CN104946248B - Water-soluble sulphurous acid hydrogen root ratiometric fluorescent probe and application thereof - Google Patents
Water-soluble sulphurous acid hydrogen root ratiometric fluorescent probe and application thereof Download PDFInfo
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- CN104946248B CN104946248B CN201510400148.9A CN201510400148A CN104946248B CN 104946248 B CN104946248 B CN 104946248B CN 201510400148 A CN201510400148 A CN 201510400148A CN 104946248 B CN104946248 B CN 104946248B
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- probe
- acid hydrogen
- sulphurous acid
- hydrogen root
- benzimidazole
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Abstract
The invention discloses a water-soluble sulphurous acid hydrogen root ratiometric fluorescent probe. The probe is 3-((3'-benzimidazole)-carbonyl propylene)-7-diethylin coumarin containing alpha, beta-unsaturated ketone, and the chemical structural formula is displayed as a first formula. The probe and a sulphurous acid hydrogen root can be selectively acted, red color is changed into blue color, and naked eye distinguishment can be achieved, a fluorescent ratio response effect is obtained, and analysis can be conducted through ultraviolet absorption and the fluorospectrophotometry. Sulphurous acid hydrogen root ions can be detected in a ratio mode, ratio imaging inside cells is achieved, and the content of the sulphurous acid hydrogen root in a liquid sample can rapidly detected by made test strips. The probe is expected to play a role in industrial manufacture and food detection and biological science, a wide application prospect is obtained.
Description
Technical field
The present invention relates to a kind of bisulfite fluorescence probe, more particularly to a kind of water-soluble bisulfite ratio fluorescent
Probe and its application；Belong to organic molecule fluorescence probe field.
Background technology
Coumarin fluorescent group itself has larger molar extinction coefficient and quantum yield, can reach after modification near red
It is outer luminous, can be used for luminescent material, chemical sensor and mark biomolecule.It is sub- using ratio fluorescent effect detection in recent years
Bisulfate ion, and the research of imaging is constantly developed in active somatic cell.
Sulphite is worldwide widely used food additives, can serve as food preservative and bleaching
Agent, is highly effective antiseptic.Human body in metabolic process from can also produce sulfurous acid ion, but the sulfurous of high concentration
Hydrochlorate can cause people's diarrhoea, allergy, blood pressure reduction, asthma etc., WHO to specify that the permission intake of everyone daily sulphite is 0-
0.7mg/kg (in terms of sulfur dioxide).Substantial amounts of sulfur dioxide, the acid rain heavy corrosion of formation are now discharged in the industrial production
Building and crops, and rivers and lakes can be caused excessively to be acidified.Therefore convenient and swift detection is sub- in biological and actual sample
Sulfate needs keeping punching for scientific researcher.
The detection method of the sulphite for currently existing has chromatography, sensor method, electrochemical process, Capillary Electrophoresis
With zymotechnic combination etc., but these methods have a significant limitations, such as expensive equipment, process complicated, time-consuming, be unfavorable for inspection
Survey.In the patent applied【CN102072901B】、【CN103743726A】During large-scale instrument detection food can not be used
Sulphite, but it is only used for the detection of liquid-like.And emerging fluorescence method is the features such as have easy to operate, sensitivity is high.
The patent for having authorized【CN102924530B】、【CN102659677B】The probe of specific detection sulphite is illustrated, but
Be respectively present mono signal detection and cannot cell imaging shortcoming.Based on this, exploitation has cell imaging ability, and can be with
Ratio test, the bisulfite fluorescence probe of bore hole identification become problem urgently to be resolved hurrily at present.
The content of the invention
For the deficiencies in the prior art, it is glimmering that the problem to be solved in the present invention is to provide a kind of water-soluble bisulfite ratio
Light probe and its application.
Water-soluble bisulfite ratio fluorescent probe of the present invention, it is characterised in that：The fluorescence probe is to contain
α, 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins of alpha, beta-unsaturated ketone, its chemical structural formula such as formula (I)
It is shown,
The preparation method of above-mentioned water-soluble bisulfite ratio fluorescent probe is：By known method synthesize 3- aldehyde radicals-
7- diethylaminocoumarins and 5- acetyl group benzimidazoles, then the two reaction obtains 3- ((3 '-benzimidazole)-carbonyls third
Alkene) -7- diethylaminocoumarins, mono-crystalline structures such as Fig. 1.
Application of the above-mentioned water-soluble bisulfite ratio fluorescent probe in detection sample containing bisulfite.
In above-mentioned application：The sample containing bisulfite is preferably biological cell, the food containing bisulfite or contains
The solution of bisulfite.
Water-soluble bisulfite ratio fluorescent probe of the present invention is preparing bisulfite open hole detection test paper
In application.
Probe 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins of the present invention can be with sulfurous
There is in aqueous mikey addition reaction in sour hydrogen radical, block conjugated system, as shown in Fig. 2 cumarin blue-fluorescence is discharged,
So as to reach the effect of detection.
Specifically：Prepare the PBS of 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins
The solution of (CTAB 1mM, pH=7.4), is quantitatively adding respectively the Cys of micro updating, GSH, mercaptoethanol, PA,
HSO3 -,CH3CO2 -,ClO3 -,CN-,CO3 2-,H2PO4 -,HCO3 2-,HPO4 2-,F-,Cl-,Br-,I-,IO3-,NO2-,NO3 -,S2-,
S2O3 2-,SO3 2-,SO4 2-The aqueous solution.It is tried to different sulphur by ultraviolet-visible spectrophotometry and Tissue with Spectrofluorometric
Alcohol, the selectivity of Common Anions and responding ability.As a result probe 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethyls are shown
Amido cumarin has good selectivity to bisulfite, adds bisulfite cross-reference to show that fluorescence emission wavelengths are sent out
The blue shift of 147nm is given birth to, with extraordinary rate responsive.See Fig. 3,4.
In the A549 living cells of 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins for adding 1.0 μM
In be added without NaHSO3With addition NaHSO3Compared with control cells fluorescent staining micro-imaging changes.Fluorescence imaging shows 3- ((3 '-benzos
Imidazoles)-carbonyl propylene) -7- diethylaminocoumarins penetrate into it is intracellular.Control group A cell blue channel unstressed configuration, redness is logical
Road light tone fluorescence.Control group B blue channels show fluorescence, red channel fluorescent quenching.See Fig. 5.
In variable concentrations NaHSO33- ((3 '-benzimidazole)-carbonyl propylene) -7- of 0.01mg/mL is added in the aqueous solution
Diethylaminocoumarin filter paper, change in fluorescence under observation visible ray and uviol lamp.As a result show and increase with concentration of sodium bisulphite,
Test paper is changed into light blue from pale red under visible ray, and test paper is changed into blue from redness under uviol lamp.See Fig. 6.
Water-soluble bisulfite ratio fluorescent probe 3- ((3 '-benzimidazole)-carbonyl propylene) -7- that the present invention is provided
Diethylaminocoumarin can optionally with bisulfite act on, be changed into light blue from redness, can discernable by eye, with glimmering
Light rate responsive effect, it is also possible to be analyzed by UV absorption and fluorimetry.Not only can ratio test sulfurous acid
Hydrogen radical ion, realizes intracellular ratio imaging, and the test strips made can open hole detection liquid-like sulfite rapidly
Hydrogen radical content.It is expected to be played a role in industrial production and food inspection and bioscience, has broad application prospects.
Description of the drawings
Fig. 1 is 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarin mono-crystalline structures schematic diagrames.
Fig. 2 is 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins and sodium hydrogensulfite addition compound product
High resolution mass spectrum.
Fig. 3 for 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins PBS (CTAB 1mM,
PH=7.4 solution) adds the ultraviolet absorpting spectrum of Common Anions and various mercaptan.
Fig. 4 for 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins PBS (CTAB 1mM,
PH=7.4 solution) adds the fluorescent emission collection of illustrative plates of Common Anions and various mercaptan.
Fig. 5 is that 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins are micro- in A549 intracellular Fluorescences
Image.
Wherein：A groups are that under the conditions of 37 DEG C, A549 cells are in 3- ((3 '-benzimidazole)-carbonyls third for adding 1.0 μM
Alkene) -7- diethylaminocoumarins cell culture fluid in cultivate 1 hour；B groups are A549 cells in 2- (the 3- second for adding 1.0 μM
Acyl group) cumarin base acrylate cell culture fluid in cultivate 1 hour, PBS cushioning liquid wash three times after, then containing again
There are 5mM NaHSO3Cell culture fluid in soak the image of 0.5 hour.
Fig. 6 is 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins as detection bisulfite
The photo that test paper is obtained under visible ray and uviol lamp.
Wherein：Soaked with 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins solution of 0.01mg/mL
Test strips are made in filter paper, drying, are then immersed in the aqueous solution of sodium bisulfite (a of variable concentrations:0,b:1×10-5M,c:1×10-4M,d:1×10-3M,e:1×10-2M,f:1×10-1M), take pictures under visible ray and uviol lamp.
Specific embodiment
Embodiment 1
3- aldehyde radical -7- diethylaminocoumarins and 5- acetyl group benzimidazoles are synthesized by known method, then the two reaction
Obtain 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins with red fluorescence.Specifically：
By 3- aldehyde radicals -7- diethylaminocoumarins (1,5.0mmol), 5- acetyl group benzimidazoles (2,7.5mmol) and 1mL
Piperidines is added in 100mL absolute ethyl alcohols, is heated to backflow, and reaction is cooled to room temperature, has red solid to separate out after 9 hours, suction filtration,
Ethanol is washed three times, and crude product ethyl alcohol recrystallization obtains bright red solid and is 3- ((3 '-benzimidazole)-carbonyl propylene) -7-
Diethylaminocoumarin (BICO), fusing point：287-292 DEG C, yield 61%.Monocrystalline is shown in Fig. 1.
Infrared analysis (KBr, cm-1):3250(NH),3063(ArH),2969(CH2CH3), 1721 (C=O), 1563 (C=
C)。
Nuclear magnetic resonance hydrogen spectruming determining：1H NMR(400MHz,DMSO-d6):δ=1.09 (t, J=7.0Hz, 6H, CH3),
3.47 (q, J=7.0Hz, 4H, CH2), 6.60 (d, J=2.0Hz, 1H, ArH), 6.78 (dd, J=2.0and 9.0Hz, 1H,
), ArH 7.35-7.37 (m, 2H, ArH), 7.54 (d, J=9.0Hz, 1H, ArH), 7.59 (s, 1H, ArH), 7.83 (d, J=
15.8Hz, 1H ,-CO-CH=), 7.88 (s, 1H, ArH), 8.40 (d, J=15.8Hz, 1H ,=CH-Ar), 8.45 (s, 1H, 4-H
of coumarin),13.39(s,1H,NH)。
Carbon-13 nmr spectra is determined：13C NMR(100MHz,DMSO-d6):12.83,44.85,96.67,108.99,
110.43,113.46,121.27,131.43,140.31,147.25,149.83,152.62,175.04,160.15,181.51。
High resolution mass spectrum：m/z[M+H]+calcd for C23H22N3O3:388.1661,found 388.1670。
3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins can be with bisulfite in aqueous
Generation mikey addition reaction, blocks conjugated system, discharges cumarin blue-fluorescence.Its course of reaction is shown below：
Embodiment 2
With micro syringe respectively to filling 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylin that 10mL is prepared
In the solution of the PBS (CTAB 1mM, pH=7.4) of cumarin, the Cys of 10 equivalents, GSH, sulfydryl are quantitatively adding respectively
Ethanol, PA, HSO3 -,CH3CO2 -,ClO3 -,CN-,CO3 2-,H2PO4 -,HCO3 2-,HPO4 2-,F-,Cl-,Br-,I-,IO3-,
NO2-,NO3 -,S2-,S2O3 2-,SO3 2-,SO4 2-The aqueous solution, effect carries out ultraviolet-visible spectrophotometry and fluorescence point after 1 hour
Light photometry is tested, and shows probe 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins to bisulfite
There is good selectivity, add bisulfite cross-reference to show that fluorescence emission wavelengths there occurs the blue shift of 147nm, with non-
Often good rate responsive.See Fig. 3,4.
Embodiment 3
Intracellular Fluorescence imaging test：
Two groups of A, B are control group, control group A：Under the conditions of 37 DEG C, A549 cells are in 3- ((the 3 '-benzos for adding 1.0 μM
Imidazoles)-carbonyl propylene) -7- diethylaminocoumarins cell culture fluid in cultivate 1 hour；Control group B：A549 cells are adding
Cultivate 1 hour in the cell culture fluid of 2- (3- acetyl group) the cumarin base acrylate for entering 1.0 μM, the washing of PBS cushioning liquid
After three times, then again containing 5mM NaHSO3Cell culture fluid in soak 0.5 hour.
It is intracellular that fluorescence imaging shows that 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins are penetrated into.
Control group A cell blue channel unstressed configuration, red channel light tone fluorescence.Control group B blue channels show fluorescence, red channel fluorescence
It is quenched.See Fig. 5.
Embodiment 4
The probe of the present invention is tested as bisulfite test paper：
Filter paper is soaked with 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins solution of 0.01mg/mL,
Test strips are made in drying, are then immersed in the aqueous solution of sodium bisulfite (a of variable concentrations:0,b:1×10-5M,c:1×10-4M,d:
1×10-3M,e:1×10-2M,f:1×10-1M), take pictures under visible ray and uviol lamp.
As a result：Increase with concentration of sodium bisulphite, it is seen that test paper is changed into light blue from pale red under light, test paper under uviol lamp
It is changed into blue from redness.See Fig. 6.
Claims (1)
1. application of a kind of water-soluble bisulfite ratio fluorescent probe in bisulfite open hole detection test paper is prepared, its
In：The fluorescence probe is to contain α, 3- ((3 '-benzimidazole)-carbonyl propylene) -7- diethylaminocoumarins of alpha, beta-unsaturated ketone,
Shown in its chemical structural formula such as formula (I),
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| CN106749142B (en) * | 2016-11-15 | 2019-01-25 | 山西大学 | A kind of SO32-/HSO3-Detection reagent and its synthetic method and application |
| CN108623575B (en) * | 2017-03-21 | 2020-04-10 | 山东第一医科大学(山东省医学科学院) | Simple and effective fluorescent probe for detecting sulfite |
| CN106946902B (en) * | 2017-04-11 | 2019-03-01 | 济南大学 | A kind of sulfur dioxide near-infrared-two-photon ratio fluorescent probe and preparation method thereof |
| CN107474044B (en) * | 2017-08-10 | 2019-12-06 | 河北大学 | Coumarin derivative, preparation method and application thereof, and bisulfite detection kit |
| CN108285448A (en) * | 2017-10-12 | 2018-07-17 | 泰山医学院 | Pyrido [1,2-a] the benzimidazole SO of benzimidazole modification32-Ratio fluorescent probe and its application |
| CN108102647A (en) * | 2018-03-06 | 2018-06-01 | 泰山医学院 | Imidazopyridine cyanofuran class inferior sulfate radical fluorescence probe and its application |
| CN108484625A (en) * | 2018-05-30 | 2018-09-04 | 济南大学 | A kind of colorimetric fluorescence probe of quick high-selectivity sensitive analysis bisulfite |
| CN109187455B (en) * | 2018-08-13 | 2021-10-15 | 天津理工大学 | Kit for detecting content of sulfite in food and application thereof |
| CN110655510B (en) * | 2019-09-12 | 2021-12-10 | 山东大学 | Sulfite ratiometric fluorescent probe targeting lipid droplets and application thereof |
| CN111380845A (en) * | 2020-03-06 | 2020-07-07 | 谭锐 | Application of coumarin probe in quantitative detection of sulfur dioxide |
| CN113372282B (en) * | 2021-07-01 | 2023-03-24 | 齐齐哈尔大学 | Fluorescent probe for detecting bisulfite ions in real time and preparation method and application thereof |
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| CN102659677B (en) * | 2012-04-28 | 2014-09-10 | 中南大学 | Fluorescent molecular probe for detecting sulfite ions through naked eyes and fluorescence ratio as well as synthesis and application thereof |
| CN102924530B (en) * | 2012-11-22 | 2015-03-11 | 中山大学 | Iridium complex as well as preparation method and application thereof to detection of sulfur dioxide |
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