CN103630521A - Method based on fluorescence silver nano-clusters for detecting cysteamine in blood serum - Google Patents
Method based on fluorescence silver nano-clusters for detecting cysteamine in blood serum Download PDFInfo
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- CN103630521A CN103630521A CN201310652621.3A CN201310652621A CN103630521A CN 103630521 A CN103630521 A CN 103630521A CN 201310652621 A CN201310652621 A CN 201310652621A CN 103630521 A CN103630521 A CN 103630521A
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- cysteamine
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- silver nanoclusters
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- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 229960003151 mercaptamine Drugs 0.000 title claims abstract description 57
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 229910052709 silver Inorganic materials 0.000 title claims abstract description 35
- 239000004332 silver Substances 0.000 title claims abstract description 35
- 210000002966 serum Anatomy 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000000243 solution Substances 0.000 claims abstract description 32
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 claims description 6
- 235000019136 lipoic acid Nutrition 0.000 claims description 6
- 229960002663 thioctic acid Drugs 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 13
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000002131 composite material Substances 0.000 abstract 1
- 238000007865 diluting Methods 0.000 abstract 1
- 239000003085 diluting agent Substances 0.000 abstract 1
- 239000011550 stock solution Substances 0.000 abstract 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 8
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 5
- 108010024636 Glutathione Proteins 0.000 description 4
- 229960003180 glutathione Drugs 0.000 description 4
- 229910000033 sodium borohydride Inorganic materials 0.000 description 3
- 239000012279 sodium borohydride Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000002848 electrochemical method Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 229940100228 acetyl coenzyme a Drugs 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
A method based on fluorescence silver nano-clusters for detecting cysteamine in blood serum is characterized by comprising the following steps: step 1), diluting a composite fluorescence silver nano-cluster stock solution by 400 times, taking 900 MuL of the diluent as probe solutions, adding 10 MuL of a blood serum sample containing cysteamine of a certain concentration and 10 MuL of a blood serum sample containing cysteamine of another concentration respectively, detecting the fluorescence intensity ratio between the silver nano-cluster solutions after 5 minutes' hybrid reaction and a blank control group with a fluorescence detector, and drawing a linear relational graph of the fluorescence intensity ratio and the cysteamine concentrations, wherein the cysteamine concentrations range from 5 to 120 Mum; step 2 ), adding 10MuL of blood serum solution containing cysteamine of an unknown concentration into another probe solution, and detecting the fluorescence intensity ratio between the silver nano-cluster solution after 5 minutes' reaction and the blank control group. The concentrations of cysteamine in the blood serum samples can be obtained according to the relational graph in the step 2. The method has the advantages that the concentrated recognition capability to cysteamine molecules is excellent; the operation is simple; the reactions are fast; the required sample quantity is low; the detection sensitivity is relatively high.
Description
[technical field]
The present invention relates to a kind of analytical approach that detects cysteamine in serum based on fluorescence silver nanoclusters.
[technical background]
Cysteamine (Cysteamine, CS) HSCH
2cH
2nH
2, claiming again β-mercaptoethylmaine, soluble in water and alcohol, is equivalent to the decarboxylate of halfcystine, is the ingredient of acetyl coenzyme A, is a kind of natural bioactivator that is present in animal, plant and human body.The sulfydryl of cysteamine molecule and amino rich active, is bearing important physiologic function in vivo.
Cysteamine can reduce growth inhibition cellulose content, improves level of growth hormone, promotes growth of animal.Cysteamine can be used to feed to be added and clinical practice.Therefore a kind of method that develops concentration of simple, quick, accurate, special, sensitive mensuration serum cysteamine is necessary.
In current mensuration serum, the method for cysteamine mainly be take high performance liquid chromatography and electrochemical method as main.High performance liquid chromatography is sensitive, and accuracy is high, but apparatus expensive.Electrochemical method comparatively speaking sensitivity is low, and need prepare electrode, and operation is comparatively complicated.Therefore develop a kind of easy and simple to handlely, highly sensitive, and the new method that does not rely on expensive device is very significant.
[summary of the invention]
Object of the present invention: the method that is to provide cysteamine in a kind of novel detection serum based on fluorescence silver nanoclusters, not high enough to overcome the method medium sensitivity of existing detection cysteamine, detection required time is long, or need complicated step and expensive large-scale instrument, be difficult to realize defect quick, accurate, special, high-sensitive mensuration.
Thinking of the present invention: the silver nanoclusters that lipoic acid is modified, there is fluorescence, maximum excitation wavelength and maximum emission wavelength are respectively 430nm and 640nm.This fluorescence can, by the quencher of cysteamine molecule, therefore can be determined the concentration of cysteamine by its fluorescence in the variation before and after quencher.
Concrete technical scheme of the present invention is: a kind of method of cysteamine in novel detection serum based on fluorescence silver nanoclusters, the silver nanoclusters that first synthetic lipoic acid is modified.Synthetic silver nanoclusters adopts Chem.Mater.2010, and the synthetic method described in 22,4364 – 4371, slightly revises.The silver nanoclusters size of synthesized is 1-3nm, and maximum excitation wavelength is 430nm, and maximum emission wavelength is 640nm, and fluorescence quantum efficiency is 1.7%.Synthetic concrete operation method is: in the little flask of 51.5mg lipoic acid powder dissolution in having 19mL ultrapure water, 4.8mg sodium borohydride solids adds in flask, lipoic acid and NaBH
4mol ratio be 2:1, stirring reaction 15min.The liquor argenti nitratis ophthalmicus that adds 1mL25mM, continues to stir 5min, adds excessive sodium borohydride 95mg,, continue stirring reaction 120min.In reaction, the mol ratio of sodium borohydride, lipoic acid, silver nitrate is for being 100:10:1.Synthetic 4 ℃ of preservations of fluorescence silver nanoclusters solution lucifuge.Its detection method comprises the following steps successively:
1) synthetic fluorescence silver nanoclusters stoste dilution is 400 times, gets 990 μ L as probe solution.In probe solution, add 10 μ L containing the blood serum sample of variable concentrations cysteamine respectively, react 5 minutes.Cysteamine concentration range is 5-120 μ M.With the silver nanoclusters solution after fluorescence detector mensuration hybrid reaction and the fluorescence intensity ratio of blank group, make the linear relationship chart of fluorescence intensity ratio and cysteamine concentration.
2) synthetic fluorescence silver nanoclusters stoste dilution is 400 times, gets 990 μ L as probe solution.Add 10 μ L containing the serum solution of unknown concentration cysteamine, react after 5 minutes, record the fluorescence intensity ratio of reacted silver nanoclusters solution and blank group.According to step 2) in graph of a relation calculate the concentration of cysteamine in blood serum sample.
The present invention has good single-minded recognition capability to cysteamine molecule, simple to operate, is swift in response, and sample demand is few, and detection sensitivity is higher.
[accompanying drawing explanation]
Fig. 1: the exciting and transmitting figure of fluorescence silver nanoclusters solution.
Fig. 2: dilute the variation that the fluorescence silver nanoclusters solution of 10 times adds fluorescence intensity after 1mM cysteamine aqueous solution.
Fig. 3: silver nanoclusters solution is the cysteamine of 500 μ M determinands respectively, halfcystine, glutathione, ordinate is add determinand fluorescence F afterwards and add previous fluorescence F
0ratio.
Fig. 4: the fluorescence intensity change that adds silver nanoclusters solution after variable concentrations cysteamine blood serum sample.
Fig. 5: the ratio F/F of the fluorescence before adding variable concentrations cysteamine fluorescence afterwards and adding
0linear relationship curve with cysteamine concentration.
[embodiment]
Following instance is further elaborated the present invention in connection with accompanying drawing, to foundation is provided for a better understanding of the present invention.
Embodiment 1 selectivity test
Configure respectively cysteamine, halfcystine, the glutathione aqueous solution of 500 μ M, respectively get the fluorescence silver nanoclusters solution that 0.5mL joins respectively 10 times of 0.5mL dilutions, mix reaction 5min.Only add as shown in Figure 3 cysteamine solution, the fluorescence of silver nanoclusters solution just can disappear.Halfcystine and glutathione are almost on the not impact of the fluorescence of silver nanoclusters.This illustrates halfcystine, and cysteamine analog common in these biosomes of glutathione can not cause interference to measuring, so the selectivity of this method is relatively good.
The mensuration of embodiment 2 sensing ranges and linear relationship
Prepare respectively the cysteamine cow's serum solution of 0,5,10,20,40,60,80,100,120 μ M, get respectively in the silver nanoclusters solution that 10 μ L join 400 times of 990 μ L dilutions, mix reaction 5min, fluorescence detector is surveyed its reacted fluorescence intensity F, and adding not the reacted fluorescence intensity of cow's serum containing cysteamine is F
0.The parameter of concrete fluoroscopic examination setting is for exciting 430nm, and sweep velocity 1200nm/min, excites width 10nm, transmitting width 20nm.Make F/F
0with the graph of a relation of cysteamine concentration, as shown in Figure 5.When known cysteamine concentration is 5-120 μ M, linear relationship is better, and equation of linear regression is F/F
0=-0.0067c+1.0064, c is cysteamine concentration, R
2be 0.9986.
This method can be used for detecting the concentration of cysteamine in actual blood serum sample, and the recovery is as shown in table 1.
Table 1
Claims (4)
1. a method for cysteamine in the detection serum based on fluorescence silver nanoclusters, is characterized in that the fluorescence of the silver nanoclusters that synthetic lipoic acid is modified can be by the quencher of cysteamine molecule, and therefore the logical change in fluorescence of relatively crossing quencher front and back is measured the concentration of cysteamine; Measuring cysteamine concentration realizes by following steps:
Step 1: add the serum solution that contains concentration known cysteamine in fluorescence silver nanoclusters solution, with the solution after fluorescence detector mensuration hybrid reaction and the fluorescence intensity ratio of blank group, make the graph of a relation of fluorescence intensity ratio and cysteamine concentration;
Step 2: add cysteamine concentration serum solution to be measured to fluorescence silver nanoclusters solution, record the fluorescence intensity ratio of itself and blank group, calculate the concentration of cysteamine solution in serum solution to be measured according to the graph of a relation in step 1.
2. the method for cysteamine in the detection serum based on fluorescence silver nanoclusters described in claim 1, is characterized in that in cysteamine concentration determination, and fluorescence silver nanoclusters solution is 400 times of stoste dilutions, and volume is 990 μ L.
3. the method for cysteamine in the detection serum based on fluorescence silver nanoclusters described in claim 1, is characterized in that, in cysteamine concentration determination, in serum solution to be measured, the scope of application of cysteamine concentration is 5-120 μ M, and volume is 10 μ L.
4. the method for cysteamine in the detection serum based on fluorescence silver nanoclusters described in claim 1, is characterized in that in cysteamine concentration determination, and serum solution to be measured and fluorescence silver nanoclusters solution reaction time are 5 minutes, and temperature of reaction is room temperature.
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| CN201310652621.3A CN103630521B (en) | 2013-12-05 | 2013-12-05 | A kind of method based on cysteamine in the detection serum of fluorescence silver nanoclusters |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103913443A (en) * | 2014-04-23 | 2014-07-09 | 安徽师范大学 | Aptamer sensor based on DNA-Ag NCs (deoxyribonucleic acid-silver nanoclusters) as well as preparation method, application and detection method thereof |
| CN104345053A (en) * | 2014-10-11 | 2015-02-11 | 汕头大学 | Gold nanoparticle biological sensor for detecting serum creatinine and preparation method of gold nanoparticles biological sensor |
| CN106862584A (en) * | 2015-12-13 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of synthetic method of atom number and the controllable silver nanoclusters of particle size |
| CN114594151A (en) * | 2022-02-25 | 2022-06-07 | 北京工业大学 | Application of cysteamine as electrochemiluminescence co-reactant |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103913443A (en) * | 2014-04-23 | 2014-07-09 | 安徽师范大学 | Aptamer sensor based on DNA-Ag NCs (deoxyribonucleic acid-silver nanoclusters) as well as preparation method, application and detection method thereof |
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| CN104345053A (en) * | 2014-10-11 | 2015-02-11 | 汕头大学 | Gold nanoparticle biological sensor for detecting serum creatinine and preparation method of gold nanoparticles biological sensor |
| CN104345053B (en) * | 2014-10-11 | 2017-01-11 | 汕头大学 | Gold nanoparticle biological sensor for detecting serum creatinine and preparation method of gold nanoparticles biological sensor |
| CN106862584A (en) * | 2015-12-13 | 2017-06-20 | 中国科学院大连化学物理研究所 | A kind of synthetic method of atom number and the controllable silver nanoclusters of particle size |
| CN114594151A (en) * | 2022-02-25 | 2022-06-07 | 北京工业大学 | Application of cysteamine as electrochemiluminescence co-reactant |
| CN114594151B (en) * | 2022-02-25 | 2023-12-01 | 北京工业大学 | Application of cysteamine as electrochemiluminescence coreactant |
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