CN106520114B - A kind of preparation method and application of fluorescence gold nano cluster probe - Google Patents
A kind of preparation method and application of fluorescence gold nano cluster probe Download PDFInfo
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- CN106520114B CN106520114B CN201610835262.9A CN201610835262A CN106520114B CN 106520114 B CN106520114 B CN 106520114B CN 201610835262 A CN201610835262 A CN 201610835262A CN 106520114 B CN106520114 B CN 106520114B
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
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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
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
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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
- B82Y40/00—Manufacture or treatment of nanostructures
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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
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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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
Abstract
The present invention provides a kind of preparation method and application of fluorescence gold nano cluster probe, belong to fluorescent nano material field.The probe is in alkaline environment, to utilize the fluorescence gold nano cluster probe that " one kettle way " is made uniform in size, stability is good, is responded to glutaraldehyde (GA) using fibroin albumen as template and protective agent；This method raw material is easy to get extensively, and cost is relatively low, easy to operate, has good repeatability；And fluorescence gold nano cluster probe good water solubility obtained, stability are strong, can be applied to the detection of GA in actual water sample.
Description
Technical field
The present invention relates to fluorescent nano material preparing technical fields, and in particular to a kind of system of fluorescence gold nano cluster probe
Preparation Method and prepared probe can be used for detecting the glutaraldehyde in actual water sample.
Background technology
Metal nanometer cluster typically refers to a kind of novel fluorescence nano material being made of several to dozens of metallic atoms
Material.It has strong, anti-light Bleachability good and high quantum yield of strong fluorescent emission, good biocompatibility, stability etc. excellent
Put and receive the concern of numerous researchers.In metal nanometer cluster, most study is gold nanoclusters, due to its water solubility
Good, the excellent feature such as toxicity is low, photostability is strong and luminous efficiency is high, be widely used in detection ion, biological micromolecule,
In terms of biomarker and imaging.
Glutaraldehyde (GA) is a kind of colourless, oiliness and non-flammable liquid at room temperature.Being of wide application for it is general, packet
Include the fixative of leather industry and the sterilizer of medical instrument etc..But GA steam exposure in air, can cause skin,
Respiratory tract and ocular allergies, and since it is in clinical and environmentally importance, we expect, therefore, that a kind of letter can be looked for
Single, sensitivity and the good method of selectivity detect GA.
Large biological molecule such as peptide and protein has good biocompatibility, itself has a variety of biological functions, easily
In the functionalization for realizing gold nano cluster, it is also commonly used for the good template of synthesis fluorescence gold nano cluster probe.Document (Water-
soluble gold nanoclusters prepared by protein-ligand interaction as
fluorescent probe for real-time assay of pyrophosphatase activity,H.H.Deng,
F.F.Wang,X.Q.Shi,H.P.Peng,A.L.Liu,X.H.Xia,W.Chen,Biosensors and
Bioelectronics 83 (2016) 1-8), with a kind of commercialized common protein ----bovine serum albumin(BSA), 3- mercaptos are added
Base propionic acid has synthesized high luminous gold nano cluster, to Fe as template3+There is response, PPi, which is added, can be such that its fluorescence restores.
But this method building-up process needs two kinds of templates, cost is higher, therefore we synthesize simply there is an urgent need to a kind of, at low cost
Honest and clean new method.
Invention content
Present invention aims at a kind of preparation method of fluorescence gold nano cluster probe is provided, this method is easy to operate, instead
Mild condition is answered, gained fluorescence gold nano cluster probe grain size is smaller, and good dispersion can be used for the detection of GA in actual water sample.
To achieve the above object, a kind of preparation method of fluorescence gold nano cluster probe provided by the invention, step include：
(1) silk cocoon is cut into 1-2cm2Small pieces, silk cocoon small pieces are placed in 0.01-0.03mol/L sodium carbonate liquors,
Degumming 0.5-2h is heated at 90-110 DEG C, and fibroin albumen is made；
(2) fibroin albumen that step (1) obtains is cleaned 2-4 times with deionized water, in CaCl2:Water:Ethyl alcohol=1:8:2
Mixed solution in, heat 1-3h at 70-90 DEG C, fibroin albumen made to dissolve；
(3) silk fibroin protein solution obtained to step (2) is cooled to room temperature, filtering, and dialyse 48h, is placed in standby in 4 DEG C of refrigerators
With；
(4) silk fibroin protein solution that step (3) obtains is measured, is stirred continuously down, 0.5- is added into silk fibroin protein solution
1.5mmol/L gold chlorides, continuing stirring makes the two mix well, and the volume ratio of chlorauric acid solution and silk fibroin water solution is 1-
4:1；
(5) sodium hydroxide solution of 60 a concentration of 1mol/L of μ L is added in the mixed solution obtained to step (4), in 25-
Continue to stir 4-10h at 95 DEG C；
(6) mixed solution for obtaining step (5) finally obtains fluorescence copper nanocluster probe solution by centrifugation.
The concentration of sodium carbonate liquor is preferably 0.02mol/L in step (1).
Silk cocoon small pieces preferably heat 1h with sodium carbonate liquor at 100 DEG C in step (1).
Fibroin albumen after the degumming obtained in step (2) is cleaned preferably 3 times with deionized water, in CaCl2:Water：Ethyl alcohol
=1:8:In 2 mixed solution, 2h preferably is heated at 80 DEG C, is made it dissolve.
In step (3) dialysis be 8000-14000Da with molecular cut off bag filter.
The concentration of chlorauric acid solution in step (4) is preferably 1mmol/L.
The volume ratio of chlorauric acid solution and silk fibroin water solution in step (4) is 2:1.
In step (5), preferably 8h is stirred at 37 DEG C.
Centrifugation is to centrifuge 10min with 13000r/min rotating speeds in step (6).
Fluorescence gold nano cluster probe prepared by the method for the present invention can be used for detecting the GA in actual water sample.
Compared with the prior art, the advantages of the present invention are as follows：
(1) using fibroin albumen as template, raw material is easy to get extensively, environmentally protective, and preparation method is simple, of low cost.
(2) fluorescence gold nano cluster probe made from has good blue luminescent properties, uses it for building practical water
The sensing system that GA is detected in sample, can be to avoid the interference of other organic matters.Fluorescence gold nano cluster probe prepared by the present invention
GA in detectable actual water sample.
(3) fluorescence gold nano cluster probe size is small made from, photostability is strong, toxic side effect is small, good water solubility, fluorescence
Intensity is high, has broad application prospects in fields such as bio-imaging, biomarkers.
Description of the drawings
Fig. 1 is the mechanism of action schematic diagram of fluorescence gold nano cluster probe prepared by the present invention
Fig. 2 is that prepare fluorescence gold nano cluster probe solution be respectively that 365nm is ultraviolet in fluorescent lamp (1) and wavelength to the present invention
Photo under lamp (2) irradiation
Fig. 3 prepares the ultraviolet figure of fluorescence-of fluorescence gold nano cluster probe solution for the present invention, and a is that ultraviolet-visible is inhaled in figure
Spectrogram is received, b is fluorescence spectra
Fig. 4 is the change of fluorescence peak intensity when different organic matters are added in fluorescence gold nano cluster probe solution prepared by the present invention
Change
Solution after fluorescence gold nano cluster probe solution and addition GA prepared by Fig. 5 present invention is with its fluorescence of the variation of pH
The variation of peak intensity
The solution that fluorescence gold nano cluster probe prepared by Fig. 6 present invention is added after GA changes with time its fluorescence peak intensity
The variation of degree
Fluorescence gold nano cluster probe solution prepared by Fig. 7 present invention with ionic strength (concentration of sodium chloride) variation its
The variation of fluorescence peak intensity
Fluorescence gold nano cluster probe solution prepared by Fig. 8 present invention with GA concentration variation its fluorescence peak intensity variation
Fig. 9 (a) is fluorescence gold nano cluster probe solution and concentration range is linear relationship between 0-80 μM of GA
Fig. 9 (b) is fluorescence gold nano cluster probe solution and concentration range is linear relationship between 0-10 μM of GA
Fig. 9 (c) is fluorescence gold nano cluster probe solution and concentration range is linear relationship between 20-80 μM of GA
Specific implementation mode
The present invention is that in alkaline environment, fluorescence gold nano cluster is prepared by " one kettle way " using fibroin albumen as template
Probe solution, and for the detection of GA in actual water sample.Below by embodiment combination attached drawing, the invention will be further described.
Embodiment 1
Using fibroin albumen as the preparation of the fluorescence gold nano cluster probe of template：
(1) degumming silkworm cocoons prepare fibroin albumen：Silk cocoon is cut into 1-2cm2Small pieces, silk cocoon small pieces are placed in 0.02mol/
In L sodium carbonate liquors, 1h is heated at 100 DEG C；
(2) fibroin albumen after degumming that step (1) obtains is cleaned 3 times with deionized water, in CaCl2:Water:Ethyl alcohol=
1:8:In 2 mixed solution, 2h is heated at 80 DEG C, is made it dissolve；
(3) silk fibroin protein solution obtained to step (2) is cooled to room temperature, filtering, and dialyse 48h, is placed in standby in 4 DEG C of refrigerators
With；
(4) silk fibroin protein solution that step (3) obtains is measured, is stirred continuously down, is added into silk fibroin protein solution
1mmol/L gold chlorides, continuing stirring makes the two mix well, and the volume ratio of chlorauric acid solution and silk fibroin water solution is 2:1；
(5) sodium hydroxide solution of 60 a concentration of 1mol/L of μ L is added in the mixed solution obtained to step (4), at 37 DEG C
Under continue stir 8h；
(6) solution for obtaining step (5) centrifuges 10min with 13000r/min rotating speeds, finally obtains fluorogold nanoclusters
Aggregate probe solution.
The mechanism of action schematic diagram of the fluorescence gold nano cluster probe of preparation is shown in Fig. 1.
The fluorescence gold nano cluster probe solution of the preparation photograph in the case where fluorescent lamp and wavelength are 365nm ultra violet lamps respectively
Piece is shown in Fig. 2, and 1 is picture of the fluorescence gold nano cluster probe solution under daylight light irradiation in figure, and color is pink colour, and 2 be wavelength
For the picture under 365nm ultra violet lamps, color is blue.
In addition, the ultraviolet figure of fluorescence-of the fluorescence gold nano cluster probe solution prepared is shown in that Fig. 3, wherein fluorogram (b) show
For the fluorescence gold nano cluster probe of preparation under the conditions of fixed excitation wavelength is 331nm, emission peak positions are in 420nm or so.
Embodiment 2
The influence of the fluorescence peak intensity for the fluorescence gold nano cluster probe solution that organic matter prepares embodiment 1 is tested：
It is configured to a concentration of 800 μ respectively with the BR buffer solutions and formaldehyde of pH=8, acetaldehyde, methanol, ethyl alcohol, acetone
mol·L-1Solution, GA is configured to 80 μm of olL-1Solution, the fluorescence gold nano for respectively preparing 0.1mL embodiments 1
Cluster probe solution is added in the above-mentioned solution containing different organic matters of 0.9mL.Fixed excitation wavelength is 331nm, at room temperature
Fluorescence spectrum detection is carried out, according to the fluorescence peak intensity of 420nm or so, detection organic matter is to fluorescence gold nano cluster probe solution
Fluorescence peak intensity influence.
Fig. 4 is shown in influence of the organic matter to the fluorescence peak intensity of fluorescence gold nano cluster probe solution：Under 331nm excitations,
From the fluorescence intensity F of the fluorescence gold nano cluster probe solution without containing machine object0It is visited with the fluorescence gold nano cluster containing organic matter
The ratio of the fluorescence intensity F of needle solution obtains：GA variations are maximum, and the variation of other organic matters is relatively small, illustrates preparation of the present invention
Fluorescence gold nano cluster probe solution being capable of qualitative detection GA.
Embodiment 3
Fluorescence gold nano cluster probe solution that pH value prepares embodiment 1 and that the fluorescence gold nano cluster after GA is added is molten
The influence of the fluorescence intensity of liquid is tested：
The fluorescence gold nano cluster probe solution respectively prepared by 100 μ L embodiments 1 and the fluorescence gold nano being added after GA
Cluster solution is added separately in the BR buffer solutions of 900 μ L difference pH value, and fixed excitation wavelength is 331nm, at room temperature into
Row fluorescence spectrum detects, and according to the fluorescence peak intensity of 420nm or so, detection pH value is to fluorescence gold nano cluster probe solution and adds
Enter the influence of the fluorescence intensity of the fluorescence gold nano cluster solution after GA.
Fluorescence intensity of the pH value to the fluorescence gold nano cluster solution after fluorescence gold nano cluster probe solution and addition GA
Influence see Fig. 5：Under 331nm excitations, fluorescence gold nano cluster probe solution and the fluorescence gold nano cluster after addition GA are molten
For the fluorescence intensity of liquid when pH is 8, fluorescence intensity change is larger, thus fluorescence gold nano cluster probe solution prepared by the present invention
Select pH=8.
Embodiment 4
Fluorescence gold nano cluster probe solution prepared by embodiment 1 is added the influence experiment after GA in time：
After GA is added in fluorescence gold nano cluster probe solution prepared by 100 μ L embodiments 1, it is added to 100 μ L BR bufferings
In solution (pH=8.0), then add 800 μ L deionized waters, fixed excitation wavelength is 331nm, is carried out in 0-10min at room temperature glimmering
Light spectral detection, according to the fluorescence peak intensity of 420nm or so, fluorescence peak of the detection time to fluorescence gold nano cluster probe solution
The influence of intensity.Fig. 6 is shown in influence of the time to the fluorescence intensity of fluorescence gold nano cluster probe solution：In 10min, fluorogold
The fluorescence intensity of nanocluster probe is held essentially constant.
Embodiment 5
The influence of the fluorescence intensity for the fluorescence gold nano cluster probe solution that ionic strength prepares embodiment 1 is tested：
Fluorescence gold nano cluster probe solution prepared by 100 μ L embodiments 1 is added to 100 μ L BR buffer solutions (pH=
8.0) in, then add 800 μ L deionized waters, fixed excitation wavelength is 331nm, be added various concentration sodium chloride solution (0~
300mmol/L), according to the fluorescence intensity of 420nm or so, fluorescence of the detection ionic strength to fluorescence gold nano cluster probe solution
The influence of intensity.
Fig. 7 is shown in influence of the ionic strength to the fluorescence intensity of fluorescence gold nano cluster probe solution：Under 331nm excitations,
For fluorescence gold nano cluster probe solution in sodium chloride solution (0~300mmol/L) range of various concentration, fluorescence intensity is basic
It is constant, illustrate that the anti-ion interference of fluorescence gold nano cluster probe solution prepared by the present invention is strong.
Embodiment 6
The experiment that fluorescence gold nano cluster probe solution prepared by embodiment 1 detects GA：
Fluorescence gold nano cluster probe solution prepared by embodiment 1 dilutes 10 times, takes the fluorogold nanoclusters after dilution
100 μ L of aggregate probe solution are added in 900 solution of the μ L containing GA, and fixed excitation wavelength is 331nm, carries out fluorescence light at room temperature
Spectrum detection, according to the fluorescence intensity of 420nm or so, influences of the detection GA to the fluorescence intensity of gold nano cluster probe solution.
Fig. 8 is shown in influences of the GA to the fluorescence intensity of fluorescence gold nano cluster probe solution：Under 331nm excitations, fluorogold
Nanocluster probe solution is after the GA that various concentration is added, and fluorescence intensity is gradually reduced, and last fluorescence peak is substantially smooth-out；
Wherein 0-80 μM is the GA of 0,1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80 μm of ol/L respectively to fluorogold
The fluorescence spectra that nanocluster probe solution fluorescence intensity influences illustrates that fluorescence gold nano cluster probe prepared by the present invention is molten
Liquid energy enough realizes the detection to GA.
In addition, the concentration of the variation of the fluorescence intensity of fluorescence gold nano cluster probe solution prepared by the present invention and GA are in line
Sexual intercourse, as shown in Fig. 9 (a), the fluorescence intensity of gold nano cluster and the concentration of GA are linear at two sections.As shown in Fig. 9 (b), GA
Linear equation be F0/ F=0.976+0.07C (R2=0.994)；As shown in Fig. 9 (c), the linear equation of GA is F0/ F=
1.303+0.0247C(R2=0.994)
Embodiment 7
The test experience of fluorescence gold nano cluster probe solution prepared by embodiment 1 for GA in actual water sample：
The fluorescence gold nano cluster probe solution prepared for embodiment 1 using standard addition method is biological in actual water sample
The experiment of GA detection applications.As shown in table 1, it is respectively the GA solution of 3,6,9 μm of ol/L with actual water sample compound concentration, will implements
Fluorescence gold nano cluster probe solution prepared by example 1 dilutes 10 times, takes the 100 μ L of fluorescence gold nano cluster probe solution after dilution
It is added separately in 900 solution of the μ L containing GA, fixed excitation wavelength is 331nm, carries out fluorescence spectrum detection at room temperature, and remember
Record corresponding fluorescence intensity.
The rate of recovery of GA in actual water sample is calculated using the linear equation in Fig. 9 (b).Multigroup parallel determination in this experiment
And the rate of recovery of GA is calculated, as shown in table 1, illustrate that fluorescence gold nano cluster probe solution prepared by embodiment 1 can be used in water
The detection of GA in sample.
Table 1 is the detection that fluorescence gold nano cluster probe solution prepared by the present invention is used for GA in actual water sample
Claims (9)
1. a kind of fluorescence gold nano cluster probe is used for the detection of glutaraldehyde in water, the fluorescence gold nano cluster probe is to pass through
The method included the following steps is prepared：
(1) silk cocoon is cut into 1-2cm2Small pieces, silk cocoon small pieces are placed in 0.01-0.03mol/L sodium carbonate liquors, in 90-
Degumming 0.5-2h is heated at 110 DEG C, and fibroin albumen is made；
(2) fibroin albumen that step (1) obtains is cleaned 2-4 times with deionized water, in CaCl2:Water:Ethyl alcohol=1:8:2 mixing
In solution, 1-3h is heated at 70-90 DEG C, fibroin albumen is made to dissolve；
(3) silk fibroin protein solution obtained to step (2) is cooled to room temperature, filtering, and dialyse 48h, is placed in spare in 4 DEG C of refrigerators；
(4) silk fibroin protein solution that step (3) obtains is measured, is stirred continuously down, 0.5- is added into silk fibroin protein solution
1.5mmol/L gold chlorides, continuing stirring makes the two mix well, and the volume ratio of chlorauric acid solution and silk fibroin water solution is 1-
4:1；
(5) sodium hydroxide solution of 60 a concentration of 1mol/L of μ L is added in the mixed solution obtained to step (4), at 25-95 DEG C
Under continue stir 4-10h；
(6) mixed solution for obtaining step (5) finally obtains fluorescence gold nano cluster probe solution by centrifugation.
2. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
A concentration of 0.02mol/L of sodium carbonate liquor in the step (1).
3. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
Silk cocoon small pieces heat 1h with sodium carbonate liquor at 100 DEG C in the step (1).
4. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
Fibroin albumen after the degumming obtained in the step (2) is cleaned 3 times with deionized water, in CaCl2:Water：Ethyl alcohol=1:8:2
Mixed solution in, heat 2h at 80 DEG C, make it dissolve.
5. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
Dialysis is that the bag filter for being 8000-14000Da with molecular cut off is dialysed in the step (3).
6. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
A concentration of 1mmol/L of chlorauric acid solution in the step (4).
7. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
The volume ratio of gold chloride and silk fibroin water solution in the step (4) is 2:1.
8. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
In the step (5), 8h is stirred at 37 DEG C.
9. a kind of detection of the fluorescence gold nano cluster probe as described in claim 1 for glutaraldehyde in water, which is characterized in that
Centrifugation is to centrifuge 10min with 13000r/min rotating speeds in the step (6).
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| CN108031857A (en) * | 2017-11-21 | 2018-05-15 | 东华大学 | A kind of preparation method for the gold nano cluster for sending out red fluorescence |
| CN108031859A (en) * | 2017-11-29 | 2018-05-15 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for synthesizing gold nanoclusters and products thereof and application |
| CN109705841B (en) * | 2018-12-24 | 2021-07-02 | 山西大学 | Gold nanocluster with transferrin as template and preparation method and application thereof |
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