CN106525785B - The cancer cell targeting fluorescent probe of copper ion in a kind of detection cancer cell - Google Patents
The cancer cell targeting fluorescent probe of copper ion in a kind of detection cancer cell Download PDFInfo
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- CN106525785B CN106525785B CN201610902353.XA CN201610902353A CN106525785B CN 106525785 B CN106525785 B CN 106525785B CN 201610902353 A CN201610902353 A CN 201610902353A CN 106525785 B CN106525785 B CN 106525785B
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- 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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- 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
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Abstract
The invention discloses a kind of cancer cell targeting fluorescent probes of copper ion in detection cancer cell, the fluorescence probe is the Rhodamine Derivatives containing biotin group, fluorescence probe of the invention is easily prepared low in cost, and there is high specific, the copper ion of solution environmental can quickly be detected, detection process will not be by the interference of other components, and the measurement of copper ion, has broad application prospects in the cancer cell that can be used for living.
Description
Technical field
The present invention relates to a kind of novel fluorescence probe detected for copper ion in cancer cell and its applications, belong to analysisization
Learn technical field.
Background technique
Content of the copper in biosystem is only second to zinc and iron, is a kind of micro heavy member necessary to biosystem
Element and nutrient, play a crucial role the basic physiological process of a variety of organisms.Important gold as life system
Belong to element, copper ion is often with metals such as cytochrome oxidase, Catalase, TYR enzyme, dopamine β-reinforcing enzymes
The catalytic cofactor form of enzyme, a series of physiology courses such as electron transmission, redox in wide participation life system,
The processes such as the intracorporal enzyme reaction of biology, enzyme transcription play an important role.It, will when copper ion concentration exception in biosystem
Lead to 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 plays an important role in life architectural study and medical diagnosis.
Recent research report, opposite normal cell, copper ion can generate obvious enrichment in tumour cell, have 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.Internal content of copper ion is reduced by copper ion chelator tetrathiomolybdate (TM), tumor group can be significantly slowed
Angiogenesis in knitting, and then inhibit tumour growth.Therefore, detection real-time, quickly is carried out to tumour to the copper ion in cancer cell
Prevention and diagnosis be of great significance.
Traditional detection method mainly has atomic absorption spectrography (AAS), inductively coupled plasma mass spectrometry method, inductive coupling etc.
The method that gas ions-atomic emission spectrometry, spectrophotometric determination method and cyclic voltammetry etc. detect copper ion, but these are examined
Survey method needs detection device costly, and relatively complicated in detection process, is not suitable for mass detection and real-time detection.
Compared to fluorescence imaging analysis method have high sensitivity, selectivity be good, response rapidly, simple operation and other advantages, and to cell base
This is not damaged, and is widely used for the detection of various biological micromolecules.Currently, the fluorescence for detecting intracellular copper ion is visited
Needle is also rapidly developed.But the copper ion fluorescence probe reported at present does not have cancer cell targeting mostly, cannot distinguish between
Cancer cell and normal cell generally have accordingly all cells.What therefore exploitation was new has highly selective, highly sensitive, light
Stability and permeable membrane are good, and the copper ion fluorescence probe with cancer cell targeting has great importance.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of novel fluorescence spies of copper ion in novel detection cancer cell
Needle and its application.
The fluorescence probe of copper ion is the rhodamine containing biotin group in novel detection cancer cell of the present invention
Derivative, it is characterised in that: the general formula of the chemical structure of the fluorescence probe such as formula () shown in, wherein biotin moiety has cancer
Cell targeted, hydrazides group is that copper ion responds site:
Formula ()
In detection biosystem of the present invention the fluorescence probe of copper ion the preparation method comprises the following steps: by shown in formula (II) change
It closes object a (1 mmol), hydrazine hydrate (2 mmol) and ethyl alcohol (5 mL) to be added in 50 mL single-necked flasks, heats 5 at 80 DEG C
Hour.It is cooled to room temperature, is added to the water, filter, drying.Obtained solid is subjected to column chromatographic purifying, obtains white product i.e.
For the fluorescence probe of copper ion in detection cancer cell of the present invention.
Formula (II)
The application of fluorescence probe of the present invention copper ion in detection cancer cell.
Fluorescence probe of the present invention can be applied to the detection evaluation of copper ion in cancer cell.Wherein biotin moiety makes
Obtaining the probe has cancer cell targeting.Specific probe of the probe as cancer cell copper ion, the ring existing for copper ion
Under border, it can be carboxyl by hydrazides catalyzing hydrolysis, generate the fluorescent material with high fluorescent emission ability, it is different by detecting
Fluorescence intensity measures the copper ion in cancer cell.
Specific measuring method are as follows: under room temperature, prepare 5 μM of fluorescence probe buffer solutions (containing 5% acetonitrile), be added to
In copper ion solution system with various concentration, evaluation index of the solution fluorescence intensity as copper ion concentration is measured.
Under the conditions of fluorescence probe of the present invention is existing for the hydrogen sulfide, the hydrazides part of rhodamine fluorogen will be urged
Change is hydrolyzed to carboxyl, and fluorescence probe can emit the red fluorescence of rhodamine fluorogen at this time (peak value is about 580 nm).Meanwhile
Since biotin has cancer cell targeting, cancer cell and normal cell can be distinguished, therefore the fluorescence probe can be used for
Detect the copper ion in cancer cell.The rapid sensitive detection that fluorescence detector realizes product can be used；Testing conditions are as follows: excitation wave
A length of 530 nm carries out the detection of fluorescence emission spectrum between 550 ~ 700 nm.
The main contents include probe is thin to the toxicity of living cells, colouring power, work in bio-imaging application study for fluorescence probe
Born of the same parents and living tissue fluorescence imaging.It is discussed using MTT colorimetric method by survival rate of the analysis cell after fluorescence probe is added
Toxicity of the fluorescence probe to cell.On this basis, using fluorescence probe of the present invention to the copper ion in cancer cell living
It is used for quickly detecting.
The invention has the benefit that
The fluorescence probe of copper ion can be obtained through chemical synthesis in detection cancer cell of the present invention, and synthesis technology is simple
Easy, raw material is cheap and easy to get, and preparation cost is low, easy to spread.
The fluorescence probe of copper ion has high specific in detection cancer cell of the present invention, is carrying out corresponding copper ion
Not by the interference of other components in detection process, the real time measure of copper ion in the cancer cell that can be used for living has wide application
Prospect.
The fluorescence probe high sensitivity of detection intracellular copper ion of the present invention, has good fluorescence emission spectrum
Characteristic (550 ~ 700 nm), may be implemented the purpose quick and precisely detected to the copper ion in cell.
Detailed description of the invention
Fig. 1 is fluorescence probe1H NMR spectra.
Fig. 2 is fluorescence spectrum of fluorescence probe under the conditions of various concentration copper ion.
Fig. 3 is the linear relationship data of fluorescence probe and copper ion concentration.
Fig. 4 is the fluorescence spectrum after fluorescence probe is reacted with different material.
Fig. 5 is imaging applications of the fluorescence probe in living cells.
A figure: only add the cell photograph via bright field of 10 μM of probes；B figure: only plus the cell red channel fluorescence of 10 μM of probes shines
Piece；C figure: the superposition photo of A figure and B figure；D figure: the cell photograph via bright field of 10 μM of probes and 50 μM of copper ions is added；E figure: add
Enter the cell red channel fluorescence photo of 10 μM of probes and 50 μM of copper ions；F figure: the superposition photo of D figure and E figure.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention protects content to be not limited only to this.
Embodiment 1
The preparation of the fluorescence probe of copper ion in the detection cancer cell
Formula (II) compound represented a (1 mmol), hydrazine hydrate (2 mmol) and ethyl alcohol (5 mL) are added to 50 mL
In single-necked flask, heated 5 hours at 80 DEG C.It is cooled to room temperature, is added to the water, filter, drying.Obtained solid is subjected to column
Chromatograph (methylene chloride: methanol=10:1) purifying, obtain yellow product, in detection cancer cell as of the present invention copper from
The fluorescence probe of son, yield 73%.
The fluorescence probe of copper ion in above-mentioned detection cancer cell1H NMR spectra is shown in Fig. 1.
Embodiment 2
Fluorescence spectrum of the fluorescence probe under different copper ion concentrations
The present invention supplies copper ion using sulfuric acid in aqueous solution.The 5 μM of probes buffering for preparing 10 parts of 5mL in advance is molten
Liquid, contains 5% acetonitrile, and the copper ion concentration being added is 0 to 125 μM.Then fluorescence detection (λ is carried outEx=530 nm)；It calculates
Fluorescence intensity in each system；By the relationship of fluorescence intensity and copper ion concentration at 581 nm of analysis, the probe is assessed to copper
The response performance of ion (see Fig. 2 and Fig. 3).Fig. 2 shows the increase with copper ion concentration, and the fluorescence intensity of solution gradually increases
By force.Fig. 3 shows that the fluorescence intensity of solution and the concentration of copper ion are in preferable when copper ion concentration is within the scope of 0 ~ 40 μM
Linear relationship, can be used the * of formula Y=19.95 X+23.27 expression, wherein Y be 581nm place fluorescence intensity, X for copper from
The concentration of son.
Embodiment 3
Fluorescence probe reacted with different material after fluorescence spectrum
The 5 μM of probe buffer solutions (containing 5 % acetonitriles, pH=7.4) for preparing 10 parts of 5mL in advance, then respectively to this
Hcys, Na that 50 μ L concentration are 200 μM are sequentially added in system2S、Na2SO3、Cys、GSH、VC、Zn2+、Fe2+、Fe3+、Hg2+、
Mg2+、Co2+Etc. analyte of interest buffer solution.Then fluorescence detection (λ is carried outEx=530 nm)；Calculate fluorescence in each system
Intensity；The different material is assessed to the interference (see figure 4) of fluorescence probe solution.As seen from the figure, add in certain probe solution
Enter Hcys, Na2S、Na2SO3、Cys、GSH、VC、Zn2+、Fe2+、Fe3+、Hg2+、Mg2+、Co2+Etc. analyte of interest when, only copper from
Son can cause solution to generate significant fluorescence, and the fluorescence of solution does not change substantially when other small molecules are added, this expression
The probe only has response to copper ion, without the interference by other small molecules.
Embodiment 4
Imaging applications of the fluorescence probe in living cells
Mouse mastopathy cell (4T1 cell) is placed in culture medium (DMEM culture solution and 10% fetal calf serum), is placed in
Condition is 37 DEG C, 5% CO2With 20% O2Incubator in cultivate 24-48h.Fluorescence of the present invention is drawn with microsyringe
In (10 μM) culture mediums of the injection containing 4T1 cell of probe, 30 min are cultivated in continuation in the incubator.Be added 100 μM of copper from
Sub- solution continues to cultivate 30 min.Culture cell 3 times is rinsed with PBS (phosphate buffer solution) later, carries out fluorescence imaging.
As a result see Fig. 5.Find out from A, B and C figure, when addition probe, cell is substantially without fluorescence；From D, E and F figure
Find out, be added after 10 μM of probes and 50 μM of vulcanized sodium, cell generates apparent fluorescence, this indicates that the probe can be used for detecting
Copper ion in cancer cell.
Claims (2)
1. the cancer cell targeting fluorescent probe of copper ion in a kind of detection cancer cell, it is characterised in that: it contains biotin group
Rhodamine Derivatives, shown in the general formula of the chemical structure of the fluorescence probe such as formula (I):
Formula (I).
2. application of the cancer cell targeting fluorescent probe described in claim 1 in detection cancer cell in copper ion.
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Non-Patent Citations (5)
| Title |
|---|
| A Long-Wavelength Fluorescent Chemodosimeter Selective for Cu(Ⅱ)Ion in Water;Virginie Dujols et al.;《J.Am.Chem.Soc.》;19971231;全文 |
| A New Highly Selective,Ratiometric and Colorimetric Fluorescence Sensor for Cu2+ with a Remarkable Red Shift in Absorption and Emission Spectra Based on Internal Charge Transfer;Shyamaprosad Goswami et al.;《Organic Letters》;20100127;全文 |
| A new rhodamine-based single molecule multianalyte(Cu2+,Hg2+)sensor and its application in the biological system;Lina Wang et al.;《Dyes and Pigments》;20120823;全文 |
| Ratiometric CdSe/ZnS Quantum Dot Protein Sensor;Christina M.Tyrakowski and Preston T. Snee;《Analytical Chemistry》;20140120;全文 |
| 铜离子荧光探针的研究进展;向雨秘 等;《浙江化工》;20131231;第44卷(第2期);全文 |
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