to the sodium polystyrene sulfonate is controlled to be 3.5:6.5, namely the mass percent of the hydrolyzed casein sodium fragment is 35 percent, and the rest is the sodium polystyrene sulfonate.
The conventional casein used at present has low solubility in water, is not easy to dissolve and is difficult to prepare, so the hydrolyzed casein fragment used is sodium salt of casein after treatment, and is dissolved when preparing a solution.
Example 7: a protein labeling method using sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring and standing for 14 minutes to fully finish the reaction, then adding citric acid or potassium carbonate to adjust the pH value to the isoelectric point +1 of the marked protein, adjusting the pH value to the isoelectric point of the marked staphylococcus aureus protein A to be 5.5, adjusting the pH value of the prepared sea urchin-shaped nano gold particles to be 6.5 according to the isoelectric point of the marked protein to be equal to the isoelectric point +1, carrying out ultrasonic treatment for 15 minutes when adding citric acid or potassium carbonate, stirring at 200rpm, adding the marked protein while stirring, reducing the stirring speed to 100rpm after the addition is finished, reacting for 6 hours, then adding hydrolyzed casein sodium fragment and polystyrene sodium sulfonate, continuing the ultrasonic stirring reaction for 20 minutes, and centrifuging and collecting the protein functional sea urchin-shaped nano gold particles.
Adding hydrolyzed casein sodium fragment with final concentration of 0.5%, namely adding 0.5g of hydrolyzed casein sodium fragment into 100ml of the above solution to dissolve to obtain a solution with concentration of 0.5%; the final concentration of the sodium polystyrene sulfonate is 0.2 percent, namely the concentration of the dissolved sodium polystyrene sulfonate is 0.2 percent after 0.2g of sodium polystyrene sulfonate is added into 100ml of the solution, the molecule of the sodium polystyrene sulfonate is 35000, the concentration ratio of the hydrolyzed casein sodium fragment to the sodium polystyrene sulfonate is controlled to be 4:6, namely the mass percentage of the hydrolyzed casein sodium fragment is 40 percent, and the rest is the sodium polystyrene sulfonate.
The conventional casein used at present has low solubility in water, is not easy to dissolve and is difficult to prepare, so the hydrolyzed casein fragment used is sodium salt of casein after treatment, and is dissolved when preparing a solution.
Example 8: a protein labeling method using sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring and standing for 21 minutes to fully finish the reaction, then adding citric acid or potassium carbonate to adjust the pH value to the isoelectric point +1 of the marked protein, adjusting the pH value to the isoelectric point of the marked staphylococcus aureus protein A to be 5.5, adjusting the pH value of the prepared sea urchin-shaped nano gold particles to be 6.5 according to the isoelectric point +1 of the marked protein, carrying out ultrasonic treatment for 11 minutes when adding citric acid or potassium carbonate, stirring at 300rpm, adding the marked protein while stirring, reducing the stirring speed to 80rpm after the addition is finished, reacting for 4 hours, then adding hydrolyzed casein sodium fragment and polystyrene sodium sulfonate, continuing the ultrasonic stirring reaction for 30 minutes, and centrifuging and collecting the protein functional sea urchin-shaped nano gold particles.
Adding hydrolyzed casein sodium fragment with final concentration of 1.5%, namely adding 0.5g of hydrolyzed casein sodium fragment into 100ml of the above solution to dissolve to obtain a solution with concentration of 0.5%; the final concentration of the sodium polystyrene sulfonate is 0.1 percent, namely the concentration of the dissolved sodium polystyrene sulfonate is 0.2 percent after 0.2g of sodium polystyrene sulfonate is added into 100ml of the solution, the molecule of the sodium polystyrene sulfonate is 40000, the concentration ratio of the hydrolyzed casein sodium fragment to the sodium polystyrene sulfonate is controlled to be 5:5, namely the mass percentage of the hydrolyzed casein sodium fragment is 50 percent, and the rest is the sodium polystyrene sulfonate.
The conventional casein used at present has low solubility in water, is not easy to dissolve and is difficult to prepare, so the hydrolyzed casein fragment used is sodium salt of casein after treatment, and is dissolved when preparing a solution.
Example 9: a protein labeling method using sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring and standing for 12 minutes to fully finish the reaction, then adding citric acid or potassium carbonate to adjust the pH value to the isoelectric point +1 of the marked protein, adjusting the pH value to the isoelectric point of the marked staphylococcus aureus protein A to be 5.5, adjusting the pH value of the prepared sea urchin-shaped nano gold particles to be 6.5 according to the isoelectric point +1 of the marked protein, carrying out ultrasonic treatment for 10 minutes when adding citric acid or potassium carbonate, stirring at 230rpm, adding the marked protein while stirring, reducing the stirring speed to 120rpm after the addition is finished, reacting for 5 hours, then adding hydrolyzed casein sodium fragment and polystyrene sodium sulfonate, continuing the ultrasonic stirring reaction for 25 minutes, and centrifuging and collecting the protein functional sea urchin-shaped nano gold particles.
Adding hydrolyzed casein sodium fragment with final concentration of 1.25%, namely adding 0.5g of hydrolyzed casein sodium fragment into 100ml of the above solution to dissolve to obtain a solution with concentration of 0.5%; the final concentration of the sodium polystyrene sulfonate is 0.4 percent, namely the concentration of the dissolved sodium polystyrene sulfonate is 0.2 percent after 0.2g of sodium polystyrene sulfonate is added into 100ml of the solution, the molecule of the sodium polystyrene sulfonate is 30000, the concentration ratio of the hydrolyzed casein sodium fragment to the sodium polystyrene sulfonate is controlled to be 3:7, namely the mass percentage of the hydrolyzed casein sodium fragment is 30 percent, and the rest is the sodium polystyrene sulfonate.
The conventional casein used at present has low solubility in water, is not easy to dissolve and is difficult to prepare, so the hydrolyzed casein fragment used is sodium salt of casein after treatment, and is dissolved when preparing a solution.
Example 10: a protein labeling method using sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, fully stirring and uniformly mixing, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring and standing for 23 minutes to fully finish the reaction, then adding citric acid or potassium carbonate to adjust the pH value to the isoelectric point +1 of the marked protein, adjusting the pH value to the isoelectric point of the marked staphylococcus aureus protein A to be 5.5, adjusting the pH value of the prepared sea urchin-shaped nano gold particles to be 6.5 according to the isoelectric point +1 of the marked protein, carrying out ultrasonic treatment for 9 minutes when adding citric acid or potassium carbonate, stirring at 270rpm, adding the marked protein while stirring, reducing the stirring speed to 90rpm after the addition is finished, reacting for 3 hours, then adding hydrolyzed casein sodium fragment and polystyrene sodium sulfonate, continuing the ultrasonic stirring reaction for 40 minutes, and centrifuging and collecting the protein functional sea urchin-shaped nano gold particles.
Adding hydrolyzed casein sodium fragment with final concentration of 1.0%, namely adding 0.5g of hydrolyzed casein sodium fragment into 100ml of the above solution to dissolve to obtain a solution with concentration of 0.5%; the final concentration of the sodium polystyrene sulfonate is 0.3 percent, namely the concentration of the dissolved sodium polystyrene sulfonate is 0.2 percent after 0.2g of sodium polystyrene sulfonate is added into 100ml of the solution, the molecule of the sodium polystyrene sulfonate is 25000, the concentration ratio of the hydrolyzed casein sodium fragment to the sodium polystyrene sulfonate is controlled to be 4.5:5.5, namely the mass percent of the hydrolyzed casein sodium fragment is 45 percent, and the rest is the sodium polystyrene sulfonate.
The conventional casein used at present has low solubility in water, is not easy to dissolve and is difficult to prepare, so the hydrolyzed casein fragment used is sodium salt of casein after treatment, and is dissolved when preparing a solution.
Claims (2)
1. A protein labeling method using sea urchin-shaped gold nanoparticles is characterized by mainly comprising the following steps: adding chloroauric acid solution into N- (2-hydroxyethyl) piperazine-N '-2-ethanesulfonic acid solution, fully and uniformly stirring, slowly stirring until the color is changed from colorless transparency to dark blue, stopping stirring, standing for 10-25 minutes to fully complete the reaction, then adding citric acid or potassium carbonate to adjust the pH value to 5.5-8.5, adding 1-5 parts by weight of N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid solution, wherein the concentration is 0.06-0.1mol/L, and the pH is 7.2-7.5; adding 0.5-1.5 parts by weight of chloroauric acid solution with the concentration of 0.01-0.03 mol/L, adjusting the pH value to the isoelectric point +1 of the marked protein, adding citric acid or potassium carbonate, performing ultrasonic treatment for 8-15 minutes at the stirring speed of 200-300 rpm, adding the marked protein while stirring, reducing the stirring speed to 80-120 rpm after the addition is finished, reacting for 2-6 hours, adding hydrolyzed casein sodium fragment and sodium polystyrene sulfonate, continuing the ultrasonic stirring reaction for 20-40 minutes, and performing centrifugal collection to obtain protein functional echinoid nano gold particles;
adding hydrolyzed casein sodium segment to the mixture until the final concentration is 0.5-1.5%; the final concentration of the sodium polystyrene sulfonate is 0.1 to 0.5 percent, the molecule of the sodium polystyrene sulfonate is 20000-40000, and the concentration ratio of the hydrolyzed casein sodium fragment to the sodium polystyrene sulfonate is controlled to be 3: 7-5: 5, namely the mass percent of the hydrolyzed casein sodium fragment is 30-50%, and the rest is sodium polystyrene sulfonate.
2. The protein labeling method using sea urchin-like gold nanoparticles according to claim 1, characterized in that: 10-15ug of protein was added to 1ml of the sea urchin-like gold particle solution.
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| CN102019179A (en) * | 2009-09-17 | 2011-04-20 | 国家纳米科学中心 | Gold-core/platinum-shell nano bar analogue enzyme solution and preparation method thereof |
| CN103252504A (en) * | 2013-04-17 | 2013-08-21 | 浙江师范大学 | Novel method for synthesizing porous flower-like nano palladium at room temperature |
| CN105461920A (en) * | 2015-12-16 | 2016-04-06 | 华南师范大学 | Method for synthesizing sea-urchin-shaped gold nanoparticles and spherical polyaniline through one-pot method and applications |
| CN105665741A (en) * | 2016-02-06 | 2016-06-15 | 北京工业大学 | Simple small-size gold nanoparticle star with regulatable branch lengths and preparing method of gold nanoparticle star |
| CN106117356A (en) * | 2016-06-30 | 2016-11-16 | 天津大学 | A kind of golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody |
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| KR20120095111A (en) * | 2011-02-18 | 2012-08-28 | 부산대학교 산학협력단 | Microchip for detecting trace phenolic endocrine disruptors and detection method using the same |
| CN102328093B (en) * | 2011-08-30 | 2013-03-20 | 吉林大学 | Method for preparing gold nano particles with echinoid structures by seed medium approach |
| CN103357887B (en) * | 2013-07-01 | 2015-12-02 | 西安交通大学 | Hollow gold-silver alloy nano particle of a kind of sea urchin shape and its preparation method and application |
| CN103433484B (en) * | 2013-08-22 | 2015-06-17 | 福建医科大学 | Bovine serum albumin-platinum composite nanomaterial mimetic peroxidase, preparation and application thereof |
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| CN102019179A (en) * | 2009-09-17 | 2011-04-20 | 国家纳米科学中心 | Gold-core/platinum-shell nano bar analogue enzyme solution and preparation method thereof |
| CN103252504A (en) * | 2013-04-17 | 2013-08-21 | 浙江师范大学 | Novel method for synthesizing porous flower-like nano palladium at room temperature |
| CN105461920A (en) * | 2015-12-16 | 2016-04-06 | 华南师范大学 | Method for synthesizing sea-urchin-shaped gold nanoparticles and spherical polyaniline through one-pot method and applications |
| CN105665741A (en) * | 2016-02-06 | 2016-06-15 | 北京工业大学 | Simple small-size gold nanoparticle star with regulatable branch lengths and preparing method of gold nanoparticle star |
| CN106117356A (en) * | 2016-06-30 | 2016-11-16 | 天津大学 | A kind of golden nanometer particle coupling horseradish peroxidase and the preparation method of alpha-fetoprotein traget antibody |
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