CN103529009A - Method for detecting hydrogen sulfide in serum through near-infrared fluorescent probe Cy-Cl - Google Patents
Method for detecting hydrogen sulfide in serum through near-infrared fluorescent probe Cy-Cl Download PDFInfo
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- CN103529009A CN103529009A CN201310533325.1A CN201310533325A CN103529009A CN 103529009 A CN103529009 A CN 103529009A CN 201310533325 A CN201310533325 A CN 201310533325A CN 103529009 A CN103529009 A CN 103529009A
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Abstract
The invention discloses a method for detecting hydrogen sulfide in serum through a near-infrared fluorescent probe Cy-Cl, and relates to a fluorescent probe. The method can detect the content of the hydrogen sulfide in the serum in a high-sensitivity and a high-selectivity mode. The method includes the steps of preparing a standard solution of the near-infrared fluorescent probe Cy-Cl, then drawing a standard curve used for detecting Na2S, and finally detecting the content of the hydrogen sulfide in a serum sample to be detected.
Description
Technical field
The present invention relates to a kind of fluorescence probe, especially relate to a kind of near infrared fluorescent probe Cy-Cl for the detection method of serum sulfuretted hydrogen.
Background technology
Sulfuretted hydrogen (H
2s) be a kind of poisonous gas, it is known to have rotten egg taste.In recent years, increasing research shows, the sulfuretted hydrogen critical function that serving as in cell signal transmission first messenger the same as CO, NO in cell.Sulfuretted hydrogen is considered to mediating a large amount of physiology and pathologic function in cell, as cell proliferation, protect cardiovascular, stimulate angiogenesis and antioxidation etc.In human body and other biological system, also have Endogenous Hydrogen Sulfide, in blood, the concentration of endogenous sulfuretted hydrogen is 10～100 μ M according to reports.Therefore the method that develops the detection blood hydrogen sulfide content of highly sensitive, high selectivity all has great significance to aspects such as seeking of the diagnosis of disease, life process.
According to reports, the method that is applied at present sulfurated hydrogen detection mainly contains: colourimetry (D.Jimenez, R.Martinez-Manez, F.Sancenon, J.V.Ros-Lis, A.Benito, J.Soto, J.Am.Chem.Soc.2003,125,9000), electrochemical methods (D.G.Searcy, M.A.Peterson, Anal.Biochem.2004,324,269; C.J.Richardson, E.A.M.Magee, J.H.Cummings, Clin.Chim.Acta.2000,293,115), gas chromatography (J.Radford-Knoery, G.A.Cutter, Anal.Chem.1993,65,976) and fluorometry etc.Find according to the study, the metabolism of sulfuretted hydrogen in body is exceedingly fast, and causes its concentration change in body very fast, thereby makes the content of accurately monitoring sulfuretted hydrogen in body become an important difficult problem.Yet traditional method all can not reach accurately, detect fast and in real time the object of hydrogen sulfide content in body.In addition, fluorometry is with highly sensitive, simple to operate and can Real-Time Monitoring and enjoy numerous scientific workers' favor.A lot of seminars, all doing one's utmost to study various fluorescence probes to be applied to sulfurated hydrogen detection, had obtained certain achievement (A.R.Lippert, E.J.New, C.J.Chang, J.Am.Chem.Soc.2011,133,10078 in recent years; H.Peng, Y.Cheng, C.Dai, A.L.King, B.L.Predmore, D.J.Lefer, B.Wang, Angew.Chem.2011,123,9846; C.Liu, J.Pan, S.Li, Y.Zhao, L.Y.Wu, C.E.Berkman, A.R.Whorton, M.Xian, Angew.Chem.2011,123,10511).Yet these probes all have shortcoming more or less and make them can not meet the demand (W.-M.Xuan, C.-Q.Sheng, Y.-T.Cao, W.-H.He, W.Wang, Angew.Chem.Int.Ed.2012,51,2282 – 2284) of biology or medical science.Therefore keep punching and seek new energy accurately, fast and the fluorescence probe of Real-Time Monitoring body sulfuretted hydrogen mobile equilibrium becomes the target of a lot of scientific workers struggles.
Found at present a kind of near infrared fluorescent probe Cy-Cl can be highly sensitive, the content of sulfuretted hydrogen in the detection serum of high selectivity.Described probe Cy-Cl belongs to anthocyanidin series, and its absorption value in hyclone is 790nm, and absorption value is always very stable, illustrate Cy-Cl not with serum in range protein, amino acid etc. there is chemical reaction.Described probe Cy-Cl be take the excitation of wavelength 760nm and can be sent the fluorescence that wavelength is 795nm in hyclone.Probe Cy-Cl is obvious downtrending at the serum medium ultraviolet absorption spectrum that contains sulfuretted hydrogen and fluorescence spectrum figure along with the increase of concentration of hydrogen sulfide.Therefore probe Cy-Cl can be used for detecting the content of sulfuretted hydrogen in serum.
Summary of the invention
The object of this invention is to provide a kind of can be highly sensitive, highly selective detects the near infrared fluorescent probe Cy-Cl of hydrogen sulfide content in serum for the detection method of serum sulfuretted hydrogen.
The present invention includes following steps:
1) standard solution of preparation near infrared fluorescent probe Cy-Cl, described near infrared fluorescent probe Cy-Cl is anthocyanidin molecule type, molecular formula is C
32h
36clN
2 +, structural formula is:
2) make and detect Na
2the typical curve of S: prepare a series of sodium sulphide standard serum solution, add respectively the standard solution of Cy-Cl in a series of sodium sulphide standard serum solution, survey OD after reaction on ultra-violet absorption spectrum instrument
790nmvalue, draws the first typical curve; Or in fluorescence spectrophotometer, survey Em after reaction
795nm(exciting Ex:760nm) value, draws the second typical curve;
3) hydrogen sulfide content in detection test serum sample: add the standard solution of Cy-Cl in test serum sample, according to step 2) method of described drafting the first typical curve or the second typical curve records the OD of test serum sample
790nmvalue or Em
795 nm(exciting Ex:760nm) value, by OD
790nmvalue or Em
795nmbring in the first typical curve or the second typical curve, can calculate the content of sulfuretted hydrogen in test serum sample.
Compared with the prior art, beneficial effect of the present invention is as follows:
Near infrared fluorescent probe Cy-Cl is the visible cyan of naked eyes and the anthocyanidin molecule that sends near-infrared fluorescent, can highly sensitive, high selectivity occur fast with sulfuretted hydrogen in serum and react, and generates colourless and does not send the anthocyanidin derivative molecular of near-infrared fluorescent.This detection can be mainly used in the quantitative detection of sulfuretted hydrogen in serum, thereby reaches some signal path in monitoring cell.By the present invention, can realize: (1) is by the existence of sulfuretted hydrogen in visual measurement sample; (2) by analytical instrument, to the measurement of the absorption intensity of the fluorescent emission intensity of reaction system or visible ray, can carry out quantitative measurment to the content of sulfuretted hydrogen in sample; (3) can be applicable to the Real-Time Monitoring of sulfuretted hydrogen in cell.
Accompanying drawing explanation
Fig. 1 be the near infrared fluorescent probe Cy-Cl of the embodiment of the present invention in hyclone in uv absorption 790nm place's value and Na
2s reaction kinetics figure.In Fig. 1, horizontal ordinate is time (second), and ordinate is absorbance.
Fig. 2 is the near infrared fluorescent probe Cy-Cl of the embodiment of the present invention excitation with 760nm in hyclone, the fluorescence intensity level at fluorescent emission 795nm place and Na
2s reaction kinetics figure.In Fig. 2, horizontal ordinate is time (second), and ordinate is fluorescence intensity.
Fig. 3 is that the near infrared fluorescent probe Cy-Cl of the embodiment of the present invention detects Na in hyclone
2the spectrogram of the uv absorption sensitivity of S.In Fig. 3, horizontal ordinate is wavelength (nm), and ordinate is absorbance.
Fig. 4 be the near infrared fluorescent probe Cy-Cl of the embodiment of the present invention in hyclone in uv absorption 790nm place and various dose Na
2the proportional curve of S reaction.In Fig. 4, horizontal ordinate is Na
2s concentration (μ m), ordinate is absorbance.
Fig. 5 is that the near infrared fluorescent probe Cy-Cl of the embodiment of the present invention detects Na in hyclone
2the fluorescence emission spectrogram of S sensitivity.In Fig. 5, horizontal ordinate is wavelength (nm), and ordinate is fluorescence intensity.
Fig. 6 is the near infrared fluorescent probe Cy-Cl of the embodiment of the present invention excitation with 760nm in hyclone, the fluorescence intensity level at fluorescent emission 795nm place and various dose Na
2s reaction ratio curve.In Fig. 6, horizontal ordinate is Na
2s concentration (μ m), ordinate is fluorescence intensity.
Fig. 7 is near infrared fluorescent probe Cy-Cl selectivity and Na in hyclone of the embodiment of the present invention
2s reaction uv absorption figure.In Fig. 7, horizontal ordinate is wavelength (nm), and ordinate is absorbance.
Fig. 8 is near infrared fluorescent probe Cy-Cl selectivity and Na in hyclone of the embodiment of the present invention
2s reaction fluorescence emission spectrogram.In Fig. 8, horizontal ordinate is wavelength (nm), and ordinate is fluorescence intensity.
Embodiment
The embodiment of the present invention comprises the following steps:
1) standard solution of preparation near infrared fluorescent probe Cy-Cl
In step 1), described near infrared fluorescent probe Cy-Cl is anthocyanidin molecule type, and molecular formula is C
32h
36clN
2 +, structural formula is:
2) make and detect Na
2the typical curve of S
Prepare a series of sodium sulphide standard serum solution, in a series of sodium sulphide standard serum solution, add respectively the standard solution of Cy-Cl, after reaction, on ultra-violet absorption spectrum instrument, survey OD
790nmvalue, is depicted as the first typical curve; Or in fluorescence spectrophotometer, survey Em after reaction
795nm(exciting Ex:760nm) value, is depicted as the second typical curve;
3) detect the hydrogen sulfide content in test serum sample
The standard solution that adds Cy-Cl in test serum sample, according to step 2) method of described making the first typical curve or the second typical curve records the OD of hyclone sample to be measured
790nmvalue or Em
795nm(exciting Ex:760nm) value, by OD
790nmvalue or Em
795nmbring in the first typical curve or the second typical curve, can calculate the content of sulfuretted hydrogen in hyclone sample to be measured.
Provide the specific operation process in the step 3) of the present embodiment below:
To the Cy-Cl that adds 10 L1mmol/L in 1mL hyclone, add successively the Na of variable concentrations respectively
2s (0,10,100,150mol/L).This reactant liquor is collected uv absorption OD at once in microplate reader
790nmtemporal evolution kinetic curve and fluorescent emission Em
795nm(exciting Ex:760nm) temporal evolution kinetic curve.Fig. 1 is Cy-Cl OD in hyclone
790nmwith Na
2s reaction kinetics figure.Fig. 2 is Cy-Cl Em in hyclone
795nm(exciting Ex:760nm) and Na
2s reaction kinetics figure.
Provide below the present embodiment in hyclone with Na
2test process and the test result of S reaction sensitivity and reaction selectivity test:
Near infrared fluorescent probe Cy-Cl in hyclone with Na
2s reaction sensitivity
To the Cy-Cl that adds 10L1mmol/L in 1mL hyclone, add successively the Na of variable concentrations respectively
2s (0-200mol/L).This reactant liquor is collected fluorescence emission spectrogram after collecting uv absorption spectra and 20min after respectively at 10min in microplate reader.Fig. 3 is that Cy-Cl detects Na in hyclone
2the spectrogram of the uv absorption sensitivity of S, Na used
2s concentration is followed successively by: 0,2,5,10,25,50,100,150, and 200 mol/L, from top to bottom).Fig. 4 is Cy-Cl OD in hyclone
790nmwith various dose Na
2the proportional curve of S reaction.Fig. 5 is that Cy-Cl detects Na in hyclone
2the fluorescence emission spectrogram of S sensitivity, Na used
2s concentration is followed successively by: 0,2,5,10,20,40,60,80,120mol/L, from top to bottom).Fig. 6 is Cy-Cl Em in hyclone
795nm(exciting Ex:760nm) and various dose Na
2s reaction ratio curve.
Near infrared fluorescent probe Cy-Cl in hyclone with Na
2s reaction selectivity
To the Cy-Cl that adds 10L1mmol/L in 1mL hyclone, add successively 5L0.1mol/L reduced glutathione, 5L0.1mol/L halfcystine, 5L0.1mol/L homocysteine, 10L0.1mol/L arginine, 10L0.1mol/L lysine, 10L0.1mol/L NaOH and 10L10mmol/L Na respectively
2s.This reactant liquor is collected fluorescence emission spectrogram after collecting uv absorption spectra and 20min after respectively at 10min in microplate reader.Fig. 7 is Cy-Cl selectivity and Na in hyclone
2s reaction uv absorption figure.Fig. 8 is Cy-Cl selectivity and Na in hyclone
2s reaction fluorescence emission spectrogram.
Claims (1)
1. near infrared fluorescent probe Cy-Cl, for the detection method of serum sulfuretted hydrogen, is characterized in that comprising the following steps:
1) standard solution of preparation near infrared fluorescent probe Cy-Cl, described near infrared fluorescent probe Cy-Cl is anthocyanidin molecule type, molecular formula is C
32h
36clN
2 +, structural formula is:
2) make and detect Na
2the typical curve of S: prepare a series of sodium sulphide standard serum solution, add respectively the standard solution of Cy-Cl in a series of sodium sulphide standard serum solution, survey OD after reaction on ultra-violet absorption spectrum instrument
790nmvalue, draws the first typical curve; Or in fluorescence spectrophotometer, survey Em after reaction
795nmvalue, draws the second typical curve;
3) hydrogen sulfide content in detection test serum sample: add the standard solution of Cy-Cl in test serum sample, according to step 2) method of described drafting the first typical curve or the second typical curve records the OD of test serum sample
790nmvalue or Em
795nmvalue, by OD
790nmvalue or Em
795nmbring in the first typical curve or the second typical curve, can calculate the content of sulfuretted hydrogen in test serum sample.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805170A (en) * | 2014-01-26 | 2014-05-21 | 大连理工常熟研究院有限公司 | Specific fluorescent probe for identifying hydrogen sulfide and application of probe |
| CN106588906A (en) * | 2016-12-14 | 2017-04-26 | 南开大学 | Near infrared fluorescent probe and preparation method and application of near infrared fluorescent probe |
| CN107033111A (en) * | 2017-04-21 | 2017-08-11 | 台州学院 | A kind of synthesis and application of the near infrared fluorescent probe for detecting hydrogen sulfide |
| CN107286186A (en) * | 2016-04-12 | 2017-10-24 | 中国科学院化学研究所 | Hydrogen sulfide fluorescence probe and its preparation method and application |
| CN113189093A (en) * | 2021-04-28 | 2021-07-30 | 西南石油大学 | Method for detecting and monitoring hydrogen sulfide gas |
| CN113358289A (en) * | 2021-04-28 | 2021-09-07 | 西南石油大学 | Characteristic response material for monitoring and detecting hydrogen sulfide gas leakage and preparation method thereof |
| CN114354593A (en) * | 2022-01-07 | 2022-04-15 | 西南石油大学 | Composite test paper for detecting hydrogen sulfide gas leakage |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805170A (en) * | 2014-01-26 | 2014-05-21 | 大连理工常熟研究院有限公司 | Specific fluorescent probe for identifying hydrogen sulfide and application of probe |
| CN107286186A (en) * | 2016-04-12 | 2017-10-24 | 中国科学院化学研究所 | Hydrogen sulfide fluorescence probe and its preparation method and application |
| CN106588906A (en) * | 2016-12-14 | 2017-04-26 | 南开大学 | Near infrared fluorescent probe and preparation method and application of near infrared fluorescent probe |
| CN107033111A (en) * | 2017-04-21 | 2017-08-11 | 台州学院 | A kind of synthesis and application of the near infrared fluorescent probe for detecting hydrogen sulfide |
| CN107033111B (en) * | 2017-04-21 | 2019-08-06 | 台州学院 | A kind of synthesis and application of the near infrared fluorescent probe detecting hydrogen sulfide |
| CN113189093A (en) * | 2021-04-28 | 2021-07-30 | 西南石油大学 | Method for detecting and monitoring hydrogen sulfide gas |
| CN113358289A (en) * | 2021-04-28 | 2021-09-07 | 西南石油大学 | Characteristic response material for monitoring and detecting hydrogen sulfide gas leakage and preparation method thereof |
| CN113358289B (en) * | 2021-04-28 | 2022-04-15 | 西南石油大学 | Characteristic response material for monitoring and detecting hydrogen sulfide gas leakage and preparation method thereof |
| CN114354593A (en) * | 2022-01-07 | 2022-04-15 | 西南石油大学 | Composite test paper for detecting hydrogen sulfide gas leakage |
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Application publication date: 20140122 |