CN106083888B - The fluorescence probe of hydrogen sulfide in a kind of detection cancer cell - Google Patents
The fluorescence probe of hydrogen sulfide in a kind of detection cancer cell Download PDFInfo
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- CN106083888B CN106083888B CN201610541898.2A CN201610541898A CN106083888B CN 106083888 B CN106083888 B CN 106083888B CN 201610541898 A CN201610541898 A CN 201610541898A CN 106083888 B CN106083888 B CN 106083888B
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- hydrogen sulfide
- fluorescence probe
- cancer cell
- fluorescence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
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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
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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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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- 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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
Abstract
The invention discloses a kind of fluorescence probe for detecting hydrogen sulfide in cancer cell, the fluorescence probe is the naphthalimide derivative containing biotin group.The easily prepared cost of fluorescence probe of the present invention is cheap, and has high specific, can quick detection solution environmental sulphion, detection process will not be disturbed by other components, available for the measure of hydrogen sulfide in cancer cell of living, be had broad application prospects.
Description
Technical field
The present invention relates to a kind of novel fluorescence probe for hydrogen sulfide in cancer cell and its application, belong to analytical chemistry skill
Art field.
Background technology
The third new endogenous gas signal that hydrogen sulfide is found after nitric oxide and carbon monoxide transmits molecule,
The gaseous signal molecule of important physiological action can be played in life system, in cardiovascular system, nervous system and immune system
It is middle to participate in a variety of pathology, physiology course.Hydrogen sulfide can produce greatly injury to the health of human body and animal body, easily cause
The damage of the tissue such as human's nervous system and brain, suck excessive hydrogen sulfide and the symptom such as headache, dizzy, weak, nauseous occur,
Death will seriously be caused.Vascular relaxing factor and blood pressure regulatory factors of the hydrogen sulfide as cardiovascular system physiological, pass through
Adjust antiotasis, suppress vascular remodeling and cardioactive negative inotropic action, have emphatically in the generation development of hypertension
The meaning wanted, while ischemic damage and reperfusion damage can also be alleviated, negativity adjusts cardiac metabolism consumption and improve cardiac muscle oxidation and answered
Swash damage, play a part of cardioprotection.Exception and Alzheimer's disease, angiocardiopathy, cancer of internal concentration of hydrogen sulfide etc.
Major disease is closely related, therefore concentration of hydrogen sulfide tool has very important significance in detection organism real-time.
Recent research reports that the growth of many cancer cells such as colon cancer cell relies on hydrogen sulfide, without hydrogen sulfide, tumour life
Length will become slow.By taking colon cancer cell as an example, most colon cancer hydrogen sulfide generation is all called CBS with one kind（Guang sulphur
Ether-beta-synthetase, cystathionine- β-synthase）Albumen it is relevant.Compared to non-cancerous tissue, in colon cancer cell
The level of CBS albumen is much higher.When blocking CBS activity using chemical method, the growth of colon cancer cell can be by obvious
Containment, and normal cell growth is then unaffected.When the colon cancer tumours cell in patient source is implanted in nude mouse, together
Sample observed the inhibitor against colon carcinoma cells effect blocked caused by CBS.Under the conditions of not having hydrogen sulfide existing, tumour growth becomes non-
Often slowly.Therefore, it is significant that prevention and diagnosis of the detection real-time to tumour are carried out to the hydrogen sulfide in cancer cell.
Fluorescence imaging analysis method has high sensitivity, selectively good, response is rapid, simple operation and other advantages, and to thin
Born of the same parents do not damage substantially, are widely used for the detection of various biological micromolecules.It is presently used for detecting the glimmering of intracellular hydrogen sulfide
Light probe is also developed rapidly.But the hydrogen sulfide fluorescence probe reported at present does not possess cancer cell targeting mostly, it is impossible to
Cancer cell and normal cell are distinguished, is typically had accordingly to all cells.Therefore developing new has high selectivity, highly sensitive
Degree, photostability and permeable membrane are good, and the hydrogen sulfide fluorescence probe with cancer cell targeting has great importance.
The content of the invention
In view of the shortcomings of the prior art, the problem to be solved in the present invention is to provide hydrogen sulfide in a kind of new detection cancer cell
Type fluorescence probe and its application.
The fluorescence probe of hydrogen sulfide is sub- for the naphthoyl containing biotin group in new detection cancer cell of the present invention
Amine derivative, it is characterised in that：The chemical structure of general formula of the fluorescence probe such as formula（I）Shown, wherein biotin moiety has
Cancer cell targeting, azido group are that hydrogen sulfide responds site：
Formula（I）
The preparation method of fluorescence probe of the present invention is：By formula（Ⅱ）Shown compound a (1 mmol), nitrine
Change sodium（1 mmol）It is added in 50 mL single-necked flasks with DMF (5 mL), is heated 5 hours at 90 DEG C.It is cold
But to room temperature, it is added to the water, filters, drying.Gained solid is subjected to column chromatography purifying, it is this hair to obtain light yellow product
The fluorescence probe of hydrogen sulfide in the bright detection cancer cell.
Formula（Ⅱ）
The application of fluorescence probe of the present invention hydrogen sulfide in detection cancer cell.
Fluorescence probe of the present invention can be applied to the detection evaluation of hydrogen sulfide in cancer cell.The probe is as cancer cell
The specific probe of hydrogen sulfide, in the environment of hydrogen sulfide presence, nitrine can be reduced to amino, generation has high fluorescence hair
The fluorescent material of ability is penetrated, passes through the hydrogen sulfide for detecting different fluorescence intensities to determine in cancer cell.
Specifically assay method is：Under room temperature condition, 5 μM of fluorescence probe ethanol solutions are prepared, are added to various concentrations
Sodium sulfide solution system in, evaluation index of the measure solution fluorescence intensity as sulphion concentration.
For fluorescence probe of the present invention under the conditions of existing for hydrogen sulfide, the azide moiety of naphthalimide fluorogen will be by
Amino is reduced to, now fluorescence probe can launch the green fluorescence of naphthalimide fluorogen（Peak value is about 544 nm）.Meanwhile
Because biotin has cancer cell targeting, cancer cell and normal cell can be distinguished, therefore the fluorescence probe can be used for
Detect the hydrogen sulfide in cancer cell.Fluorescence detector can be used to realize the rapid sensitive detection of product；Testing conditions are：Excitation wave
A length of 440 nm, the detection of fluorescence emission spectrum is carried out between 520 ~ 700 nm.
Fluorescence probe has probe thin to the toxicity of living cells, colouring power, work in bio-imaging application study main contents
Born of the same parents and living tissue fluorescence imaging.Using MTT colorimetric methods, by analyzing survival rate of the cell after fluorescence probe is added, discuss
Toxicity of the fluorescence probe to cell.On this basis, using fluorescence probe of the present invention to the hydrogen sulfide in cancer cell living
It is used for quickly detecting.
Beneficial effects of the present invention are：
The fluorescence probe of hydrogen sulfide can obtain through chemical synthesis in detection cancer cell of the present invention, and synthesis technique is simple
Easy, raw material is cheap and easy to get, and preparation cost is low, easy to spread.
The fluorescence probe of hydrogen sulfide has high specific in detection cancer cell of the present invention, is carrying out corresponding hydrogen sulfide
Do not disturbed in detection process by other components, available for the real time measure of hydrogen sulfide in cancer cell of living, there is wide application
Prospect.
The fluorescence probe high sensitivity of detection intracellular hydrogen sulfide of the present invention, has good fluorescence emission spectrum
Characteristic（520~700 nm）, it is possible to achieve the purpose quick and precisely detected to the hydrogen sulfide in cell.
Brief description of the drawings
Fig. 1 is fluorescence probe1H NMR spectras.
Fig. 2 is fluorescence spectrum of the fluorescence probe under the conditions of various concentrations vulcanized sodium.
Fig. 3 is the linear relationship data of fluorescence probe and vulcanization na concn.
Fig. 4 is fluorescence probe and the reacted fluorescence spectrum of different material.
Fig. 5 is imaging applications of the fluorescence probe in living cells.
A schemes：Only add the cell photograph via bright field of 10 μM of probes；B schemes：The cell green channel fluorescence of 10 μM of probes is only added to shine
Piece；C schemes：A schemes and the superposition photo of B figures；D schemes：Add the cell photograph via bright field of 10 μM of probes and 50 μM of vulcanized sodium；E schemes：Add
Enter the cell green channel fluorescence photo of 10 μM of probes and 50 μM of vulcanized sodium；F schemes：D schemes and the superposition photo of E figures.
Embodiment
With reference to embodiment and accompanying drawing, the present invention will be further described, but present invention protection content is not limited only to this.
Embodiment 1
Detect the preparation of the fluorescence probe of hydrogen sulfide in cancer cell
By formula（Ⅱ）Shown compound a（1 mmol）, sodium azide（1mmol）And N,N-dimethylformamide（5
mL）It is added in 50 mL single-necked flasks, is heated 5 hours at 90 DEG C.Room temperature is cooled to, is added to the water, is filtered, drying.By institute
Obtain solid and carry out column chromatography (dichloromethane:Methanol=10:1) purify, obtain yellow product, detection cancer as of the present invention
The fluorescence probe of intracellular hydrogen sulfide, yield 63%.
The fluorescence probe of hydrogen sulfide in above-mentioned detection cancer cell1H NMR spectras are shown in Fig. 1.
Embodiment 2
Fluorescence spectrum of the fluorescence probe under different vulcanization na concns
This patent supplies hydrogen sulfide in aqueous using vulcanized sodium.The 5 μM of probes buffering for preparing 10 parts of 5mL in advance is molten
Liquid, containing 5% methanol, the vulcanization na concn added is 0 to 150 μM.Then fluoroscopic examination is carried out（λEx= 440 nm）；Calculate each
Fluorescence intensity in system；By analyzing the relation of fluorescence intensity and concentration of hydrogen sulfide at 544nm, the probe is assessed to vulcanized sodium
Response performance（See Fig. 2 and Fig. 3）.Fig. 2 shows that, with the increase of vulcanization na concn, the fluorescence intensity of solution gradually strengthens.Figure
3 show, when vulcanization na concn is in the range of 40 ~ 90 μM, the fluorescence intensity of solution and the concentration of vulcanized sodium are closed in preferably linear
System, the * X -28.0 of formula Y=6.07 can be used to represent, wherein Y is the fluorescence intensity that 544nm goes out, and X is the dense of vulcanized sodium
Degree.
Embodiment 3
Fluorescence probe and the reacted fluorescence spectrum of different material
10 parts of 5mL 5 μM of probe cushioning liquid (containing 5% methanol, pH=7.4) are prepared in advance, then respectively to the body
Hcys, Na that 50 μ L concentration are 200 μM are sequentially added in system2S、Na2SO3、NO、H2O2、HClO4、Cys、GSH、NaNO2、VC
Etc. the PBS solution of small molecule.Then fluoroscopic examination is carried out（λEx= 440 nm）；Calculate fluorescence intensity in each system；Assess this not
Interference of the commaterial to fluorescence probe solution（See Fig. 4）.As seen from the figure, Hcys, Na are added in certain probe solution2S、
Na2SO3、NO、H2O2、HClO4、Cys、GSH、NaNO2, the small molecule such as VC when, only vulcanized sodium can cause solution to produce notable
Fluorescence, and the fluorescence of solution does not change substantially when adding other small molecules, and this represents that the probe only has response to vulcanized sodium,
Without being disturbed by other small molecules.
Embodiment 4
Imaging applications of the fluorescence probe in living cells
Hela cells are placed in culture medium (DMEM nutrient solutions and 10% hyclone), be positioned over condition for 37 DEG C, 5%
CO2With 20% O2Incubator in cultivate 24-48h.(10 μM) injections of fluorescence probe of the present invention are drawn with microsyringe
In culture medium containing Hela cells, continue to cultivate 30 min in incubator.100 μM of sodium sulfide solutions are added, continue to cultivate
30 min.Culture cell is rinsed with PBS (phosphate buffer solution) 3 times, carry out fluorescence imaging afterwards.
As a result Fig. 5 is seen.Find out from A, B and C figure, during addition probe, cell does not have fluorescence substantially；From D, E and F figure
Find out, add after 10 μM of probes and 50 μM of vulcanized sodium, cell produces obvious fluorescence, and this represents that the probe can be used for detecting
Hydrogen sulfide in cancer cell.
Claims (1)
1. a kind of fluorescence probe for detecting hydrogen sulfide in cancer cell, it is characterised in that it is sub- for the naphthoyl containing biotin group
Amine derivative, shown in the chemical structure of general formula such as formula (I) of the fluorescence probe：
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106753337B (en) * | 2016-12-01 | 2018-06-29 | 济南大学 | A kind of near-infrared and two-photon Double-mode imaging fluorescence probe and its preparation and application |
| CN107298654A (en) * | 2017-05-19 | 2017-10-27 | 济南大学 | The fluorescence probe of hydrogen sulfide in a kind of detection endoplasmic reticulum |
| CN109265429B (en) * | 2018-08-31 | 2023-03-31 | 河南师范大学 | Naphthalene derivative fluorescent dye and preparation method and application thereof |
| CN110372738A (en) * | 2019-07-30 | 2019-10-25 | 济南大学 | A kind of fluorescence probe and its preparation method and application of positioning fat drips detection hydrogen sulfide |
| CN110658172A (en) * | 2019-11-27 | 2020-01-07 | 济南大学 | Application of ratio type two-photon fluorescent probe in rapid and sensitive detection of hydrogen sulfide |
| CN113402530B (en) * | 2021-06-22 | 2023-02-03 | 济南大学 | Hypochlorous acid fluorescent probe with function of distinguishing cancer cells from normal cells, preparation method and application |
| CN114702447B (en) * | 2022-04-26 | 2023-07-11 | 苏州大学 | Naphthalimide derivative and preparation method and application thereof |
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| CN103160274B (en) * | 2012-11-30 | 2014-08-06 | 山东师范大学 | Fluorescence probe for detecting intracellular hydrogen sulfide and preparation method and application of fluorescence probe |
| CN104031637B (en) * | 2014-06-24 | 2015-08-12 | 浙江大学 | A kind of azo fluorescent probe and application thereof detecting biological hydrogen sulfide |
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