CN110202128A - A kind of gold and silver composite Nano cluster, preparation process and the application in biological thiol detection - Google Patents
A kind of gold and silver composite Nano cluster, preparation process and the application in biological thiol detection 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
- B22F1/0545—Dispersions or suspensions of nanosized particles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/17—Metallic particles coated with metal
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- B—PERFORMING OPERATIONS; TRANSPORTING
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention belongs to field of biotechnology, are related to the detection of biological thiol, and in particular to a kind of gold and silver composite Nano cluster solution, preparation process and the application in biological thiol detection.Preparation method provided by the invention is to mix the cytidine of 50mM, the gold chloride of 5mM and citrate buffer, the volume ratio of the cytidine, gold chloride and citrate buffer is 10-15:1-3:90-110, then it is heated under conditions of 100-120 DEG C, obtains solution A；The silver nitrate solution of 5mM is added in toward solution A, it is cooling, obtain the gold and silver composite Nano cluster.Gold and silver composite Nano cluster preparation method provided by the invention is simple, easy to operate, easy to industrialized production.
Description
Technical field
The invention belongs to field of biotechnology, are related to the detection of biological thiol, and in particular to a kind of gold and silver composite Nano
Cluster solution, preparation process and the application in biological thiol detection.
Background technique
Biological thiol is the general name of compounds containing thiol groups in organism, most representative universally present in organism
Biological thiol be three kinds of sulfhydryl compounds: cysteine (Cys), homocysteine (Hcy) and glutathione (GSH).Mercapto
Based compound is the signal of interest molecule after organism enzyme plays a role and in physiological activity, while also adjustable cell is being just
Normal redox state has the function of important in the physiological activity of organism.Research has shown that, sulfhydryl compound in organism
Concentration abnormality it is related with many kinds of diseases, therefore, detect organism in sulfhydryl compound concentration to research cell function and into
The early diagnosis of row disease has great importance.
Metal nanometre cluster is the metastable nanostructure being made of several to dozens of metallic atoms, with size
Dependence and adjustable fluorescence and many other excellent performances.Wherein, and with the fluorescent characteristic of gold and silver nano-cluster, biocompatibility most
Good, these characteristics are that it provides the foundation in fields such as fluorescence detection metal ion, cell imaging, biomarkers.
The present invention provides a kind of gold and silver composite Nano clusters (AuAg NCs), and preparation process is simple, is easy to batch metaplasia
It producing, prepared gold and silver composite Nano cluster micro can efficiently detect biological thiol molecule, existing detection technique is compared, this
It is strong to the selectivity of biological thiol molecule to invent the gold and silver composite Nano cluster provided, specificity is high, and trace may be implemented in high sensitivity
The detection of the biological thiol of magnitude, is with a wide range of applications and commercial value.
Summary of the invention
In view of this, an object of the present invention, is to provide a kind of preparation method of gold and silver composite Nano cluster.
A kind of preparation method of gold and silver composite Nano cluster, the preparation method include the following steps:
1) cytidine of 50mM, the gold chloride of 5mM and citrate buffer are mixed, the cytidine,
The volume ratio of gold chloride and citrate buffer is 10-15:1-3:90-110, is then heated under conditions of 100-120 DEG C,
Obtain solution A；
2) cooling toward the silver nitrate solution of addition 5mM in solution A, obtain the gold and silver composite Nano cluster.
Further, the volume ratio of the cytidine, gold chloride and citrate buffer is 10:1:100.
Further, the pH value of citrate buffer is 5.8-6.2 in step 1)；Heating time is 5-10min.
As a preference, the pH value of lemon phthalate buffer is 6.0 in step 1)；Heating time is 5min；Heating temperature
It is 100 DEG C.
The second object of the present invention is to provide a kind of gold and silver composite Nano cluster solution.
A kind of gold and silver composite Nano cluster solution takes the preparation method of above-mentioned gold and silver composite Nano cluster to be made.
The third object of the present invention is to provide the applications of gold and silver composite Nano cluster solution produced by the present invention.
Using the method for above-mentioned gold and silver composite Nano cluster solution detection biological thiol, include the following steps:
1) the biological thiol solution of 50mM is prepared, and is diluted to 0.5 μM of -10mM as needed；
2) the biological thiol solution of various concentration is continuously added to gold and silver composite Nano cluster solution using micro syringe
In, and be uniformly mixed；
3) fluorescence spectrum of mixed solution is recorded with sepectrophotofluorometer, until fluorescence spectrum is no longer reduced.
Further, fluorescence probe of the gold and silver composite Nano cluster solution as detection.
Biological thiol of the present invention refers in particular to cysteine (Cys) and glutathione (GSH).
Above-mentioned gold and silver composite Nano cluster solution is preparing the application in biosensor；Especially preparing optical bio biography
Application in sensor.
Above-mentioned gold and silver composite Nano cluster solution is preparing the application in fluorescence detection test.
The diagnosis of detection human blood sample product is prepared in the present invention using gold and silver composite Nano cluster as test paper decorative material for the first time
Kit, to realize biological thiol Visual retrieval, the invention can be achieved to Parkinson's disease caused by biological thiol horizontal abnormality,
The Diseases physical signs such as Alzheimer's disease and autoimmune deficiency syndrome it is not damaged, quick, highly sensitive, easy,
Visual inspection, further combined with ultrasound, Computed tomography and nuclear-magnetism etc., can carry out it is polymorphic with it is multi-modal synchronous
Diagnosis and accurate targeting positioning and treatment, have wide medical application prospect.
The method of above-mentioned gold and silver composite Nano cluster solution Visual retrieval biological thiol on paper, includes the following steps:
1) the gold and silver composite Nano cluster solution that mass concentration is 0.01-10mg/mL is added on non-blooming test paper, and
Air drying；
2) by the biological thiol solution drop of 5 μM of -1mM with the pretreated test paper surface of gold and silver composite Nano cluster solution；
3) photo of test paper is shot under 360-365nm ultraviolet light.
As a preference, the photo of test paper is shot in step 3) under 365nm ultraviolet light.
Application of the above-mentioned gold and silver composite Nano cluster solution in the diagnostic kit of preparation detection human blood sample product.
The utility model has the advantages that
1) gold and silver composite Nano cluster preparation method provided by the invention is simple, easy to operate, easy to industrialized production.It is made
Gold and silver composite Nano cluster solution fluorescence stablize, can micro efficient detection biological thiol molecule, selectivity is strong, specific
Height, is with a wide range of applications and commercial value.
2) examining for detection human blood sample product is prepared in the present invention using gold and silver composite Nano cluster as test paper decorative material for the first time
Disconnected kit, to realize biological thiol Visual retrieval.The present invention can be achieved to disease caused by a variety of biological thiol horizontal abnormalities
Quick, efficient, highly sensitive, the simple, visual inspection of disease.
3) variation of biological thiol molecule content in organism is precisely and quickly detected for preferably developing and utilizing
Sulfhydryl compound and Related product, such as exploitation biological thiol relevant healthcare product, have very important scientific value and research
Meaning.
4) foundation of biological thiol detection method provided by the invention also has weight to market monitoring mercaptan product quality
Want meaning.
Detailed description of the invention
Fig. 1 AuAg NCs solution detects Cys, and ((A) Cys is added continuously to after AuAg NCs solution to its fluorescence spectrum
It influences；(B) fit linear relationship of fluorescence intensity and Cys concentration).
Fig. 2 AuAg NCs solution detects GSH, and ((A) GSH is added continuously to after AuAg NCs solution to its fluorescence spectrum
It influences；(B) fit linear relationship of fluorescence intensity and GSH concentration).
((A) AuAg NCs fluorescence response is under 365nm ultraviolet lamp for the selective enumeration method test of Fig. 3 AuAg NCs solution
Photo, upper figure is that other amino acid are added in AuAg NCs solution；Middle figure and the following figure are added into every kind of solution respectively
Cys or GSH.(B) influence that other amino acid respond AuAg NCs, the upper figure of black histogram corresponding A, red and blue column
Shape figure respectively corresponds the middle figure and the following figure of A).
The paper Visual retrieval of Fig. 4 AuAg NCs solution tests the (paper of (A), (B) AuAg NCs to Cys and GSH
Photo of the visualization measurement under 365nm ultraviolet lamp, (C) respectively after GSH, Cys, glutamic acid and glycine is added, in 365nm
Photo under ultraviolet lamp.(D) after Cys, GSH and other amino acid being added, the photo under 365nm ultraviolet lamp)
Fig. 5 AuAg NCs detect Cys and GSH fluorescent quenching test (the transmission electron microscope TEM photo (A) of AuAg NCs and
Diameter is distributed (B) phenogram, and the transmission electron microscope TEM photo of Cys (C) and GSH (D) is added into AuAg NCs respectively).
Fig. 6 AuAg NCs detects the Fourier transform infrared spectroscopy FT-IR figure of Cys and GSH.AuAg NCs, Cys and
FT-IR after the two effect schemes (A)；FT-IR after AuAg NCs, GSH and the two act on schemes (B).
Specific embodiment
Hereinafter reference will be made to the drawings, and the preferred embodiment of the present invention is described in detail.Illustrated embodiment is in order to more preferable
Ground is illustrated the contents of the present invention, but is not that the contents of the present invention are only limitted to illustrated embodiment.So being familiar with this field
Technical staff nonessential modifications and adaptations are carried out to embodiment according to foregoing invention content, still fall within protection of the invention
Range.
Embodiment 1
Preparation process
1. by the cytidine of 50mM, the gold chloride (HAuCl of 5mM4) and pH be 6.0 citrate buffer with 10:1:100
Concentration than mixed, heated at 100 DEG C 5 points arrive solution A.
2. 5mM silver nitrate (AgNO is added in toward solution A3) solution, finally, just forming fluorogold silver nanoparticle after solution is cooling
Cluster.
Embodiment 2
Detection of the AuAg NCs to Cys and GSH under solution state
1. preparing the Cys of 50mM and being diluted to required concentration as needed.
2. continuously the Cys of various concentration is added in the AuAg NCs solution of 3mL using micro syringe, mixing is equal
It is even.
3. the fluorescence spectrum of recording solution, until fluorescence spectrum no longer subtracts.
The detection of 4.GSH is identical as Cys.
Experimental result:
The fluorescence intensity of AuAg NCs is very sensitive to Cys, reduces as Cys concentration increases fluorescence.Minimum detection limit
It (LOD) is 0.67pM, the range of linearity is 0.67pM to 110 μM, coefficient R2=0.994.
When GSH is added in AuAg NCs solution, fluorescent emission intensity is also sensitive to GSH, and with GSH concentration
Increase and proportionally reduces.The range of linearity is 0.67pM to 63 μM, R2=0.986, LOD 0.67pM.
Result above clearly illustrates that AuAg NCs prepared by the present invention can be realized in solution state to Cys and GSH
Highly sensitive detection.See Fig. 1, Fig. 2.
Embodiment 3
Choice tests of the AuAg NCs to Cys and GSH
1. Cys and GSH are added separately in identical AuAg NCs solution, make 200 μ of ultimate density.
2. other amino acid are added in another AuAg NCs solution, concentration is 10 times of Cys and GSH.Other ammonia
Base acid includes Serine, L-Aspartic acid, L (+)-glutamic acid, L- tryptophan, L-Leu, glycine, l-tyrosine, L-
Lysine, L-arginine, L-phenylalanine, L-PROLINE, l-Alanine, L-threonine, L-Glutamine, Valine, L-
Isoleucine.
3. detecting the fluorescence wide spectrum of above-mentioned two parts of solution respectively.
4. Cys or GSH is added into the sample containing other amino acid, fluorescence spectrum is measured again.
Experimental result:
The purpose of this experiment is to detect in the presence of other amino acid, and AuAg NCs detects biological thiol point
Whether son has interference.
The results show that in the presence of Cys, GSH and other 16 kinds of amino acid, it has been found that only Cys and GSH can be sudden
Go out the fluorescence of AuAg NCs, and other amino acid have not significant impact AuAg NCs Quenching of fluorescence.Importantly, other
In the presence of amino acid, Cys and GSH can also significantly quench the fluorescence of AuAg NCs.These results further prove
AuAg NCs is better than other amino acid to the selectivity of Cys and GSH.AuAg NCs has high selectivity to Cys and GSH.See
Fig. 3.
Embodiment 4
AuAg NCs Visual retrieval Cys and GSH on paper
1. the AuAg NCs sample of 0.01-10mg/mL is added on the test paper that diameter is 5mm, and dry in air.
2. by Cys the or GSH drop of 2 μ L various concentrations on the test paper surface with AuAg NCs sample pretreatment.
3. shooting the photo of test paper under 365nm ultraviolet light.
Experimental result:
As Cys concentration increases to 1mM from 0, the fluorescence intensity of paper sensor is gradually dimmed.Its detection range is from 5 μM
It is 5 μM to 1mM, LOD.Compared with Cys detection, GSH also has identical phenomenon.The concentration of GSH increases to 1mM paper sensing from 0
The fluorescence intensity of device is gradually dimmed.For its detection range from 5 μM to 1mM, LOD is 5 μM.As control, paper sensor is for examining
Survey the other parts (glutamic acid and glycine) of GSH.Glutamic acid and glycine will not quench the fluorescence of paper sensor, be shown to be
Due to the presence of Cys, GSH can quench the fluorescence of AuAg NCs.These results indicate that paper fluorescent optical sensor may be implemented
Simplicity, highly sensitive detection to Cys and GSH.In addition, we have also investigated paper sensors to the selectivity of Cys and GSH,
Similar result is obtained.In the presence of Cys and GSH, almost without fluorescence is observed, but for other all interference amino
Acid has macroscopic fluorescence.See Fig. 4
Embodiment 5
The detection of Cys in human serum sample
Cys is the main component of biological thiol in human serum, and other biological thiols are similar with the reactive mode of AuAg NCs
In Cys, selection recovery experiment is to verify the feasibility of fluorescent optical sensor in practical applications.Due to the chemical group of biological sample
At changeability, be measured using standard adding method.
1. fresh serum is centrifuged, 700 times are diluted, the solvent of Cys is then used as.
2. the Cys of various concentration is added into serum, the fluorescence spectrum of AuAg NCs and serum interaction are then measured.
Experimental result:
By Standard entertion method using Cys as the concentration of biological thiol in standard test human serum.It measures in serum
The rate of recovery of the amount of knowing Cys is 90.9%~108.3%, it was demonstrated that the reliability of biological thiol in AuAg NCs detection human serum.
Embodiment 6
Principle of the AuAg NCs to Cys and GSH detection fluorescent quenching
1. in the copper mesh that the carbon that certain density dilute solution is coated in 300 mesh is coated and dry in laboratory environment
It is dry, then characterization of size is obtained with JEM-2100F transmission electron microscope (TEM).
2. carrying out Fourier transform infrared spectroscopy (FT-IR) measurement on 5700 instrument of Nicolet using KBr method.
Experimental result:
It is characterized by TEM, has studied the quenching principle that AuAg NCs measures Cys and GSH.AuAg NCs has uniform
Partial size, about 2.4nm.After Cys and GSH interaction, particle is significantly built up, and partial size increases respectively to 200nm and 50nm.Add
The reunion for entering AuAg NCs after Cys and GSH results in bulky grain, eventually leads to fluorescent quenching.
The conjugated group of AuAg NCs Yu Cys and GSH are further studied by FT-IR.With AuAg NCs phase interaction
With rear, the peak-SH disappears, this is attributed to the combination of the Ag atom on the sulfydryl on Cys and GSH and AuAg NCs, finally causes glimmering
Optical quenching.See Fig. 5, Fig. 6.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Claims (10)
1. a kind of preparation method of gold and silver composite Nano cluster, which is characterized in that the preparation method includes the following steps:
1) cytidine of 50mM, the gold chloride of 5mM and citrate buffer are mixed, the cytidine, chlorine gold
The volume ratio of acid and citrate buffer is 10-15:1-3:90-110, then heats, obtains under conditions of 100-120 DEG C
Solution A；
2) cooling toward the silver nitrate solution of addition 5mM in solution A, obtain the gold and silver composite Nano cluster.
2. preparation method according to claim 1, spy is being, the cytidine, gold chloride and citrate
The volume ratio of buffer is 10:1:100.
3. preparation method according to claim 1, spy is being that the pH value of citrate buffer is in step 1)
5.8-6.2；Heating time is 5-10min.
4. a kind of gold and silver composite Nano cluster solution, which is characterized in that take the described in any item gold and silver of claim 1-3 are compound to receive
The preparation method of rice cluster is made.
5. feature exists, including such as using the method for gold and silver composite Nano cluster solution as claimed in claim 4 detection biological thiol
Lower step:
1) the biological thiol solution of 50mM is prepared, and is diluted to 0.5 μM of -10mM as needed；
2) continuously the biological thiol solution of various concentration is added in gold and silver composite Nano cluster solution using micro syringe, and
It is uniformly mixed；
3) fluorescence spectrum of mixed solution is recorded with sepectrophotofluorometer, until fluorescence spectrum is no longer reduced.
6. the method according to claim 5 using gold and silver composite Nano cluster solution detection biological thiol, which is characterized in that
Fluorescence probe of the gold and silver composite Nano cluster solution as detection.
7. gold and silver composite Nano cluster solution as claimed in claim 4 is preparing the application in biosensor.
8. gold and silver composite Nano cluster solution as claimed in claim 4 is preparing the application in fluorescence detection test.
9. the method for gold and silver composite Nano cluster solution Visual retrieval biological thiol on paper as claimed in claim 4, special
Sign is including the following steps:
1) the gold and silver composite Nano cluster solution that mass concentration is 0.01-10mg/mL is added on non-blooming test paper, and in air
Middle drying；
2) by the biological thiol solution drop of 5 μM of -1mM with the pretreated test paper surface of gold and silver composite Nano cluster solution；
3) photo of test paper is shot under 360-365nm ultraviolet light.
10. gold and silver composite Nano cluster solution as claimed in claim 4 answering in the diagnostic kit of preparation detection human blood sample product
With.
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| CN111739997A (en) * | 2020-07-03 | 2020-10-02 | 青岛科技大学 | White light LED (light emitting diode) capable of emitting light by exciting gold-silver alloy clusters through blue light and preparation method thereof |
| CN111739996A (en) * | 2020-07-03 | 2020-10-02 | 青岛科技大学 | White light LED based on gold-silver alloy cluster and preparation method thereof |
| CN113695585A (en) * | 2021-08-23 | 2021-11-26 | 南通大学 | Preparation method of gold and silver nanocluster protected by casein and application of gold and silver nanocluster in aureomycin detection |
| CN113695585B (en) * | 2021-08-23 | 2023-07-28 | 南通大学 | Preparation method of casein-protected gold and silver nanoclusters and application of casein-protected gold and silver nanoclusters in aureomycin detection |
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