CN105802608A - Preparation method of fluorescent probe and application of fluorescent probe in aluminum ion detection - Google Patents
Preparation method of fluorescent probe and application of fluorescent probe in aluminum ion detection Download PDFInfo
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- CN105802608A CN105802608A CN201610128435.3A CN201610128435A CN105802608A CN 105802608 A CN105802608 A CN 105802608A CN 201610128435 A CN201610128435 A CN 201610128435A CN 105802608 A CN105802608 A CN 105802608A
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
- C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
- C07C281/08—Compounds containing any of the groups, e.g. semicarbazides the other nitrogen atom being further doubly-bound to a carbon atom, e.g. semicarbazones
- C07C281/14—Compounds containing any of the groups, e.g. semicarbazides the other nitrogen atom being further doubly-bound to a carbon atom, e.g. semicarbazones the carbon atom being further bound to a carbon atom of a six-membered aromatic ring
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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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- C—CHEMISTRY; METALLURGY
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1011—Condensed systems
Abstract
The invention provides a preparation method of a fluorescent probe and an application of the fluorescent probe in aluminum ion detection. The fluorescent probe is obtained by virtue of a reaction in a heating reflux manner between 2-hydroxyl-1-naphthaldehyde and carbohydrazide. In the structure of the fluorescent probe provided by the invention, the N atoms on the C=N double bonds are influenced by a photo-induced electron transfer effect, and after the N atoms are combined with aluminum ions, the photo-induced electron transfer effect is inhibited, and planar conjugation is enhanced at the same time; two mechanisms together induce obvious enhancement of fluorescence emission; the fluorescent probe has good selectivity and sensitivity to the aluminum ions. High-selectivity identification of the aluminum ions is realized, a relatively good linear relation exists in a range of 5*10<-6> to 4*10<-5> mol/L, and the limit of detection is as low as 1.89*10<-8> mol/L, so that the fluorescent probe has a chromogenic sensing function.
Description
Technical field
The present invention relates to metal ion analysis detection technique field, particularly to the preparation of a kind of fluorescent probe
Method and the application in aluminium ion detects thereof.
Background technology
Aluminum is the metallic element that in the earth's crust, content is the abundantest, is widely used in our daily life simultaneously,
But, if the aluminium ion that human body absorbs excess may result in alzheimer's disease, parkinson and renal failure
A series of diseases such as exhaust.Therefore, the most easily and fast, in high sensitivity and dynamic measurement cell aluminum from
The concentration of son, it has also become the forward position heat that the subjects such as current chemistry, biology, basis and clinical medicine are important
Point research topic.Traditional metal ion inspection, such as cold-vapour atomic absorption method, XRF luminosity
Method, mass spectrum ICP-AES, Cold Atomic Fluorescent Mercury photometry and anode are molten
Go out voltammetry, all there is the shortcomings such as expensive equipment, analytical procedure is complicated, operating cost is high, it is difficult to meet
Being actually needed of field quick detection.And fluorescent molecular probe, due to have highly sensitive, selectivity good,
Without reference, do not affected by electric field, magnetic field, can be used in situ detection and utilize optical fiber to carry out long distance
Greatly concern is received from monitoring and other advantages.
Owing to fluorimetry has highly sensitive, selectivity is good, to sample without destroying, to cell injury
The advantage such as little, therefore fluorescent molecular probe has been widely used for the detection of metal ion.Nearly ten years, inspection
The small-molecule fluorescent probe of check weighing metal and transition metal ions has obtained great development.Wherein enhancement mode
Ion fluorescence probe has obtained very big concern, because compared to fluorescent quenching type probe, enhancement mode probe is more
It is suitably applied living things system.But much metal ion (such as mercury ion and copper ion) is to fluorescent dye
There is stronger quenching effect.Therefore, design synthesis Fluorescence Increasing type metalfluorescent probe has become and has ground at present
The focus studied carefully and difficult point.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of fluorescent probe and in aluminium ion detects
Application.The fluorescent probe that the present invention provides has good selectivity and sensitivity to aluminium ion, it is achieved that
To aluminum ions high Selective recognition, it it is a kind of fluorescent probe with the sensing function that adds lustre to.
The invention provides the application in aluminium ion detects of a kind of fluorescent probe, described fluorescent probe is tool
There is trans 2-((2 hydroxy naphthalene-1-base) methylene) the hydrazine carbonyl hydrazide of structure shown in Formulas I,
Preferably, the detection of described aluminium ion comprises the following steps:
(1) by fluorescent probe and aqueous solution to be measured, or cultivate after mix with cell culture medium, detect
Solution to be measured or the fluorescence intensity of cell to be measured；
(2) fluorescence intensity obtained according to step (1) and predetermined linearity curve, obtain to be measured water-soluble
Aluminum ions concentration in liquid or cell to be measured.
Preferably, described linearity curve is the linearity curve of aluminium ion concentration and fluorescence intensity.
Present invention also offers the preparation method of the fluorescent probe of structure shown in a kind of Formulas I, including following step
Rapid:
2-hydroxyl-1-naphthaldehyde and carbohydrazide are carried out in the mixed solvent of second alcohol and water back flow reaction had
There is trans 2-((2 hydroxy naphthalene-1-base) methylene) the hydrazine carbonyl hydrazide of structure shown in Formulas I.
Preferably, the reaction system of described back flow reaction is by 2-hydroxyl-1-naphthaldehyde ethanol solution and carbohydrazide water
Solution is mixed to get.
Preferably, described 2-hydroxyl-1-naphthaldehyde ethanol solution is prepared by following methods:
In a heated condition, 2-hydroxyl-1-naphthaldehyde being dissolved in dehydrated alcohol, the temperature of described heating is
40～50 DEG C, the time of described heating is 25～35min.
Preferably, the molar concentration of described 2-hydroxyl-1-naphthaldehyde ethanol solution is 0.04～0.08mol/L.
Preferably, the molar concentration of described carbohydrazide aqueous solution is 0.2～0.3mol/L.
Preferably, described 2-hydroxyl-1-naphthaldehyde is 1: 3～5 with the mol ratio of carbohydrazide.
Preferably, the temperature of described back flow reaction is 40～50 DEG C, and the time of back flow reaction is 4～5h.
The invention provides the preparation method of a kind of fluorescent probe and the application in aluminium ion detects thereof.This
In the fluorescent probe structure that invention provides, the atom N in C=N double bond is by photoinduced electron transfer effect
Impact, inhibits photoinduced electron transfer effect with aluminium ion after being combined, and enhances plane altogether simultaneously
Yoke, two kinds of mechanism jointly cause fluorescent emission to be remarkably reinforced, have good selectivity and spirit to aluminium ion
Sensitivity, it is achieved that to aluminum ions high Selective recognition, is that a kind of fluorescence with the sensing function that adds lustre to is visited
Pin.Test result indicate that, the fluorescent probe that the employing present invention prepares is when detecting aluminium ion, 5
×10-6～4 × 10-5Mol/L scope has preferable linear relationship, detection limit as little as 1.89 × 10-8mol/L。
The present invention passes through 2-hydroxyl-1-naphthaldehyde and carbohydrazide in the mixed solvent of second alcohol and water to be heated to reflux
Mode is reacted, and obtains trans 2-((2 hydroxy naphthalene-1-base) methylene) hydrazine carbonyl hydrazide.The present invention provides
Preparation method reaction condition gentle, simple and effective, productivity is up to 81%.
Accompanying drawing explanation
Fig. 1 is 2-hydroxyl-1-naphthaldehyde and the chemical equation of carbohydrazide reaction；
Fig. 2 is the fluorescence spectrum figure of fluorescent probe and different metal ionization；
Fig. 3 is fluorescent probe and the fluorescence intensity change bar diagram of different metal ionization at 445nm；
Fig. 4 is variable concentrations Al in fluorescent probe solution3+Ultra-violet absorption spectrum variation diagram；
Fig. 5 is variable concentrations Al in fluorescent probe solution3+Fluorogram；
Fig. 6 is that fluorescent probe is to Al3+Detection by quantitative schematic diagram；
Fig. 7 is the intracellular Al of PC-12 in embodiment 13+The cell imaging figure of detection.
Detailed description of the invention
The invention provides the preparation method of a kind of fluorescent probe, comprise the following steps:
2-hydroxyl-1-naphthaldehyde and carbohydrazide are carried out in the mixed solvent of second alcohol and water back flow reaction had
There is trans 2-((2 hydroxy naphthalene-1-base) methylene) the hydrazine carbonyl hydrazide of Formulas I structure,
In the present invention, the reaction system of described back flow reaction is by 2-hydroxyl-1-naphthaldehyde ethanol solution and phosphinylidyne
Hydrazine aqueous solution obtains.2-hydroxyl-1-naphthaldehyde is preferably mixed by the present invention with dehydrated alcohol, after heating
To 2-hydroxyl-1-naphthaldehyde ethanol solution.The present invention does not has special restriction to the operation of described mixing and heating,
Use the technical scheme of mixing well known to those skilled in the art and heating.In the present invention, described
Heating is preferably heating in water bath；The temperature of described heating is preferably 40～50 DEG C, more preferably 43～47 DEG C；
Described heat time heating time is preferably 25～35min.The heating rate that the present invention is warming up to described heating-up temperature is preferred
It is 3～5 DEG C of min.In the present invention, the molar concentration of described 2-hydroxyl-1-naphthaldehyde ethanol solution is preferably
0.04～0.08mol/L, more preferably 0.05～0.07mol/L.
The preferred carbohydrazide of the present invention mixes with water, obtains carbohydrazide aqueous solution.The present invention is to described mixing
Operation does not has special restriction, uses the technical scheme of mixing well known to those skilled in the art.?
In the present invention, the molar concentration of described carbohydrazide aqueous solution is preferably 0.2～0.3mol/L, more preferably
0.22～0.26mol/L.
The present invention is by described 2-hydroxyl-1-naphthaldehyde ethanol solution and carbohydrazide aqueous solution, and back flow reaction obtains
To trans 2-((2 hydroxy naphthalene-1-base) methylene) hydrazine carbonyl hydrazide, structural formula is shown in formula I.This
The bright operation to described mixing does not has special restriction, uses the skill of mixing well known to those skilled in the art
Art scheme.Described carbohydrazide aqueous solution is preferably dropped to 2-hydroxyl-1-naphthaldehyde ethanol solution by the present invention
In；Described drop rate is preferably 2～4mL/min.In the present invention, described 2-hydroxyl-1-naphthaldehyde and carbon
The mol ratio of hydrazides is 1: 3～5.
In the present invention, described back flow reaction is preferably carried out under water bath with thermostatic control.The temperature of described back flow reaction
Degree is preferably 40～50 DEG C, more preferably 43～47 DEG C；The time of described back flow reaction is preferably 4～5h, more
It is preferably 1.3～4.7h.In the present invention, the chemical reaction occurred in described back flow reaction is as shown in Figure 1.
By the carbohydrazide shown in the 2-hydroxyl-1-naphthaldehyde shown in Formula II and formula III in the mixed solvent of second alcohol and water
Carry out back flow reaction, obtain trans 2-((2 hydroxy naphthalene-1-base) methylene) the hydrazine carbonyl shown in Formulas I
Hydrazine.
After back flow reaction completes, reacted mixed solution is preferably cooled to room temperature by the present invention, through filtering,
Wash and be dried to obtain product.In the present invention, the speed of described cooling is preferably 0.5～1.5 DEG C/min.
The present invention does not has special restriction to the described operation being filtered, washed and dried, and uses people in the art
The technical scheme being filtered, washed and dried known to Yuan.The present invention is preferably molten to the mixing after cooling
Liquid carries out sucking filtration, obtains yellow mercury oxide.The present invention preferably washes described yellow mercury oxide with water 3～4 times.
In the present invention, described dry temperature is preferably 75～85 DEG C, more preferably 78～82 DEG C；Described dry
Time be preferably 40～50min.
Dried product is preferably carried out recrystallization purification by the present invention, obtains trans 2-((2 hydroxy naphthalene-1-
Base) methylene) hydrazine carbonyl hydrazide.The present invention does not has special restriction to the operation of described recrystallization, uses
The operation of recrystallization well known to those skilled in the art.In the present invention, the solvent of described recrystallization
It is preferably methanol；The temperature of described recrystallization is preferably 30～40 DEG C, more preferably 33～37 DEG C.
Present invention also offers the application in aluminium ion detects of the fluorescent probe of structure shown in a kind of Formulas I.?
In the present invention, the detection of described aluminium ion preferably includes in aqueous solution aluminium ion in aluminum ions detection and cell
Detection.The present invention does not has special restriction to the operation that described aluminium ion detects, and uses art technology
Fluorescent probe known to personnel detects aluminum ions technical scheme.
In the present invention, described aluminium ion detects and preferably includes following steps:
(1) described fluorescent probe is mixed with liquid to be measured, or cultivate after mixing with cell culture medium, detection
Solution to be measured or the fluorescence intensity of cell to be measured；
(2) fluorescence intensity obtained according to step (1) and linear equation, obtain aqueous solution to be measured or thin
Aluminum ions concentration in born of the same parents.
When described aluminium ion is detected as aluminum ions detection in aqueous solution, the present invention is preferably by described fluorescence
After probe is configured to mother solution and aqueous solution to be measured with DMF for solvent, carry out glimmering
The detection of light intensity.In the present invention, in described mother solution, the concentration of fluorescent probe is preferably 1mmol/L.
In the present invention, in described aqueous solution to be measured, the concentration of fluorescent probe is preferably 0.5～1.5 × 10-5mol/L.This
Invent the detection method to described fluorescence intensity and there is no special restriction, use those skilled in the art to know
Fluorescence intensity detection operation.In the present invention, the detection of described fluorescence intensity is preferably in Shimadzu
Measuring on PF-3501PC spectrofluorophotometer, excitation wavelength is 309nm.
After obtaining the fluorescence intensity of solution to be measured, the present invention is according to described fluorescence intensity and predetermined linear song
Line, obtains aluminum ions concentration in solution to be measured.In the present invention, described linearity curve is fluorescence intensity
Linearity curve with aluminium ion concentration；Described linearity curve obtains the most as follows:
The aluminium ion standard solution of gradient concentration is provided；
Described aluminium ion standard solution is mixed with the DMF solution of fluorescent probe, obtains
Standard solution to be measured；
Described standard solution to be measured is carried out fluorescence intensity detection, obtains the fluorescence intensity of standard solution to be measured；
Fluorescence intensity according to described standard solution to be measured and corresponding aluminium ion concentration, obtain linearity curve.
In the present invention, the range of linearity of described linearity curve is preferably 5 × 10-6～4 × 10-5mol/L。
When described aluminium ion is detected as aluminum ions detection in cell, described fluorescence is preferably visited by the present invention
Pin mixes with cell culture medium, carries out aluminium ion detection in cell after cultivation.Described cell is not had by the present invention
There is special restriction, use zooblast well known to those skilled in the art.In the present invention, institute
State cell and be preferably PC-12 cell and/or fibroblasts of adult human dermis.The present invention operation to described cultivation
There is no special restriction, use the technical scheme that cell well known to those skilled in the art is cultivated.?
In the present invention, described cultivation is preferably carried out in the DMEM culture medium containing 10% hyclone；Described
The temperature cultivated is preferably 36～38 DEG C；The gas concentration lwevel of described cultivation is preferably 4～6%；Described training
The time supported is preferably 25～35min.
The present invention the most after incubation, removes culture medium, washed cell, observes and measure.This
The bright operation to described removal culture medium does not has special restriction, uses well known to those skilled in the art carrying
Take the technical scheme of cell.In the present invention, it is preferred to culture medium is absorbed with suction pipe.The present invention couple
The washing of described cell, the operation observed and measure do not have special restriction, use those skilled in the art ripe
The cell washing known, the technical scheme observed and measure.In the present invention, it is preferred to buffer with PBS
Liquid washed cell；Described washing times is preferably 3 times.Present invention preferably employs fluorescence microscope to cell
Carry out imaging.
The present invention does not has special restriction to the detection method of fluorescence intensity in described cell, uses this area
The operation of fluorescence intensity detection in cell known to technical staff.In the present invention, described fluorescence is strong
The detection of degree preferably employs after confocal fluorescent microscope takes pictures, with Image-Pro software analysis picture, system
Meter fluorescence intensity.
In order to further illustrate the present invention, below in conjunction with the embodiment system to the fluorescent probe that the present invention provides
Preparation Method and aluminium ion measure in application be described in detail, but they can not be interpreted as right
The restriction of scope.
In an embodiment of the present invention, the chemical constitution of fluorescent probe uses nuclear magnetic resonance analyser to detect,
Absorption spectrum is measured on TU-1901 type ultraviolet-visible spectrophotometer, and sweep limits is 200-600nm,
Fluorescence spectrum is measured on Shimadzu PF-3501PC spectrofluorophotometer, and excitation wavelength is 309nm.
Embodiment 1:
The preparation of fluorescent probe:
(1) use 10mL dehydrated alcohol as solvent, 50mg 2-hydroxyl-1-naphthaldehyde is dissolved in reaction
In Ping, with the ramp of 4 DEG C/min to 45 DEG C, temperature retention time 30min；
(2) employing 10mL water is as solvent, after being dissolved by 108mg carbohydrazide, with the speed of 3mL/min
Rate is added dropwise over reaction bulb, heating reflux reaction 4.5 hours at 45 DEG C；
(3) reacted mixed liquor is cooled to room temperature, sucking filtration with the speed of 1 DEG C/min, washs with water
3 times, in 80 DEG C of baking ovens, it is dried 45min；
(4) it is heated to 35 DEG C of recrystallization with methanol for solvent, obtains fluorescent probe, productivity 81%.
Proton nmr spectra result is as follows:
1H NMR (600MHz, DMSO-d6) δ 11.03 (s, 1H), 9.19 (s, 1H), 8.36 (d, J=8.6
Hz, 1H), 7.89 (t, J=7.8Hz, 3H), 7.60 (t, J=7.6Hz, 1H), 7.41 (t, J=7.4Hz, 1H),
7.23 (d, J=8.9Hz, 1H).
Carbon spectrum result is as follows:
13C NMR (500MHz, DMSO-d6) δ (ppm): 156.76,156.39,141.85,131.54,
131.46,128.86,127.88,127.47,123.39,121.09,118.88,109.31.
Infrared results is as follows:
IR(KBr)νmax(cm-1): 3324,3211,3093,2969,1676,1620,1600,1538,1466,
1416,1385,1345,1288,1241,1193,1162,1044,936,821,736,701；
Mass spectral results is as follows:
ESI-MS m/z:[M+H+]245.25。
Structural Identification data show, fluorescent probe is the trans 2-((2 hydroxy naphthalene-1-of structure shown in Formulas I
Base) methylene) hydrazine carbonyl hydrazide.
The fluorescent probe obtained is configured to DMF for solvent the mother solution of 1mmol/L；
The concentration that the mother solution obtained is diluted with water to fluorescent probe is 1 × 10-5The solution of mol/L (1 times of equivalent).
The test of Absorption and fluorescence spectrum, uses 1cm cuvette respectively, pipettes the fluorescence of 3mL every time
Probe aqueous solution, in cuvette, is separately added into different volumes 1 × 10 with pipettor-4Mol/L (10 times of equivalents)
K+, Na+, Ni2+, Mg2+, Ca2+, Mn2+, Fe3+, NH4 +, Co2+, Li+, Al3+, Cu2+,
Fe2+, Ba2+, Pb2+, Cr3+Deng the aqueous solution of metal ion, glimmering with Absorption and fluorescence spectrum research
Light probe and the spectral quality of different metal ionic interaction.
The fluorescence spectrum figure of fluorescent probe and different metal ionization is as shown in Figure 2.Fluorescence from figure
Curve is it can be seen that fluorescent probe itself is almost without fluorescence, when 5 × 10-6～4 × 10-5Mol/L (0.5 to 4
Times equivalent) aluminium ion of 4 times of equivalents adds fashionable, and fluorescence intensity is remarkably reinforced, the alkali metal of 10 times of equivalents
(K+, Na+, Li+), alkaline-earth metal (Ca2+) and transition metal (Mn2+, Cu2+, Co2+, Pb2+)
Addition all do not cause the significant change of spectrum, thus, the fluorescent probe that the present invention prepares is to Al3+
Ion has shown narrow spectrum recognition effect.
At 445nm, the fluorescence intensity change of fluorescent probe and different metal ionization is as shown in Figure 3.
It can be seen that the aluminum ions addition of only 4 times amount, fluorescent probe fluorescence at 445nm
Intensity enhancing 225 times, this is owing to the atom N in C=N double bond in the structure of fluorescent probe is by photoinduction
The impact of electron transfer effect, after being combined, inhibit photoinduced electron transfer effect with aluminium ion,
Enhancing planar conjugate, two kinds of mechanism jointly cause fluorescent emission to be remarkably reinforced, reach aluminium ion simultaneously
Excellent recognition effect.
The absorption spectrum of variable concentrations aluminium ion and fluorescent probe effect is as shown in Figure 4.Can from figure
Going out, the absworption peak at 378nm gradually strengthens, and the absworption peak at 355nm is gradually lowered, and can
With see an obvious equilibrium point at 365nm, solution colour is also from the colourless yellow that becomes, this phenomenon
Show that the fluorescent probe of synthesis defines new coordination compound with aluminium ion.
The fluorescence spectrum of variable concentrations aluminium ion and fluorescent probe effect is as shown in Figure 5.Can from figure
Go out, with Al3+Concentration increases, and at 445nm, emission peak intensity gradually strengthens, and works as Al3+Concentration reaches
4×10-5During mol/L (4 times of equivalents), fluorescence intensity tends towards stability, and solution becomes under 365nm uviol lamp
For navy blue.
The relation of aluminium ion concentration and emission peak intensity is as shown in Figure 6.It can be seen that at Al3+Concentration is
5×10-6～4 × 10-5Mol/L scope has preferable linear relationship, and linear equation is: y=22.849X+1.5023,
R2=0.9939, y are aluminium ion concentration, and X is fluorescence intensity, and detection is limited to 1.89 × 10-8mol/L.Fluorescence
Probe is to Al3+Detection by quantitative can be tested by fluorescence titration and linear relatively directrix curve is studied.
Embodiment 2:
The most aluminum ions detection of fluorescent probe that embodiment 1 prepares:
(1) for solvent, described fluorescent probe being configured to concentration with DMF is
The mother solution of 1mmol/L；
(2) mother solution step (1) obtained and concentration are 2 × 10-5The aluminium ion solution mixing inspection of mol/L
Survey fluorescence intensity；
(3) according to step (2) described fluorescence intensity and linear equation determine aluminium ion concentration be 1.982 ×
10-5Mol/L, error is 0.90%.
Embodiment 3:
The fluorescent probe that embodiment 1 prepares is aluminum ions detection in cell:
(1) PC-12 cell is placed in containing 10% hyclone and the DMEM of 10 μMs of fluorescent probes
In the culture dish of culture medium, under 37 DEG C and 5% carbon dioxide conditions, train 30min；
(2) absorb culture medium with suction pipe, with PBS washed cell 3 times, use fluorescence microscope
Take pictures, observe aluminium ion distribution, with Image-Pro software analysis picture, add up fluorescence intensity；
(3) according to step (2) described fluorescence intensity and linear equation determine aluminium ion concentration be 3.951 ×
10-5Mol/L, error is 1.23%.
In cell, aluminium ion is distributed as shown in Figure 7.From Fig. 7, (a, d figure is that comparison (is added without Al3+And spy
Pin), b, e figure adds probe cell imaging after 30 minutes, and c, f figure is to add aluminium ion to cultivate 30
The cell imaging figure after probe is cultivated 30 minutes is added after minute) it can be seen that the present embodiment is prepared into
To fluorescent probe distribution aluminum ions in cell can be carried out Accurate Determining.
Embodiment 4:
The preparation of fluorescent probe:
(1) use 10mL dehydrated alcohol as solvent, 35mg 2-hydroxyl-1-naphthaldehyde is dissolved in reaction
In Ping, with the ramp of 4 DEG C/min to 43 DEG C, temperature retention time 35min；
(2) employing 10mL water is as solvent, after being dissolved by 90mg carbohydrazide, with the speed of 3mL/min
Rate is added dropwise over reaction bulb, heating reflux reaction 5 hours at 40 DEG C；
(3) reacted mixed liquor is cooled to room temperature, sucking filtration with the speed of 1 DEG C/min, washs with water
3 times, in 80 DEG C of baking ovens, it is dried 50min；
(4) it is heated to 34 DEG C of recrystallization with methanol for solvent, obtains fluorescent probe, productivity 80.4%.
Proton nmr spectra result is as follows:
1H NMR (600MHz, DMSO-d6) δ 11.03 (s, 1H), 9.19 (s, 1H), 8.36 (d, J=8.6
Hz, 1H), 7.89 (t, J=7.8Hz, 3H), 7.60 (t, J=7.6Hz, 1H), 7.41 (t, J=7.4Hz, 1H),
7.23 (d, J=8.9Hz, 1H).
Carbon spectrum result is as follows:
13C NMR (500MHz, DMSO-d6) δ (ppm): 156.76,156.39,141.85,131.54,
131.46,128.86,127.88,127.47,123.39,121.09,118.88,109.31.
Infrared results is as follows:
IR(KBr)νmax(cm-1): 3324,3211,3093,2969,1676,1620,1600,1538,1466,
1416,1385,1345,1288,1241,1193,1162,1044,936,821,736,701；
Mass spectral results is as follows:
ESI-MS m/z:[M+H+]245.25。
Structural Identification data show, fluorescent probe is the trans 2-((2 hydroxy naphthalene-1-of structure shown in Formulas I
Base) methylene) hydrazine carbonyl hydrazide.
Embodiment 5:
The most aluminum ions detection of fluorescent probe that embodiment 4 prepares:
(1) for solvent, described fluorescent probe being configured to concentration with DMF is
The mother solution of 1mmol/L；
(2) mother solution step (1) obtained and concentration are 2 × 10-5The aluminium ion solution mixing inspection of mol/L
Survey fluorescence intensity；
(3) according to step (2) described fluorescence intensity and linear equation determine aluminium ion concentration be 2.022 ×
10-5Mol/L, error is 1.1%.
Embodiment 6:
The fluorescent probe that embodiment 4 prepares is aluminum ions detection in cell:
(1) fibroblasts of adult human dermis is placed in containing 10% hyclone and 10 μMs of fluorescent probes
In the culture dish of DMEM culture medium, under 37 DEG C and 5% carbon dioxide conditions, cultivate 30min；
(2) absorb culture medium with suction pipe, with PBS washed cell 3 times, use fluorescence microscope
Take pictures, observe aluminium ion distribution, with Image-Pro software analysis picture, add up fluorescence intensity；
(3) according to step (2) described fluorescence intensity and linear equation determine aluminium ion concentration be 2.968 ×
10-5Mol/L, error is 1.07%.
Embodiment 7:
The preparation of fluorescent probe:
(1) use 10mL dehydrated alcohol as solvent, 66mg 2-hydroxyl-1-naphthaldehyde is dissolved in reaction
In Ping, with the ramp of 4 DEG C/min to 48 DEG C, temperature retention time 25min；
(2) employing 10mL water is as solvent, after being dissolved by 135mg carbohydrazide, with the speed of 3mL/min
Rate is added dropwise over reaction bulb, heating reflux reaction 4 hours at 50 DEG C；
(3) reacted mixed liquor is cooled to room temperature, sucking filtration with the speed of 1 DEG C/min, washs with water
3 times, in 80 DEG C of baking ovens, it is dried 40min；
(4) it is heated to 35 DEG C of recrystallization with methanol for solvent, obtains fluorescent probe, productivity 82%.
Proton nmr spectra result is as follows:
1H NMR (600MHz, DMSO-d6) δ 11.03 (s, 1H), 9.19 (s, 1H), 8.36 (d, J=8.6
Hz, 1H), 7.89 (t, J=7.8Hz, 3H), 7.60 (t, J=7.6Hz, 1H), 7.41 (t, J=7.4Hz, 1H),
7.23 (d, J=8.9Hz, 1H).
Carbon spectrum result is as follows:
13C NMR (500MHz, DMSO-d6) δ (ppm): 156.76,156.39,141.85,131.54,
131.46,128.86,127.88,127.47,123.39,121.09,118.88,109.31.
Infrared results is as follows:
IR(KBr)νmax(cm-1): 3324,3211,3093,2969,1676,1620,1600,1538,1466,
1416,1385,1345,1288,1241,1193,1162,1044,936,821,736,701；
Mass spectral results is as follows:
ESI-MS m/z:[M+H+]245.25。
Structural Identification data show, fluorescent probe is the trans 2-((2 hydroxy naphthalene-1-of structure shown in Formulas I
Base) methylene) hydrazine carbonyl hydrazide.
Embodiment 8:
The most aluminum ions detection of fluorescent probe that embodiment 7 prepares:
(1) for solvent, described fluorescent probe being configured to concentration with DMF is
The mother solution of 1mmol/L；
(2) mother solution step (1) obtained and concentration are 2 × 10-5The aluminium ion solution mixing inspection of mol/L
Survey fluorescence intensity；
(3) according to step (2) described fluorescence intensity and linear equation determine aluminium ion concentration be 1.971 ×
10-5Mol/L, error is 1.45%.
Embodiment 9:
The fluorescent probe that embodiment 7 prepares is aluminum ions detection in cell:
(1) PC-12 cell and fibroblasts of adult human dermis are placed in containing 10% hyclone and 10 μMs
In the culture dish of the DMEM culture medium of fluorescent probe, cultivate under 37 DEG C and 5% carbon dioxide conditions
30min；
(2) absorb culture medium with suction pipe, with PBS washed cell 3 times, use fluorescence microscope
Take pictures, observe aluminium ion distribution, with Image-Pro software analysis picture, add up fluorescence intensity；
(3) according to step (2) described fluorescence intensity and linear equation determine aluminium ion concentration be 2.023 ×
10-5Mol/L, error is 1.15%.
As can be seen from the above embodiments, fluorescent probe prepared by the preparation method that the present invention provides is to Al3+
Ion has shown narrow spectrum recognition effect, and detection is limited to 1.89 × 10-8Mol/L, linearity curve linear
Scope is 5 × 10-6～4 × 10-5Mol/L, detects error as little as 0.9%.
The above is only the preferred embodiment of the present invention, and the present invention not makees any pro forma limit
System.It should be pointed out that, for those skilled in the art, without departing from the principle of the invention
On the premise of, it is also possible to making some improvements and modifications, these improvements and modifications also should be regarded as the present invention's
Protection domain.
Claims (10)
1. a fluorescent probe application in aluminium ion detects, described fluorescent probe is for having shown in Formulas I
Trans 2-((2 hydroxy naphthalene-1-base) methylene) the hydrazine carbonyl hydrazide of structure,
Application the most according to claim 1, it is characterised in that the detection of described aluminium ion includes following
Step:
(1) by fluorescent probe and aqueous solution to be measured, or cultivate after mix with cell culture medium, detect
Solution to be measured or the fluorescence intensity of cell to be measured；
(2) fluorescence intensity obtained according to step (1) and predetermined linearity curve, obtain to be measured water-soluble
Aluminum ions concentration in liquid or cell to be measured.
Application the most according to claim 2, it is characterised in that described linearity curve is that aluminium ion is dense
Degree and the linearity curve of fluorescence intensity.
4. a preparation method for the fluorescent probe of structure shown in Formulas I, comprises the following steps:
2-hydroxyl-1-naphthaldehyde and carbohydrazide are carried out in the mixed solvent of second alcohol and water back flow reaction had
There is trans 2-((2 hydroxy naphthalene-1-base) methylene) the hydrazine carbonyl hydrazide of structure shown in Formulas I.
Preparation method the most according to claim 4, it is characterised in that the reaction of described back flow reaction
System is obtained by 2-hydroxyl-1-naphthaldehyde ethanol solution and carbohydrazide aqueous solution.
Preparation method the most according to claim 5, it is characterised in that described 2-hydroxyl-1-naphthaldehyde second
Alcoholic solution is prepared by following methods:
In a heated condition, 2-hydroxyl-1-naphthaldehyde being dissolved in dehydrated alcohol, the temperature of described heating is
40～50 DEG C, the time of described heating is 25～35min.
7. according to the preparation method described in claim 5 or 6, it is characterised in that described 2-hydroxyl-1-
The molar concentration of naphthaldehyde ethanol solution is 0.04～0.08mol/L.
Preparation method the most according to claim 5, it is characterised in that described carbohydrazide aqueous solution
Molar concentration is 0.2～0.3mol/L.
9. according to the preparation method described in claim 4 or 5, it is characterised in that described 2-hydroxyl-1-
Naphthaldehyde is 1: 3～5 with the mol ratio of carbohydrazide.
Preparation method the most according to claim 4, it is characterised in that the temperature of described back flow reaction
Degree is 40～50 DEG C, and the time of back flow reaction is 4～5h.
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