CN107748257A - Preparation method and application based on gold nanoclusters chitosan complexes membrane reagent box - Google Patents
Preparation method and application based on gold nanoclusters chitosan complexes membrane reagent box Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and application based on gold nanoclusters chitosan complexes membrane reagent box.The present invention is mainly：Prepare gold nanoclusters chitosan solution；Gold nanoclusters chitosan solution is evaporated in microwell plate gold nanoclusters chitosan complexes film is made；Gold nanoclusters on gold nanoclusters chitosan complexes film are combined with the sulfydryl of PSA aptamers end by golden sulfide linkage, then the PSA in testing sample is combined with PSA aptamers with PSA specific effect by PSA aptamers；Citric acid three sodium solution, polyglycol solution, hydrogen peroxide solution and chlorauric acid solution are added into system, according to the difference of PSA concentration, amount from the connection of PSA aptamers also can be different, it is different to the level of coverage on gold nanoclusters chitosan complexes film surface, the color that gold grain is presented is also different, reaches detection prostate antigen i.e. PSA purpose with this.Raw material sources of the present invention are extensive, and phenomenon is obvious, and detection sensitivity visualization is high.
Description
Technical field
The invention belongs to chemical-biological sensing and technical field of biological, and in particular to one kind is based on gold nanoclusters shell
The preparation method and application of glycan complexes membrane kit.
Background technology
Chitosan (chitosan) has good bioadhesion with promoting absorption, and it is also known as deacetylated crust
Element, it is to be obtained by the chitin (chitin) being widely present in nature by deacetylation.Chitosan is as a kind of day
The premium properties such as right macromolecule, its blood compatibility, biocompatibility, nontoxicity causes the extensive concern of every field,
In biological medicine, food processing, chemical enterprise, cosmetics synthesis, water resource processing, METAL EXTRACTION and recovery, biochemistry and biology
The application study of the various fields such as medical science achieves breakthrough progress.Early there is research report to prove display, chitosan has drop
The effect of blood pressure and blood lipoid.In addition, chitosan has been put into as thickener and fruit glaze agent.It is free due to containing in chitosan molecule
Amino, therefore in an acidic solution easily into salt, in cationic property.As amino quantity increases in chitosan molecule, its ammonia
Base characteristic is more prominent, and this is exactly the place of its peculiar property, has thus established many biological characteristicses for chitosan.
Metal nanometre cluster is generally made up of several to 100 atoms, a diameter of 1nm-2nm, and its property is different from single original
Son and nano particle.Nano material has skin effect, small-size effect and macro quanta tunnel effect.Noble metal nano group
The catalytic performance of cluster is a up-and-coming application.Gold is initially considered to have catalytically inactive, but the gold of Nano grade
It has been proved to that there is good catalytic activity in an extensive chemical reaction at present.Another of gold nanoclusters is important
Application be exactly to be used as artificial mimic enzyme, due to native enzyme there is stability difference and catalytic activity sensitivity easily by environment
The defects of influence, people are slowly directed to the research of artificial mimic enzyme, and metal nanometre cluster possesses the inherent enzyme similar to native enzyme
Activity, because the small volume of its ultra micro, it is less toxic the advantages that have in molecular imaging, bio-sensing, medicine and catalytic field it is potential
Application value.
In recent years, the development of tumour is in rising trend, and the early diagnosis of tumour is that most important medical science urgently to be resolved hurrily is asked
One of topic.China's tumour registration area prostate-cancer incidence is 9.92/10 ten thousand within 2012, and prostate cancer occupies male malignancy
The 6th of the incidence of disease, Specific marker of the prostate specific antigen (PSA) as prostate cancer, the diagnosis to prostate cancer
Specificity reachesIt is considered as the tumor markers of the prostate cancer of most worthy, is widely used in prostate
Monitoring after screening, diagnosis and the treatment of cancer.
The content of the invention
An object of the present invention is to provide a kind of preparation side based on gold nanoclusters chitosan complexes membrane reagent box
Method.
The preparation method based on gold nanoclusters chitosan complexes membrane reagent box, comprises the following steps：
(1) gold nanoclusters chitosan solution is prepared as the gold nanoclusters analogue enztme with peroxidase activity；
(2) gold nanoclusters chitosan solution prepared by step (1) is evaporated in microwell plate and gold nanoclusters chitosan is made
Complexes membrane；
(3) be by the gold nanoclusters on gold nanoclusters chitosan complexes film and prostate antigen PSA aptamers end
Sulfydryl combined by golden sulfide linkage, the PSA and PSA in testing sample are then made by the specific effect of PSA aptamers and PSA
Aptamers combine；
(4) gold grain development step：Citric acid three sodium solution, polyglycol solution, dioxygen are added into step (3) system
The aqueous solution and chlorauric acid solution, according to the difference of PSA concentration, the amount from the connection of PSA aptamers also can be different, to gold nanoclusters shell
The level of coverage on glycan complexes membrane surface is different, i.e., gold nanoclusters chitosan complexes film shows different catalytic capabilities,
The color that gold grain is presented is also different, reaches detection prostate antigen i.e. PSA purpose with this.
Preferably, the gold nanoclusters chitosan solution for preparing described in step (1) comprises the following steps：
(a) chitosan solution is prepared：By volumetric concentration be 0.1% acetic acid as solvent, chitosan is dissolved as concentration
For 5mg/mL solution, chitosan solution pH is adjusted to 6.4 by the NaOH for adding 10mol/L；
(b) chitosan solution for taking 7.5mL steps (a) to prepare, 60mL distilled water is added, and under intense agitation,
600 μ L 0.11mol/L mercaptopropionic acid is added, and 1.5mL 10mmol/L HAuCl is added dropwise4, the Jenner that now obtains
Rice cluster chitosan solution shows orange fluorescence under uv analyzer.
Preferably, step (2) the gold nanoclusters chitosan complexes film is ultraviolet by gold nanoclusters chitosan solution
Under conditions of light irradiation 4h, film forming is evaporated under the conditions of 40 DEG C of the temperature inside the box is dried.
Preferably, step (3) the PSA aptamers base sequence is：
5’-HS-C6-TTTTTAATTAAAGCTCGCCATCAAATAGCTTT-3’。
Preferably, in step (3), PSA aptamers and gold nanoclusters chitosan complexes film by golden sulfide linkage combined when
Between be 12h.
Preferably, in step (3), PSA aptamers and gold nanoclusters chitosan complexes film by golden sulfide linkage combination 12h it
Afterwards, flushed three times with 0.02mol/L PBS cushioning liquid, then add the sample solution containing PSA, PSA aptamers and PSA antigens
The time of specific effect is 20min.
Preferably, in step (3), the sample solution containing PSA is that the PBS for being 0.01mol/L with pH=7.2, concentration is buffered
Obtained from solution dilution.
Preferably, in step (4) the gold grain development step, developer includes citric acid three sodium solution, polyethylene glycol
Solution, hydrogen peroxide solution and chlorauric acid solution, citric acid three sodium solution concentration are 4mmol/L, and polyglycol solution concentration is
22.4 μ g/mL, hydrogen peroxide solution concentration are 400mmol/L, and chlorauric acid solution concentration is 1mmol/L, in process color, lemon
After sour three sodium solutions, polyglycol solution, hydrogen peroxide solution and gold nanoclusters chitosan complexes film reaction 30min, add
1mmol/L chlorauric acid solution.
Specifically, in step (4), prostate antigen is PSA detectable concentration range 10-13~10-19g/mL。
The second object of the present invention is to provide a kind of preparation side based on gold nanoclusters chitosan complexes membrane reagent box
The application of method, i.e.,：Quick detection applied to prostate cancer antigen in blood serum sample.
The present invention is using chitosan, the gold nanoclusters of gold chloride and mercaptopropionic acid fast reaction generation with fluorescence, in purple
Under the irradiation of outer light, the fluorescence of gold nanoclusters disappears and shows the property of peroxidase, so that gold nanoclusters chitosan
Complexes membrane also shows the property of peroxidase, i.e. complexes membrane and H2O2Effect forms hydroxyl radical free radical, hydroxyl radical free radical
Further reaction can form water and oxygen.By the aptamers specific effect on PSA and nano-cluster chitosan complexes film surface,
Be covered in complexes membrane surface PSA hinder film Mimetic Peroxidase catalytic activity, i.e. H2O2It can not be decomposed into immediately
Water and oxygen.The size of PSA concentration directly affects effective catalysis area of gold nanoclusters chitosan complexes film, so as to influence to show
H in toner2O2Catalytic decomposition, make developer reaction generation different colours aurosol, reach visual to PSA high sensitivity
Change detection.
Brief description of the drawings
Fig. 1 is the UV-Vis spectra figure before and after gold nanoclusters chitosan solution ultraviolet irradiation in the embodiment of the present invention 1
With fluorescence spectra.
Fig. 2 is that gold nanoclusters chitosan film is changed over time to hydrogen peroxide before and after ultraviolet irradiation in the embodiment of the present invention 1
Decomposition difference causes gold grain color developing effect different, the absorbance change figure determined by ultraviolet-uisible spectrophotometer.
Fig. 3 is the gold grain colour developing figure that gold nanoclusters chitosan detects various concentrations PSA in the embodiment of the present invention 2.
Fig. 4 is PSA quantitative measurement standard curve maps in the embodiment of the present invention 2.
Embodiment
With reference to specific embodiments and the drawings, the present invention is described in further detail.It is raw materials used in embodiment to be
Conventional chemical raw material, instrument are conventional chemical instrument.
Embodiment 1：
Prepare gold nanoclusters chitosan complexes film：
350mg Chitosan powder is weighed, by the use of the acetic acid that volumetric concentration is 0.1% as solvent, prepares 70mL5mg/L's
Chitosan solution, the pH value of the 5mg/L prepared chitosan solution is then adjusted to 6.4 with 10mol/L NaOH solution and treated
With.In addition, weighing the mercaptopropionic acid that 0.0467g mercaptopropionic acids are configured to 0.11mol/L, added in 100mL beaker
60mL distilled water, the above-mentioned chitosan solution 7.5mL for mixing up pH value is added, 0.11mol/L is added after being stirred vigorously 1min
The μ L of mercaptopropionic acid 600,1.5mL10mmol/L HAuCL is then added dropwise4Solution, now solution need continuing vigorous
Stir more than 3min, you can obtain clear and show fluorescent orange but the gold nanoclusters chitosan solution without catalytic.Will
This gold nanoclusters chitosan solution, which is placed under uviol lamp, irradiates 4h, you can obtaining yellow has catalytic but non-blooming gold nano
Cluster chitosan solution.The gold nanoclusters chitosan solution of the catalytic activity is added in microwell plate, is put into drying box temperature adjustment extremely
40 DEG C are evaporated, and obtain the gold nanoclusters chitosan film of catalytic.
Gold nanoclusters chitosan solution is changed in the ultraviolet front and rear ultraviolet and fluorescence intensity of irradiation, catalytic capability
Change therewith, ultraviolet-visible spectrogram and fluorescence spectra are as shown in Figure 1 before and after its ultraviolet irradiation.Gold nanoclusters shell gathers
The ultraviolet front and rear catalytic capability of sugar juice irradiation can be by carrying out quantitative explanation to decomposing hydrogen dioxide solution.Select different time of ultraviolet irradiation
Gold nanoclusters chitosan solution and hydrogen peroxide act on, then reacted again with gold chloride；When the timing of gold chloride concentration one, hydrogen peroxide
The nanogold particle of claret is generated when concentration is sufficiently large, corresponding surface plasma absorption intensity is big, conversely, hydrogen peroxide is dense
Spend smaller, the aggregation of the gold nano grain of generation is serious and smaller in blueness, corresponding surface plasma absorption intensity.Do not irradiate
Ultraviolet gold nanoclusters chitosan solution is not because have catalytic, no matter how it changes over time, to hydrogen peroxide all
Without decomposition, now the content of hydrogen peroxide is identical with the hydrogen peroxide content being initially added, therefore the table of gained gold nano grain
Surface plasma absorption intensity has almost no change, as shown in Figure 2.
Embodiment 2：
PSA detection in serum：
PSA aptamers are diluted to concentration as 10 with 0.01mol/L PBS solution first-14G/mL, PSA aptamers alkali
Motif is classified as：5’-HS-C6-TTTTTAATTAAAGCTCGCCATCAAATAGCTTT-3’.Then aptamers are added into gold nano
In cluster chitosan complexes film, treat aptamers and after gold nanoclusters chitosan complexes film reaction 12h hours, outwell, use
0.02mol/L PBS is flushed three times.PSA standard liquids are to delay the PBS that PSA is dissolved in pH=7.2, concentration is 0.01mol/L
Rush in solution and be formulated.The gold nanoclusters chitosan that the PSA standard liquids of various concentrations are added to connection PSA aptamers is answered
Outwell, flushed three times with 0.01mol/L PBS, then to gold nanoclusters chitosan complexes film after reacting 20min in compound film
The middle μ L of hydrogen peroxide 330 added with 400mmol/L, after hydrogen peroxide and gold nanoclusters chitosan complexes film effect 30min, to
4mmol/L trisodium citrate, 22.4 each 200 μ L of μ g/mL polyglycol solutions are added in centrifuge tube, then add 200 μ thereto
The above-mentioned hydrogen peroxide for having reacted 30min of L, the chlorauric acid solution that 200 μ L concentration are 1mmol/L is eventually adding, after 30min, its
Color change is as shown in figure 3, diminishing with prostate antigen PSA concentration, and the color that gold nano grain is shown is gradually by red
It is changed into bluish violet.The mapping of the absorbance at 520nm of the gold nano grain after the PSA colour developings of finite concentration scope is taken, can be obtained
The good standard curve of linear relationship, as shown in Figure 4.
PSA detection in actual blood serum sample：The μ L of male patient's serum 500 of prostate cancer are taken, with 10mmo/L PBS
Cushioning liquid dilutes, and takes the blood serum sample after 100 μ L dilutions to be added to the gold nanoclusters chitosan complexes of connection PSA aptamers
After reacting 20min in film, flushed three times with 0.01mol/L PBS, then add and use into gold nanoclusters chitosan complexes film
The 400mmol/L μ L of hydrogen peroxide 330, after hydrogen peroxide and gold nanoclusters chitosan film effect 30min, added into centrifuge tube
4mmol/L trisodium citrate, 22.4 each 200 μ L of μ g/mL polyglycol solutions, then the 200 above-mentioned reactions of μ L are added thereto
30min hydrogen peroxide, the chlorauric acid solution that 200 μ L concentration are 1mmol/L is eventually adding, after 30min, surveys its absorbance, root
The content of PSA marks in human serum can be calculated according to its standard curve, PSA measured values are with curing in serum of patients with prostate cancer
Institute's testing result comparative result is as shown in table 1.
Table 1
Claims (10)
1. a kind of preparation method based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that comprise the following steps：
(1) gold nanoclusters chitosan solution is prepared as the gold nanoclusters analogue enztme with peroxidase activity；
(2) gold nanoclusters chitosan solution prepared by step (1) is evaporated to that gold nanoclusters chitosan is made is compound in microwell plate
Thing film；
(3) be by the gold nanoclusters on gold nanoclusters chitosan complexes film and prostate antigen PSA aptamers end mercapto
Base is combined by golden sulfide linkage, then the PSA in testing sample is adapted to PSA with PSA specific effect by PSA aptamers
Body combines；
(4) gold grain development step：It is water-soluble that citric acid three sodium solution, polyglycol solution, dioxygen are added into step (3) system
Liquid and chlorauric acid solution, according to the difference of PSA concentration, the amount from the connection of PSA aptamers also can be different, to gold nanoclusters chitosan
The level of coverage on complexes membrane surface is different, i.e., gold nanoclusters chitosan complexes film shows different catalytic capabilities, gold
The color that grain is presented is also different, reaches detection prostate antigen i.e. PSA purpose with this.
2. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
The gold nanoclusters chitosan solution for preparing described in step (1) comprises the following steps：
(a) chitosan solution is prepared：By volumetric concentration be 0.1% acetic acid as solvent, chitosan is dissolved as into concentration is
5mg/mL solution, chitosan solution pH is adjusted to 6.4 by the NaOH for adding 10mol/L；
(b) chitosan solution for taking 7.5mL steps (a) to prepare, 60mL distilled water is added, and under intense agitation, added
600 μ L 0.11mol/L mercaptopropionic acid, and 1.5mL 10mmol/L HAuCl is added dropwise4, the gold nanoclusters that now obtain
Chitosan solution shows orange fluorescence under uv analyzer.
3. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
Step (2) the gold nanoclusters chitosan complexes film be by gold nanoclusters chitosan solution ultra violet lamp 4h condition
Under, it is evaporated film forming under the conditions of 40 DEG C of the temperature inside the box is dried.
4. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
Step (3) the PSA aptamers base sequence is：
5’-HS-C6-TTTTTAATTAAAGCTCGCCATCAAATAGCTTT-3’。
5. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
In step (3), PSA aptamers are 12h with the time that gold nanoclusters chitosan complexes film is combined by golden sulfide linkage.
6. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
In step (3), after PSA aptamers and gold nanoclusters chitosan complexes film are by golden sulfide linkage combination 12h, 0.02mol/L is used
PBS cushioning liquid flushes three times, and then adds the sample solution containing PSA, PSA aptamers and the effect of PSA antigentic specificities when
Between be 20min.
7. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
In step (3), the sample solution containing PSA is to be obtained with pH=7.2, concentration for 0.01mol/L PBS cushioning liquid dilution
's.
8. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
In step (4) the gold grain development step, developer includes citric acid three sodium solution, polyglycol solution, hydrogen peroxide solution
And chlorauric acid solution, citric acid three sodium solution concentration are 4mmol/L, polyglycol solution concentration is 22.4 μ g/mL, hydrogen peroxide
Solution concentration is 400mmol/L, and chlorauric acid solution concentration is 1mmol/L, in process color, citric acid three sodium solution, poly- second
After glycol solution, hydrogen peroxide solution and gold nanoclusters chitosan complexes film reaction 30min, 1mmol/L gold chloride is added
Solution.
9. the preparation method according to claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, it is characterised in that：
In step (4), prostate antigen is PSA detectable concentration range 10-13~10-19g/mL。
10. a kind of application of preparation method as claimed in claim 1 based on gold nanoclusters chitosan complexes membrane reagent box, its
It is characterised by：Quick detection applied to prostate cancer antigen in blood serum sample.
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| CN108489976A (en) * | 2018-03-21 | 2018-09-04 | 湖南科技大学 | A kind of preparation method of gold nanorods gold nanoclusters compound quick detection kit |
| CN110514716A (en) * | 2019-09-09 | 2019-11-29 | 山东省农业科学院农业质量标准与检测技术研究所 | For detecting the preparation method of the current type aptamer sensor of pesticide residue |
| CN115598118A (en) * | 2022-11-08 | 2023-01-13 | 西南石油大学（Cn） | Colorimetric sensing reagent, intelligent label, colorimetric method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108489976A (en) * | 2018-03-21 | 2018-09-04 | 湖南科技大学 | A kind of preparation method of gold nanorods gold nanoclusters compound quick detection kit |
| CN110514716A (en) * | 2019-09-09 | 2019-11-29 | 山东省农业科学院农业质量标准与检测技术研究所 | For detecting the preparation method of the current type aptamer sensor of pesticide residue |
| CN110514716B (en) * | 2019-09-09 | 2022-06-28 | 山东省农业科学院农业质量标准与检测技术研究所 | Preparation method of current type aptamer sensor for detecting pesticide residue |
| CN115598118A (en) * | 2022-11-08 | 2023-01-13 | 西南石油大学（Cn） | Colorimetric sensing reagent, intelligent label, colorimetric method and application |
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