CN106525785A - Cancer cell targeted fluorescent probe for detecting copper ions in cancer cells - Google Patents
Cancer cell targeted fluorescent probe for detecting copper ions in cancer cells Download PDFInfo
- Publication number
- CN106525785A CN106525785A CN201610902353.XA CN201610902353A CN106525785A CN 106525785 A CN106525785 A CN 106525785A CN 201610902353 A CN201610902353 A CN 201610902353A CN 106525785 A CN106525785 A CN 106525785A
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- copper ion
- cancerous cell
- detection
- copper ions
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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"
-
- 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
- G01N2021/6417—Spectrofluorimetric devices
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a cancer cell targeted fluorescent probe for detecting copper ions in cancer cells. The fluorescent probe is a rhodamine derivative containing biotin groups. The fluorescent probe is easy to prepare, low in cost and high in specificity, can be used for quickly detecting the copper ions in the solution environment, can not be interfered by other components in the detection process, can be used for determining the copper ions in live cancer cells, and has wide application prospects.
Description
Technical field
The present invention relates to it is a kind of for the novel fluorescence probe of copper ion detection and its application in cancerous cell, belong to analysisization
Learn technical field.
Background technology
Content of the copper in living things system is only second to zinc and ferrum, a kind of micro heavy unit necessary to be living things system
Various organic basic physiological processes are played vital effect by element and nutrient.As the important gold of life system
Category element, copper ion is often with metals such as cytochrome oxidase, Catalase, TYR enzyme, dopamine β-reinforcing enzymes
A series of physiological process such as the catalytic cofactor form of enzyme, electron transmission, oxidoreduction in wide participation life system,
The processes such as biological internal enzyme reaction, enzyme transcription play an important role.When copper ion concentration in living things system is abnormal, will
Cause the generation of the diseases such as this syndrome of face gram, Wilson formula syndrome, familial amyotrophic disease and alzheimer's disease.Cause
This, the copper ion detection method for seeking a kind of rapid sensitive has important effect in life architectural study and medical diagnosiss.
Recent research is reported, with respect to normal cell, copper ion can produce substantially enrichment in tumor cell, with higher
Concentration.Clinical research finds that the copper ion concentration in cancer patient's blood plasma is reacted with tumor progression degree and Drug therapy
It is closely related.By chelating copper ions agent tetrathiomolybdate（TM）Internal content of copper ion is reduced, tumor group can be significantly slowed
Angiogenesis in knitting, and then suppress tumour growth.Therefore, detection real-time is carried out to the copper ion in cancerous cell to tumor
Prevention and diagnose it is significant.
Traditional detection method mainly has atomic absorption spectrography (AAS), inductively coupled plasma mass spectrometry method, inductive etc.
The method that gas ions-atomic emission spectrometry, spectrophotometric determination method and cyclic voltammetry etc. detect copper ion, but these inspections
Survey method needs testing equipment costly, and relatively complicated in detection process, is not suitable for mass detection and real-time detection.
Compare fluorescence imaging analysis method and have that sensitivity is high, selectivity is good, response is rapid, simple operation and other advantages, and to cell base
This does not damage, and is widely used for the detection of various biological micromolecules.It is presently used for detecting that the fluorescence of intracellular copper ion is visited
Pin is also developed rapidly.But, the copper ion fluorescence probe of report does not mostly possess cancerous cell targeting at present, it is impossible to distinguish
All cells are typically had accordingly by cancerous cell and normal cell.Therefore develop new with high selectivity, high sensitivity, light
Stability and permeable membrane are good, and the copper ion fluorescence probe with cancerous cell targeting has great importance.
The content of the invention
For the deficiencies in the prior art, the invention provides the novel fluorescence of copper ion is visited in a kind of new detection cancerous cell
Pin and its application.
In new detection cancerous cell of the present invention, the fluorescent probe of copper ion is the rhodamine containing biotin group
Derivant, it is characterised in that：The chemical structure of general formula of the fluorescent probe such as formula（）Shown, wherein biotin moiety has cancer
Cell targeted, hydrazides group is that copper ion responds site：
Formula（）
In detection living things system of the present invention, the preparation method of the fluorescent probe of copper ion is：By formula（Ⅱ）Shown compound
A (1 mmol), hydrazine hydrate（2 mmol）It is added in 50 mL single-necked flasks with ethanol (5 mL), heats 5 hours at 80 DEG C.
Room temperature is cooled to, is added to the water, filtered, drying.Gained solid is carried out into column chromatography purification, white product is obtained and is this
The fluorescent probe of copper ion in the bright detection cancerous cell.
Formula（Ⅱ）
Fluorescent probe of the present invention is detecting the application of copper ion in cancerous cell.
Fluorescent probe of the present invention can be applicable to the detection evaluation of copper ion in cancerous cell.Wherein biotin moiety makes
Obtain the probe and there is cancerous cell targeting.Specific probe of the probe as cancerous cell copper ion, in the ring that copper ion is present
Under border, can be carboxyl by hydrazides catalyzing hydrolysis, generate the fluorescent material with high fluorescent emission ability, it is different by detecting
Fluorescence intensity is determining the copper ion in cancerous cell.
Specifically assay method is：Under room temperature condition, 5 μM of fluorescent probe buffer solution are prepared（Containing 5% acetonitrile）, it is added to
In copper ion solution system with variable concentrations, solution fluorescence intensity is determined as the evaluation index of copper ion concentration.
Fluorescent probe of the present invention under conditions of hydrogen sulfide presence, will be urged by the hydrazides part of rhodamine fluorogen
Change is hydrolyzed to carboxyl, and now fluorescent probe can launch the red fluorescence of rhodamine fluorogen（Peak value is about 580 nm）.Meanwhile,
As biotin has cancerous cell targeting, cancerous cell and normal cell can be distinguished, therefore the fluorescent probe can be used for
Copper ion in detection cancerous cell.The rapid sensitive detection of product can be realized using fluorescence detector；Testing conditions are：Excitation wave
A length of 530 nm, carries out the detection of fluorescence emission spectrum between 550 ~ 700 nm.
Fluorescent probe has probe to the toxicity of living cells, colouring power, living thin in bio-imaging applied research main contents
Born of the same parents and living tissue fluorescence imaging.Using MTT colorimetrys, by analyzing survival rate of the cell after fluorescent probe is added, discuss
Toxicity of the fluorescent probe to cell.On this basis, using fluorescent probe of the present invention to the copper ion in cancerous cell living
It is used for quickly detecting.
Beneficial effects of the present invention are：
In detection cancerous cell of the present invention the fluorescent probe of copper ion can Jing chemosynthesis obtain, synthesis technique is simply easy
OK, raw material is cheap and easy to get, and preparation cost is low, it is easy to promote.
In detection cancerous cell of the present invention, the fluorescent probe of copper ion has high specific, is carrying out corresponding copper ion
Do not disturbed by other components in detection process, the real time measure of copper ion in the cancerous cell that can be used to living, with wide application
Prospect.
The fluorescent probe sensitivity of detection intracellular copper ion of the present invention is high, with good fluorescence emission spectrum
Characteristic（550~700 nm）, it is possible to achieve the purpose quick and precisely detected by the copper ion in cell.
Description of the drawings
Fig. 1 is fluorescent probe1H NMR spectras.
Fig. 2 is fluorescence spectrum of the fluorescent probe under the conditions of variable concentrations copper ion.
Fig. 3 is the linear relationship data of fluorescent probe and copper ion concentration.
Fig. 4 is fluorescent probe and the reacted fluorescence spectrum of different material.
Fig. 5 is imaging applications of the fluorescent probe in living cells.
A schemes：Only add the cell photograph via bright field of 10 μM of probes；B schemes：The cell red channel fluorescence of 10 μM of probes is only added to shine
Piece；C schemes：A schemes the superposition photo with B figures；D schemes：Add the cell photograph via bright field of 10 μM of probes and 50 μM of copper ions；E schemes：Plus
Enter the cell red channel fluorescence photo of 10 μM of probes and 50 μM of copper ions；F schemes：D schemes the superposition photo with E figures.
Specific 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
The preparation of the fluorescent probe of copper ion in the detection cancerous cell
By formula（Ⅱ）Shown compound a（1 mmol）, hydrazine hydrate（2 mmol）And ethanol（5 mL）It is added to 50 mL single port
In flask, heat 5 hours at 80 DEG C.Room temperature is cooled to, is added to the water, filtered, drying.Gained solid is carried out into column chromatography
(dichloromethane:Methanol=10:1) purification, obtains yellow product, copper ion in detection cancerous cell as of the present invention
Fluorescent probe, yield 73%.
The fluorescent probe of copper ion in above-mentioned detection cancerous cell1H NMR spectras are shown in Fig. 1.
Embodiment 2
Fluorescence spectrum of the fluorescent probe under different copper ion concentrations
The present invention supplies copper ion in aqueous using sulphuric acid.Prepare 5 μM of probe buffer solution of 10 parts of 5mL in advance, contain
5% acetonitrile, the copper ion concentration for being added are 0 to 125 μM.Then carry out fluoroscopic examination（λEx= 530 nm）；Calculate each system
Middle fluorescence intensity；By analyzing the relation of the fluorescence intensity at 581 nm and copper ion concentration, the probe is assessed to copper ion
Response performance（See Fig. 2 and Fig. 3）.Fig. 2 shows that, with the increase of copper ion concentration, the fluorescence intensity of solution gradually strengthens.Fig. 3
Show, when copper ion concentration is in the range of 0 ~ 40 μM, the concentration of the fluorescence intensity of solution and copper ion is closed in preferably linear
System, can be represented using formula Y=19.95 * X+23.27, and wherein Y is the fluorescence intensity at 581nm places, and X is the dense of copper ion
Degree.
Embodiment 3
Fluorescent probe and the reacted fluorescence spectrum of different material
(containing 5 % acetonitriles, pH=7.4), then respectively to the system to prepare 5 μM of probe buffer solution of 10 parts of 5mL in advance
In sequentially add Hcys, Na that 50 μ L concentration are 200 μM2S、Na2SO3、Cys、GSH、VC、Zn2+、Fe2+、Fe3+、Hg2+、Mg2 +、Co2+Etc. the buffer solution of analyte of interest.Then carry out fluoroscopic examination（λEx= 530 nm）；In calculating each system, fluorescence is strong
Degree；Assess interference of the different material to fluorescent probe solution（See Fig. 4）.As seen from the figure, add in certain probe solution
Hcys、Na2S、Na2SO3、Cys、GSH、VC、Zn2+、Fe2+、Fe3+、Hg2+、Mg2+、Co2+During etc. analyte of interest, only copper ion
Solution can be caused to produce significant fluorescence, and the fluorescence of solution is not changed in substantially when adding other small molecules, this represents should
Probe only has response to copper ion, and is not disturbed by other small molecules.
Embodiment 4
Imaging applications of the fluorescent probe in living cells
By mouse mastopathy cell（4T1 cells）It is placed in culture medium (DMEM culture fluid and 10% hyclone), is positioned over condition
For 37 DEG C, 5% CO2With 20% O2Incubator in cultivate 24-48h.Fluorescent probe of the present invention is drawn with microsyringe
In (10 μM) culture medium of the injection containing 4T1 cells, 30 min are cultivated in continuation in incubator.Add 100 μM of copper ions molten
Liquid, continues 30 min of culture.Cultured cells 3 times is rinsed with PBS (phosphate buffered solution) afterwards, fluorescence imaging is carried out.
As a result see Fig. 5.Find out from A, B and C figure, during addition probe, the substantially no fluorescence of cell；From D, E and F figure
Find out, after adding 10 μM of probes and 50 μM of sodium sulfide, cell produces obvious fluorescence, and this represents that the probe can be used to detect
Copper ion in cancerous cell.
Claims (3)
1. it is a kind of detection cancerous cell in copper ion cancerous cell targeting fluorescent probe, it is characterised in that：Which contains biotin group
Rhodamine Derivatives, the chemical structure of general formula such as formula of the fluorescent probe（）It is shown：
Formula（）.
2. it is a kind of it is according to claim 1 detection cancerous cell in copper ion cancerous cell targeting fluorescent probe preparation side
Method, it is characterised in that：
By 1 mmol formulas（Ⅱ）Shown compound, 2 mmol hydrazine hydrates and 5 mL ethanol are added in 50 mL single-necked flasks, and 80
Heat 5 hours at DEG C；Room temperature is cooled to, is added to the water, filtered, drying；Gained solid is carried out into column chromatography purification, obtains white
Color product is the fluorescent probe of copper ion in detection cancerous cell of the present invention；
Formula（Ⅱ）.
3. application of the cancerous cell targeting fluorescent probe described in claim 1 or 2 in copper ion in detection cancerous cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610902353.XA CN106525785B (en) | 2016-10-17 | 2016-10-17 | The cancer cell targeting fluorescent probe of copper ion in a kind of detection cancer cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610902353.XA CN106525785B (en) | 2016-10-17 | 2016-10-17 | The cancer cell targeting fluorescent probe of copper ion in a kind of detection cancer cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106525785A true CN106525785A (en) | 2017-03-22 |
| CN106525785B CN106525785B (en) | 2019-07-02 |
Family
ID=58332529
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610902353.XA Expired - Fee Related CN106525785B (en) | 2016-10-17 | 2016-10-17 | The cancer cell targeting fluorescent probe of copper ion in a kind of detection cancer cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106525785B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113402530A (en) * | 2021-06-22 | 2021-09-17 | 济南大学 | Hypochlorous acid fluorescent probe with function of distinguishing cancer cells from normal cells, preparation method and application |
-
2016
- 2016-10-17 CN CN201610902353.XA patent/CN106525785B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113402530A (en) * | 2021-06-22 | 2021-09-17 | 济南大学 | Hypochlorous acid fluorescent probe with function of distinguishing cancer cells from normal cells, preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106525785B (en) | 2019-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | A near-infrared fluorescent probe for imaging of endogenous hydrogen sulfide in living cells and mice | |
| CN109266332B (en) | Preparation method of ratiometric fluorescent probe for quantitatively detecting AChE and BChE in blood | |
| CN105524612B (en) | A kind of isophorone class fluorescence probe and its preparation and application | |
| CN106083888B (en) | The fluorescence probe of hydrogen sulfide in a kind of detection cancer cell | |
| Zhou et al. | A naphthalimide-indole fused chromophore-based fluorescent probe for instantaneous detection of thiophenol with a red emission and a large Stokes shift | |
| Luo et al. | Molecular engineering of a colorimetric two-photon fluorescent probe for visualizing H2S level in lysosome and tumor | |
| CN105693703B (en) | A kind of novel Ratiometric fluorescent probe for the imaging of intracellular lysosomal pH | |
| Gan et al. | Turn-on fluorescent probe for sensing exogenous and endogenous hypochlorous acid in living cells, zebrafishes and mice | |
| CN106967102B (en) | A kind of enhanced fluorescence probe of hydrogen peroxide based on Rhodamine Derivatives | |
| CN112939957B (en) | Benzoindole derivative In-XY1, and synthesis method and application thereof | |
| CN106117241B (en) | The fluorescence probe of lysosomal pH in a kind of detection cancer cell | |
| CN107216270A (en) | A kind of application for detecting hydrogen sulfide high selectivity response type fluorescence probe | |
| CN108148573A (en) | A kind of fluorescence probe for detecting lysosomal pH and its synthetic method and application | |
| CN108117544A (en) | A kind of reversible sulfur dioxide/sulfurous acid（Hydrogen）The fluorescence probe of salt | |
| Li et al. | Pyridine functionalized carbon dots for specific detection of tryptophan in human serum samples and living cells | |
| Rong et al. | Double-channel based fluorescent probe for differentiating GSH and H2Sn (n> 1) via a single-wavelength excitation with long-wavelength emission | |
| CN109776564A (en) | The ferrous ion fluorescence probe and its synthetic method of a kind of xanthene structure and application | |
| CN106632212A (en) | Fluorescent probe for detecting cysteine in cell | |
| CN109651249A (en) | A kind of fluorescence probe detecting endocytoplasmic reticulum cysteine and its synthesis and application | |
| CN107383078B (en) | Phenylboric acid ester compounds and benzoyl peroxide detection kit comprising the compound | |
| Li et al. | Ratiometric fluorescence and chromaticity dual-readout assay for β-glucuronidase activity based on luminescent lanthanide metal-organic framework | |
| CN113563279B (en) | Two-photon fluorescent probe for detecting nitroreductase and preparation method and application thereof | |
| Zhang et al. | Based on theoretical calculations designed a novel dual-channel chemo-sensor for Mg2+ and Zn2+ detection and bioimaging applications | |
| Tian et al. | A dual-mode portable platform with spatiotemporal temperature–pressure signal readouts for ultrasensitive quantitative determination of cancer cells | |
| CN106525785A (en) | Cancer cell targeted fluorescent probe for detecting copper ions in cancer cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190702 Termination date: 20201017 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |