CN104498579A - Fluorescent probe method for monitoring Zn<2+> in cancer cells - Google Patents
Fluorescent probe method for monitoring Zn<2+> in cancer cells Download PDFInfo
- Publication number
- CN104498579A CN104498579A CN201410565454.3A CN201410565454A CN104498579A CN 104498579 A CN104498579 A CN 104498579A CN 201410565454 A CN201410565454 A CN 201410565454A CN 104498579 A CN104498579 A CN 104498579A
- Authority
- CN
- China
- Prior art keywords
- cell
- probe
- fluorescence
- solution
- cancer cells
- 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
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a fluorescent probe method for monitoring Zn<2+> in cancer cells. The fluorescent probe method utilizes bis-(7-hydroxy-coumarin-8-aldehyde)ethylenediamine as a fluorescent probe of a trace amount of Zn<2+>. Through the selectively-formed probe-Zn<2+> complex characteristic of emitting 457nm wavelength strong fluorescence, the probe and Zn<2+> are respectively permeated into active cancer cells so that fusion with the cells is realized. After Zn<2+> in cells is dyed by the probe, a distinct blue fluorescent cell image is detected and showed by an inverted fluorescence microscope. The probe is used as a fluorescent imaging reagent of Zn<2+> in active cancer cells. The probe has a chemical structural formula shown in the following description.
Description
Technical field
The invention belongs to analytical chemistry field, specifically a kind of for Zn in live body cancer cells
2+the fluorescent probe method detected.
Background technology
Cell is the fundamental unit of vital movement, and be the base unit that all organic life entities are formed, cell research is all starting points based on life science, and its periodic phenomena such as growth, aging will directly affect the vital movement of organism.Some heavy metals and transition metal have very important physiological action and function because participating in the processes such as cell development, genetic transcription, neurotransmission, but when content exceedes its corresponding threshold value, they to the growing of organism, physiological metabolism even morphological structure etc. can cause obvious damage.Therefore will study the impact of various ion pair organism, a very necessary step is exactly the online visualization real-time monitoring of ion in active somatic cell.Viable cell imaging technique is research means important in technical field of life science, combines with fixed cell research, can explain the multiple biological phenomena in viable cell.In recent years, along with the development of life science, the research of people to aspects such as intracellular active specy, cell signaling and apoptosis is more and more deep.
Induced Fluorescence Microscopy is a kind of method that can be implemented in the visual real-time detection intracellular ion of line, this kind of method not only has that method is easy, highly sensitive, selectivity is good and the advantage such as fast response time than other means, more importantly can observe active somatic cell when not killing cell, maintain the most real the most complete information in cell, become the most applicable research means carrying out ion quantitative assay and qualitative analysis in biomass cells.Meanwhile, fluorescent microscope is also determined to be identified ion in intracellular distribution and dynamic change by the fluorescence of specific position in observation of cell, carries out cellular localization and the dynamics research of biomolecules or subcellular structure.The Rhodamine Derivatives that Ghost etc. report is applied to Hg in live body HeLa cell
2+optical image technology, its selectivity is better than general sensor, when adding Hg in sensor solution
2+time can produce strong fluorescence, and when adding finite concentration KI in this system, fluorescent signal disappears.Wang etc. report a kind of salicylic aldehyde phenyl hydrazones zinc ion fluorescent, only have Zn
2+ion can cause the remarkable enhancing of ligand fluorescence, and is applied to Zn in transitional cell bladder carcinoma cell line
2+the fluorescence imaging analysis of ion, indicates the application prospect of this probe in biological chemistry system.Although the optical pickocff molecule of report is more, and there is higher selectivity, but due to water-soluble, the reason such as cell-penetrating ability and cytotoxicity, the sensor molecule that can be applied to tissue and cell levels heavy metal and transition metal ion optical imagery is still less, how to obtain cytotoxicity little, energy penetration cell wall or cytolemma, it is one of Major Difficulties of research that heavy metal and transition metal ion have optionally optical pickocff.
Zinc is only second to the transition metal of iron as people's in-vivo content, in cytobiology and cytotrophy process, play very important effect, genetic expression, protein structure and function, cellular metabolism, the synthesis of enzyme and the generation of neurotransmission and disease etc. are all closely related with the content of zinc element.At present atomic absorption spectrometry, spectrophotometry, inductively coupled plasma atomic emission spectrometry, electrochemical process and fluorescent spectrometry etc. are mainly contained to the measuring method of zine ion, wherein, fluorescent spectrometry transmits as nondestructive because having, and fast, responsive feature and receiving much concern.The major objective of fluorescent optical sensor is to appearing in the various tissue of human body, comprises the detection of brain, enteron aisle, pancreas and amphiblestroid zine ion.Up to the present, to the fluorescent optical sensor of zine ion successful Application at the zine ion fluorescence imaging of active somatic cell, hippocampal slices.Utilize the relevant report of probe in detecting zine ion existing many, but really can be applied to the few of imaging in viable cell.The probe that bibliographical information is a kind of is Material synthesis with thiosemicarbazide and cinnamophenone, and use it for Zn in neuronal cell
2+imaging analysis.
Summary of the invention
The object of the invention is to study a kind of energy highly sensitive, high select to Zn in live body cancer cells
2+the fluorescent probe method of monitoring.The one of being synthesized by contriver is to Zn
2+highly sensitive, highly selective detects the fluorescent probe identified, realizes trace Zn in active somatic cell
2+fluorescent dye and imaging.
One of the present invention is used for Zn in cancer cells
2+the fluorescent probe method of monitoring be with compound two-(7-hydroxyl-tonka bean camphor-8-aldehyde) contracting quadrol is for trace Zn in monitoring cell
2+fluorescent probe, utilize probe and Zn
2+selectivity forms title complex and launches the characteristic of intense fluorescence, probe, Zn
2+can penetrate into respectively in cancer cells alive, with cytocompatibility, by Zn in probe and cell
2+in conjunction with emitting fluorescence, detect blue-fluorescence cell image clearly with fluorescence inverted microscope, probe is used as Zn in live body cancer cells
2+fluorescence imaging reagent.
Above-mentioned one is used for Zn in cancer cells
2+the fluorescent probe method of monitoring is to Zn in cell with probe
2+dye, with the fluoroscopic image of microscopic cell, concrete grammar is:
1) cell cultures: active prostate cancer (PC3) cell is inoculated in containing 10% foetal calf serum with containing in 1% dual anti-substratum (RPMI 1640) through recovery, is 37 DEG C in temperature, 5% CO
2and saturated humidity is cultivate in the incubator of 100%, goes down to posterity 1 time every 2-3 days, the full cell of growth selection is inoculated in 12 orifice plates to be cultivated, and density is 2 × 10
4individual/ml;
2) probe and cell are hatched: secondary daily fresh culture cleans cell twice, and cell being immersed concentration is 10 μm of olL
-1probe solution { 900 μ L substratum+100 μm of olL
-1dMF/the H of probe
2o(DMF/H
2o, v/v, 1/4) in mixing solutions 100 μ L}, at 5% CO of 37 DEG C
2, saturated humidity 100% incubator in hatch 30 min, sucking-off, containing the nutrient solution of probe, with fresh culture cleaning cell three times, is observed under fluorescence inverted microscope and carries out bright field and details in a play not acted out on stage, but told through dialogues is taken pictures;
3) Zn
2+to cell dyeing: immerse again containing 50 μm of olL in above-mentioned cell of hatching through probe
-1zn
2+solution (900 μ L substratum+500 μm of olL
-1zn
2+solution 100 μ L), at 5% CO of 37 DEG C
2, saturated humidity 100% incubator in hatch 30 min, carry out intracellular probes to Zn
2+dyeing, sucking-off is containing Zn
2+the nutrient solution of solution, the cell after dyeing cleans three times with fresh culture again, observes probe to Zn in cell under fluorescence inverted microscope
2+fluorescence imaging after dyeing, takes pictures to cell image and obtains cell outline image clearly.
Above-mentioned one is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, by concentration 1 × 10
-5~ 1 × 10
-4molL
-1zn in scope
2+live body PC3 cell is dyeed, Zn
2+can penetrate in cell, cell is round and smooth full shape; By concentration 1 × 10
-5~ 2 × 10
-5molL
-1probe in scope dyes to cell, and probe can penetrate in cell, and cell is round and smooth full shape; Probe, Zn
2+with cell, there is good consistency, to cytotoxic, can not apoptosis be made; In above-mentioned concentration range, through probe solution and Zn
2+the cell that solution-dyed is crossed can observe blue-fluorescence cell distribution image by fluorescence inverted microscope, and dyeing has selective aggregation ability.
Above-mentioned one is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, reagent used is analytical pure or biochemical reagents, and water is redistilled water or physiological saline; Live body PC3 cell is human body prostate cancer cell; Photographing device is fluorescence inverted microscope.
Above-mentioned one is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, described probe chemical structural formula is:
Molecular formula: C
22h
16n
2o
6
Molecular weight: 404.10
Fusing point: be greater than 300 DEG C
Solvability: be slightly soluble in chloroform, methyl-sulphoxide, DMF etc., be insoluble to ethanol.
Spectral quality: at DMF/ H
2o(v/v, 1/4), in mixed solvent, 360nm wavelength excites down, Zn
2+add make probe 457nm place produce sharp fluorescent emissions, 360nm wavelength produce maximal ultraviolet absorption.
Above-mentioned one is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, as Zn in cell
2+the fluorescence imaging reagent detected is two-(7-hydroxyl-tonka bean camphor-8-aldehyde) contracting quadrol; its synthetic method is for raw material with umbelliferone, hexamethylenetetramine and quadrol; be solvent at methylene dichloride, ethanol; take trifluoroacetic acid as solvent and catalyzer, through acetylize, formylation, condensation reaction and obtaining.
The present invention's fluorescent probe used, as intracellular image probe, meets (1) good water-soluble; (2) there is good thermostability in physiological conditions; (3) good membrane penetrating, and be nontoxic to cell; And Zn (4)
2+selective, high-sensitive Fluorescence Enhancement, has longer excitation wavelength and higher luminous productive rate.Utilize probe in active somatic cell and Zn
2+dyeing, clearly can obtain with fluorescence inverted microscope and launch the cell image of blue-fluorescence.Invent a kind of Imaging-PAM of active somatic cell of novelty.
The structure of the fluorescent probe of the present invention's synthesis characterizes through NMR (Nuclear Magnetic Resonance) spectrum, mass spectrum and infrared spectra.Structural characterization data list in table 1.
The structural characterization data of table 1 fluorescent probe
Accompanying drawing explanation
Fig. 1. the fluorescence spectrum of probe and different metal ionization.
Concentration is 1.00 × 10
-5molL
-1probe DMF/H
2o(v/v, 1/4) solution, do not add metal ion respectively or add 2.00 × 10
-4molL
-1configuration metal ions Zn
2+, Li
+, Na
+, K
+, Mg
2+, Ca
2+, Ba
2+, Sr
2+, Hg
2+, Co
2+, Ni
2+, Cu
2+, Cd
2+, Pb
2+, Ag
+, Al
3+, Fe
3+, Cr
3+after fluorescence spectrum.Zn
2+add probe significantly strengthened at the fluorescence at 457nm place.And other above-mentioned Experiment Metal ions add the fluorescence intensity that can not change probe.The excitation wavelength of test is 360nm, and emission wavelength is 457nm.
Fig. 2 coexistent metallic ion is to probe in detecting Zn
2+fluorescence intensity impact.
Be 1.00 × 10 in concentration
-5molL
-1the DMF/H of probe
2o(v/v, 1/4), in solution, 2.00 × 10 are added
-4molL
-1zn
2+after, measure the fluorescence intensity level that probe is 457nm place at wavelength.Again respectively to probe
-zn
2+adding concentration in solution is Zn
2+other metal ions of concentration 10 times amount: Li
+, Na
+, K
+, Mg
2+, Ca
2+, Ba
2+, Sr
2+, Ag
+, Hg
2+, Co
2+, Ni
2+, Cu
2+, Cd
2+, Pb
2+, Al
3+, Cr
3+, Fe
3+after fluorescence intensity level.White bars represents at probe
-zn
2+solution add respectively other metal ions above-mentioned coexist after fluorescence intensity at 457nm place.Probe in detecting Zn
2+fluorescence intensity except slightly by Co
2+, Cr
3+, Ni
2+, Cu
2+and Hg
2+impact outside, other Experiment Metal ion coexist can not cause probe
s1-Zn
2+the change of solution fluorescence intensity.Show probe in detecting Zn
2+fluorescence intensity by the impact that other metal ions except above-mentioned metal ion coexist.The excitation wavelength detected is 360nm, and fluorescence emission wavelengths is 457nm.
The Zn of Fig. 3 different concns
2+to the fluorometric titration spectrogram of probe.
Be 1.00 × 10 in concentration
-5molL
-1the DMF/H of probe
2o(v/v, 1/4) adding concentration respectively in solution is 1.00 × 10
-5~ 2.00 × 10
-4molL
-1zn
2+to in probe solution, along with Zn
2+add, fluorescence intensity increases gradually.Show probe in detecting Zn
2+fluorescence intensity and Zn
2+the linear relation of concentration.The excitation wavelength of test is 360nm, and fluorescence emission wavelengths is 457nm.
In order to only have probe in PC3 cell, (concentration is 10 μm of olL to Fig. 4-1
-1) or only have Zn
2+(concentration is 50 μm of olL to solution
-1) time bright field Photomicrograph, cell is full state, proves that this probe does not have toxicity to PC3 cell in test concentrations;
In order to only have probe in PC3 cell, (concentration is 10 μm of olL to Fig. 4-2
-1) or only have Zn
2+(concentration is 50 μm of olL to solution
-1) time details in a play not acted out on stage, but told through dialogues fluorescence micrograph, display unstressed configuration phenomenon;
In order to have probe in PC3 cell simultaneously, (concentration is 10 μm of olL to Fig. 4-3
-1) and Zn
2+(concentration is 50 μm of olL
-1) details in a play not acted out on stage, but told through dialogues fluorescence micrograph when existing, observe the cell distribution of blue-fluorescence clearly with the fluorescence inverted microscope of IX-71 type, prove probe and Zn
2+ion achieves dyeing in cell, presents cell dyeing fluorescence imaging.
In order to only have probe in PC3 cell, (concentration is 10 μm of olL to Fig. 5-1
-1) or only have Zn
2+(concentration is 50 μm of olL to solution
-1) time bright field Photomicrograph, cell is full state, proves that this probe does not have toxicity to PC3 cell in experimental concentration;
In order to only have probe in PC3 cell, (concentration is 10 μm of olL to Fig. 5-2
-1) or only have Zn
2+(concentration is 50 μm of olL to solution
-1) time details in a play not acted out on stage, but told through dialogues fluorescence micrograph, display unstressed configuration phenomenon;
In order to have probe in PC3 cell simultaneously, (concentration is 10 μm of olL to Fig. 5-3
-1) and Zn
2+(concentration is 50 μm of olL
-1) details in a play not acted out on stage, but told through dialogues fluorescence micrograph when existing, observe blue-fluorescence cell distribution clearly with the fluorescence inverted microscope of Ti model, prove probe and Zn
2+ion achieves dyeing in cell, presents cell dyeing fluorescence imaging.
Embodiment
Embodiment one: the compound method of each solution, reagent in the present invention
(1) preparation of probe solution: the probe taking 40.4mg, dissolve with DMF, be mixed with 100mL solution, concentration is 1.00 × 10
-3molL
-1; Use physiological saline stepwise dilution to suitable concentration as required.
(2) Zn
2+standardized solution: take 59.5mg analytical pure Zn (NO
3)
2, with physiological saline solution, be mixed with 100mL solution, concentration is 2.00 × 10
-3molL
-1; Use physiological saline stepwise dilution to suitable concentration as required.
(3) preparation of other coexisting ion solution: nitrate or the hydrochloride of getting analytically pure various metal, uses second distillation water dissolution, and to be mixed with concentration be 2.00 × 10
-3molL
-1the second distillation aqueous solution.
The ethanolic soln of (4) 75%: dehydrated alcohol 75mL adding distil water is to 100mL, and mixing, room temperature preservation is for subsequent use.
(5) phosphate buffer soln (D-hanks balanced salt solution): 0.4gKCl, 0.06g KH
2pO
4, 8.0gNaCl, 1.0g C
6h
12o
6, 0.35gNaHCO
3, 0.152g Na
2hPO
412H
2o, 100,000 IU are dual anti-, and adjustment pH is 7.2 ~ 7.4, and deionized water is settled to 1000mL, and pin type filter (0.22um import millipore filtration) filtration sterilization, packing is for subsequent use.
(6) 1 ten thousand unit (IU)/dual anti-solution of mL: benzylpenicillin sodium (800,000 unit) is dissolved in 40mL D-hanks solution, is made into final concentration 20,000 units/mL; Vetstrep (1,600,000 unit) is dissolved in 80mL D-hanks solution, is made into final concentration 20,000 units/mL.Get isopyknic benzylpenicillin sodium solution and the mixing of Vetstrep solution respectively, the final concentration obtaining benzylpenicillin sodium and Vetstrep is the solution of 10,000 units/mL; Pin type filter (0.22um import millipore filtration) filtration sterilization, packing 1mL/ props up, and-20 DEG C save backup.
(7) 0.25% trypsinase: take 0.25g trypsinase, are dissolved in the D-hanks liquid of 100mL, and pin type filter (0.22um import millipore filtration) filtration sterilization, packing 1mL/ props up, and-20 DEG C save backup.
(8) 0.02% ethylenediamine tetraacetic acid (EDTA)s (EDTA): by 0.02gEDTA, are dissolved in the D-hanks liquid of 100mL, and pin type filter (0.22um import millipore filtration) filtration sterilization, packing 1mL/ props up, and-20 DEG C save backup.
(9) nutrient solution: with Sterile pipette measure 10mL the foetal calf serum of deactivation, 90mL substratum (modified form RPMI-1640) and the dual anti-liquid of 1mL be mixed in the sterile culture flask of 100mL, 2 ~ 8 DEG C save backup.
The present invention's fluorescent microscope used is respectively: IX-71 type fluorescence inverted phase contrast microscope, Japanese Olympus company; The fluorescence inverted microscope of Ti model, Japanese Nikon company; Cary Eclipse spectrophotofluorometer, VARIAN company of the U.S.; ThermoFisher 8000 water storage type CO
2cell culture incubator; AR1530/C electronic balance; 25cm
2tissue Culture Flask, Corning company of U.S. vertical pressure steam sterilizer (LS-B75); DHG-9230A electric heating constant-temperature blowing drying box, the grand experimental installation company limited of upper Nereid.
Embodiment two: probe is to Zn
2+the selectivity fluoroscopic examination of ion
The DMF storing solution (1.00 × 10 of probe is added in 10.0 mL volumetric flasks
-4molL
-1, 1mL), configuration metal ions Zn
2+(2.00 × 10
-3molL
-1, 1 mL), with DMF/H
2o(v/v, 1/4) solution dilution is to scale, and shake up, the quartz colorimetric utensil moving into 1cm carries out fluorescence spectrometry.
Arranging fluorescence exciting wavelength is 360nm, adds the probe (1.00 × 10 of about 3ml in the cuvette of 1cm
-5molL
-1) DMF/H
2o(v/v, 1/4) solution carries out fluorescence spectrum test, and probe is launched at 457nm wavelength place unstressed configuration.Add Zn
2+(2.00 × 10
-4molL
-1) after, probe solution launches intense blue fluorescence.Under the same terms, in probe solution, add Li respectively
+, Na
+, K
+, Mg
2+, Ca
2+, Ba
2+, Sr
2+, Hg
2+, Co
2+, Ni
2+, Cu
2+, Ag
+, Pb
2+, Cd
2+, Al
3+, Cr
3+, Fe
3+after metal ion, fluorescence spectrum and the intensity of probe can not be changed.Probe is only to Zn
2+selective fluoroscopic examination response performance, selection wavelength is that the fluorescence intensity level at 457nm place carries out measuring (accompanying drawing 1).
With under above-mentioned fluorescent method same test condition, probe in detecting Zn
2+probe-Zn is present at above-mentioned metal ion respectively as coexisting ion at the fluorescent value at 457nm wavelength place
2+in solution, when coexistent metallic ion concentration is Zn
2+during ionic concn 10 times amount, except being slightly subject to Co
2+, Cr
3+, Ni
2+, Cu
2+and Hg
2+impact outside, probe in detecting Zn
2+fluorescence intensity do not coexist by other metal ion and affect (accompanying drawing 2).
Embodiment three: the fluorescent microscopic imaging of live body PC3 cell
(1) recovery cell
PC3 cell is taken out in-80 DEG C of refrigerators, the water being placed in 37 DEG C rocks cell cryopreservation tube fast, thawed completely in 1-2 minute, in aseptic operating platform, sucked in centrifuge tube, add about 11ml nutrient solution (containing 10% foetal calf serum, 1% dual anti-liquid) and be mixed, and by this cell suspension centrifugal 5min on 1000r/min whizzer, remove supernatant liquid
,the cell of bottom settlings is added nutrient solution featheriness to break up to be mixed and transfer in culturing bottle, to make in culturing bottle nutrient solution volume in 5-7mL, and insert 37 DEG C, containing 5% CO
2incubator in cultivate.
(2) observe → go down to posterity → fishplate bar
Change a nutrient solution every day, and examine under a microscope cell growth status, until PC3 cell attachment covers with on whole culturing bottle wall, can go down to posterity, old nutrient solution is outwelled in aseptic operating platform, outwell after adding the EDTA liquid intrusion cell 30s of 1mL, the trypsin solution adding 1mL again digests, examine under a microscope until after cell size change circle, pat culturing bottle to make cell detachment and add appropriate nutrient solution immediately to stop digestion, it is divided into two and is incubated in 2 culturing bottles, when cell attachment after going down to posterity is paved with on whole culturing bottle wall, with go down to posterity operate identical, after adding EDTA and trypsinase, cell dissociation is come off and add immediately 3mL nutrient solution stop digestion, preparation is inoculated in 12 orifice plates.In each orifice plate, add the enchylema stoping digestion of 200mL, then add after appropriate new nutrient solution is mixed and orifice plate is inserted 37 DEG C, containing 5% CO
2incubator in cultivate.
(3) cell dyeing
Observe the cell growth condition in orifice plate next day, treat that cell attachment generates, discard old nutrient solution, wash 2 times with new nutrient solution, for subsequent use.
A group: add 1 × 10 respectively in 3 holes of cell cultures orifice plate
-5molL
-1zn
2+solution (1 × 10
-5molL
-1zn
2+preparation: nutrient solution+100mL 10 of the normal saline of 900mL
-4molL
-1zn
2+storing solution), 5 × 10
-5molL
-1zn
2+solution (nutrient solution+100uL 5 × 10 of the normal saline of 900uL
-4molL
-1zn
2+storing solution), 10 × 10
-5molL
-1zn
2+solution (nutrient solution+100mL 10 of the normal saline of 900mL
-3molL
-1zn
2+storing solution), at 37 DEG C, containing 5% CO
2incubator in hatch within 30 minutes, be placed on fluorescence inverted microscope under carry out light field and details in a play not acted out on stage, but told through dialogues is taken pictures, cell display unstressed configuration.(accompanying drawing 4-1,4-2,5-1,5-2).
B group: add 1 × 10 in 3 holes of Tissue Culture Plate
-5molL
-1probe
s1liquid (1 × 10
-5molL
-1probe
s1: the nutrient solution+100mL DMF/ (DMF+H of 900 mL
2o) (v/v, 20%) mixed solvent preparation 10
-4molL
-1storing solution); 2 × 10 are added again in another 3 holes of Tissue Culture Plate
-5molL
-1probe
s1, at 37 DEG C, containing 5%CO
2incubator in hatch 30 minutes, wash twice with fresh modified form RPMI-1640 substratum, carry out light field and dark-field imaging is taken pictures under being placed in fluorescence inverted microscope, cell there is no fluorescence display.(accompanying drawing 4-1,4-2,5-1,5-2).
C group: through the PC3 cell in 6 holes of step B process, it is 1 × 10 that one_to_one corresponding immerses concentration respectively
-5molL
-1, 5 × 10
-5molL
-1, 10 × 10
-5molL
-1zn
2+in solution, at 37 DEG C, containing 5% CO
2incubator in hatch 30 minutes after, sucking-off nutrient solution, the fresh modified form RPMI-1640 substratum of gained cell washes twice, and carry out details in a play not acted out on stage, but told through dialogues fluorescence imaging under being placed in fluorescence inverted microscope and take pictures, result shows, at Zn
2+concentration is 1 × 10
-5~ 1 × 10
-4molL
-1scope, concentration and probe concentration is 1 × 10
-5~ 2 × 10
-5molL
-1after cell being dyeed in scope, all observe blue-fluorescence cell image by fluorescence inverted microscope.Through 1 × 10
-5molL
-1probe solution and 5 × 10
-5molL
-1zn
2+the cell that solution-dyed is crossed presents blue-fluorescence image (accompanying drawing 4-3,5-3) clearly.Illustrate that probe is to the intracellular Zn of PC3
2+dye successfully.
Claims (6)
1. one kind for Zn in cancer cells
2+the fluorescent probe method of monitoring, it is characterized in that with compound two-(7-hydroxyl-tonka bean camphor-8-aldehyde) contracting quadrol is for trace Zn in monitoring cell
2+fluorescent probe, utilize probe and Zn
2+selectivity forms title complex and launches the characteristic of intense fluorescence, probe, Zn
2+can penetrate into respectively in cancer cells alive, with cytocompatibility, by Zn in probe and cell
2+in conjunction with emitting fluorescence, detect blue-fluorescence cell image clearly with fluorescence inverted microscope, probe is used as Zn in live body cancer cells
2+fluorescence imaging reagent.
2. one according to claim 1 is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, is characterized in that with probe Zn in cell
2+dye, with the fluoroscopic image of microscopic cell, concrete grammar is:
1) cell cultures: active prostate cancer (PC3) cell is inoculated in containing 10% foetal calf serum with containing in 1% dual anti-substratum (RPMI 1640) through recovery, is 37 DEG C in temperature, 5% CO
2and saturated humidity is cultivate in the incubator of 100%, goes down to posterity 1 time every 2-3 days, the full cell of growth selection is inoculated in 12 orifice plates to be cultivated, and density is 2 × 10
4individual/ml;
2) probe and cell are hatched: secondary daily fresh culture cleans cell twice, and cell being immersed concentration is 10 μm of olL
-1probe solution { 900 μ L substratum+100 μm of olL
-1dMF/the H of probe
2o(DMF/H
2o, v/v, 1/4) in mixing solutions 100 μ L}, at 5% CO of 37 DEG C
2, saturated humidity 100% incubator in hatch 30 min, sucking-off, containing the nutrient solution of probe, with fresh culture cleaning cell three times, is observed under fluorescence inverted microscope and carries out bright field and details in a play not acted out on stage, but told through dialogues is taken pictures;
3) Zn
2+to cell dyeing: immerse again containing 50 μm of olL in above-mentioned cell of hatching through probe
-1zn
2+solution (900 μ L substratum+500 μm of olL
-1zn
2+solution 100 μ L), at 5% CO of 37 DEG C
2, saturated humidity 100% incubator in hatch 30 min, carry out intracellular probes to Zn
2+dyeing, sucking-off is containing Zn
2+the nutrient solution of solution, the cell after dyeing cleans three times with fresh culture again, observes probe to Zn in cell under fluorescence inverted microscope
2+fluorescence imaging after dyeing, takes pictures to cell image and obtains cell outline image clearly.
3. one according to claim 2 is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, is characterized in that by concentration 1 × 10
-5~ 1 × 10
-4molL
-1zn in scope
2+live body PC3 cell is dyeed, Zn
2+can penetrate in cell, cell is round and smooth full shape; By concentration 1 × 10
-5~ 2 × 10
-5molL
-1probe in scope dyes to cell, and probe can penetrate in cell, and cell is round and smooth full shape; Probe, Zn
2+with cell, there is good consistency, to cytotoxic, can not apoptosis be made; In above-mentioned concentration range, through probe solution and Zn
2+the cell that solution-dyed is crossed can observe blue-fluorescence cell distribution image by fluorescence inverted microscope, and dyeing has selective aggregation ability.
4. one according to claim 2 is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, it is characterized in that reagent used is analytical pure or biochemical reagents, water is redistilled water or physiological saline; Live body PC3 cell is human body prostate cancer cell; Photographing device is fluorescence inverted microscope.
5. one according to claim 1 is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, is characterized in that described probe chemical structural formula is:
Molecular formula: C
22h
16n
2o
6
Molecular weight: 404.10
Fusing point: be greater than 300 DEG C
Solvability: be slightly soluble in chloroform, methyl-sulphoxide, DMF etc., be insoluble to ethanol
Spectral quality: at DMF/ H
2o(v/v, 1/4), in mixed solvent, 360nm wavelength excites down, Zn
2+add make probe 457nm place produce sharp fluorescent emissions, 360nm wavelength produce maximal ultraviolet absorption.
6. one according to claim 1 is used for Zn in cancer cells
2+the fluorescent probe method of monitoring, is characterized in that as Zn in cell
2+the fluorescence imaging reagent detected is two-(7-hydroxyl-tonka bean camphor-8-aldehyde) contracting quadrol; its synthetic method is for raw material with umbelliferone, hexamethylenetetramine and quadrol; be solvent at methylene dichloride, ethanol; take trifluoroacetic acid as solvent and catalyzer, through acetylize, formylation, condensation reaction and obtaining.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410565454.3A CN104498579B (en) | 2014-10-22 | 2014-10-22 | One kind is for Zn in cancer cell2+The fluorescence probe method of monitoring |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410565454.3A CN104498579B (en) | 2014-10-22 | 2014-10-22 | One kind is for Zn in cancer cell2+The fluorescence probe method of monitoring |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104498579A true CN104498579A (en) | 2015-04-08 |
| CN104498579B CN104498579B (en) | 2017-06-16 |
Family
ID=52940029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410565454.3A Active CN104498579B (en) | 2014-10-22 | 2014-10-22 | One kind is for Zn in cancer cell2+The fluorescence probe method of monitoring |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104498579B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105859706A (en) * | 2016-04-29 | 2016-08-17 | 辽宁科技大学 | Organic compound capable of fluorescently recognizing zinc ions and preparing and using method thereof |
| CN112812036A (en) * | 2021-01-19 | 2021-05-18 | 安徽医科大学第二附属医院 | Zn2+Ion-activated fluorescent probe |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101439191A (en) * | 2008-12-31 | 2009-05-27 | 南京大学 | Visible light induced zinc ion fluorescence angiographic reagent capable of being applied in cell/tissue/living body as well as synthesizing method and applications |
| CN101613344A (en) * | 2008-06-25 | 2009-12-30 | 中国科学院理化技术研究所 | The fluorescent probe of selectively detecting zinc ions in cells and preparation method and use thereof |
| CN102154005A (en) * | 2011-03-01 | 2011-08-17 | 韶关学院 | Stilbene two-photon fluorescence probe for detecting zinc ion in cell |
| CN103467455A (en) * | 2013-09-17 | 2013-12-25 | 安徽大学 | Zinc complex two-photon absorption material with living tissue developing function and preparation method thereof |
| WO2014011005A1 (en) * | 2012-07-13 | 2014-01-16 | 이화여자대학교 산학협력단 | Cyanine fluorescent probe, method for detecting zinc ion using same and method for preparing same |
| CN104004513A (en) * | 2014-06-03 | 2014-08-27 | 兰州大学 | Fluorescence molecular probe for detecting zinc ions and preparation method thereof |
-
2014
- 2014-10-22 CN CN201410565454.3A patent/CN104498579B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613344A (en) * | 2008-06-25 | 2009-12-30 | 中国科学院理化技术研究所 | The fluorescent probe of selectively detecting zinc ions in cells and preparation method and use thereof |
| CN101439191A (en) * | 2008-12-31 | 2009-05-27 | 南京大学 | Visible light induced zinc ion fluorescence angiographic reagent capable of being applied in cell/tissue/living body as well as synthesizing method and applications |
| CN102154005A (en) * | 2011-03-01 | 2011-08-17 | 韶关学院 | Stilbene two-photon fluorescence probe for detecting zinc ion in cell |
| WO2014011005A1 (en) * | 2012-07-13 | 2014-01-16 | 이화여자대학교 산학협력단 | Cyanine fluorescent probe, method for detecting zinc ion using same and method for preparing same |
| CN103467455A (en) * | 2013-09-17 | 2013-12-25 | 安徽大学 | Zinc complex two-photon absorption material with living tissue developing function and preparation method thereof |
| CN104004513A (en) * | 2014-06-03 | 2014-08-27 | 兰州大学 | Fluorescence molecular probe for detecting zinc ions and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 敬静等: "发光ZnSalen配合物在分子荧光成像中的应用进展", 《中国科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105859706A (en) * | 2016-04-29 | 2016-08-17 | 辽宁科技大学 | Organic compound capable of fluorescently recognizing zinc ions and preparing and using method thereof |
| CN105859706B (en) * | 2016-04-29 | 2018-04-27 | 辽宁科技大学 | A kind of organic compound and its preparation and application of fluorescence identifying zinc ion |
| CN112812036A (en) * | 2021-01-19 | 2021-05-18 | 安徽医科大学第二附属医院 | Zn2+Ion-activated fluorescent probe |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104498579B (en) | 2017-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Glutathione-stabilized copper nanoclusters mediated-inner filter effect for sensitive and selective determination of p-nitrophenol and alkaline phosphatase activity | |
| CN104198449B (en) | A kind of fluorescent probe method for living cells imaging | |
| US10264976B2 (en) | Biocompatible flavonoid compounds for organelle and cell imaging | |
| Shi et al. | In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe | |
| Zhu et al. | Highly water-soluble BODIPY-based fluorescent probes for sensitive fluorescent sensing of zinc (II) | |
| CN102746313A (en) | Rhodamine-B hydrazide derivative containing 1,2,4-triazole structural unit, preparation method and application thereof | |
| Kumar et al. | Interplay between silica deposition and viability during the life span of sorghum silica cells | |
| CN104819966B (en) | Calixarenes fluorescence probe is applied to Zn in living cells2+、F-The method of fluorescence imaging | |
| He et al. | The ICT-based fluorescence and colorimetric dual sensing of endogenous hypochlorite in living cells, bacteria, and zebrafish | |
| CN108117544A (en) | A kind of reversible sulfur dioxide/sulfurous acid（Hydrogen）The fluorescence probe of salt | |
| CN108844931A (en) | LZQ fluorescence probe detects SO at the same time2With the application in HSA | |
| Bertman et al. | A fluorescent flavonoid for lysosome detection in live cells under “wash free” conditions | |
| Danish et al. | Two‐Photon Probes for Zn2+ Ions with Various Dissociation Constants. Detection of Zn2+ Ions in Live Cells and Tissues by Two‐Photon Microscopy | |
| CN104498579A (en) | Fluorescent probe method for monitoring Zn<2+> in cancer cells | |
| Kuntam et al. | Characterization of a new class of blue-fluorescent lipid droplet markers for live-cell imaging in plants | |
| CN104962279B (en) | A kind of NO3‑The detection reagent of ion and application | |
| CN112500386B (en) | Near-infrared HClO fluorescent probe based on piroctone olamine, preparation and application thereof | |
| Lim et al. | In Vivo Imaging of Near‐Membrane Calcium Ions with Two‐Photon Probes | |
| CN105548097B (en) | A kind of competent cell imaging method using fluorescence probe under extreme ph values | |
| CN115855893A (en) | Application of silver nanoclusters in glyphosate detection, detection method of glyphosate in plants and dynamic monitoring method | |
| CN112694469A (en) | HOCl fluorescent probe based on pyrrazone and red hydrazine, preparation method and application | |
| CN107449761B (en) | Double-channel fluorescence imaging detection of trace Cu in active cancer cells2+And Hg2+Method (2) | |
| CN108181272A (en) | Micro Fe in a kind of binary channels fluorescence imaging detection living cells3+、Hg2+And Cu2+Method | |
| CN108548804B (en) | Single-channel fluorescence imaging detection of trace Ca in active cancer cells2+、Sr2+And Ba2+Method (2) | |
| CN113817010B (en) | Water-soluble multifunctional glycosyl fluorescent probe and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201112 Address after: No.3, pingzhong Road, Matou Town, Pingguo City, Baise City, Guangxi Zhuang Autonomous Region Patentee after: Guangxi Pingguo Runmin Poverty Alleviation Development Co., Ltd Address before: 550025 Huaxi, Guiyang, Guizhou University (North) Technology Department Patentee before: Guizhou University |
|
| TR01 | Transfer of patent right |