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WO2014011005A1 - Sonde fluorescente cyaninique, procédé permettant de détecter des ions zinc l'utilisant et procédé de préparation de la sonde - Google Patents

Sonde fluorescente cyaninique, procédé permettant de détecter des ions zinc l'utilisant et procédé de préparation de la sonde Download PDF

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WO2014011005A1
WO2014011005A1 PCT/KR2013/006288 KR2013006288W WO2014011005A1 WO 2014011005 A1 WO2014011005 A1 WO 2014011005A1 KR 2013006288 W KR2013006288 W KR 2013006288W WO 2014011005 A1 WO2014011005 A1 WO 2014011005A1
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cyanine
ctmpa
fluorescent probe
based fluorescent
formula
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Korean (ko)
Inventor
윤주영
궈즈첸
신인재
김건희
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Ewha Womans University
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Ewha Womans University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • 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"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • the present application relates to a cyanine-based fluorescent probe, a zinc ion detection method using the cyanine-based fluorescent probe, a method for producing the cyanine-based fluorescent probe, a fluorescent sensor including the cyanine-based fluorescent probe, and a cyanine-based compound.
  • Zinc ions are essential components in various biological processes and are often present in tightly bound forms in proteins.
  • mobile intracellular zinc ions are also present in various human tissues, including the brain, intestine, pancreas, and retina.
  • zinc ions are known to play an important role in the regulation of apoptosis and are released from intracellular metal proteins during this process.
  • Diseases resulting from free zinc metabolism are closely associated with many physiological conditions such as Alzheimer's disease, epilepsy, Parkinson's disease, stroke, and infant diarrhea.
  • the molecular basis of the involvement of mobile zinc ions in pathophysiological processes has been extensively studied, the fundamental questions remain, in particular, questions regarding the role played by mobile zinc ions in the regulation of apoptosis. have. Therefore, the development of improved methods for quantifying and visualizing the mobile zinc ions is very important.
  • Fluorescent sensors have been a powerful tool for observing in vitro and in vivo levels of biologically related species and for understanding their function.
  • Zn 2+ selective fluorescent sensors have been used to obtain a great deal of information about zinc biology.
  • fluorophores such as fluorescein, coumarin, and 4-nitrobenzooxadiazole.
  • conjugating specific receptors There has been a focus on conjugating specific receptors.
  • Some fluorescent zinc sensors have been used to image endogenous Zn 2+ in living cells and hippocampus slices, and more interestingly, visible light active fluorescent probes have been used to track endogenous Zn 2+ during zebrafish development. .
  • the ratiometric method where the analytical concentration is determined by measuring the ratio of absorbance or fluorescence intensity at two wavelengths, has a built-in correction that eliminates ambient effects and increases signal fidelity. ).
  • ratiometric fluorescent probes that emit at long wavelengths have been designed for in vivo detection of endogenous Zn 2+ .
  • the present application is a zinc-selective probe that emits light at a long wavelength, and has a high sensitivity and high selectivity to zinc, and reacts with endogenous zinc in living cells and organisms to show a change from blue to red at the maximum emission wavelength and a large short wavelength shift.
  • a cyanine derivative for detecting zinc ions a cyanine fluorescent probe, a zinc ion detection method using the cyanine fluorescent probe, and a method of manufacturing the cyanine fluorescent probe are provided.
  • the first aspect of the present application can provide a cyanine-based fluorescent probe comprising a compound represented by the following formula (1):
  • the second aspect of the present application can provide a method for detecting zinc ions in vivo using the cyanine-based fluorescent probe.
  • a method of preparing a cyanine-based fluorescent probe which includes reacting TMPA represented by the following Chemical Formula 2 with tricarbocyanine chloride, may be:
  • the fourth aspect of the present application can provide a fluorescent sensor including the cyanine-based fluorescent probe.
  • the fifth aspect of the present application can provide a cyanine compound represented by the following Chemical Formula 1:
  • Highly sensitive and highly selective cyanine-based fluorescent sensors can be prepared according to the present invention, which can be used to detect endogenous Zn 2+ ions in living cells and organisms.
  • cyanine-based fluorescent probe included in the fluorescent sensor according to the present invention when Zn 2+ is added to a solution of CTMPA, a color change from blue to bright red, which can be easily determined by the naked eye, occurs, and at the same time, the maximum emission of CTMPA Significantly large hyperchromic shifts from about 730 nm to about 590 nm, with a wavelength change of about 140 nm, and cell or neural globules can be detected.
  • the CTMPA according to the present disclosure is capable of absorbing zinc ions in living cells and organisms because of useful features including large spectral shift induced by Zn 2+ , low fluorescence background, and high sensitivity to Zn 2+ . Can be used to detect.
  • the CTMPA can be used to observe endogenous zinc ions released during apoptosis, and the probe can be used to track intact Zn 2+ during zebrafish development. Due to the low background and high sensitivity of the CTMPA, the CTMPA according to the present application is the first fluorescence detection probe of neuromasts in zebrafish, and can be very useful in zinc biology research.
  • a strategy that relies on the change in the ⁇ -electron conjugation length of cyanine molecules promoted by metal ion coordination may show great potential for the creation of new cyanine probes.
  • FIG. 1 is a schematic diagram illustrating the synthesis of fluorescent Zn 2+ probe CTMPA according to one embodiment of the present disclosure.
  • Absorbance and emission spectra of the appropriate CTMPA (5.0 ⁇ M) are shown using ⁇ M, 2.5 ⁇ M, 3.0 ⁇ M, 3.5 ⁇ M, 4.0 ⁇ M, 4.5 ⁇ M, and 5.0 ⁇ M).
  • FIG. 3 is a schematic diagram showing a given binding mode of CTMPA for Zn 2+ according to one embodiment of the present disclosure.
  • FIG. 4A shows the pH profile of CTMPA in a mixture of CH 3 CN-H 2 O (1: 9) according to one embodiment of the present application
  • FIG. 4B shows CH 3 CN-H 2 O according to one embodiment of the present application.
  • FIG. 4C shows the change in fluorescence intensity at 730 nm of CTMPA in a mixture of CH 3 CN-H 2 O (1: 9) according to an embodiment of the present application.
  • FIG. 5 is a schematic diagram of a given pH-response mode of CTMPA according to one embodiment of the present disclosure.
  • FIG. 6A illustrates hydrogen numbering according to the presence or absence of Zn 2+ of CTMPA according to an embodiment of the present application
  • FIG. 6B illustrates 1 of CTMPA in CD 3 CN during titration using Zn 2+ according to an embodiment of the present application.
  • H NMR spectrum is shown.
  • (i) is the spectrum of the glass CTMPA
  • (iii) the [Zn 2+] / a [CTMPA] 1 The spectrum of the mixture of 1 is shown.
  • Figure 7 shows a 1 H NMR spectrum of CTMPA during Zn 2+ titration in CD 3 CN according to an embodiment of the present application.
  • Figure 9a is a graph showing the absorption ratio ( A 510 nm / A 670 nm ) of CTMPA (1 ⁇ M) to log 10 C [Zn2 +] free in the buffer according to an embodiment of the present application
  • Figure 9b is one embodiment of the present application of a buffer solution according to example CTMPA (1 ⁇ M) versus log 10 C [Zn2 +] is a graph showing the fluorescence ratio (I 590 nm / I 730 nm ) of the free.
  • CTMPA 10 is a bar graph showing metal ion selectivity of CTMPA according to one embodiment of the present disclosure.
  • FIG. 11 shows fluorescence images of exogenous zinc ions in (a) C2C12 and (b) NIH3T3 cells using CTMPA according to one embodiment of the present application.
  • FIG. 12 shows fluorescence images of intact zinc ions in (a) C2C12 and (b) NIH3T3 cells released during apoptosis using CTMPA according to one embodiment of the present application.
  • FIG. 13 shows fluorescence images of intact zinc ions released into dead cells in (a) C2C12 and (b) NIH3T3 cells using CTMPA according to one embodiment of the present application.
  • 15 is a photograph showing detection of endogenous zinc ions in zebrafish using CTMPA according to an embodiment of the present application.
  • FIG. 16 is an image showing distribution of neurospheres along grouped living bodies in (A) side and (B) dorsal region according to one embodiment of the present disclosure.
  • the supraorbital region includes preoptic (PO), and supraorbital (SO) neurospheres.
  • the caudal-cranial region includes the ear (otic, O), the occipital (OC), the dorsal (D), and the middle (MI) neurospheres, and the posterior (P) Neurospheres are placed in the trunk area.
  • 17A-17H show NMR and mass spectrometer spectra of compounds according to one embodiment of the present application, respectively.
  • the term "combination (s) thereof" included in the representation of a makushi form refers to one or more mixtures or combinations selected from the group consisting of the components described in the representation of makushi form, It means to include one or more selected from the group consisting of the above components.
  • the first aspect of the present application can provide a cyanine-based fluorescent probe comprising a compound represented by the following formula (1):
  • the cyanine-based fluorescent probe according to the present invention has high selectivity and high sensitivity to endogenous zinc ions in living cells and organisms, and exhibits a change from blue to red and a large short wavelength shift.
  • tricarbocyanine was chosen as the chromophore / fluorophore because it absorbs light with a large extinction coefficient, usually in the near infrared region (about 650 nm to about 900 nm).
  • a novel strategy can be adopted such that significant wavelength shifts occur in the maximum absorption and emission of the cyanine sites that depend on changes in the degree of conjugation of the ⁇ -electron system.
  • Cyanine-based Zn 2+ Two factors can be considered in designing selective probes. As a first element, the probe is Zn 2+ It must contain a site showing strong and selective binding. For this purpose, the large Zn of the tris-pyridine moiety (TMPA) 2+ Tris-pyridine sites (TMPA) can be introduced into the probe due to binding affinity (see FIG. 1).
  • the sensor is Zn 2+
  • the binding should include features that allow alteration of the effect of meso-substituents on the polymethine ⁇ -electron field of the tricarbocyanine chromophore / fluorophore. In this way, Zn 2+ The combination of will result in a large shift in maximum absorption and luminescence, resulting in a low fluorescence background.
  • the cyanine-based fluorescent probe may be used for detecting zinc ions (Zn 2+ ), but may not be limited thereto.
  • the compound represented by Chemical Formula 1 may be reacted with zinc ions (Zn 2+ ) to form a complex, but may not be limited thereto.
  • the cyanine-based fluorescent probe including the compound represented by Formula 1 may detect endogenous zinc ions (Zn 2+ ) in vivo, but may not be limited thereto.
  • the compound represented by Chemical Formula 1 may be a color change from blue to red at the maximum emission wavelength in response to Zn 2+ , but may not be limited thereto.
  • the compound represented by Chemical Formula 1 may represent short wavelength shift in the emission wavelength and the absorption wavelength by reaction with Zn 2+ , but may not be limited thereto.
  • the compound represented by Chemical Formula 1 may exhibit short wavelength shift from about 730 nm to about 590 nm at the maximum emission wavelength by reaction with Zn 2+ , but may not be limited thereto. have.
  • pyridine and meso-substituted nitrogen in the compound represented by Chemical Formula 1 may be coordinated by reacting with Zn 2+ , but may not be limited thereto.
  • the compound represented by Formula 1 (CTMPA) and the Zn 2+ may be to form a complex in a ratio of about 1: about 1, but may not be limited thereto.
  • the cyanine-based fluorescent probe may detect endogenous zinc ions (Zn 2+ ) in vivo, but may not be limited thereto.
  • the cyanine-based fluorescent probe may detect zinc ions within the nM range, but may not be limited thereto.
  • the cyanine-based fluorescent probe may be, for example, about 0.1 nM to about 1,000 nM, about 0.1 nM to about 500 nM, about 0.1 nM to about 100 nM, about 0.1 nM to about 80 nM, about 0.1 nM to about 50 nM , About 0.1 nM to about 30 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, about 0.1 nM to about 1 nM, about 1 nM to about 1,000 nM, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 80 nM, about 1 nM to about 50 nM, about 1 nM to about 30 nM, about 1 nM to about 10 nM,
  • the second aspect of the present application can provide a method for detecting zinc ions in vivo using the cyanine-based fluorescent probe.
  • the detection method may be one capable of imaging the distribution of endogenous zinc ions in vivo, but may not be limited thereto.
  • a method of preparing a cyanine-based fluorescent probe which includes reacting TMPA represented by the following Chemical Formula 2 with tricarbocyanine chloride, may be:
  • the TMPA and the tricarbocyanide chloride may be reacted by heating at about 70 ° C. to about 100 ° C. under an argon atmosphere, but may not be limited thereto.
  • it may further include purifying the cyanine-based fluorescent probe, but may not be limited thereto.
  • the fourth aspect of the present application can provide a fluorescent sensor including the cyanine-based fluorescent probe.
  • the fifth aspect of the present application can provide a cyanine compound represented by the following Chemical Formula 1:
  • the second to fifth aspects of the present application respectively, using the cyanine-based fluorescent probe according to the first aspect of the present invention, zinc ion detection method in vivo, a method for producing a cyanine-based fluorescent probe according to the first aspect of the present application, the cyanine-based A fluorescent sensor including a fluorescent probe, and a cyanine compound represented by Chemical Formula 1, and detailed descriptions of portions overlapping with the first aspect of the present disclosure are omitted, but the descriptions of the first aspect of the present disclosure The same may be applied to each of the second to fifth aspects of the present disclosure even if the description thereof is omitted.
  • the fluorescence spectrometer was used to determine the apparent dissociation constant ( K d ) calculated by using the following equation;
  • R is the absorption ratio or fluorescence intensity ratio at two wavelengths
  • R max is the maximum ratio
  • R 0 is the ratio without addition of Zn 2+
  • [M 2+ ] free is the ratio of free Zn 2+ Concentration.
  • Example 2 Detection of zinc ions in living cells and organisms
  • C2C12 cells mouse myoblast cell line
  • NIH3T3 cells mouse embryonic fibroblast cell line
  • culture medium 10% fetal bovine serum (FBS), penicillin 50 units / mL and 50 ⁇ g / mL of streptomycin were maintained in Dulbecco's Modified Eagle Medium (DMEM).
  • C2C12 cells and NIH3T3 cells were attached in 24-well plates at a density of 1 ⁇ 10 4 cells per well in culture medium. After 24 hours, cells were incubated at 37 ° C. for 1 hour in the presence of 250 ⁇ M H 2 O 2 . After 6 hours, cells were treated with 2 ⁇ M CTMPA at 37 ° C. for 1 hour. After washing twice with Dulbecco's phosphate buffered saline (DPBS), cells were imaged by confocal microscopy. For TPEN experiments, cells treated in the presence of 250 ⁇ M H 2 O 2 for 6 hours were incubated with 50 ⁇ M TPEN at 37 ° C. for 0.5 hours. After washing twice with DPBS to remove the remaining TPEN, cells were treated with 2 ⁇ M CTMPA at 37 ° C. for 1 hour. The treated cells were washed and imaged by confocal microscopy.
  • DPBS Dulbecco's phosphate buffered saline
  • the zebrafish was kept at 28 ° C. and optimal reproductive conditions were maintained. For mating, the male and female zebrafish were kept in a tank at 28 ° C. with a 12 hour / 12 hour light cycle, and then scattering was induced by providing photostimulation in the morning. Nearly all eggs were fertilized immediately. All steps of zebrafish were performed in E3 embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO 4 , 1 mM CaCl 2 , 0.15 mM KH 2 PO 4 , 0.05 mM Na 2 HPO 4 , 0.7 mM NaHCO 3 , 5% to 10% methylene blue; pH 7.5).
  • Zebrafish embryos 24, 36, 48, 72, and 96 hours after fertilization were subjected to 1 hour in E3 medium containing 0.1% (v / v) DMSO containing 2 ⁇ M CTMPA at 28 ° C. While incubated.
  • E3 medium containing 0.1% (v / v) DMSO containing 2 ⁇ M CTMPA at 28 ° C. While incubated.
  • zebrafish was exposed to 100 ⁇ M TPEN in E3 medium at 28 ° C. for 1 hour to remove intact zinc ions in zebrafish. After washing with E3 medium to remove the remaining TPEN, the zebrafish was incubated in E3 medium containing 2 ⁇ M CTMPA at 28 ° C. for 1 hour. The treated zebrafish were washed and imaged by confocal microscopy.
  • CTMPA Due to the fact that the TMPA site of CTMPA contains three pyrimidine groups that contribute as zinc coordination sites, CTMPA has this enhanced Zn 2+ binding capacity.
  • the primary amine moiety in TMPA is linked to the center of the polymethine chain of tricarbocyanine in the probe, and as a result, the coordination to Zn 2+ will affect the degree of conjugation of the ⁇ -electron system. This phenomenon results in observable shifts of maximum wavelengths in the absorption and emission spectra. Based on this reason, the Zn 2+ -specific fluorescent probe CTMPA was prepared according to the procedure shown in FIG. 1. In brief, TMPA was synthesized from 2,6-dichloromethylpyridine in 22% yield. CTMPA was prepared in 32% yield by reacting tricarbocyanine IR-780 with chloride in DMF and TMPA.
  • the near infrared band at about 730 nm in the emission spectrum decreases with a sharp increase in the band at about 590 nm.
  • the observation is that the characteristic emission band of CTMPA is Zn 2+ It demonstrates that a large short wavelength shift (about 140 nm) proceeds with binding to.
  • CTMPA-Zn 2+ Of about 1 a complex of about 1 is produced and I 590 nm Of I 730 nm And A 510nm Of A 670 nm Ratio of in vitro Zn 2+ It can be used to measure the concentration.
  • Figure 2a shows an absorption spectrum [inset is Zn 2+ Before and after addition of Zn 2+ Ratio of absorbance as a function of the concentration and color change of A 510nm Of A 670 nm );
  • 2B shows the emission spectrum ( ⁇ ) in the region of about 670 nm or more.
  • ex 670 nm);
  • 2D shows the emission spectrum ( ⁇ ) in the region of about 560 nm or more.
  • ex 560 nm) (inset is Zn 2+ Before and after addition of Zn 2+ Ratio of fluorescence intensity as a function of concentration and fluorescence change of ( I 590 nm Of I 730 nm )).
  • FIG. 4a shows the pH profile of CTMPA in a mixture of CH 3 CN-H 2 O (1: 9) according to this example
  • FIG. 4b shows CH 3 CN-H 2 O (1: 9) according to this example
  • Deprotonation of NH in the meso-substituents of CTMPA according to this example shortens the ⁇ -conjugated system in the tricarbocyanine chromophore / fluorophore, which shows short wavelength shift at maximum absorption and emission.
  • This result is because a large blue shift of the CTMPA increased by a combination of Zn 2+, Zn 2+ in a coordination bond to the NH of the probe to lower the NH of p K a, at about pH 7.4 the CTMPA-Zn 2 + Suggestion caused by deprotonation of NH in the complex (see FIG. 3).
  • Absorption and luminescence spectra of CTMPA according to the present embodiment remain almost unchanged between about pH 2.8 and about pH 9.7, and therefore, the probe may be applied to in vivo detection of Zn 2+ . It is expected.
  • FIGS. 6 and 7 show hydrogen numbering according to the presence or absence of Zn 2+ of CTMPA according to the present embodiment
  • FIG. 6b shows a 1 H NMR spectrum of CTMPA in CD 3 CN during titration using Zn 2+ according to the present embodiment.
  • (i) is the spectrum of the glass CTMPA, (ii) the [Zn 2+] / 0.5 of [CTMPA]: a mixture of a spectrum of the 1, (iii) the [Zn 2+] / [CTMPA]
  • the spectrum of the mixture of 1: 1 is shown.
  • Figure 7 shows the 1 H NMR spectrum of CTMPA during the Zn 2+ titration process of CD 3 CN according to the present embodiment.
  • Detailed confirmation of the 1 H NMR signals is shown in Table 1.
  • the apparent dissociation constant ( K d ) of the CTMPA-Zn 2+ complex plots the fluorescence intensity ratio I 590 nm / I 730 nm or the absorption ratio A 510 nm / A 670 nm as a function of Zn 2+ concentration.
  • K d The apparent dissociation constant
  • FIG. 9B shows CTMPA (1 ⁇ M) versus log 10 C [in buffer according to this Example. It is a graph showing the fluorescence ratio ( I 590 nm / I 730 nm ) of Zn2 +] free .
  • CTMPA Previously developed Zn 2+ Unlike the receptors, CTMPA has three pyridine groups and Zn 2+ It contains one meso-nitrogen that acts as a coordination site. The presence of multiple coordination sites in the probe is Cd 2+ And Zn of CTMPA relative to other metal ions 2+ It is expected to increase binding selectivity.
  • Gray bars show various competing metal ions (Na in a solution of CTMPA (5 ⁇ M) + , K + , Mg 2+ And Ca 2+ 1,000 ⁇ M for ; CD 2+ 10 ⁇ M for; After addition of 25 ⁇ M) for all other metal ions
  • I 590 nm Of I 730 nm Indicates a ratio.
  • Black bars are Zn in a solution of CTMPA (5 ⁇ M) in the presence of the competing metal ions. 2+ After addition of I 590 nm Of I 730 nm Indicates a ratio.
  • CTMPA will be suitable for detecting zinc ions in living cells and organisms.
  • the Zn 2+ bioimaging ability of the CTMPA was measured using live mammalian cells.
  • Mouse C2C12 myoblasts and mouse NIH3T3 embryonic fibroblasts were incubated with 2 ⁇ M CTMPA for 1 hour (see FIG. 11).
  • FIG. 11 shows fluorescence images of exogenous zinc ions in (a) C2C12 and (b) NIH3T3 cells using CTMPA according to this Example.
  • FIG. 11 shows fluorescence images of exogenous zinc ions in (a) C2C12 and (b) NIH3T3 cells using CTMPA.
  • FIG. 12 shows fluorescence images of intact zinc ions in (a) C2C12 and (b) NIH3T3 cells released during apoptosis using CTMPA according to this Example, and FIG. 13 shows CTMPA according to this Example.
  • Fluorescence images of intact zinc ions released in dead cells in (a) C2C12 and (b) NIH3T3 cells are shown.
  • H 2 O 2 -treated cells were treated with 50 ⁇ M TPEN for 0.5 h prior to treatment with the probe. Were incubated. In this case, the fluorescence intensity of the cells was significantly reduced (FIG. 12), indicating that the probe senses the zinc ions actually released during apoptosis.
  • Figure 12 shows fluorescence imaging of intact zinc ions in (a) C2C12 and (b) NIH3T3 cells released during apoptosis using CTMPA.
  • Cells were cultured in the absence (left) or presence (center) of 250 ⁇ M H 2 O 2 for 6 hours, causing apoptosis and then treated for 1 hour with 2 ⁇ M CTMPA.
  • CTMPA CTMPA
  • FIG. 16 is an image showing the distribution of neurospheres according to the grouped living bodies in (A) side and (B) dorsal regions according to the present embodiment, and the upper eye and the supraorbital region are in front of the visual cross (preoptic, PO). ), And supraorbital (SO) neurospheres.
  • the caudal-cranial region includes the ear (otic, O), the occipital (OC), the dorsal (D), and the middle (MI) neurospheres, and the posterior (P) Neurospheres are placed in the trunk area. It should be noted that this phenomenon was not observed when using different zinc probes such as NBD-TPEA and ZTRS. Since CTMPA has a very high binding affinity for Zn 2+ , and CTMPA exhibits long wavelength luminescence that inhibits autofluorescence, these probes have a unique ability to detect intact zinc ions in zebrafish neurospheres. .
  • CTMPA as a novel fluorescent Zn 2+ sensor with long wavelength emission maximum.
  • CTMPA has high sensitivity and high selectivity for Zn 2+ relative to other metal ions, and a detection limit in the nanomolar range.
  • the significant absorption and emission wavelength shifts that occur when CTMPA forms a complex with Zn 2+ make the sensor ideally suited for bioimaging of endogenous free zinc ions.
  • This advantageous feature was demonstrated by using CTMPA for fluorescence detection of intact zinc ions in the zebrafish neurospheres.
  • the strategy devised by the inventors of the present invention is the high sensitivity and high selectivity, ratiometric cyanine-based metal ion sensor, which is dependent on the modulation of the conjugation length of the ⁇ -electron of the cyanine chromophore / fluorophore by metal ion coordination bonds. It may be applicable to the formation. In particular, an approach based on apparent changes in the ⁇ -electron system of cyanine dyes will serve as the basis for devising other types of cyanine-based fluorescent probes for in vivo imaging applications.

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PCT/KR2013/006288 2012-07-13 2013-07-12 Sonde fluorescente cyaninique, procédé permettant de détecter des ions zinc l'utilisant et procédé de préparation de la sonde Ceased WO2014011005A1 (fr)

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CN104498579A (zh) * 2014-10-22 2015-04-08 贵州大学 一种用于癌细胞中Zn2+监测的荧光探针方法
CN106867511A (zh) * 2015-12-11 2017-06-20 中国科学院大连化学物理研究所 一种开关型锌离子荧光探针及其制备方法和应用
CN107860732A (zh) * 2017-04-19 2018-03-30 南京晓庄学院 一种多信号探针的用途
CN112209871A (zh) * 2020-10-29 2021-01-12 西北师范大学 一种基于四苯乙烯的锌离子荧光探针及其制备方法与应用
CN112858233A (zh) * 2019-11-28 2021-05-28 天津科技大学 一种新型的水体中锌离子的检测方法

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CN104498579A (zh) * 2014-10-22 2015-04-08 贵州大学 一种用于癌细胞中Zn2+监测的荧光探针方法
CN106867511A (zh) * 2015-12-11 2017-06-20 中国科学院大连化学物理研究所 一种开关型锌离子荧光探针及其制备方法和应用
CN106867511B (zh) * 2015-12-11 2019-01-22 中国科学院大连化学物理研究所 一种开关型锌离子荧光探针及其制备方法和应用
CN107860732A (zh) * 2017-04-19 2018-03-30 南京晓庄学院 一种多信号探针的用途
CN107860732B (zh) * 2017-04-19 2019-05-24 南京晓庄学院 一种多信号探针的用途
CN112858233A (zh) * 2019-11-28 2021-05-28 天津科技大学 一种新型的水体中锌离子的检测方法
CN112209871A (zh) * 2020-10-29 2021-01-12 西北师范大学 一种基于四苯乙烯的锌离子荧光探针及其制备方法与应用

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