CN102838984A - Preparation method of chymotrypsin protected fluorescent au nanocluster material - Google Patents
Preparation method of chymotrypsin protected fluorescent au nanocluster material Download PDFInfo
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- CN102838984A CN102838984A CN2012103363671A CN201210336367A CN102838984A CN 102838984 A CN102838984 A CN 102838984A CN 2012103363671 A CN2012103363671 A CN 2012103363671A CN 201210336367 A CN201210336367 A CN 201210336367A CN 102838984 A CN102838984 A CN 102838984A
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Abstract
The invention discloses a preparation method of a chymotrypsin protected fluorescent au nanocluster material, which comprises the following steps that a chymotrypsin solution is dropwise added to a chloroauric acid solution and mixed uniformly; under a lucifugal condition, the pH of the mixed solution is adjusted to be alkaline; and the mixed solution reacts under a water bath condition at 20-37 DEG C for 1-48 h, and then is separated and purified to obtain the chymotrypsin protected fluorescent au nanocluster material. The preparation method is simple and convenient to operate; reaction conditions are mild; adopted reagents are environmental-friendly; generated au nanoclusters are aggregated difficultly; the prepared chymotrypsin protected fluorescent au nanocluster material has high-yield fluorescence quanta and is good in biocompatibility.
Description
Technical field
The present invention relates to a kind of gold nano cluster, relate in particular to a kind of Quimotrase protective money nanocluster Preparation of Fluorescent Material method.
Background technology
Up to now, the investigator has invented multiple fluorescent nano material, like semiconductor-quantum-point, coating doped with nanometer particle, mix lanthanon nanoparticle and carbon nano dot etc.Compare with GFP with the small molecules dyestuff, above-mentioned fluorescent nano material has high optical physics property, big specific surface area, is easy to carry out characteristics such as finishing and color tunable.Therefore, they might supply the not enough even alternative conventional fluorescent probe of small molecules dyestuff, on bio-sensing, small molecules imaging, photoelectronics and nanosecond medical science, great application prospect are arranged.
Metal nanometer cluster is a novel nano fluorescent material that receives much concern, and it is made up of to a hundreds of atom several, and general diameter is less than 2nm, and character is between the precious metal of monatomic and nanoparticle or big volume.The size of metal nanometer cluster is near Fermi's wavelength of electronics, and the successive energy state is split into discrete energy state, the optics different with nanoparticle, electricity and chemical property.Its most outstanding characteristic is the character of pl-, and has bigger Stokes shift and high emissivity.The metal nanometer cluster fluorescent material is mainly used in biomarker and photoemissivity.
The scientific research personnel is devoted to study the route of synthesis of water soluble fluorescence gold nano cluster recently.Because nanocluster is easy to reunite, so the metals ion in the reducing solution is easy to obtain macromole nanoparticle rather than micromolecular nanocluster.In addition, the part that is wrapped in particle surface has very big influence to its emission characteristic.So, select suitable reagent to be used to stablize extra small nanoparticle submanifold and can prevent that not only its reunion from can also improve fluorescence quantum yield.
Protein can be as the metal nanometer cluster template of synthetic hyperfluorescence as biomacromolecule.Contain a large amount of binding sites in the protein structure, these binding sites can be stablized even the reducing metal ion, thereby set up the support of synthesized micromolecule metal nanometer cluster.2009, as reductive agent and protective material, synthesized first with the gold nano cluster fluorescent material (Au NCs) of protein as stablizer with bovine serum albumin (BSA).It should be noted that this gold nano cluster fluorescent material original position in BSA and chlorauric acid solution is synthetic.Therefore, BSA is not only the reductive agent of hydrochloro-auric acid, or the protective material of gold nano cluster.Receive the inspiration of this discovery, investigators begin report and remove synthetic fluorescence Au NCs such as N,O-Diacetylmuramidase and ferritin etc. as biological support.Enzyme is the protein of the special chemical reaction of catalysis, and at present, existing bibliographical information horseradish peroxidase (HRP) is used for synthetic fluorescence Au NCs as reductive agent and protective material.
Summary of the invention
The invention provides a kind of Quimotrase protective money nanocluster Preparation of Fluorescent Material method, this method can stop the generation of golden nanometer particle, prevents the reunion of gold nano cluster, and the material that makes has better biocompatibility.
A kind of Quimotrase protective money nanocluster Preparation of Fluorescent Material method may further comprise the steps:
The Quimotrase drips of solution is added in the chlorauric acid solution, stirs; Under the lucifuge condition, the pH value of regulating mixing solutions is an alkalescence, and after reacting 1～48h under 20～37 ℃ of water bath condition, separation and purification makes described Quimotrase protective money nanocluster fluorescent material.
Quimotrase is a protein molecule; In the present invention as the metal nanometer cluster template of synthesizing hyperfluorescence; Contain the amino acid binding site that contains sulfydryl, disulfide linkage, amino in a large number in its molecule; These sites combine with hydrochloro-auric acid through gold-sulfide linkage or electrostatic interaction in the preparation process, form Quimotrase-hydrochloro-auric acid mixture, when pH value of solution is transferred to alkalescence; But the tyrosine in the Quimotrase and contain Methionin, l-arginine in-situ reducing hydrochloro-auric acid under alkaline condition of primary amino obtains Quimotrase protective money nanocluster.
The schematic diagram of the inventive method as shown in Figure 1, on the one hand, Quimotrase makes the hydrochloro-auric acid location; Can reduce the content of hydrochloro-auric acid in the solution; The generation of the golden nanometer particle that prevention is bigger than gold nano cluster particle diameter, on the other hand, Quimotrase can prevent the gold nano cluster reunion.With the existing material compared that is wrapped in the gold nano cluster surface, Quimotrase is less to the emission characteristic influence of gold nano cluster, can improve the output of fluorescent quantum; And Quimotrase is the green bio preparation, and amino is rich on the surface, and the fluorescent material of generation has excellent biological compatibility.
Described Quimotrase can be the Quimotrase that derives from animal, like pig Quimotrase, BCTR etc., is preferably BCTR.
Under the certain condition of hydrochloro-auric acid concentration; The Quimotrase strength of solution influences the amount of single enzyme molecule combined chloride auric acid; And then influence generates the amount of gold nano cluster and the luminous intensity of fluorescent material; Said Quimotrase strength of solution is preferably 15～90mg/mL, and more preferably 40～60mg/mL most preferably is 50mg/mL.
Chlorauric acid solution concentration is too high, and with the reunion that causes gold nano cluster and the generation of gold particle, the concentration of said chlorauric acid solution is preferably 0.1%～2%, and more preferably 0.5%～1%, most preferably be 1%.
Single Quimotrase binding site molecule point is limited; Therefore the mol ratio between Quimotrase and the hydrochloro-auric acid can finally influence the performance of fluorescent material; Like luminous quantity, particle diameter or the like; Between Quimotrase and the hydrochloro-auric acid mol ratio be preferably 0.068: 1～0.410: 1, more preferably 0.1～0.3: 1.
At alkaline condition, help the Methionin that improves tyrosine and contain primary amino, arginic reductibility, the pH value of said mixing solutions is 10～12, most preferably is 12.
The method of said separation and purification is dialysis, and this method is simple to operate, and mild condition is less to the product influence.
The said reaction times is preferably 4h.
Quimotrase protective money nanocluster fluorescent material provided by the invention since its excellent biological compatibility with have no side effect, therefore in aspect tool wide application prospect such as the detection of cell imaging, albumen and bacterium, immunoassay, biosensors.Like a kind of Quimotrase protective money nanocluster fluorescent material provided by the invention; Owing to the existence of Quimotrase has abundant carboxyl; Can form amido linkage and further fixedly contain amino folic acid through catalysis; Obtain having the composite fluorescence nano-probe of specificity selection function,, can carry out the fluorescent mark imaging of cell through the specific recognition of folic acid and cancer cells surface folacin receptor.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention has introduced Quimotrase as reductive agent and protective material, and the reductibility under alkaline condition of the reductibility functional group on the Quimotrase is strong, helps the synthetic of gold nano cluster;
(2) simple operating steps of the present invention, reaction conditions is gentle, the agents useful for same environmental friendliness, the gold nano cluster of generation is difficult for reuniting;
(3) fluorescent quantum of the Quimotrase protective money nanocluster fluorescent material of the present invention preparation with high yield also has excellent biological compatibility.
Description of drawings
The preparation synoptic diagram of Fig. 1 Quimotrase protective money of the present invention nanocluster.
The high-resolution-ration transmission electric-lens figure of the Quimotrase protective money nanocluster of Fig. 2 embodiment of the invention 1.
The Quimotrase protective money nanocluster (left side, red fluorescence) of the embodiment of the invention 1 and the photo of Quimotrase (right side, no fluorescence) solution under Fig. 3 UV-light (365nm, purple background) irradiation.
The Quimotrase protective money nanocluster (A) of Fig. 4 embodiment of the invention 1 and the fluorescence emission spectrogram of Quimotrase (B), excitation wavelength is 475nm.
The fluorescence excitation spectrum and the emmission spectrum figure of the Quimotrase protective money nanocluster of Fig. 5 embodiment of the invention 1.
The visible light spectrogram of the Quimotrase protective money nanocluster of Fig. 6 embodiment of the invention 1.
The x-ray photoelectron power spectrum (Au4f) of the Quimotrase protective money nanocluster of Fig. 7 embodiment of the invention 1.
The energy-dispersive spectroscopy of the Quimotrase protective money nanocluster of Fig. 8 embodiment of the invention 1.
Embodiment
Embodiment 1
A) hydrochloro-auric acid is dissolved in to make concentration in the ultrapure water be 1% chlorauric acid solution; BCTR solution with 50mg/mL under magnetic agitation dropwise joins in the chlorauric acid solution; Quimotrase/hydrochloro-auric acid mol ratio is 0.212: 1, magnetic agitation 10min;
B) under the lucifuge condition; In a), add the 1mol/L sodium hydroxide solution in the gained solution; Regulator solution pH to 12; Behind 37 ℃ stirred in water bath reaction 4h, with gained solution pack in the dialysis tubing with distill water dialysis to dialyzate specific conductivity constant till, solution lyophilize in the dialysis tubing is obtained Quimotrase protective money nanocluster fluorescent material.
Subsequently, the Quimotrase protective money nanocluster through step (a)～(b) preparation is carried out operations such as fluorescent spectroscopy, transmission electron microscope scanning, the Measurement results that obtains is shown in Fig. 2～8.
Among the high-resolution-ration transmission electric-lens figure shown in Figure 2, only observe particle diameter, prove that the gained material is a nanocluster at the following particle of 2nm.Quimotrase protective money nanocluster exhibit red fluorescence (left side) and pure chymotrypsin protein enzyme solution (right side) then do not have fluorescence under Fig. 3 medium ultraviolet light (365nm, purple background) irradiation.In the fluorescence emission spectrogram shown in Figure 4; During with the optical excitation of 475nm; The fluorescence emission peak that Quimotrase protective money nanocluster has; And Quimotrase does not have fluorescence peak, and the fluorescence excitation spectrum of figure five Quimotrase protective money nanoclusters and emmission spectrum figure show that the maximum excitation wavelength is about 475nm, and maximum emission wavelength is about 628nm.Among Fig. 6, do not occur the plasma body absorption peak of golden nanometer particle characteristic in the visible spectrum, prove that once more synthetic is a nanocluster.Among Fig. 7, the x-ray photoelectron power spectrum characterizes provides the valence state information (Au (4f of gold in the gold nano cluster
7/2)) and Au (4f
5/2)).Energy-dispersive spectroscopy characterizes the existence proved C, N, O, Au element among Fig. 8, has proved the compound of Quimotrase and gold.These results show effective preparation of Quimotrase protective money nanocluster fluorescent material.Select for use with the similar rhodamine B of Quimotrase protective money nanocluster fluorescent material photoluminescent property be reference material, calculating Quimotrase protective money nanocluster fluorescent materials quantum yield is 2.3%.
Embodiment 2
A) hydrochloro-auric acid is dissolved in to make concentration in the ultrapure water be 0.5% chlorauric acid solution; BCTR solution with 20mg/mL under magnetic agitation dropwise joins in the chlorauric acid solution; Quimotrase/hydrochloro-auric acid mol ratio is 0.170: 1, magnetic agitation 10min;
B) under the lucifuge condition; In a), add the 1mol/L sodium hydroxide solution in the gained solution; Regulator solution pH to 10; Behind 25 ℃ stirred in water bath reaction 8h, with gained solution pack in the dialysis tubing with distill water dialysis to dialyzate specific conductivity constant till, solution lyophilize in the dialysis tubing is obtained Quimotrase protective money nanocluster fluorescent material.
Equally effectively made Quimotrase protective money nanocluster fluorescent material through the above-mentioned embodiment of experiment showed, 2.
Claims (10)
1. Quimotrase protective money nanocluster Preparation of Fluorescent Material method may further comprise the steps:
The Quimotrase drips of solution is added in the chlorauric acid solution, stirs; Under the lucifuge condition, the pH value of regulating mixing solutions is an alkalescence, and after reacting 1～48h under 20～37 ℃ of water bath condition, separation and purification makes described Quimotrase protective money nanocluster fluorescent material.
2. preparation method according to claim 1 is characterized in that, described Quimotrase is a BCTR.
3. preparation method according to claim 1 is characterized in that, the concentration of said chymotrypsin protein enzyme solution is 15～90mg/mL.
4. preparation method according to claim 1 is characterized in that, the concentration of said chlorauric acid solution is 0.1%～2%.
5. preparation method according to claim 4 is characterized in that, the concentration of said chlorauric acid solution is 0.5%～1%.
6. preparation method according to claim 1 is characterized in that, the mol ratio between Quimotrase and the hydrochloro-auric acid is 0.068: 1～0.410: 1.
7. preparation method according to claim 1 is characterized in that, the mol ratio between Quimotrase and the hydrochloro-auric acid is 0.1～0.3: 1.
8. preparation method according to claim 1 is characterized in that, the pH value of said mixing solutions is 10～12.
9. preparation method according to claim 8 is characterized in that, the pH value of said mixing solutions is 12.
10. preparation method according to claim 1 is characterized in that, the method for said separation and purification is dialysis.
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Cited By (8)
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| CN105328203A (en) * | 2015-09-26 | 2016-02-17 | 福建医科大学 | 1-H-1,2,4-triazole-3-thiol-bovine serum albumin-gold nanocluster fluorescent material and preparation method thereof |
| CN105606684A (en) * | 2016-02-01 | 2016-05-25 | 盐城工学院 | Preparation method and application of protein-based graphene-single walled carbon nanotube-nanogold compound |
| CN105823775A (en) * | 2016-03-22 | 2016-08-03 | 中国石油大学(华东) | Kit for detecting residual thiophosphate organophosphorus pesticides and application method of kit |
| CN105860963A (en) * | 2016-04-16 | 2016-08-17 | 福建医科大学 | Guanidine hydrochloride/6-aza-2-sulfo-thymine-gold nano cluster and preparation method thereof |
| CN105860959A (en) * | 2016-04-16 | 2016-08-17 | 福建医科大学 | Arginine/6-aza-2-sulfo-thymine-au nanocluster and preparing method thereof |
| CN110082329A (en) * | 2019-01-24 | 2019-08-02 | 安徽师范大学 | A kind of the fluorescence platinum cluster and preparation method and application of bromelain package |
| CN111925790A (en) * | 2020-08-17 | 2020-11-13 | 安徽师范大学 | Preparation method of fluorescent gold nanocluster and application of fluorescent gold nanocluster in ampicillin detection |
| CN114309634A (en) * | 2021-12-20 | 2022-04-12 | 中山大学 | Preparation of gold quantum clusters and application of gold quantum clusters in gastrointestinal tract radiography and inflammation treatment |
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| CN105606684A (en) * | 2016-02-01 | 2016-05-25 | 盐城工学院 | Preparation method and application of protein-based graphene-single walled carbon nanotube-nanogold compound |
| CN105606684B (en) * | 2016-02-01 | 2019-08-06 | 盐城工学院 | A kind of graphene based on protein-single-walled carbon nanotube-nano-Au composite preparation method and applications |
| CN105823775A (en) * | 2016-03-22 | 2016-08-03 | 中国石油大学(华东) | Kit for detecting residual thiophosphate organophosphorus pesticides and application method of kit |
| CN105860963B (en) * | 2016-04-16 | 2018-06-22 | 福建医科大学 | Guanidine hydrochloride/6- azepines -2- thio-thymines-gold nano cluster and preparation method thereof |
| CN105860959B (en) * | 2016-04-16 | 2018-05-18 | 福建医科大学 | Arginine/6- azepines -2- thio-thymines-gold nano cluster and preparation method thereof |
| CN105860959A (en) * | 2016-04-16 | 2016-08-17 | 福建医科大学 | Arginine/6-aza-2-sulfo-thymine-au nanocluster and preparing method thereof |
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| CN110082329A (en) * | 2019-01-24 | 2019-08-02 | 安徽师范大学 | A kind of the fluorescence platinum cluster and preparation method and application of bromelain package |
| CN110082329B (en) * | 2019-01-24 | 2022-03-15 | 安徽师范大学 | Bromelain-coated fluorescent platinum nanocluster and preparation method and application thereof |
| CN111925790A (en) * | 2020-08-17 | 2020-11-13 | 安徽师范大学 | Preparation method of fluorescent gold nanocluster and application of fluorescent gold nanocluster in ampicillin detection |
| CN111925790B (en) * | 2020-08-17 | 2023-03-24 | 安徽师范大学 | Preparation method of fluorescent gold nanocluster and application of fluorescent gold nanocluster in ampicillin detection |
| CN114309634A (en) * | 2021-12-20 | 2022-04-12 | 中山大学 | Preparation of gold quantum clusters and application of gold quantum clusters in gastrointestinal tract radiography and inflammation treatment |
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