CN107367495A - A kind of sedimentation type alkaline phosphatase fluorescence probe and its synthetic method and application - Google Patents
A kind of sedimentation type alkaline phosphatase fluorescence probe and its synthetic method and application Download PDFInfo
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- CN107367495A CN107367495A CN201710592026.3A CN201710592026A CN107367495A CN 107367495 A CN107367495 A CN 107367495A CN 201710592026 A CN201710592026 A CN 201710592026A CN 107367495 A CN107367495 A CN 107367495A
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- 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
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Abstract
The invention discloses a kind of sedimentation type alkaline phosphatase fluorescence probe and its synthetic method and application.The synthetic method of the fluorescence probe, comprises the following steps：(1) the solid state fluorescence dyestuff (HTPQ) of synthesizing new；(2) dyestuff is designed to the probe (HTPQA) of alkaline phosphatase.The application includes：The fluorescence probe is in cushioning liquid to the response performance of alkaline phosphatase；The fluorescence probe stablizes indiffusible ability to endogenous cellular alkaline phosphatase in situ detection and fluorescence signal.The present invention has synthesized the sedimentation type solid state fluorescence dyestuff first, solves the problems, such as that conventional fluorescent probe is unable in situ detection and signal easily spreads；And the fluorescence probe has solid state fluorescence property, background fluorescence signal can be effectively reduced, improves the sensitivity of probe；The fluorescence probe is fine to living cells Color, and dyeing time is shorter, and staining efficiency is high, can carry out in situ detection to endogenous cellular alkaline phosphatase.
Description
Technical field
The invention belongs to fluorescent probe technique field, is related to a kind of sedimentation type alkaline phosphatase fluorescence probe and its synthesis side
Method and application.
Background technology
Small-molecule fluorescent probe can effectively monitor the dependent event with imaging cells vital movement, in the past few decades
In, small-molecule fluorescent probe is widely used for the detection of biological targets thing.However, existing fluorescence probe is based on thin mostly
Kytoplasm-soluble dye design, such as cumarin, rhodamine and fluorescein.These fluorescence probes, if not being positioned at spy
Fixed organelle, the information in situ of biological event can not be typically provided, because fluorescence signal molecule can be quickly spread away from target
Thing reaction site.The meeting of fluorescence signal molecule and then cell is diffused out, reduce fluorescence signal, cause low signal to noise ratio and low spirit
Sensitivity.
" self-positioning " type protease-activated fluorescent probe of commercialization at present, i.e.,97 phosphatase substrates (
97phosphatase substrate from ThermoFisher), the probe is with that after phosphatase enzymes, can react position
Point produces the precipitation with green fluorescence.However, the maximum excitation of the probe, in 350nm, positioned at ultraviolet region, this is very not
Beneficial to bio-imaging, and the most short excitation wavelength of fluorescence co-focusing imaging and flow cytometry etc. is usually 405nm (than them
The big 50nm of maximum excitation), this can cause the fluorescence signal of probe to weaken significantly.
Alkaline phosphatase (ALP) is catalytic proteins and nonprotein substrate hydrolysis and the hydrolase of transphosphorylation, is wrapped
A series of isodynamic enzymes being widely distributed in mammalian tissues are included.ALP has expression in many tissues, such as liver, bone
Tissue and kidney, its activity is frequently as the important biomolecule mark in medical diagnosis.In many diseases, ALP activity can all raise,
Such as disease of bone (scleromalacia, osteomalacia, Gegenbaur's cell bone tumour) and liver disease (hepatitis, obstructive jaundice, liver cancer),
And ALP Activity determinations have been the conventional meanses in clinically medical diagnosis on disease.Therefore, can not be former for current fluorescence probe
Position detection of alkaline phosphatase activity, fluorescence signal easily spreads and the problem such as fluorescence imaging effect difference, and working out one kind in situ can examine
Survey the high fluorescence probe of living cells endogenous alkaline phosphatase activity, fluorescence imaging contrast and seem very significant.
The content of the invention
It is an object of the invention to provide a kind of sedimentation type alkaline phosphatase fluorescence probe and its synthetic method and application, with
Solve in situ detection living cells endogenous alkaline phosphatase activity, fluorescence probe in cell imaging signal easily diffusion and fluorescence into
As technical problems such as effect differences.
The present invention provides this sedimentation type alkaline phosphatase fluorescence probe, and its structural formula is as follows：
The synthetic method of sedimentation type alkaline phosphatase fluorescence probe of the present invention, comprises the following steps：
(1) it is that 4- hydroxyls-tetraphenyl ethylene and methenamine are dissolved in glacial acetic acid by compound 1, is heated to reflux lower 3h, dichloro
Methane and petroleum ether cross post separation for eluant, eluent and obtain faint yellow intermediate；
(2) above-mentioned faint yellow intermediate, 2-Amino-5-chlorobenzamide and p-methyl benzenesulfonic acid are flowed back in absolute ethyl alcohol
5h is stirred, DDQ is added after cooling, suction filtration obtains the solid of yellow, washs solid with ethanol, drying arrives
The New Solid fluorescent dye HTPQ of yellow is the chloro- 2- of 6- (2- hydroxyls -5- (1,2,2- triphenyl vinyls) phenyl) -4 (3
Hydrogen)-quinazolinone；
(3) under nitrogen protection, HTPQ and pyridine are dissolved in solvent, react 10min, Ran Houhuan under being stirred in ice bath
It is slow to add POCl3, stirring reaction 2h under normal temperature.Reaction solution is subjected to extraction and collects organic phase, organic phase depressurizes rotation after drying
Extractant is boiled off, again mistake post separation after dissolving crude product, the mixed solution of dichloromethane and ethanol is eluant, eluent, is obtained
Alkaline phosphatase fluorescence probe HTPQA.
The ratio between 4- hydroxyls-tetraphenyl ethylene, Wu Luotuo amount of material is 1 in the step (1):(1~1.5).
The volume ratio of dichloromethane and petroleum ether is 1 in the step (1):(2~10).
Faint yellow intermediate, 2-Amino-5-chlorobenzamide, p-methyl benzenesulfonic acid and dichloro dicyano in the step (2)
The ratio between amount of material of benzoquinones is 1:(1~1.5):(0.1~0.5):(1.5~3).
The ratio between HTPQ, pyridine and POCl3 amount of material is 1 in the step (3):(1~1.5):(1~1.3).
Extractant is dichloromethane, the mixture of second alcohol and water in the step (3), and its volume ratio is 2:(1~2):
2。
The volume ratio of dichloromethane and ethanol is 1 in step (3) eluant, eluent:(1~50).
A kind of sedimentation type alkaline phosphatase fluorescence probe of present invention detection of alkaline phosphatase activity in cushioning liquid in vitro
Application.
A kind of application of sedimentation type alkaline phosphatase fluorescence probe of present invention detection of alkaline phosphatase activity in living cells.
The present invention is one as a result of New Solid fluorescent dye HTPQ, HTPQ and is based on intermolecular proton transfer mechanism
Solid state fluorescence dyestuff, its photoluminescent property can be by regulating and controlling to its hydroxyl protection and deprotection.Institute has been synthesized using HTPQ
The sedimentation type alkaline phosphatase fluorescence probe HTPQA stated, the fluorescence probe have preferably water-soluble, fluorescence probe and alkalescence
After phosphatase enzymes, fluorogen HTPQ is discharged, due to strong hydrophobic effect and then aggregate and precipitate, produces very strong solid state fluorescence letter
Number.The fluorescence probe is difficult diffusion with that can generate dye precipitated, fluorescence signal after alkaline phosphatase enzyme reaction in the original location, realizes alkali
The in situ detection and high-resolution of acid phosphatase, the fluorescence imaging of high-contrast.
Compared with prior art, the advantageous effects having are the present invention：
1) synthetic method of the fluorescence probe is simple, and the fluorescence probe has solid luminescent property, can effectively reduce
Background fluorescence signal, improve the sensitivity of probe；
2) fluorescence probe can respond saturation to the response quickly of alkaline phosphatase in 20min；
3) fluorescence probe is good to the selectivity of alkaline phosphatase；
4) fluorescence probe is fine to living cells Color, and dyeing time is shorter, and staining efficiency is high；
5) fluorescence probe can be to endogenous cellular alkaline phosphatase in situ detection；
6) the fluorescence probe good light stability, fluorescence signal is stable in living cells imaging, indiffusion.
Brief description of the drawings
Fig. 1 is the reaction equation of fluorescence probe of the present invention synthesis.
Fig. 2 is the fluorescence emission spectrum of fluorescence probe of the present invention with the increased change curve of alkaline phosphatase activities, horizontal stroke
Coordinate is wavelength (nm), and ordinate is fluorescent emission intensity.
Fig. 3 is the fluorescence probe and alkaline phosphatase polymerization kinetics curves of the present invention, and abscissa is the reaction time
(min), ordinate is fluorescent emission intensity.
Fig. 4 is that (dyeing liquor is to alkaline phosphatase co-focusing imaging detection figure on living cells film for the fluorescence probe of the present invention
TBS buffer solutions, pH 7.4).
Fig. 5 is the real-time co-focusing imaging figure after the fluorescence probe of the present invention dyes to living cells.
Fig. 6 is the fluorescence probe of the present invention to alkaline phosphatase co-focusing imaging detection figure (dyeing liquor in living cells endochylema
For NaH containing 1mM2PO4TBS buffer solutions, pH 7.4).
Fig. 7, which is that the fluorescence probe light of the present invention is stable, investigates figure.
Embodiment
With reference to instantiation, the present invention is further elaborated, but the present invention is not limited to following examples.It is described
Method is conventional method unless otherwise instructed, and the raw material such as can obtain without explanation from open commercial sources.
The sedimentation type alkaline phosphatase fluorescence probe HTPQA of embodiment 1 synthesis
(1) 4- hydroxyls-tetraphenyl ethylene (3.482g) and methenamine (2.102g) are dissolved in glacial acetic acid (50mL), heated
The lower 3h of backflow, dichloromethane and petroleum ether are that (volume ratio of dichloromethane and petroleum ether is 1 to eluant, eluent:5) post separation is crossed to obtain
Faint yellow intermediate (1.316g, 35%)；
(2) by faint yellow intermediate (0.3761g), 2-Amino-5-chlorobenzamide (0.2050g) and p-methyl benzenesulfonic acid
(10mg) return stirring 5h in absolute ethyl alcohol, adds DDQ (0.5g) after cooling, suction filtration obtains consolidating for yellow
Body, solid, drying to pure yellow compound (0.43g, 81.5%), i.e. New Solid fluorescent dye are washed with ethanol
That is the chloro- 2- of 6- (2- hydroxyls -5- (1,2,2- triphenyl vinyl) phenyl) -4 (3 hydrogen)-quinazolinones, are named as HTPQ；
HTPQ mass spectrum and nuclear-magnetism characterizes：
1HNMR(DMSO-d6,400MHz)δ(ppm):δ 13.22 (s, 1H), 12.49 (s, 1H), 8.05 (d, J=8.04,
1H), 7.93 (d, J=7.93,1H), 7.86 (dd, J=7.85,1H), 7.76 (d, J=7.76,1H), 7.15-7.13 (m, J=
7.14Hz, 8H), 7.08-6.99 (m, J=7.02Hz, 8H), 6.80 (d, J=6.79Hz, 1H)
13CNMR(101MHz,Chloroform-d):δ159.63,154.57,148.01,144.92,143.87,
141.92,137.61,135.63,134.75,133.03,132.00-130.37(m),129.61-127.38(m),127.28-
125.50(m),123.91,118.42.
MS(EI):m/z 526.1,calcd 526.1.
(3) under nitrogen protection, by the chloro- 2- of compound 6- (2- hydroxyls -5- (1,2,2- triphenyl vinyl) phenyl) -4
(3 hydrogen)-quinazolinone (HTPQ, 0.2631g) is dissolved in 50mL dry methylene chloride, adds anhydrous pyridine (0.15mL),
10min is stirred in ice bath, is then slowly added into POCl3 (0.12mL), stirring reaction 2 hours under normal temperature.After completion of the reaction,
Vacuum rotary steam removes solvent, is dissolved again with ethanol, column chromatography for separation (eluant, eluent：The volume ratio of dichloromethane and ethanol is 30:
1) white solid (0.24g, 39%) is obtained, is named as HTPQA, the reaction equation that it is synthesized is as shown in Figure 1.
Mass spectrum and nuclear-magnetism characterize：
1HNMR(DMSO-d6,400MHz)δ(ppm):δ 14.4 (s, 1H), 8.02 (s, 1H), 7.80 (d, J=7.79,
1H), 7.62 (d, J=7.61,1H), 7.43 (s, 1H), 7.20 (d, J=7.19,1H), 7.015-7.03 (m, J=7.09Hz,
14H), 6.99 (d, J=6.99Hz, 2H), 4.37 (br s, 2H)；
13CNMR(101MHz,DMSO-d6):δ161.07,153.00,148.21,144.67-142.44(m),141.41,
139.72,138.59,134.61 (d, J=29.9Hz), 133.64,131.93-130.62 (m), 130.10,128.85-
127.74 (m), 127.06 (d, J=17.2Hz), 125.20,123.78,122.63,99.98；
HRMS(ESI):m/z 637.2984,[M+CH3OH-H]-,calcd.606.1111.
Detection of the embodiment 2HTPQA probes in test tube to alkaline phosphatase
HTPQA probes are made into 1mM DMSO stock solutions, preserved at -20 DEG C.Detection architecture is TBS cushioning liquid
(10mM, pH 8.0, containing 5% DMSO).The reaction system of HTPQA probes and alkaline phosphatase is swayed 30 points at 37 DEG C
Clock, then measure its fluorescence emission spectrum.The excitation wavelength for setting luminoscope is 410nm, and launch wavelength range of receiving is 500-
700nm.Its result is as shown in Fig. 2 figure it is seen that the HTPQA probes have good response to alkaline phosphatase.
The kinetics of embodiment 3HTPQA probes and alkaline phosphatase is investigated
Reaction system is TBS cushioning liquid (10mM, pH8.0, containing 5% DMSO).By HTPQA probes and different activities
Alkaline phosphatase measures first order fluorescence emission spectrum at intervals of two minutes at 37 DEG C, until fluorescence intensity no longer changes.Then with
The intensity of maximum emission peak is ordinate, and the reaction time maps for abscissa, obtains polymerization kinetics curves as shown in Figure 3,
From figure 3, it can be seen that response quickly of the HTPQA probes to phosphatase.The excitation wavelength for setting luminoscope is 410nm, hair
The long range of receiving of ejected wave is 500-700nm.
Embodiment 4HTPQA probes dye imaging experiment to living cells
A) HeLa and HepG2 cells are seeded in optics culture dish in advance, 40,000 cells is inoculated with each ware, be incubated
24h, original culture medium (DMEM, dual anti-containing 5%FBS and 10%) is then sucked, add TBS buffer solutions (10mM, pH 7.4).
By the HTPQA probes with after cell incubation 40 minutes, sucking original culture medium, and cleaned 3 times with TBS, be copolymerized with laser
Focusing microscope detects its fluorescence signal.Its result is as shown in figure 4, from fig. 4, it can be seen that the HTPQA probes in situ can be examined
The alkaline phosphatase surveyed on cell membrane.
B) HeLa and HepG2 cells are seeded in optics culture dish in advance, 40,000 cells is inoculated with each ware, be incubated
24h, original culture medium (DMEM, dual anti-containing 5%FBS and 10%) is then sucked, add TBS buffer solutions (10mM, pH 7.4).
The probe is added in cell, its fluorescence signal is detected with laser confocal microscope.Its result is as shown in figure 5, can from Fig. 5
To find out, the stable indiffusion of fluorescence signal of the HTPQA probes.
C) HeLa and HepG2 cells are seeded in optics culture dish in advance, 40,000 cells is inoculated with each ware, be incubated
24h, then sucks original culture medium (DMEM, dual anti-containing 5%FBS and 10%), add TBS buffer solutions (10mM, pH 7.4,
NaH containing 1mM2PO4).By the HTPQA probes with after cell incubation 40 minutes, sucking original culture medium, and 3 are cleaned with TBS
It is secondary, detect its fluorescence signal with laser confocal microscope.Its result is as shown in fig. 6, from fig. 6, it can be seen that the HTPQA is visited
Pin being capable of the intracytoplasmic alkaline phosphatase of in situ detection.
Embodiment 5HTPQA probe light study on the stability
A) by solid luminescent dyestuff (HTPQ) and commercial fluorescence dyestuff (MitoTracker Deep Red) in identical
Under the conditions of carry out real-time fluorescence scanning, as a result as shown in Figure 7 A, as can be seen from Figure 7A, the solid luminescent dyestuff (HTPQ) tool
There is good photostability.
B) by HTPQA probes with utilizing the fluorescence that commercial fluorescence dyestuff (MitoTracker Deep Red) is prepared
Probe is incubated with cell under the same conditions, and real-time fluorescence imaging is carried out after being incubated 1 hour, as a result as shown in Figure 7 B,
As can be seen from Figure 7B, the fluorescence signal photobleaching of the HTPQA probes is few, good light stability.
Claims (10)
1. a kind of sedimentation type alkaline phosphatase fluorescence probe, it is characterised in that the structural formula of the fluorescence probe is as follows：
2. a kind of synthetic method of sedimentation type alkaline phosphatase fluorescence probe, comprises the following steps：
(1) it is that 4- hydroxyls-tetraphenyl ethylene and methenamine are dissolved in glacial acetic acid by compound 1, is heated to reflux lower 3h, dichloromethane
Post separation is crossed for eluant, eluent obtain faint yellow intermediate with petroleum ether；
(2) by above-mentioned faint yellow intermediate, 2-Amino-5-chlorobenzamide and the p-methyl benzenesulfonic acid return stirring in absolute ethyl alcohol
5h, adds DDQ after cooling, suction filtration obtains the solid of yellow, solid, drying to yellow are washed with ethanol
New Solid fluorescent dye HTPQ；
(3) under nitrogen protection, HTPQ and pyridine are dissolved in solvent, react 10min under being stirred in ice bath, then slowly added
Enter POCl3, stirring reaction 2h under normal temperature.Reaction solution is subjected to extraction and collects organic phase, vacuum rotary steam is gone after organic phase is dried
Fall extractant, again mistake post separation after dissolving crude product, the mixed solution of dichloromethane and ethanol is eluant, eluent, obtains alkalescence
Phosphatase fluorescence probe HTPQA.
3. the synthetic method of sedimentation type alkaline phosphatase fluorescence probe according to claim 2, it is characterised in that the step
Suddenly the ratio between 4- hydroxyls-tetraphenyl ethylene, Wu Luotuo amount of material is 1 in (1):(1~1.5).
4. the synthetic method of sedimentation type alkaline phosphatase fluorescence probe according to claim 2, it is characterised in that the step
Suddenly the volume ratio of dichloromethane and petroleum ether is 1 in (1):(2~10).
5. the synthetic method of sedimentation type alkaline phosphatase fluorescence probe according to claim 2, it is characterised in that the step
Suddenly the amount of the material of faint yellow intermediate in (2), 2-Amino-5-chlorobenzamide, p-methyl benzenesulfonic acid and DDQ
The ratio between be 1:(1~1.5):(0.1~0.5):(1.5~3).
6. the synthetic method of sedimentation type alkaline phosphatase fluorescence probe according to claim 2, it is characterised in that the step
Suddenly the ratio between HTPQ, pyridine and POCl3 amount of material is 1 in (3):(1~1.5):(1~1.3).
7. the synthetic method of sedimentation type alkaline phosphatase fluorescence probe according to claim 2, it is characterised in that the step
Suddenly extractant is dichloromethane, the mixture of second alcohol and water in (3), and its volume ratio is 2:(1~2):2.
8. the synthetic method of sedimentation type alkaline phosphatase fluorescence probe according to claim 2, it is characterised in that the step
Suddenly the volume ratio of dichloromethane and ethanol is 1 in (3) eluant, eluent:(1~50).
9. a kind of sedimentation type alkaline phosphatase fluorescence probe according to claim any one of 1-8 is in vitro in cushioning liquid
The application of detection of alkaline phosphatase activity.
10. a kind of sedimentation type alkaline phosphatase fluorescence probe according to claim any one of 1-8 detects in living cells
The application of alkaline phosphatase activities.
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| CN108948081A (en) * | 2018-07-12 | 2018-12-07 | 青岛科技大学 | A kind of Ratiometric fluorescent probe measuring alkaline phosphatase and its synthetic method and application |
| CN109053802A (en) * | 2018-09-01 | 2018-12-21 | 青岛科技大学 | A kind of Ratio-type near infrared fluorescent probe and its synthetic method and application |
| CN109369719A (en) * | 2018-11-05 | 2019-02-22 | 深圳大学 | A kind of molecular probe and preparation method and application for alkaline phosphatase detection |
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| CN108948081B (en) * | 2018-07-12 | 2020-07-07 | 青岛科技大学 | Ratiometric fluorescent probe for determining alkaline phosphatase, and synthetic method and application thereof |
| CN109053802A (en) * | 2018-09-01 | 2018-12-21 | 青岛科技大学 | A kind of Ratio-type near infrared fluorescent probe and its synthetic method and application |
| CN109053802B (en) * | 2018-09-01 | 2020-06-23 | 青岛科技大学 | Ratio type near-infrared fluorescent probe and synthetic method and application thereof |
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| CN109369719B (en) * | 2018-11-05 | 2021-06-01 | 深圳大学 | Molecular probe for alkaline phosphatase detection and preparation method and application thereof |
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