CN104498579B - One kind is for Zn in cancer cell2+The fluorescence probe method of monitoring - Google Patents

Abstract

It is of the invention a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, is with double (aldehyde of 7 Hydroxycoumarin 8) contracting ethylenediamines as trace Zn²⁺Fluorescence probe, using probe and Zn²⁺Selectively formed complex can launch the characteristic of 457nm wavelength intense fluorescences, by probe and Zn²⁺Penetrate into respectively in active cancer cell, be allowed to and cytocompatibility.By probe to intracellular Zn²⁺After dyeing, detected with fluorescence inverted microscope and manifest clearly blue-fluorescence cell image, probe is used as Zn in live body cancer cell²⁺Fluorescence imaging reagent.The chemical structural formula of probe is：

Description

One kind is for Zn in cancer cell2+The fluorescence probe method of monitoring

Technical field

It is specifically a kind of for Zn in live body cancer cell the invention belongs to analytical chemistry field²⁺The fluorescence probe of detection Method.

Background technology

Cell is the base unit of vital movement, is the base unit that all organic life entities are constituted, and cell research is institute There is the starting point based on life science, the periodic phenomena such as its growth, aging will directly affect the vital movement of organism.One A little heavy metals and transition metal have very heavy because participating in the processes such as cell development, genetic transcription, neurotransmission The physiological action and function wanted, but when content exceedes its corresponding threshold value, they are to the growing of organism, physiological metabolism Even morphosis etc. can cause substantially damage.Therefore the influence of various ion pair organisms, very necessary one to be studied Step is exactly the online visualization real-time monitoring of ion in active somatic cell.Living cells imaging technique is weight in technical field of life science The research meanses wanted, combine with fixed cell research, can explain the various biological phenomenas in living cells.In recent years, with Continuing to develop for life science, people grind to the aspects such as intracellular active specy, cellular signal transduction and Apoptosis Study carefully more and more deep.

Induced Fluorescence Microscopy is a kind of method that can be realized and visualize real-time detection intracellular ion online, this kind of Method not only has the advantages that method is easy, sensitivity is high, selectivity is good and fast response time, mainly than other means Can be that active somatic cell is observed in the case where cell is not killed, maintain most complete most real information in cell, make it As the research meanses for being most appropriate for ion quantitative determination and qualitative analysis in biological cell.Meanwhile, fluorescence microscope can also The distribution in the cell of identified ion and dynamic change are determined by the fluorescence of ad-hoc location in observation of cell, biological point is carried out The cellular localization and dynamics research of son or subcellular structure.The Rhodamine Derivatives that Ghost etc. is reported are applied to live body Hg in HeLa cells²⁺Optical image technology, its selectively be better than general sensor, Hg is added in sensor solution²⁺ When can produce strong fluorescence, and during to adding finite concentration KI in the system, fluorescence signal disappears.Wang etc. reports one kind Salicylide phenyl hydrazones zinc ion fluorescent, only Zn²⁺Ion can cause significantly increasing for ligand fluorescence, and be applied to wing Zn in Guang cancer cell²⁺The fluorescence imaging analysis of ion, indicate application prospect of the probe in biochemistry system.Although report The optical pickocff molecule in road is more, and with selectivity higher, but due to water-soluble, cell-penetrating ability and cell toxicant The reasons such as property, can apply to the sensor molecule of tissue and cellular level heavy metal and transition metal ions optical imagery still compared with Few, it is small how to obtain cytotoxicity, can penetration cell wall or cell membrane, heavy metal and transition metal ions are selective Optical pickocff is one of Major Difficulties of research.

Zinc is only second to the transition metal of iron as people's in-vivo content, is risen during cell biology and cytotrophy Very important effect, gene expression, protein structure and function, cell metabolism, the synthesis of enzyme and neurotransmission and disease The content all with Zn-ef ficiency such as generation of disease is closely related.The assay method to zinc ion mainly has atomic absorption spectrum at present Method, AAS, ICP-AES, electrochemical process and fluorescent spectrometry etc., wherein, fluorescence Spectroscopic methodology quickly, receives much concern because being non-destructive with transmission signal the characteristics of sensitive.The main mesh of fluorescent optical sensor Mark be to appearing in the various tissues of human body in, including the zinc ion of brain, enteron aisle, pancreas and retina detection.To being at present Only, the fluorescent optical sensor to zinc ion has been successfully applied in the zinc ion fluorescence imaging of active somatic cell, hippocampal slices.Using spy The relevant report of pin detection zinc ion is many, but really is able to be applied to the few of the interior imaging of living cells.Document report A kind of probe with thiosemicarbazides and chalcone as Material synthesis, and use it for Zn in neuronal cell²⁺Imaging analysis.

The content of the invention

The Zn it is an object of the invention to study that a kind of energy is highly sensitive, in the high selection cancer cell to live body²⁺The fluorescence of monitoring Detecting probe method.By a kind of to Zn of inventor's synthesis²⁺Highly sensitive, high selectivity detection identification fluorescence probe, realizes to work Trace Zn in body cell²⁺Fluorescent staining and imaging.

It is of the present invention a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring be with compound it is double-(7- hydroxyls- Cumarin -8- aldehyde) contracting ethylenediamine is the intracellular trace Zn of monitoring²⁺Fluorescence probe, using probe and Zn²⁺It is selectively formed to match somebody with somebody Compound and launch the characteristic of intense fluorescence, probe, Zn²⁺Can penetrate into respectively in cancer cell living, and cytocompatibility, by probe with Intracellular Zn²⁺With reference to transmitting fluorescence, clearly blue-fluorescence cell image is detected with fluorescence inverted microscope, probe is used as to live Zn in body cancer cell²⁺Fluorescence imaging reagent.

It is above-mentioned a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring is to intracellular Zn with probe²⁺Contaminated Color, with the fluoroscopic image of microscopic cell, specific method is：

1) cell culture：Active prostate cancer（PC3）Cell is inoculated in containing 10% hyclone and dual anti-containing 1% through recovery Culture medium（RPMI 1640）In, it is 37 DEG C, 5% CO in temperature₂And cultivated in the incubator that saturated humidity is 100%, every Pass on 1 time within 2-3 days, the full cell of growth selection is cultivated in being inoculated in 12 orifice plates, density is 2 × 10⁴Individual/ml；

2) probe is hatched with cell：Cell immersion concentration is 10 μ twice by secondary daily fresh culture cleaning cell mol·L^-1Probe solution { 900+100 μm of olL of μ L culture mediums^-1N,N-dimethylformamide/the H of probe₂O（DMF/H₂O, v/ V, 1/4）The μ L of mixed solution 100 in, in 37 DEG C of 5% CO₂, saturated humidity 100% incubator in be incubated 30 min, suction out Nutrient solution containing probe, cell is cleaned with fresh culture three times, and bright field and details in a play not acted out on stage, but told through dialogues are observed and carried out under fluorescence inverted microscope Take pictures；

3) Zn²⁺To cell dyeing：Immersed again in the above-mentioned cell hatched through probe containing 50 μm of olL^-1 Zn²⁺It is molten Liquid（900+500 μm of olL of μ L culture mediums^-1 Zn²⁺The μ L of solution 100）, in 37 DEG C of 5% CO₂, saturated humidity 100% incubator 30 min of middle incubation, carry out intracellular probes to Zn²⁺Dyeing, suctions out and contains Zn²⁺The nutrient solution of solution, the cell after dyeing is used again Fresh culture is cleaned three times, and probe is observed under fluorescence inverted microscope to intracellular Zn²⁺Fluorescence imaging after dyeing, to thin Born of the same parents' image is taken pictures and obtains clearly cell outline image.

It is above-mentioned a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, with concentration 1 × 10^-5~1 × 10^-4 mol·L^-1In the range of Zn²⁺Live body PC3 cells are dyeed, Zn²⁺Can penetrate it is intracellular, cell be in round and smooth full shape； With concentration 1 × 10^-5~2 × 10^-5 mol·L^-1In the range of probe cell is dyeed, probe can penetrate cell Interior, cell is in round and smooth full shape；Probe, Zn²⁺There is good compatibility with cell, to cytotoxic, cell will not be made to wither Die；In above-mentioned concentration range, through probe solution and Zn²⁺The cell that solution was dyeed can be transferred through fluorescence inverted microscope observation To blue-fluorescence cell distribution image, dyeing is with selective aggregation ability.

It is above-mentioned a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, reagent used is to analyze pure or biochemical examination Agent, water is redistilled water or physiological saline；Live body PC3 cells are human body prostate gland cancer cell；Photographing device is inverted for fluorescence Microscope.

It is above-mentioned a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, the probe chemical structural formula is：

Molecular formula：C₂₂H₁₆N₂O₆

Molecular weight：404.10

Fusing point：More than 300 DEG C

Dissolubility：Chloroform, dimethyl sulfoxide, DMF etc. are slightly soluble in, insoluble in ethanol.

Spectral quality：In DMF/ H₂O（V/v, 1/4）In mixed solvent, 360nm wavelength is excited down, Zn²⁺Addition make spy Pin produces sharp fluorescent emissions at 457nm, and maximal ultraviolet absorption is produced in 360nm wavelength.

Above-mentioned one kind is used for Zn in cancer cell²⁺The fluorescence probe method of monitoring, as Zn in cell²⁺The fluorescence of detection Imaging agents pair-(7- hydroxyls-cumarin -8- aldehyde) contracting ethylenediamine, its synthetic method is with umbelliferone, six methines four Amine and ethylenediamine are raw material, are solvent in dichloromethane, ethanol, with trifluoroacetic acid as solvent and catalyst, through acetylation, first Acylated, condensation reaction and obtain.

Fluorescence probe used by the present invention meets as intracellular image probe（1）Good water solubility；（2） There is good heat endurance under physiological condition；（3）Good membrane penetrating, and be nontoxic to cell；（4）With Zn²⁺Have Selectivity, highly sensitive Fluorescence Enhancement, there is excitation wavelength and luminous yield higher more long.It is thin in live body using probe Intracellular and Zn²⁺Coloration, with fluorescence inverted microscope can clearly obtain transmitting blue-fluorescence cell image.Invent one Plant the Imaging-PAM of novel active somatic cell.

The structure of the fluorescence probe of present invention synthesis is characterized through NMR spectrum, mass spectrum and infrared spectrum.Knot Structure characterize data is listed in table 1.

The structural characterization data of the fluorescence probe of table 1

Brief description of the drawings

The fluorescence spectrum that Fig. 1 probes are acted on different metal ions.

Concentration is 1.00 × 10^-5 mol·L^-1Probe DMF/H₂O（V/v, 1/4）Solution, be not added with respectively metal ion or Add 2.00 × 10^-4 mol·L^-1Configuration metal ions Zn²⁺, Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Hg²⁺, Co²⁺, Ni²⁺, Cu^{2 +}, Cd²⁺, Pb²⁺, Ag⁺, Al³⁺, Fe³⁺, Cr³⁺Fluorescence spectrum afterwards.Zn²⁺Addition fluorescence of the probe at 457nm is significantly increased By force.And the addition of other above-mentioned Experiment Metal ions will not change the fluorescence intensity of probe.The excitation wavelength of test is 360nm, Launch wavelength is 457nm.

Fig. 2 coexistent metallic ions are to probe in detecting Zn²⁺Fluorescence intensity influence.

It is 1.00 × 10 in concentration^-5 mol·L^-1Probe DMF/H₂O（V/v, 1/4）In solution, addition 2.00 × 10^-4 mol·L^-1Zn²⁺Afterwards, it is the fluorescence intensity level at 457nm to determine probe in wavelength.Again respectively to probe-Zn²⁺Solution Middle addition concentration is Zn²⁺10 times of other metal ions of amount of concentration：Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Ag⁺, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Al³⁺, Cr³⁺, Fe³⁺Fluorescence intensity level afterwards.White bars are represented in probe-Zn²⁺Solution is distinguished Fluorescence intensity after adding above-mentioned other metal ions to coexist at 457nm.Probe in detecting Zn²⁺Fluorescence intensity except slightly receive Co^{2 +}, Cr³⁺, Ni²⁺, Cu²⁺And Hg²⁺Influence outside, other Experiment Metal ions coexist and will not cause probe s1-Zn²⁺Solution fluorescence is strong The change of degree.Show probe in detecting Zn²⁺The shadow that is not coexisted by other metal ions in addition to above-mentioned metal ion of fluorescence intensity Ring.The excitation wavelength of detection is 360nm, and fluorescence emission wavelengths are 457nm.

The Zn of Fig. 3 various concentrations²⁺To the fluorescence titration spectrogram of probe.

It is 1.00 × 10 in concentration^-5 mol·L^-1The DMF/H of probe₂O（V/v, 1/4）Concentration is separately added into solution is 1.00×10^-5~2.00 × 10^-4 mol·L^-1Zn²⁺To in probe solution, with Zn²⁺Addition, fluorescence intensity gradually increases Greatly.Show probe in detecting Zn²⁺Fluorescence intensity and Zn²⁺The wired sexual intercourse of concentration.The excitation wavelength of test is 360nm, fluorescence hair The a length of 457nm of ejected wave.

Fig. 4-1 is the intracellular only probes of PC3（Concentration is 10 μm of olL^-1）Or only Zn²⁺Solution（Concentration is 50 μ mol·L^-1）When bright field microphoto, cell be in full state, it was demonstrated that the probe in test concentrations to PC3 cells without poison Property；

Fig. 4-2 is the intracellular only probes of PC3（Concentration is 10 μm of olL^-1）Or only Zn²⁺Solution（Concentration is 50 μ mol·L^-1）When details in a play not acted out on stage, but told through dialogues fluorescence micrograph, show unstressed configuration phenomenon；

Fig. 4-3 is that PC3 is intracellular while there is probe（Concentration is 10 μm of olL^-1）And Zn²⁺（Concentration is 50 μm of olL^-1）In the presence of details in a play not acted out on stage, but told through dialogues fluorescence micrograph, observe the thin of clearly blue-fluorescence with the fluorescence inverted microscope of IX-71 types Born of the same parents are distributed, it was demonstrated that probe and Zn²⁺Ion realizes dyeing in the cell, and cell dyeing fluorescence imaging is presented.

Fig. 5-1 is the intracellular only probes of PC3（Concentration is 10 μm of olL^-1）Or only Zn²⁺Solution（Concentration is 50 μ mol·L^-1）When bright field microphoto, cell be in full state, it was demonstrated that the probe in experimental concentration to PC3 cells without poison Property；

Fig. 5-2 is the intracellular only probes of PC3（Concentration is 10 μm of olL^-1）Or only Zn²⁺Solution（Concentration is 50 μ mol·L^-1）When details in a play not acted out on stage, but told through dialogues fluorescence micrograph, show unstressed configuration phenomenon；

Fig. 5-3 is that PC3 is intracellular while there is probe（Concentration is 10 μm of olL^-1）And Zn²⁺（Concentration is 50 μm of olL^-1）In the presence of details in a play not acted out on stage, but told through dialogues fluorescence micrograph, observe clearly blue-fluorescence cell point with the fluorescence inverted microscope of Ti models Cloth, it was demonstrated that probe and Zn²⁺Ion realizes dyeing in the cell, and cell dyeing fluorescence imaging is presented.

Specific embodiment

Embodiment one：The compound method of each solution, reagent in the present invention

（1）The preparation of probe solution：The probe of 40.4mg is weighed, is dissolved with DMF, be configured to 100mL solution, concentration is 1.00×10^-3mol·L^-1；As needed with physiological saline stepwise dilution to suitable concentration.

（2）Zn²⁺Standard liquid：Weigh 59.5mg and analyze pure Zn (NO₃)₂, with physiological saline solution, it is configured to 100mL molten Liquid, concentration is 2.00 × 10^-3 mol·L^-1；As needed with physiological saline stepwise dilution to suitable concentration.

（3）The preparation of other coexisting ion solution：The nitrate or hydrochloride of analytically pure various metals are taken, secondary steaming is used Distilled water dissolves, and it is 2.00 × 10 to be configured to concentration^-3 mol·L^-1The second distillation aqueous solution.

（4）75% ethanol solution：Absolute ethyl alcohol 75mL adds distilled water to 100mL, mixes, and room temperature preservation is standby.

（5）PBS（D-hanks balanced salt solutions）：0.4gKCl、0.06g KH₂PO₄、8.0gNaCl、 1.0g C₆H₁₂O₆、0.35gNaHCO₃、0.152g Na₂HPO₄·12H₂O, 100,000 IU are dual anti-, adjustment pH be 7.2~7.4, go from Sub- water is settled to 1000mL, pin type filter（0.22um import miillpore filters）Filtration sterilization, dispenses standby.

（6）10000 units（IU）The dual anti-solution of/mL：By Benzylpenicillin sodium salt（800000 units）It is dissolved in 40mL D-hanks solution, It is made into 20,000 units of final concentration/mL；By streptomycin sulphate（1600000 units）It is dissolved in 80mL D-hanks solution, is made into final concentration 20000 units/mL.Isometric Benzylpenicillin sodium salt solution and streptomycin sulfate solution mixing is taken respectively, obtains Benzylpenicillin sodium salt and sulfuric acid The final concentration of streptomysin is the solution of 10,000 units/mL；Pin type filter（0.22um import miillpore filters）Filtration sterilization, packing 1mL/ branch, -20 DEG C save backup.

（7）0.25% trypsase：0.25g trypsase is weighed, is dissolved in the D-hanks liquid of 100mL, pin type filter （0.22um import miillpore filters）Filtration sterilization, dispenses 1mL/ branch, and -20 DEG C save backup.

（8）0.02% ethylenediamine tetra-acetic acid（EDTA）：By 0.02gEDTA, it is dissolved in the D-hanks liquid of 100mL, pin type Filter（0.22um import miillpore filters）Filtration sterilization, dispenses 1mL/ branch, and -20 DEG C save backup.

（9）Nutrient solution：Hyclone, the 90mL culture mediums that 10mL has been inactivated are measured with Sterile pipette（Modified form RPMI- 1640）And the dual anti-liquid of 1mL is mixed in the sterile culture flask of 100mL, 2~8 DEG C save backup.

Fluorescence microscope used of the invention is respectively：IX-71 type fluorescence inverted phase contrast microscopes, Japanese Olympus companies； The fluorescence inverted microscope of Ti models, Japanese Nikon company；Cary Eclipse sepectrophotofluorometers, U.S. VARIAN is public Department；The water storage type CO of ThermoFisher 8000₂Cell culture incubator；AR1530/C electronic balances；25cm²Tissue Culture Flask, the U.S. Corning companies vertical pressure steam sterilizer（LS-B75）；DHG-9230A electric heating constant-temperature blowing drying boxes, the upper grand reality of Nereid Test equipment Co., Ltd.

Embodiment two：Probe is to Zn²⁺The selective fluoroscopic examination of ion

The DMF storing solutions of probe are added in 10.0 mL volumetric flasks（1.00×10^-4 mol·L^-1, 1mL）, metal from Sub- Zn²⁺（2.00×10^-3 mol·L^-1, 1 mL）, with DMF/H₂O（V/v, 1/4）Solution is diluted to scale, shakes up, and moves into 1cm Quartz colorimetric utensil carry out fluorescence spectrometry.

Setting fluorescence exciting wavelength is 360nm, and the probe of about 3ml is added in the cuvette of 1cm（1.00×10^-5 mol·L^-1）DMF/H₂O（V/v, 1/4）Solution carries out fluorescence spectrum test, probe unstressed configuration transmitting at 457nm wavelength. Add Zn²⁺（2.00×10^-4 mol·L^-1）Afterwards, probe solution transmitting intense blue fluorescence.Under the same terms, in probe solution In be separately added into Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Sr²⁺, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Pb²⁺, Cd²⁺, Al³⁺, Cr³⁺, Fe³⁺After metal ion, the fluorescence spectrum and intensity of probe will not be changed.Probe is only to Zn²⁺Selective fluoroscopic examination response Can, selection wavelength is that the fluorescence intensity level at 457nm is measured（Accompanying drawing 1）.

Under the conditions of above-mentioned fluorescence method same test, probe in detecting Zn²⁺Fluorescent value at 457nm wavelength is in above-mentioned metal Ion is present in probe-Zn respectively as coexisting ion²⁺In solution, when coexistent metallic ion concentration is Zn²⁺10 times of ion concentration During amount, except slightly by Co²⁺, Cr³⁺, Ni²⁺, Cu²⁺And Hg²⁺Influence outside, probe in detecting Zn²⁺Fluorescence intensity by other metals from Son coexists influence（Accompanying drawing 2）.

Embodiment three：The fluorescent microscopic imaging of live body PC3 cells

（1）Recovery cell

PC3 cells are taken out from -80 DEG C of refrigerators, is placed in 37 DEG C of water and is quickly rocked cell cryopreservation tube, in 1-2 minutes Thaw completely, in aseptic operating platform, sucked in centrifuge tube, plus about 11ml nutrient solutions（Containing 10% hyclone, 1% is dual anti- Liquid）It is mixed, and this cell suspension is removed into supernatant liquor, by the thin of bottom precipitation in 5min is centrifuged on 1000r/min centrifuges Born of the same parents add nutrient solution featheriness to break up to be mixed and be transferred in blake bottle, and nutrient solution volume is placed into 37 in 5-7mL in making blake bottle DEG C, containing 5% CO₂Incubator in cultivated.

（2）Observation → passage → fishplate bar

Change a nutrient solution daily, and examine under a microscope cell growth status, until PC3 cell attachments cover with In whole culture bottle wall, you can passage, old nutrient solution is outwelled in aseptic operating platform, add the EDTA liquid of 1mL to invade cell 30s After outwell, the trypsin solution for adding 1mL is digested, and after examining under a microscope until cell size is rounded, pats training Foster bottle makes cell detachment and adds appropriate nutrient solution to prevent digestion immediately, it is divided into two and is incubated in 2 blake bottles, to be passed Cell attachment after generation is paved with when in whole culture bottle wall, identical with passage operation, is made after addition EDTA and trypsase thin Born of the same parents' digestion comes off and adds the nutrient solution of 3mL to prevent digestion, preparation from being inoculated in 12 orifice plates immediately.Added in each orifice plate The cell liquid for having prevented digestion of 200mL, then add appropriate new nutrient solution that orifice plate is inserted into 37 DEG C after being mixed, containing 5% CO₂Training Support and cultivated in case.

（3）Cell dyeing

Cell growth condition in next day observation orifice plate, treats that cell attachment is generated, and discards old nutrient solution, is washed with new nutrient solution Wash 2 times, it is standby.

A groups：1 × 10 is separately added into toward 3 holes of cell culture orifice plate^-5 mol·L^-1 Zn²⁺Solution（1×10^-5 mol·L^-1 Zn²⁺Prepare：Nutrient solution+the 100mL of 900mL is with the 10 of normal saline^-4 mol·L^-1 Zn²⁺Storing solution）, 5 ×10^-5 mol·L^-1 Zn²⁺Solution（Nutrient solution+the 100uL of 900uL is with the 5 × 10 of normal saline^-4 mol·L^-1 Zn²⁺ Storing solution）, 10 × 10^-5 mol·L^-1 Zn²⁺Solution（Nutrient solution+the 100mL of 900mL is with the 10 of normal saline^-3 mol· L^-1 Zn²⁺Storing solution）, at 37 DEG C, containing 5% CO₂Incubator in be incubated 30 minutes after be placed under fluorescence inverted microscope and carry out Light field and details in a play not acted out on stage, but told through dialogues are taken pictures, cell display unstressed configuration.（Accompanying drawing 4-1,4-2,5-1,5-2）.

B groups：1 × 10 is added in toward 3 holes of Tissue Culture Plate^-5 mol·L^-1Probe s1 liquid (1 × 10^-5 mol·L^-1 Probe s1：Nutrient solution+100mL DMF/ (the DMF+H of 900 mL₂O)（V/v, 20%）The 10 of mixed solvent preparation^-4 mol·L^-1 Storing solution）；Again toward addition 2 × 10 in another 3 holes of Tissue Culture Plate^-5 mol·L^-1Probe s1, at 37 DEG C, containing 5% CO₂Incubator in be incubated 30 minutes, washed twice with fresh modified form RPMI-1640 culture mediums, be placed in fluorescence be inverted it is micro- Light field being carried out under mirror and dark-field imaging being taken pictures, cell has no fluorescence display.（Accompanying drawing 4-1,4-2,5-1,5-2）.

C groups：The PC3 cells in 6 holes treated by step B, it is 1 × 10 to correspond immersion concentration respectively^-5 mol·L^-1、5×10^-5 mol·L^-1、10×10^-5 mol·L^-1 Zn²⁺In solution, at 37 DEG C, containing 5% CO₂Incubator After interior incubation 30 minutes, nutrient solution is suctioned out, gained cell is washed twice with fresh modified form RPMI-1640 culture mediums, is placed in glimmering Details in a play not acted out on stage, but told through dialogues fluorescence imaging is carried out under light inverted microscope to take pictures, and is as a result shown, in Zn²⁺Concentration is 1 × 10^-5~1 × 10^-4 mol· L^-1Scope, concentration and probe concentration is 1 × 10^-5~2 × 10^-5 mol·L^-1In the range of cell is dyeed after, can pass through fluorescence Inverted microscope observes blue-fluorescence cell image.Through 1 × 10^-5 mol·L^-1Probe solution and 5 × 10^-5 mol·L^-1 Zn²⁺The cell that solution was dyeed is presented clearly blue-fluorescence image（Accompanying drawing 4-3,5-3）.Illustrate that probe is intracellular to PC3 Zn²⁺Dye successfully.

Claims (6)

1. a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, it is characterized in that with compound pair-(7- hydroxyls-tonka-bean Element -8- aldehyde) contracting ethylenediamine is the intracellular trace Zn of monitoring²⁺Fluorescence probe, using probe and Zn²⁺Selectively formed complex And launch the characteristic of intense fluorescence, probe, Zn²⁺Can penetrate into respectively in cancer cell living, and cytocompatibility, by probe and cell Interior Zn²⁺With reference to transmitting fluorescence, clearly blue-fluorescence cell image is detected with fluorescence inverted microscope, probe is used as live body cancer Zn in cell²⁺Fluorescence imaging reagent.

2. according to claim 1 a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, it is characterized in that using probe To intracellular Zn²⁺Dyeed, with the fluoroscopic image of microscopic cell, specific method is：

1) cell culture：PC3 activity prostate gland cancer cells, abbreviation PC3 cells are inoculated in containing 10% hyclone and contain through recovery It is 37 DEG C, 5%CO in temperature in the culture medium of 1% dual anti-RPMI1640₂And cultivated in the incubator that saturated humidity is 100%, Passed on 1 time every 2-3 days, the full cell of growth selection is cultivated in being inoculated in 12 orifice plates, density is 2 × 10⁴Individual/ml；

2) probe is hatched with cell：Cell immersion concentration is 10 μm of olL twice by secondary daily fresh culture cleaning cell^-1 In probe solution, its composition is by DMF/H that the μ L volume ratios of 900 μ L culture mediums+100 are 1/4₂What O was prepared Concentration is 100 μm of olL^-1Probe is mixed, in 37 DEG C of 5%CO₂, saturated humidity 100% incubator in be incubated 30min, suctions out the nutrient solution containing probe, cell is cleaned with fresh culture three times, is observed under fluorescence inverted microscope and carried out Bright field and details in a play not acted out on stage, but told through dialogues are taken pictures；

3)Zn²⁺To cell dyeing：Immersed again in the above-mentioned cell hatched through probe containing 50 μm of olL^-1Zn²⁺Solution, its group Into by the μ L concentration of 900 μ L culture mediums+100 be 500 μm of olL^-1Zn²⁺Solution is mixed, in 37 DEG C of 5%CO₂, saturation it is wet 30min is incubated in the incubator of degree 100%, intracellular probes is carried out to Zn²⁺Dyeing, suctions out and contains Zn²⁺The nutrient solution of solution, dye Cell after color is cleaned three times with fresh culture again, and probe is observed under fluorescence inverted microscope to intracellular Zn²⁺After dyeing Fluorescence imaging, acquisition clearly cell outline image of being taken pictures to cell image.

3. according to claim 2 a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, it is characterized in that using concentration 1 × 10^-5~1 × 10^-4mol·L^-1In the range of Zn²⁺PC3 cells are dyeed, Zn²⁺Can penetrate intracellular, cell In round and smooth full shape；With concentration 1 × 10^-5~2 × 10^-5mol·L^-1In the range of probe cell is dyeed, probe energy Penetrate into it is intracellular, cell be in round and smooth full shape；Probe, Zn²⁺There is good compatibility with cell, to cytotoxic, no Apoptosis can be made；In above-mentioned concentration range, through probe solution and Zn²⁺The cell that solution was dyeed can be transferred through fluorescence inversion Microscope observes blue-fluorescence cell distribution image, and dyeing has selective aggregation ability.

4. according to claim 2 a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, it is characterized in that used To analyze pure or biochemical reagents, water is redistilled water or physiological saline to reagent；PC3 cells are human body prostate gland cancer cell；Clap It is fluorescence inverted microscope according to equipment.

5. according to claim 1 a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, it is characterized in that the spy Pin chemical structural formula is：

Molecular formula：C₂₂H₁₆N₂O₆

Molecular weight：404.10

Fusing point：More than 300 DEG C

Dissolubility：Chloroform, dimethyl sulfoxide, DMF are slightly soluble in, insoluble in ethanol

Spectral quality：In the DMF/H that volume ratio is 1/4₂In O mixed solvents, 360nm wavelength is excited down, Zn²⁺Addition make probe Sharp fluorescent emissions are produced at 457nm, maximal ultraviolet absorption is produced in 360nm wavelength.

6. according to claim 1 a kind of for Zn in cancer cell²⁺The fluorescence probe method of monitoring, it is characterized in that as thin Zn in born of the same parents²⁺Fluorescence imaging reagent pair-(7- hydroxyls-cumarin -8- aldehyde) contracting ethylenediamine of detection, its synthetic method is with 7- hydroxyls Cumarin, hexamethylenetetramine and ethylenediamine are raw material, are solvent in dichloromethane, ethanol, so that trifluoroacetic acid is as solvent and urges Agent, through acetylation, formylated, condensation reaction and obtain.