CN106317063B - A kind of preparation and application of response type mercury ion fluorescence probe - Google Patents
A kind of preparation and application of response type mercury ion fluorescence probe Download PDFInfo
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Abstract
The invention discloses a kind of response type mercury ion fluorescence probe and its preparation method and application.This method includes：(1) by 5 aminophenyl, 10,15,20 trisulfonic acid base phenyl porphyrin (PNH2) be dissolved in distilled water, the solution of 7.5mg/mL is formed, thiophosgene is dissolved in dichloromethane, the solution of 5 μ L/mL is formed, is purified after reacting 3~5h under room temperature after both isometric mixing, obtains 5 phenyl isothiocyanate bases 10,15,20 trisulfonic acid base phenyl porphyrins (PNCS)；(2) 5 phenyl isothiocyanate bases 10,15,20 trisulfonic acid base phenyl porphyrins (PNCS) are with ethylene diamine derivative with mixed solvent of the equimolar than being dissolved in acetone and distilled water after mixing, form the solution that total concentration is 20mg/mL, 6~10h is reacted at normal temperatures, and final product porphyrin-thiocarbamide (PT) is obtained after purification.The probe good water solubility, shows mercury ion high sensitivity and selectivity, and obvious color and change in fluorescence are shown when detecting mercury ion；With reference to laser confocal scanning microtechnic, mercury ion detecting imaging of the probe in living cells can be also obtained.
Description
Technical field
The present invention relates to a kind of preparation of response type mercury ion fluorescence probe, and in particular to a kind of to be derived with ethylenediamine thiocarbamide
The preparation of porphyrin-thiocarbamide (PT) class water solubility mercury ion fluorescence probe that thing is reactive group, Porphyrin Molecule is fluorescent chromophore
Method, and the probe molecule are applied to the Sensitive Detection of mercury ion in aqueous solution and cell.
Background technology
Mercury is a kind of heavy metal with serious physiological-toxicity, can cause serious harm to human body and ecological environment, i.e.,
Just in the very low situation of concentration, can also sizable toxic action be produced to the mankind and animals and plants.Most typical example is exactly to send out
The raw minamata disease in Japan, generates entire society extremely ill effect.For these reasons, mercury by dominated column in the whole world
On environmental monitoring system (GEMS) inventory, in environment the detection of mercury and the improvement of mercury-containing waste water caused people greatly to pay close attention to, also
The great attention of the Chinese government is caused, " 13 planning " key special subjects is listed in, key area, key enterprise is added conscientiously
Strong monitoring and supervision.The analysis of mercury and the basis that monitoring technology is mercury pollution prevention, but mercury ion detecting skill ripe at present
Art be mostly by large-sized analytic instrument, it is of high cost, time-consuming, it is difficult to realize quick Site Detection demand.And commercialization is fast
Fast detecting instrument and mercury test strips, its sensitivity is low (Monitoring lower-cut 0.1mg/L), does not reach the limit value of national Specification
(sewage 0.05mg/L, drinking water 0.001mg/L), and poor selectivity, easily by other ion interferences, it is sensitive, accurate to meet
Testing requirements.Therefore, high sensitivity is developed, selectivity is good, conveniently mercury ion detecting new method is extremely urgent.
Optochemical sensor detection is a kind of new mercury ion detecting method for coming into being in recent years, because its have both it is convenient,
It is sensitive, do not destroy sample and be easily achieved real-time in-situ detection and favored by people.At present, the research group of lot of domestic and foreign
It is conceived to different sensor mechanisms and reports the system much to mercury ion with optic response.Wherein, it is based on gold to make up
Belong to deficiency of the sensor of ions binding mechanism in terms of selectivity, development is based on specific between probe molecule and mercury ion, special
The response type mercury ion photochemical probe of one chemical principle structure causes the extensive concern of researcher.This probe molecule
Usually it is made of two parts：A part is to play the role of the chromophore of signal reports；Another part is recognition group, can with mercury from
Son occurs specific, single-minded chemical reaction and changes whole probe molecule structure, so that cause the change of chromophore photophysical property,
Achieve the purpose that to mercury ion specific detection.In the design of response type mercury ion photochemical probe, the selection of recognition unit
Particularly significant, it determines the combination sensitivity and selectivity of whole probe molecule and mercury ion, and further influences chromophore
Signal output.Mercury ion has thiophilic property, can pull out the sulphur atom of some thiocarbamides and 40 thione derivatives, generates corresponding de-
Sulphur product.Therefore such reaction active groups can be combined with chromophore by cleverly Molecular Design, using its with
Front and rear product of the generation with different structure of desulphurization reaction occurs for mercury ion, so as to change the optical physics of connected chromophore molecule
It can realize the detection to mercury ion.Wherein, thiourea derivative is that the most commonly used mercury promotees activity of organic reaction group.The patent No.
For the patent " one kind is used for the Nile blue fluorescent dye of mercury ion detecting " of CN103980728B, Patent No.
Patent " fluorescence probe and its system from rhodamine B, diethylenetriamines and phenyl isothiocyanate of CN201410341346.8
Preparation Method and application ", a kind of patent " Hg containing double carbon-sulfur bond structure rhodamines of Patent No. CN201510892168.22+It is glimmering
Light probe and preparation method and application " reports application of the thiourea derivative in mercury ion probe.However, these are reacted
Although type mercury ion fluorescence probe is with higher sensitivity and selectivity, in practical applications, especially in bio-imaging
With many problems are still suffered from detection：Such as some probe molecule poorly water-solubles, need to add in the detection a certain proportion of
Organic solvent, this detection for mercury ion in organism are very unfavorable；The fluorescence hair of fluorescent chromophore in some probe molecules
Ejected wave length is less than 590nm, and for biological sample, its sample matrices and some impurity also have absorption or fluorescence in this region,
More serious background interference is often produced, limits the raising of fluorescence analysis sensitivity；In addition, some probes itself are glimmering
Easy Bleachability monitored to real-time, high-resolution dynamic mercury ion of light also brings extreme difficulties.Improve water solubility, the choosing of probe molecule
Select with good stability, suitable for the near-infrared fluorescent chromophore of life system and fluorescence imaging analysis, it is final to realize biology
The high selectivity Sensitive Detection of internal mercury ion is the key point to solve the above problems.
Porphyrin is a kind of Conjugate macrocycle compound formed that is connected by four pyrrole rings by methine, has high fluorescence
Quantum yield, big Stokes displacements, are the near-infrared fluorescent chromophore signaling molecules of a kind of excellent performance, are widely used in glimmering
The structure of optical molecule probe.The easy modified of porphyrin compound can make its big ring periphery couple different substituents and active function
Group, very big side is provided to regulate and control the photophysical property of the fluorescence signal group and improving the hydrophilicity and hydrophobicity of whole probe molecule
Just.Therefore, it can be combined with mercury ion recognition unit by MOLECULE DESIGN and chemical reaction, synthesizes a series of optical physics and light
The adjustable functional fluorescence probe of chemical property, the detection applied to mercury ion in water body and organism.
The content of the invention
The present invention for present in existing fluorescence probe preparation complexity, poorly water-soluble and background interference are serious the problems such as,
Provide a kind of water-soluble for porphyrin-Thiourea of fluorescent chromophore by reactive group, Porphyrin Molecule of ethylenediamine thiourea derivative
Property mercury ion fluorescence probe and preparation method thereof, and the application of probe molecule mercury ion detecting in water body and cell.
A kind of porphyrin-Thiourea water solubility mercury ion fluorescence probe (PT), has following general structure：
Wherein, R1=R2=H, or R1=H, R2=CH3, or R1=H, R2=C2H5
A kind of preparation method of porphyrin-Thiourea water solubility mercury ion fluorescence probe (PT), includes the following steps：
By 5- aminophenyl -10,15,20- trisulfonic acid base phenyl porphyrins (PNH2) be dissolved in distilled water, thiophosgene is dissolved in
In dichloromethane, after reacting 3~5h under room temperature after both isometric mixing, 5- isothiocyanic acid bases are obtained after purification, freeze-drying
Phenyl -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS)；By 5- phenyl isothiocyanate base -10,15,20- trisulfonic acid base phenyl
Porphyrin (PNCS) is dissolved in the mixed solvent of acetone and distilled water after being mixed with ethylene diamine derivative, react 6~10h at normal temperatures
Afterwards, solvent is spin-dried for using Rotary Evaporators, dialyse 48~56h in the bag filter of 1000kDa, is obtained after being freeze-dried 36~42h
To final product porphyrin-thiocarbamide (PT).
The preparation method of the porphyrin-Thiourea water solubility mercury ion fluorescence probe (PT), specifically comprises the following steps：
(1) with commercially available tetraphenylporphyrin (TPP) for starting material, reference literature：The synthesis and reaction of nitrobenzophenone porphyrin
(R.Luguya,L.Jaquinod,F.R.Fronczek,A.G.H.Vicente,K.M.Smith, Synthesis and
60 (2004) 2757-2763 of reactions of meso- (p-nitrophenyl) porphyrins, Tetrahedron) in
The preparation method of record, by TPP by trifluoroacetic acid/sodium nitrite mixed system nitrification, stannous chloride/mixed in hydrochloric acid system also
Original, is prepared 5- aminophenyls -10,15,20- Triphenylporphyrins (ATPP).
The structural formula of TPP is：
The structural formula of ATPP is：
(2) 5- aminophenyls -10,15,20- Triphenylporphyrins (ATPP) are dissolved in the concentrated sulfuric acid, form 0.04 g/mL's
Solution, is heated to flowing back, and stops heating after reacting 6~9h；The sodium hydroxide solution that reaction solution adds 1mol/L is neutralized to solution
PH be 7, in the bag filter of 1000kDa dialyse 45~50h, be freeze-dried 22~28h after obtain 5- aminophenyls -10,15,
20- trisulfonic acid base phenyl porphyrins (PNH2)。
PNH2Structural formula be：
(3) by 5- aminophenyl -10,15,20- trisulfonic acid base phenyl porphyrins (PNH2) be dissolved in solvent one, by thiophosgene
It is dissolved in solvent two, after reacting 3~5h under room temperature after both isometric mixing, dialyse 45 in the bag filter of 1000kDa~
50h, 5- phenyl isothiocyanate bases -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS) are obtained after being freeze-dried 34~38h.
The structural formula of PNCS is：
(4) 5- phenyl isothiocyanates base -10,15,20- trisulfonic acids base phenyl porphyrin (PNCS) and ethylene diamine derivative with etc.
The mixed solvent of acetone and distilled water is dissolved in after mixed in molar ratio, forms the solution that total concentration is 20mg/mL, at normal temperatures instead
After answering 6~10h, solvent is spin-dried for using Rotary Evaporators, dialyse 48~56h in the bag filter of 1000kDa, freeze-drying 36~
Final product porphyrin-thiocarbamide (PT) is obtained after 42h.
The ethylene diamine derivative is respectively ethylenediamine (EA), N- methyl ethylenediamines (MEA), n-ethylethylenediamine
(EEA), structural formula is as follows：
The reason for selecting these types of compound is that its is cheap and easy to get, molecular volume is small, high with PNCS reaction efficiencies, and raw
Into PT it is water-soluble preferably.
The solvent one is distilled water.
The solvent two is dichloromethane.
The synthesis of 5- aminophenyl -10,15,20- Triphenylporphyrins (ATPP) is in strict accordance with document in the step (1)
Report method synthesizes.
5- aminophenyls -10,15,20- trisulfonic acid base phenyl porphyrins (PNH in the step (2)2) synthesis with reference to text
Offer：A kind of easy method synthesis and four (4- sulfonic groups) phenyl porphyrins of purifying (T.S.Srivastava, M.Tsutsui,
Preparation and purification of tetrasodium meso-tetra(p-sulfopheny1)
Porphine.An easy procedure) in report method, by optimizing the ratio of ATPP and the concentrated sulfuric acid, adjustment is heated back
The time is flowed, obtains yield and the higher 5- aminophenyls -10,15 of purity, 20- trisulfonic acid base phenyl porphyrins (PNH2)。
5- aminophenyls -10,15,20- trisulfonic acid base phenyl porphyrins (PNH in the step (3)2) with solvent one plus
It is preferably 7.5mg/mL to enter the ratio between amount, and thiophosgene can make with the ratio between the addition of solvent two preferably 5 μ L/mL, the two ratios
Obtaining 5- aminophenyls -10,15,20- trisulfonic acids base phenyl porphyrin respectively can be most in the case where solvent dosage is minimum with thiophosgene
Fully dissolving, more conducively reaction fully carry out.
5- aminophenyls -10,15,20- trisulfonic acid base phenyl porphyrins (PNH in the step (3)2) aqueous solution and sulphur
The dichloromethane solution volume ratio preferably 1 of phosgene:1, this volume ratio can cause 5- aminophenyls -10,15 in solution, 20-
The molar ratio of trisulfonic acid base phenyl porphyrin and thiophosgene is 1:7.5, the ratio can peak efficiency promote 5- aminophenyls -10,15,
20- trisulfonic acid base phenyl Porphyrin mutation is 5- phenyl isothiocyanate bases -10,15, and 20- trisulfonic acid base phenyl porphyrins (PNCS), keep away
Exempt from the generation of accessory substance.
The mixed solvent of acetone and distilled water in the step (4), wherein, acetone, the volume ratio of distilled water are 1:1.
The reason for selecting both solvents is that PNCS is soluble easily in water, and ethylene diamine derivative is soluble in acetone, and acetone is miscible with water, two
Person's mixing can reach solution and mix homogeneous, the effect fully reacted.
5- phenyl isothiocyanate base -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS) and second in the step (4)
The molar ratio of diamine derivative is 1:1.Experiment shows, when the two molar ratio is 1:When 1, reaction efficiency highest, side reaction are minimum.
Probe molecule made from the preparation method, it is characterised in that described porphyrin-thiocarbamide (PT) molecule is water
Soluble molecule.
By thiourea group and mercury ion distinctive guanidine annulation can occur for described porphyrin-thiocarbamide (PT) molecule,
Cause PT molecule structure changes, the color and fluorescent characteristic for causing porphyrin signal group change, and realize the single-minded of mercury ion
Property detection.It is shown below：
Described porphyrin-thiocarbamide (PT) molecule has good membrane penetrating, the detection available for mercury ion in cell.
The invention has the advantages that：
(1) synthetic method of porphyrin-thiocarbamide (PT) probe molecule of the invention, mild condition, step are simple, post processing letter
Just；
(2) present invention can promote thiourea derivative that special intramolecular guanidine annulation occurs using mercury ion, other
Ion realizes the specificity and accuracy of mercury ion detecting without significantly interfering with；
(3) present invention uses Porphyrin Molecule as signal reports group, which is near-infrared fluorescent chromophore molecule, light
Learn excellent performance, it is sensitive to microenvironment, larger Stocks displacements can be obtained, enhance the sensitivity of reaction signal；
(4) porphyrin-thiocarbamide (PT) probe molecule of the invention has good water solubility, and ratio can be shown to mercury ion
The Dual Sensitive property of color/fluorescence is, it can be achieved that the quickly detection, and can be to the detection of mercury ion of trace mercury in water solution ion
Show high selectivity；
It (5) can be used for intracellular by means of laser confocal scanning microtechnic, porphyrin-thiocarbamide (PT) probe of the invention
Mercury ion detecting is imaged.
Brief description of the drawings
Fig. 1 is color change figure of porphyrin-thiocarbamide (PT) probe solution to different metal ions of the preparation of embodiment 1；
Fig. 2 is the mercury ion titration fluorescence spectrogram of porphyrin-thiocarbamide (PT) probe prepared by embodiment 2；
Fig. 3 is the shows fluorescent microscopy images of the mercury ion in cell detection of porphyrin-thiocarbamide (PT) probe prepared by embodiment 3
Piece.
Embodiment
Embodiment 1
Tetraphenylporphyrin (TPP) (800mg, 1.31mmol) is dissolved in trifluoroacetic acid (50mL), adds sodium sulfite
(100mg,1.45mmol).After reacting 3min at room temperature, add 500mL water and reaction is quenched.Using dichloromethane extractive reaction
Liquid, by organic layer respectively with after saturated sodium bicarbonate aqueous solution, distillation water washing, rotary evaporation obtains violet solid.Silica gel color
Post separation is composed, dichloromethane is solvent, collects the second colour band product.Solvent evaporated is rotated, continuously adds concentrated hydrochloric acid (50mL),
Stannous chloride (1100mg, 4.875mmol).Mixed liquor is heated to 65 DEG C, after reacting 1h under argon gas protection, adds 500mL
Distilled water.Add ammonium hydroxide and be neutralized to pH as 8.Using solvent evaporated is rotated after dichloromethane extractive reaction liquid, silica gel chromatographic column divides
From dichloromethane is solvent, collects the 3rd colour band product.Obtain 5- aminophenyl -10,15,20- Triphenylporphyrins
(ATPP)。
By 5- aminophenyls -10,15,20- Triphenylporphyrins (ATPP) (320mg, 3.9mmol) are dissolved in the 8mL concentrated sulfuric acids
In, the solution of 0.04g/mL is formed, is heated to flowing back, stops heating after reacting 8h；Reaction solution adds the sodium hydroxide of 1mol/L
Solution be neutralized to solution pH be 7, dialyse 46h in the bag filter of 1000kDa, be freeze-dried 24h after obtain 5- aminophenyls-
10,15,20- trisulfonic acid base phenyl porphyrins (PNH2)。
By 5- aminophenyl -10,15,20- trisulfonic acid base phenyl porphyrins (PNH2) (150mg, 0.172mmol) be dissolved in
In 20mL distilled water, thiophosgene (100 μ L, 1.3mmol) is dissolved in 20mL dichloromethane, reacts 4h under room temperature after both mix
Afterwards, dialyse 48h in the bag filter of 1000kDa, and 5- phenyl isothiocyanate bases -10,15,20- tri- are obtained after being freeze-dried 36 h
Sulfonic group phenyl porphyrin (PNCS).
5- phenyl isothiocyanate base -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS) (40mg, 0.041 mmol) and second
In the mixed solvent (the body of acetone and distilled water of 2.1mL acetone, distilled water is dissolved in after diamines (2.46mg, 0.041mmol) mixing
Product is than being 1:1) after, reacting 6h at normal temperatures, solvent is spin-dried for using Rotary Evaporators, is dialysed in the bag filter of 1000kDa
50h, final product porphyrin-thiocarbamide (PT) is obtained after being freeze-dried 38h, is verified using nuclear magnetic resonance spectroscopy, high resolution mass spectrum
Its chemical constitution.Its structural formula is as follows：
Embodiment 2
Tetraphenylporphyrin (TPP) (800mg, 1.31mmol) is dissolved in trifluoroacetic acid (50mL), adds sodium sulfite
(100mg,1.45mmol).After reacting 3min at room temperature, add 500mL water and reaction is quenched.Using dichloromethane extractive reaction
Liquid, by organic layer respectively with after saturated sodium bicarbonate aqueous solution, distillation water washing, rotary evaporation obtains violet solid.Silica gel color
Post separation is composed, dichloromethane is solvent, collects the second colour band product.Solvent evaporated is rotated, continuously adds concentrated hydrochloric acid (50mL),
Stannous chloride (1100 mg, 4.875mmol).Mixed liquor is heated to 65 DEG C, after reacting 1h under argon gas protection, adds 500mL
Distilled water.Add ammonium hydroxide and be neutralized to pH as 8.Using solvent evaporated is rotated after dichloromethane extractive reaction liquid, silica gel chromatographic column divides
From dichloromethane is solvent, collects the 3rd colour band product.Obtain 5- aminophenyl -10,15,20- Triphenylporphyrins
(ATPP)。
By 5- aminophenyls -10,15,20- Triphenylporphyrins (ATPP) (480mg, 4.35mmol) are dissolved in the 12mL concentrated sulfuric acids
In, the solution of 0.04g/mL is formed, is heated to flowing back, stops heating after reacting 9h；Reaction solution adds the sodium hydroxide of 1mol/L
Solution be neutralized to solution pH be 7, dialyse 50h in the bag filter of 1000kDa, be freeze-dried 28h after obtain 5- aminophenyls-
10,15,20- trisulfonic acid base phenyl porphyrins (PNH2)。
By 5- aminophenyl -10,15,20- trisulfonic acid base phenyl porphyrins (PNH2) (300mg, 0.344mmol) be dissolved in
In 40mL distilled water, thiophosgene (200 μ L, 2.6mmol) is dissolved in 40mL dichloromethane, reacts 5h under room temperature after both mix
Afterwards, dialyse 50h in the bag filter of 1000kDa, and 5- phenyl isothiocyanate bases -10,15,20- tri- are obtained after being freeze-dried 38 h
Sulfonic group phenyl porphyrin (PNCS).
5- phenyl isothiocyanate base -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS) (40mg, 0.041 mmol) and N-
2.15mL acetone is dissolved in after methyl ethylenediamine (3.05mg, 0.041mmol) mixing, (acetone is with steaming for the in the mixed solvent of distilled water
The volume ratio of distilled water is 1:1) after, reacting 8h at normal temperatures, solvent is spin-dried for using Rotary Evaporators, in the bag filter of 1000kDa
Middle dialysis 48h, obtains final product porphyrin-thiocarbamide (PT), using nuclear magnetic resonance spectroscopy, high-resolution matter after being freeze-dried 40h
Spectrum verifies its chemical constitution.Its structural formula is as follows：
Embodiment 3
Tetraphenylporphyrin (TPP) (800mg, 1.31mmol) is dissolved in trifluoroacetic acid (50mL), adds sodium sulfite
(100mg,1.45mmol).After reacting 3min at room temperature, add 500mL water and reaction is quenched.Using dichloromethane extractive reaction
Liquid, by organic layer respectively with after saturated sodium bicarbonate aqueous solution, distillation water washing, rotary evaporation obtains violet solid.Silica gel color
Post separation is composed, dichloromethane is solvent, collects the second colour band product.Solvent evaporated is rotated, continuously adds concentrated hydrochloric acid (50mL),
Stannous chloride (1100 mg, 4.875mmol).Mixed liquor is heated to 65 DEG C, after reacting 1h under argon gas protection, adds 500mL
Distilled water.Add ammonium hydroxide and be neutralized to pH as 8.Using solvent evaporated is rotated after dichloromethane extractive reaction liquid, silica gel chromatographic column divides
From dichloromethane is solvent, collects the 3rd colour band product.Obtain 5- aminophenyl -10,15,20- Triphenylporphyrins
(ATPP)。
By 5- aminophenyls -10,15,20- Triphenylporphyrins (ATPP) (240mg, 2.93mmol) are dissolved in the 6mL concentrated sulfuric acids
In, the solution of 0.04g/mL is formed, is heated to flowing back, stops heating after reacting 8h；Reaction solution adds the sodium hydroxide of 1mol/L
Solution be neutralized to solution pH be 7, dialyse 49h in the bag filter of 1000kDa, be freeze-dried 28h after obtain 5- aminophenyls-
10,15,20- trisulfonic acid base phenyl porphyrins (PNH2)。
By 5- aminophenyl -10,15,20- trisulfonic acid base phenyl porphyrins (PNH2) (150mg, 0.172mmol) be dissolved in
In 20mL distilled water, thiophosgene (100 μ L, 1.3mmol) is dissolved in 20mL dichloromethane, reacts 4h under room temperature after both mix
Afterwards, dialyse 47h in the bag filter of 1000kDa, and 5- phenyl isothiocyanate bases -10,15,20- tri- are obtained after being freeze-dried 36 h
Sulfonic group phenyl porphyrin (PNCS).
5- phenyl isothiocyanate base -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS) (50mg, 0.051 mmol) and N-
2.73mL acetone is dissolved in after ethylethylenediamine (4.51mg, 0.051mmol) mixing, (acetone is with steaming for the in the mixed solvent of distilled water
The volume ratio of distilled water is 1:1) after, reacting 7.5h at normal temperatures, solvent is spin-dried for using Rotary Evaporators, in the dialysis of 1000kDa
Dialyse 46h in bag, final product porphyrin-thiocarbamide (PT) is obtained after being freeze-dried 42h, using nuclear magnetic resonance spectroscopy, high-resolution
Mass spectrum verifies its chemical constitution.Its structural formula is as follows：
Application examples 1
Selective enumeration method of porphyrin-thiocarbamide (PT) probe molecule to mercury ion in aqueous solution：Due to only having mercury ion to lure
Lead thiourea group and intramolecular guanidine annulation occurs, cause porphyrin signal group that color and spectral signal response occurs, therefore
It can will be separately added into certain density PT molecules aqueous solution in the different metal ions aqueous solution of isoconcentration, it is molten to visually observe PT
The color change of liquid.Detailed process is as follows：PT aqueous solution 0.4mL (10 μs that prepare are added in embodiment 1 in 5mL seed bottles
M), then it is separately added into the Mg of 10 times of equivalents2+,Ca2+,Ba2+, Zn2+,Ni2+,Al3+,K+, Cd2+,Na+Aqueous solution 0.4mL, use
Distilled water dilution is settled to 4.0mL, stands after five minutes, visually observes solution colour change, finds there was only Hg2+PT can be caused molten
The color of liquid is changed into yellow green by pink, other without obvious color change, illustrate PT probes to there is the higher choosing of mercury ion
Selecting property (Fig. 1).
Application examples 2
Fluorescence titration of porphyrin-thiocarbamide (PT) probe molecule to various concentrations mercury ion in aqueous solution：In 10 mL volumetric flasks
After the PT aqueous solutions 1.0mL (10 μM) prepared in middle addition embodiment 2, then it is separately added into the mercury ion aqueous solution of various concentrations
1.0mL, 10.0mL is settled to using distilled water dilution, and after standing 5 minutes, various concentrations mercury is added using fluorescence spectrometry
The change in fluorescence spectrogram of ion.As a result as shown in Fig. 2.With the enhancing of ion concentration of mercury, the fluorescence intensity at 651nm is gradual
Weaken, the fluorescence intensity at 602nm gradually strengthens, and illustrates that PT solution shows Ratio-type fluorescence response for mercury ion.
The parameter of Fluorescence Spectrometer is as follows：Excitation wave wavelength is 420nm；Received wave wave band is the nm of 525nm~775.
Application examples 3
Porphyrin-the thiocarbamide prepared by embodiment 3 of 1mM is prepared before experiment using phosphate buffer (PBS, pH 7.4)
(PT) probe molecule storing solution.Take above-mentioned 15 μ L PT molecule storing solutions to be separately added into the culture medium containing attached cell, be placed in
After being incubated 15min in 37 DEG C of 5% incubator, cleaned 3 times with phosphate buffer (PBS, pH 7.4), to remove in culture medium
The excessive probe molecule for being introduced into cell.The cell culture medium for taking one of them to be incubated through PT molecules, further adds 2 equivalents
15 μ L of mercury ion aqueous solution, be placed in 37 DEG C of 5% incubator be incubated 15min after, with phosphate buffer (PBS, pH
7.4) clean 3 times, to remove the mercury ion for being introduced into cell excessive in culture medium.Two groups of cells are separately fixed at by more than
After on 96 orifice plates, using Olympus FV1000 confocal laser scanning microscope imaging contexts.The results are shown in Figure 3.Can be with
Find out, do not add before mercury ion, into the cell without obvious fluorescence；After mercury ion adds, obvious fluorescence is showed into the cell and is increased
By force.Illustrate that synthesized PT molecules have good cell membrane penetration, and excellent detection is shown to mercury ion in cell
Performance.
The parameter of laser confocal microscope is as follows：A. laser power is 50 mW of argon ion laser gross output
20%, excitation wavelength 405nm, spectroscope are TD 488/543/633nm, and detection emission band is 575nm~620nm；B. swash
Optical scanning intensity is the 10% of laser output power；C. detecting pinhole is 1 Airy units；D. Photomultiplier tube voltage is
1000V。
Claims (5)
1. a kind of preparation method of response type mercury ion fluorescence probe, it is characterised in that the structural formula of the fluorescence probe is as follows：
Wherein, R1=R2=H, or R1=H, R2=CH3, or R1=H, R2=C2H5；
Include the following steps：
(1) by 5- aminophenyl -10,15,20- trisulfonic acid base phenyl porphyrins (PNH2) be dissolved in solvent one, thiophosgene is dissolved in molten
In agent two, after reacting 3~5h under room temperature after both isometric mixing, dialyse 45~50h in the bag filter of 1000kDa, freezing
5- phenyl isothiocyanate base -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS) are obtained after dry 34~38h；
(2) 5- phenyl isothiocyanates base -10,15,20- trisulfonic acids base phenyl porphyrin (PNCS) is molten after being mixed with ethylene diamine derivative
In acetone and the mixed solvent of distilled water, the solution that concentration is 20mg/mL is formed, after reacting 6~10h at normal temperatures, using rotation
Turn evaporimeter and be spin-dried for solvent, dialyse 48~56h in the bag filter of 1000kDa, and final production is obtained after being freeze-dried 36~42h
Thing porphyrin-thiocarbamide (PT)；
The ethylene diamine derivative is respectively ethylenediamine (EA), N- methyl ethylenediamines (MEA), n-ethylethylenediamine (EEA), is tied
Structure formula is as follows：
A kind of 2. preparation method of response type mercury ion fluorescence probe according to claim 1, it is characterised in that step
(1) solvent one described in is distilled water, and the solvent two is dichloromethane.
A kind of 3. preparation method of response type mercury ion fluorescence probe according to claim 1, it is characterised in that step
(1) the ratio between 5- aminophenyls -10,15 in, addition of 20- trisulfonic acids base phenyl porphyrin and solvent one are 75mg/mL, thiophosgene
It is 5 μ L/mL with the ratio between the addition of solvent two.
A kind of 4. preparation method of response type mercury ion fluorescence probe according to claim 1, it is characterised in that step
(2) mixed solvent of acetone and distilled water described in, wherein, acetone, the volume ratio of distilled water are 1:1.
A kind of 5. preparation method of response type mercury ion fluorescence probe according to claim 1, it is characterised in that step
(2) 5- phenyl isothiocyanates base -10,15,20- trisulfonic acid base phenyl porphyrins (PNCS) and the molar ratio of ethylene diamine derivative are in
1:1。
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| CN109211857A (en) * | 2018-09-17 | 2019-01-15 | 南京工业大学 | One kind can detect mercury, cadmium, lead, the solid phase fluorescent sensor of arsenic heavy metal ion simultaneously |
| CN112552901B (en) * | 2019-09-10 | 2022-06-21 | 浙江大学城市学院 | Ratio type zinc ion fluorescent probe and preparation and application thereof |
| CN111499552B (en) * | 2020-05-07 | 2022-03-04 | 池州学院 | Synthetic method and application of novel thiourea compound |
| CN113968864B (en) * | 2020-07-23 | 2023-04-07 | 浙大城市学院 | Cu based on zinc porphyrin axial coordination regulation + Fluorescent probe, preparation method and application |
| CN115925725A (en) * | 2022-11-25 | 2023-04-07 | 杭州珈晟生物数字技术有限公司 | Aluminum ion fluorescent probe, preparation method and application |
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