CN109761931A - A kind of Ratiometric fluorescent probe and its preparation method and application detecting internal pH - Google Patents
A kind of Ratiometric fluorescent probe and its preparation method and application detecting internal pH Download PDFInfo
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- CN109761931A CN109761931A CN201910141848.9A CN201910141848A CN109761931A CN 109761931 A CN109761931 A CN 109761931A CN 201910141848 A CN201910141848 A CN 201910141848A CN 109761931 A CN109761931 A CN 109761931A
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Abstract
The present invention provides a kind of Ratiometric fluorescent probes and its preparation method and application for detecting internal pH.The chemical structural formula of the fluorescence probe are as follows:.Acquisition can be further being reacted near amino thiophenols with the reaction product of 5- bromosalicylaldehyde by 4- cyanophenylboronic acid.Fluorescence probe of the invention has high specific, not by the interference of other components in carrying out corresponding pH detection process, can be used for the real time measure of pH in living cells.The high sensitivity of the probe has good fluorescence emission spectral property (415-700 nm), and the measurement of internal pH is carried out by drawing standard curve, the purpose quick and precisely detected to internal pH may be implemented.Fluorescence probe synthetic method provided by the invention, simple for process, raw material is cheap and easy to get, and preparation cost is low, easy to spread.This probe has broad application prospects in biological monitoring field.
Description
Technical field
The invention belongs to technical field of analytical chemistry, and in particular to it is a kind of detect internal pH Ratiometric fluorescent probe and
It is applied.
Background technique
Important parameter one of of the pH value as intracellular metabolism is maintaining cell growth, proliferation, apoptosis and signal to pass
It leads etc. in normal physiological processes and plays key effect.In addition to this, the internalization of the endocytosis of cell, phagocytosis and receptors ligand
It also will receive the influence of pH Deng internalization access.In normal organism pH value of body (pH value) it is always stable a certain range it
Interior, human body Optimal pH is in alkalescent, is 7.35-7.45, may result in cell function when fluctuation up and down occurs in it and hinder
Hinder, it is serious can be with crisis life security.If pH is lower than normal value, cell can inhibit enzyme and hormonal activity, reduction group in acidity
Function is knitted, human body immunity degradation can be also made, red blood cell and blood platelet are easily assembled.Colleague, acidic cell environment are unfavorable for
Calcium uptake, cause osteoporosis, and some researches show that cellular pH environment to be in meeting within the scope of 6.2-6.9 so that its canceration
Probability greatly increases.If pH value is higher to occur alkalosis, disturbance of acid-base balance can also cause the certain organs of human body irreversible
Damage.
Currently, the method that can be used for detecting intracellular ph value includes31P nuclear magnetic resonance method, microelectrode method, absorption spectrometry
Deng but there are mechanical damages, expensive for these methods, it is difficult to the disadvantages of precise measurement.And fluorescence spectrum rule is using glimmering
The variation such as optical parameter such as fluorescence intensity, fluorescence lifetime is measured pH value, high sensitivity, good, the fast response time etc. of selectivity
Advantage, it is often more important that be capable of the variation of real-time in-situ monitoring internal pH and become the detection internal pH being widely used at present
A kind of important method.
Summary of the invention
For the problems of the prior art, the present invention provides a kind of Ratiometric fluorescent probe for detecting pH, and monitoring range is wide,
Strong antijamming capability.
It is a further object of the present invention to provide a kind of above-mentioned fluorescence probes, and pH is answered in detection solution or in biological cell
With.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of Ratiometric fluorescent probe detecting pH, chemical structural formula are as shown in the formula (I):
Formula (I).
The preparation method of above-mentioned fluorescence probe, comprising the following steps:
(1) by 4- cyanophenylboronic acid, 5- bromosalicylaldehyde, Pd (dppf) Cl2([bis- (diphenylphosphino) ferrocene of 1,1'-] dichloro
Change palladium), potassium acetate is dissolved in Isosorbide-5-Nitrae-dioxane, and reaction solution is cooled to room to reacting sufficiently by heating stirring under nitrogen protection
Temperature is filtered to remove solid impurity, and filtrate isolates and purifies to obtain light yellow compound 1:
；
(2) compound 1, near amino thiophenols in the presence of p-methyl benzenesulfonic acid react in dimethylformamide by heating stirring；
After the reaction was completed, reaction solution is cooling rapidly at 0 DEG C or so, water is added in backward reaction solution, obtains yellow solid precipitate, separates
It precipitates and purifies to obtain compound 2, i.e. fluorescence probe:
。
In step (1), the molar ratio of 4- cyanophenylboronic acid and 5- bromosalicylaldehyde is 0.8-1:1-1.5.
In step (2), the molar ratio of compound 1 and near amino thiophenols is 1:1-1.5.
In step (1), isolate and purify process are as follows: with petroleum ether: ethyl acetate volume ratio is the leacheate of 5:1, by filtrate
(petroleum ether: ethyl acetate=5:1) is chromatographed by column.
In step (2), purification process are as follows: with petroleum ether: ethyl acetate volume ratio is the leacheate of 5:1, and precipitating is passed through
Column chromatographs to obtain probe.
A kind of application of above-mentioned fluorescence probe pH in detection solution, cell or organism.
Mechanism of the invention is as follows:
Fluorescence probe of the present invention has 2-(2 '-hydroxy phenyl) benzothiazole (HBT) group, in pH gradually to alkaline item
When part changes, the nitrogen-atoms of hydroxyl hydrogen and benzothiazole forms intramolecular hydrogen bond and forms ESIPT process on probe, blue at this time glimmering
Light enhances；And when under acidic environment, ESIPT process blocks to issue green fluorescence；It is logical that fluorescence detector can be used
The fluorescence intensity for crossing detection different wave length, realizes the detection of pH.
The invention has the following advantages that
The Ratiometric fluorescent probe of detection internal pH provided by the invention can be obtained through chemical synthesis, and synthesis technology is simply easy
Row, raw material is cheap and easy to get, and preparation cost is low, easy to spread.This fluorescence probe has high specific, detects carrying out corresponding pH
Not by the interference of other components in journey, it can be used for the real time measure of pH in living cells.The high sensitivity of the probe has good
Fluorescence emission spectral property (415-700 nm) carries out the measurement of internal pH by drawing standard curve, may be implemented to thin
The purpose that pH intracellular is quick and precisely detected.Probe of the invention has broad application prospects.
Detailed description of the invention
Fig. 1 is fluorescence probe1H NMR spectra；
Fig. 2 is fluorescence spectrum of the fluorescence probe under condition of different pH；
Fig. 3 is the fluorescence intensity of fluorescence probe and the linear relationship of pH；
Fig. 4 is the fluorescence intensity after fluorescence probe is reacted in pH6.5 and pH7.5 with different material respectively；
Fig. 5 is toxicity test of the fluorescence probe to living cells；
Fig. 6 is fluorescence imaging application of the fluorescence probe in living cells.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
The synthesis of 1 fluorescence probe of embodiment
By 4- cyanophenylboronic acid (147 mg, 1 mmol), 5- bromosalicylaldehyde (201 mg, 1 mmol), Pd (dppf) Cl2(146
Mg, 20 mol%), potassium acetate (393 mg, 4 mmol) is dissolved in 10 mL Isosorbide-5-Nitraes-dioxane that heating stirring is to 90 DEG C, in nitrogen
It is sufficiently reacted under gas shielded 12 hours.Reaction stop after, reaction solution is cooled to room temperature, solid impurity is filtered to remove, then with
Petroleum ether: ethyl acetate=5:1v/v is that the yellow solid powder that filtrate is passed through column Chromatographic purification by leacheate is compound 1:
；
Take compound 1(88 mg, 0.4 mmol), 5 mL N, N- dimethyl methyls of near amino thiophenols (50 mg, 0.4 mmol)
P-methyl benzenesulfonic acid (10 mg, 0.058 mmol) heating stirring 2h under the conditions of 90 DEG C is added after amide (DMF) dissolution.Reaction
After the completion, reaction solution is cooling rapidly at 0 DEG C or so, 5 mL water are added in backward reaction solution, obtain yellow solid precipitate, filter
Obtain crude product.Crude product is chromatographed into progress purification & isolation by column and obtains compound 2, as fluorescence probe；Its1H NMR spectra
Such as Fig. 1:
。
Response of 2 fluorescence probe of embodiment to pH
It preparing 5 μM of probe buffer solutions of 13 part of 4 mL in advance, contains 5% methanol, pH is respectively 3,3.5,4,4.5,5,5.5,6,
6.5,7,7.5,8,8.5,9.Then fluorescent scanning (λ is carried outex=405 nm)；Calculate fluorescence intensity in each system；The probe pair
The response of different pH is as shown in Figure 2: as pH gradually becomes alkalinity by acidity, the fluorescence intensity (I at 473 nm473) gradually increase
By force, the fluorescence intensity (I at 546 nm546) there is no significant change.Calculate I473/ I546Ratio, make I473/ I546With the line of pH
Sexual intercourse is as shown in Figure 3: within the scope of pH6.5-8.5, pH variation and I473/ I546It is linearly related.
Selectivity of 3 fluorescence probe of embodiment to different ions
Prepare in advance 20 part of 4 mL 5 μM of probe buffer solutions (containing 5% methanol, pH=6.5 and 7.5), then respectively to the body
The PBS solution for the different testing sample that 100 μ L concentration are 40 mM is sequentially added in system.Then fluorescence detection (λ is carried outex= 405
Nm, λem=473nm and 546nm)；Calculate I in each system473/ I546；Assess interference of the different material to fluorescence probe solution
Property, as a result as shown in Figure 4, wherein 1- blank, 2-AlCl3, 3-CaCl2, 4-NaClO, 5-CuSO4, 6-Cys, 7-NaF, 8-
FeSO4, 9-Fe2(SO4)3, 10-GSH, 11-H2O2, 12-H2S, 13-Hcy, 14-MgCl, 15-CoCl2, 16-SO2, 17-ZnCl:
Different ions are added under the same conditions, it is found that the probe is little by the influence of ion.
Toxicity of 4 fluorescence probe of embodiment to cell
Prepare the probe buffer solution (containing 5% methanol, pH=7.4) that concentration is 5-50 μM and A549 is analyzed using MTT colorimetric method
Absorbance of the cell after fluorescence probe is added calculates cell survival rate, as a result as shown in Figure 5: when concentration is 5-50 μM,
The survival rate of cell is 98%-83%, and when concentration is less than 10 μM, cell survival rate is 95% or more.
Imaging applications of 5 fluorescence probe of embodiment in living cells
A549 cell is placed in culture medium (DMEM culture solution and 10% fetal calf serum), the condition that is placed in is 37 DEG C, 5% CO2With
20% O2Incubator in cultivate 24 h.(10 μM) injection pH of fluorescence probe points prepared with microsyringe extraction embodiment 1
Wei not be in 5.5,6.5,7.5, the 8.5 processed A549 cell of PBS buffer solution, continuation cultivates 10 min in the incubator and goes forward side by side
Row carries out fluorescence imaging (λ in blue channel and green channelex=405 nm) result is shown in Fig. 6: blue channel fluorescence with alkalinity increasing
Add and significant enhancing, and the fluorescence of green channel does not have significant variation under 405 nm excitation.
Claims (5)
1. a kind of Ratiometric fluorescent probe for detecting pH, chemical structural formula are as shown in the formula (I):
Formula (I).
2. a kind of preparation method of fluorescence probe as described in claim 1, which comprises the following steps:
(1) by 4- cyanophenylboronic acid, 5- bromosalicylaldehyde, Pd (dppf) Cl2, potassium acetate is dissolved in Isosorbide-5-Nitrae-dioxane, in nitrogen protection
Reaction solution is cooled to room temperature to reacting sufficiently, is filtered to remove solid impurity, filtrate isolates and purifies to obtain pale yellow by lower heating stirring
Color compound 1:
；
(2) heating stirring is reacted in dimethylformamide in the presence of p-methyl benzenesulfonic acid for compound 1 and near amino thiophenols；
After the reaction was completed, reaction solution is cooling rapidly at 0 DEG C or so, water is added in backward reaction solution, obtains yellow solid precipitate, separates
It precipitates and purifies to obtain compound 2, i.e. fluorescence probe:
。
3. preparation method according to claim 2, which is characterized in that in step (1), 4- cyanophenylboronic acid and 5- bromine water poplar
The molar ratio of aldehyde is 0.8-1:1-1.5；In step (2), the molar ratio of compound 1 and near amino thiophenols is 1:1-1.5.
4. preparation method according to claim 2, which is characterized in that in step (1), isolate and purify process are as follows: with petroleum
Ether: ethyl acetate volume ratio is the leacheate of 5:1, and filtrate is chromatographed (petroleum ether: ethyl acetate=5:1) by column；
In step (2), purification process are as follows: with petroleum ether: ethyl acetate volume ratio is the leacheate of 5:1, and precipitating is passed through column layer
Analysis obtains probe.
5. the application of fluorescence probe as described in claim 1 pH in detection solution, cell or organism a kind of.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111995599A (en) * | 2020-09-07 | 2020-11-27 | 中南大学 | Ratio type fluorescent molecular probe and preparation method and application thereof |
| CN113234040A (en) * | 2021-05-28 | 2021-08-10 | 中国科学院新疆理化技术研究所 | Fluorescent probe molecule for detecting pH and preparation method thereof |
| CN117567370A (en) * | 2023-11-23 | 2024-02-20 | 山东大学 | pH value detection fluorescent probe and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113234040B (en) * | 2021-05-28 | 2022-03-25 | 中国科学院新疆理化技术研究所 | Fluorescent probe molecule for detecting pH and preparation method thereof |
| CN117567370A (en) * | 2023-11-23 | 2024-02-20 | 山东大学 | pH value detection fluorescent probe and preparation method and application thereof |
| CN117567370B (en) * | 2023-11-23 | 2024-05-10 | 山东大学 | PH value detection fluorescent probe and preparation method and application thereof |
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