CN106053401A - Mitochondrion-targeted long-wavelength fluorescent probe and application thereof - Google Patents
Mitochondrion-targeted long-wavelength fluorescent probe and application thereof Download PDFInfo
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- CN106053401A CN106053401A CN201610128941.2A CN201610128941A CN106053401A CN 106053401 A CN106053401 A CN 106053401A CN 201610128941 A CN201610128941 A CN 201610128941A CN 106053401 A CN106053401 A CN 106053401A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention relates to a mitochondrion-targeted long-wavelength fluorescent probe. The fluorescent probe is FAI with a structure as shown in a formula (I) which is described in the specification. The fluorescent probe can rapidly locate a mitochondrion. Moreover, the fluorescent probe has a broad fluorescence emission spectrum (600-700 nm), and good chemical and light stability, dissolvability and biological compatibility. Laser confocal microscopic experiment results show that the probe has good cell permeability and exerts no toxic and side effect on cells and living bodies.
Description
Present disclosure relates to a kind of Mitochondrially targeted type long-wavelength fluorescent probe based on fluorescein.
Background technology mitochondrion is the place of aerobic respiration in cell, and the various activities for organism provide energy, quilt
What people were vivid becomes " power factory ".Additionally, mitochondrion also take part in the physiology such as cellular metabolism, signal conduction and apoptosis
Process, is one of very important organelle in cell.Mitochondrion is present in most eukaryotic cell, not of uniform size, and one
As in (0.5-1.0) × (1-2) μm, need under an optical microscope just can see through specific stain.Mitochondrion is typically short
Bar-shaped or spherical shape, also has other shapes such as ring-type, wire, dumbbell shaped, branching shape, flat plate-like.Mitochondrial quantity and shape
Not only relevant with the kind of cell, also closely related with metabolic activity and relevant disease.The most mitochondrial form, quantity and
Distribution situation is significant with prevention relevant disease for studying mitochondrial function.
Along with the fast development of fluorescent technique, fluorescent probe has become preclinical medicine and the important hands of bio-imaging research
Section.It is high that fluorescent probe has fluorescence quantum yield, good light stability, and the advantages such as bio-compatibility is good are often applied to cell, group
Knit and in the vegeto-animal fluorescence imaging of live body that build is less, become the ideal tools of micro-imaging.It is currently used for mitochondrion
The fluorescent probe kind of imaging is a lot, and the overwhelming majority is short wavelength's fluorescent probe, and it is launched wavelength and is positioned at 400-600nm, but
Transmitted wave length is near infrared region and the fluorescent probe kind that can be positioned in mitochondrion is the most little.Long-wavelength fluorescent probe with
Short wavelength compares has obvious advantage, long-wavelength fluorescent probe have stronger tissue permeability and less biological tissue or
The interference of organism autofluorescence, less to the light damage of biological tissue, can be that the offer of short wavelength's probe positions reality altogether simultaneously
The reference probe tested, is therefore badly in need of exploitation and has targeting and be positioned mitochondrial long-wavelength fluorescent probe.
Summary of the invention the invention provides a kind of Mitochondrially targeted type long-wavelength fluorescent probe FAI.The structure of FAI such as (I)
Shown in:
Above-mentioned this fluorescent probe to prepare reaction equation as follows:
The synthetic method of above-mentioned FAI probe is carried out as follows:
The dichlorofluorescein list aldehyde of 1 mole, with the N-R-2 of 1.1 times of moles, 3,3-trimethyl-3H-indole, difference
Joining in dehydrated alcohol, under nitrogen protection heating reflux reaction 5-7 hour, reactant liquor cooled and filtered, filter cake is with anhydrous
Ethanol purge, filter cake, after dehydrated alcohol recrystallization, obtains yellow solid and is target product.
Mitochondrion can be dyeed by above-mentioned FAI probe, and described living cells is Hela cell strain, MCF-7 cell
Strain or RAW264.7 cell strain.
The invention has the beneficial effects as follows: the fluorescent probe molecule of the present invention can quickly be positioned in mitochondrion, it addition,
This probe also has longer transmitting wavelength (600-650nm), preferable chemical stability, preferable dissolubility and bio-compatible
Property, do not disturbed by other species such as active oxygen and active nitrogen.Laser confocal imaging experiment shows that this kind of probe has preferably
Cell permeability, cell and organism are had no side effect.
Accompanying drawing explanation
Fig. 1 is fluorescence spectrum and the absorption spectrum (normalization) of probe FAI-1
Fig. 2 is that the FAI for preparing of embodiment is to the fluorescence co-focusing micro-imaging in mitochondrion.
Detailed description of the invention
The preparation method of dichlorofluorescein list aldehyde:
Dichlorofluorescein (2.5g, 6.14mmol) is dissolved in 8ml NaOH 33% (mass fraction) solution and 14ml methanol,
Add 0.4ml 15-crown ether-5, drip 12ml chloroform at 0 DEG C, then be warming up to back flow reaction 10h at 55 DEG C.At 0 DEG C
Being acidified by reactant liquor concentrated hydrochloric acid, pH is adjusted to 1-2, flocculent deposit occurs.It is filtrated to get orange/yellow solid.Use silica gel column chromatography
Carry, obtain light yellow solid 0.9g, productivity 15%.MS:427.9
Embodiment (1) FAI-1
Dichlorofluorescein list aldehyde (0.59g, 1.39mmol) and N-methyl-2,3,3-trimethyl-3H-indole (0.25g,
1.56mmol), it is dissolved in 2mL dehydrated alcohol, heating reflux reaction 5-7 hour under nitrogen protection, reactant liquor cooled and filtered,
Filter cake washes of absolute alcohol, filter cake, after dehydrated alcohol recrystallization, obtains yellow solid and is target product, and productivity is
89%.MS:584.1
Embodiment (2) FAI-2
Dichlorofluorescein list aldehyde (0.6g, 1.41mmol) and N-ethyl-2,3,3-trimethyl-3H-indole (0.3g,
1.62mmol), it is dissolved in 2mL dehydrated alcohol, heating reflux reaction 5-7 hour under nitrogen protection, reactant liquor cooled and filtered,
Filter cake washes of absolute alcohol, filter cake, after dehydrated alcohol recrystallization, obtains yellow solid and is target product, and productivity is
89%.MS:584.1
Embodiment (3) FAI-1 is to mitochondrial fluorescent microscopic imaging
In the culture dish containing MCF-7 or RAW264.7 (Hela) cell strain, adding concentration is the FAI-of 0.05-0.1M
The dimethyl sulphoxide solution of 1, after mixing homogeneously with cell culture fluid, makes FAI-1 final concentration in culture fluid reach 10uM.Dye
After color 5min, it is carried out three times by the phosphate buffered solution of pH=7.2, then this culture dish is placed in 37 DEG C of constant temperature culture
Case is carried out hatch 24 hours, finally this culture dish is placed under Laser Scanning Confocal Microscope and observes.Result is as shown in Figure 2.
Experimental result finds, is infected with in the cell mitochondrial of FAI-1 and presents stronger red fluorescence, test result indicate that
FAI-1 has preferable cell-membrane permeable, it is possible to be positioned in the mitochondrion of cell.
Claims (4)
1. a Mitochondrially targeted type long-wavelength fluorescent probe, fluorescent probe is FAI.Its general structure is as shown in (I):
2. the synthetic method of FAI fluorescent probe as claimed in claim 1, its feature comprises the steps:
The dichlorofluorescein list aldehyde of 1 mole, with the N-R-2 of 1.1 times of moles, 3,3-trimethyl-3H-indole, is separately added into
In dehydrated alcohol, heating reflux reaction 5-7 hour under nitrogen protection, reactant liquor cooled and filtered, filter cake dehydrated alcohol
Cleaning, filter cake, after dehydrated alcohol recrystallization, obtains yellow solid and is target product.
3. the application of FAI as claimed in claim 1, it is characterised in that for the fluorescent microscopic imaging of intracellular mitochondrial.
Apply the most as claimed in claim 1, it is characterised in that described cell be Hela cell strain, MCF-7 cell strain,
RAW264.7 cell strain.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510167181 | 2015-04-10 | ||
| CN2015101671811 | 2015-04-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106053401A true CN106053401A (en) | 2016-10-26 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610128941.2A Pending CN106053401A (en) | 2015-04-10 | 2016-03-08 | Mitochondrion-targeted long-wavelength fluorescent probe and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333678A (en) * | 2013-06-25 | 2013-10-02 | 山东大学 | One and two-photon mitochondrion fluorescence probe and application thereof |
| CN103937490A (en) * | 2014-04-24 | 2014-07-23 | 辽宁大学 | Fluorescent probe for detecting sulfur ions in mitochondria and application thereof |
-
2016
- 2016-03-08 CN CN201610128941.2A patent/CN106053401A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333678A (en) * | 2013-06-25 | 2013-10-02 | 山东大学 | One and two-photon mitochondrion fluorescence probe and application thereof |
| CN103937490A (en) * | 2014-04-24 | 2014-07-23 | 辽宁大学 | Fluorescent probe for detecting sulfur ions in mitochondria and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| XIAOQING XIONG ET AL.: "Construction of long-wavelength fluorescein analogues and their application as fluorescent probes", 《CHEM. EUR. J》 * |
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Application publication date: 20161026 |