CN102585802A - Novel water-soluble sulfydryl fluorescent probe, and preparation method and application thereof - Google Patents
Novel water-soluble sulfydryl fluorescent probe, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a novel water-soluble sulfydryl fluorescent probe which is a 2-(2'-hydroxyphenyl) benzimidazole (HPBI) compound modified by quaternary ammonium salt. The compound has the structure of 2-[5-(trimethyl quaternary ammonium salt)-acetamido-2'-hydroxyphenyl) benzimidazole. The fluorescent probe can be used for detecting a biological sulfhydryl compound in aqueous solution and living tumor cell, i.e. the novel water-soluble sulfydryl fluorescent probe is selectively coordinated with cupric ions and an obtained coordination compound can enter the living cell to be reacted with the sulfhydryl compound, so that a fluorescent compound is changed into ionic molecules again and fluorescent light is recovered. The novel water-soluble sulfydryl fluorescent probe has the advantages that the novel water-soluble sulfydryl fluorescent probe has excellent water solubility and biocompatibility and high sulfhydryl measurement sensitivity; before and after GSH (glutathione) reaction, the fluorescent light is rapidly changed and is stable; the novel water-soluble sulfydryl fluorescent probe can be stored and used for a long time; and according to the fluorescent probe, a good research method is provided for researching a biological signal channel related to the biological sulfydryl and the significance and diagnosis of the sulfydryl in the in vivo process of diseases or organisms.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of new type water-solubility mercapto fluorescence probe.
Background technology
Sulfydryl (SH) is one of chemically reactive is the highest in the cell group.In protein, sulfydryl partly is the reactive behavior the highest functional group relevant with enzymic activity.Sulfhydryl compound in the cell, especially gsh, as a kind of important antioxidants in the organism, it can dispose the intravital radical of people, and cleaning and purification human internal environment pollute, thereby have promoted people's physical and mental health.Particularly reduced glutathion (GSH) itself is subject to some material oxidation, so it can protect sulfydryl in numerous protein and the enzyme equimolecular not by like objectionable impurities oxidations such as radicals in vivo, thereby lets protein and its physiological function of enzyme equimolecular performance.Along with mercapto functional group important physiological effect in vivo is familiar with gradually, the method for the biological sulfhydryl compound of discriminating and quantitatively determined receives publicity day by day.Traditional detection method has Nitroprusside ion method, iodimetry,iodometry, amperometry, spectrophotometry, colourimetry etc.; But there are shortcomings such as sensitivity low (the mmole level is above could to be detected), complicated operation, poor selectivity in these methods; Therefore the mensuration that can not adapt to sulfhydryl compound in some biological sample need more synthetic high-sensitivity analysis detection reagent.
Fluorometry is because its cost is low, and favorable repeatability is measured sensitivity, and selectivity is high, and can realize visual inspection, is a kind of extraordinary mensuration means.Therefore, novel sulfydryl luciferase assay reagent becomes the focus of research.Mercapto fluorescence probe is because detection sensitivity is high; And can be used as biological structure research indication mechanism, thereby be widely applied to the fields such as diagnosis, HPLC analysis sulfhydryl compound of monitoring and location, the disease of research protein structure and microenvironment character, trace detection choline enzyme or glutathione S-transferase, histochemical stain, Ag-Ab reflection.
The mercapto fluorescence probe of having reported has aryl halide, red sulphonyl aziridines etc.; They have sensitivity preferably; Generally about nmole (nmol), but these probes self all have stronger fluorescence, can cause lower SNR when being used for the viable cell imaging to the detectability of sulfydryl; Also have one type like the cumarone sulfonic acid halide, though autofluorescence a little less than, need and be higher than under the room temperature at alkaline condition and derive with sulfydryl, also can't be applied to viable cell and form images.The reagent of the most frequently used sulfydryl of deriving is the acetyl halide verivate, mainly is the iodo-acid amide verivate, and it is fast with the sulfydryl reaction, under room temperature and physiological pH, can carry out, but product loses fluorophore easily, and self is to photo-labile.These existing mercapto fluorescence probes all can not well satisfy the requirement of od-ray detection and fluorescence imaging.
We are through discovering that the small molecules fluorescent chemicals that contains 2-(2 '-hydroxy phenyl) benzoglyoxaline (HPBI) structure is one type of fluorescent chemicals with excited state prototropy (ESIPT) characteristic; It is big to have stokes (Stokes) displacement; The fluorescent quantum yield is high; Can form more stable advantages such as title complex with transition metal, can be used as the characteristic fluorophore of new function fluorescent molecular probe.As part, new bivalent cupric ion title complex has been synthesized in design, has carried out the preliminary study of transition metal complex class mercapto fluorescence probe with 2-(2 '-hydroxy phenyl) benzoglyoxaline (HPBI) compounds for we.Experimental result shows, HPBI class fluorescent chemicals with after paramagnetic cupric cooperates photoextinction has taken place.This is because excited state prototropy (ESIPT) mechanism, can under the optical excitation condition, form enol form and the stronger fluorescence of keto-acid tautomer generation based on the function fluorescent probe molecule of HPBI structure.Along with the adding of cupric ion, form new mixture and made the ESIPT process be obstructed, probe molecule fluorescence disappears.Reduced glutathion (GSH) adds the back and has formed new mixture with bivalent cupric ion, makes probe compound dissociate out, and the ESIPT process is able to recover, and produces fluorescence.We can develop the method that a kind of quick, easy being used to detects GSH based on this principle.
Summary of the invention
The objective of the invention is to above-mentioned existing problems; A kind of new type water-solubility mercapto fluorescence probe and preparation method thereof is provided; This fluorescent probe good biocompatibility can be used for GSH imaging mensuration in the viable cell, measures real sulfhydryl compound concentration change in the implantable bioartificial body; Help the diagnosis of relative disease, and provide a kind of quick, easy being used to detect the method for reduced glutathion (GSH).
Technical scheme of the present invention:
A kind of new type water-solubility mercapto fluorescence probe, compound name be called 2-[5 '-(QAE alkali)-acetamido-2 '-hydroxy phenyl] benzoglyoxaline (TMACA-HPBI), its chemical molecular formula is C
18H
21N
4O
2Cl; This compound with 2-(2 '-hydroxy phenyl) benzoglyoxaline (HPBI) structure as fluorescent chromophore; Improve the water-soluble of molecule with trimethyl ammonium chloride as hydrophilic radical; Fusing point is higher than 300 ℃, and water-soluble back is to send blue-fluorescence under the ultra violet lamp of 365nm at wavelength, and the molecular structural formula of this fluorescent probe is:
A kind of preparation method of said new type water-solubility mercapto fluorescence probe, step is following:
1) with the aniline aniline-water solution that obtains soluble in water; Adding concentrated hydrochloric acid dissolves aniline fully; Ice bath is cooled to 0-4 ℃, obtains anilinechloride solution, with the Sodium Nitrite sodium nitrite in aqueous solution that obtains soluble in water; Ice bath is cooled to be added drop-wise in the above-mentioned anilinechloride solution with the speed that p.s., 1-2 dripped after 0-4 ℃, makes diazonium salt of aniline solution;
2) the NaOH aqueous solution is added in the salicylic aldehyde obtain salicylic aldehyde solution, under 0 ℃, diazonium salt of aniline solution is added drop-wise in the salicylic aldehyde alkali aqueous solution with the speed that p.s., 3-4 dripped, drip mass percent concentration simultaneously and be 20% Na
2CO
3It is 7-8 that solution keeps pH, treat that diazonium salt of aniline solution dropwises after, throw out is used the absolute ethyl alcohol recrystallization, can make 2-hydroxyl-5-azobenzene salicylic aldehyde;
3) 2-hydroxyl-5-azobenzene salicylic aldehyde, sodium sulfite anhy 96 are dissolved in the absolute ethyl alcohol, stir down at 20-25 ℃ and obtain mixing solutions, O-Phenylene Diamine is dissolved in N; Obtain o-phenylenediamine solution in the dinethylformamide; Then o-phenylenediamine solution is added drop-wise in the above-mentioned mixing solutions, reacted 1-3 hour down, pour reactant into cold water at 78-80 ℃; Separate out yellow mercury oxide; After filtration under diminished pressure, the vacuum-drying, use acetone recrystallization twice again, make 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline;
4) with 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline, mass percentage content be that 80% Hydrazine Hydrate 80, mass percentage content are that the 5%Pd/C catalyzer mixes with absolute ethyl alcohol; Reacted 30-60 minute down at 78-80 ℃; Filtered while hot then; Filtrating has been concentrated into crystal has separated out, filter cake is used ethyl alcohol recrystallization behind the suction filtration, make 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline;
5) with 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline joins and obtains acetone soln in the acetone, under 60 ℃ of conditions, is stirred to dissolving, adds chloroacetyl chloride and triethylamine after being cooled to room temperature; Stirred 3-5 hour down at 20-25 ℃; This solution is poured in the water, faint yellow deposition occurred, left standstill 1-2 hour; The water flush cake is 2-3 time behind the suction filtration, make after the drying 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline;
6) with 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline and mass percent concentration be that 33% Trimethylamine 99 ethanolic soln joins in the absolute ethyl alcohol; Under 70-80 ℃ of temperature, stirred 24-48 hour; Decompression steams 95% of ethanol volume; Be cooled to add after 20-25 ℃ the ether of 50 times of surplus solution volumes; Obtain white precipitate, suction filtration, drying, obtain white powder be title product-2-[5 '-(QAE alkali)-acetamido-2 '-hydroxy phenyl] benzoglyoxaline (TMACA-HPBI).
The mass percent concentration of said aniline-water solution is 12-14%, and the mass percent concentration of concentrated hydrochloric acid is 37%, and aniline-water solution and concentrated hydrochloric acid volume ratio are 8: 3; The mass percent concentration of said sodium nitrite in aqueous solution is 30%, and the volume ratio of sodium nitrite in aqueous solution and anilinechloride solution is 1: 4.
The mass percent concentration of the said NaOH aqueous solution is 1%, and aqueous sodium hydroxide solution and salicylic aldehyde mass ratio are 50: 1, and salicylic aldehyde solution is 6: 1 with diazonium salt of aniline liquor capacity ratio.
The mass ratio of said 2-hydroxyl-5-azobenzene salicylic aldehyde, sodium sulfite anhy 96 and absolute ethyl alcohol is 2: 1: 150, O-Phenylene Diamine dissolving and N, and the mass ratio of dinethylformamide is 1: 10, o-phenylenediamine solution is 1: 12 with the mixed liquor volume ratio.
Said 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline, mass percentage content be that 80% Hydrazine Hydrate 80, the mass ratio that mass percentage content is 5%Pd/C catalyzer and absolute ethyl alcohol are 13: 30: 1: 320.
Said 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline and acetone mass ratio be 1: 4, the volume ratio of acetone soln and chloroacetyl chloride and triethylamine is 20: 2: 3.
Said 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline, mass percent concentration be that 33% the Trimethylamine 99 aqueous ethanolic solution and the mass ratio of absolute ethyl alcohol are 1: 2: 90.
A kind of said new type water-solubility mercapto fluorescence probe application, be used for the detection of biological sulfhydryl compound of the aqueous solution and vivo tumor cell, method is following:
1) said fluorescent probe is mixed with the aqueous solution of 10 μ M, then with Cu
2+Reaction forms the complex type fluorescent probe, adds the testing sample aqueous solution that contains different sulfhydryl compounds again, and reduced glutathion in the working sample (GSH) concentration is used for the external quick diagnosis of GSH concentration;
2) with above-mentioned title complex probe and active somatic cell co-cultivation; With no change before and after the normal cell L929 effect; After tumour cell Hela effect, can under the fluorescence electron microscope, observe in the cell and send blue-greenish colour fluorescence with cytolemma, can be used for detecting the vivo tumor cell.
Said new type water-solubility mercapto fluorescence probe preparing method's operational path is represented as follows:
Fluorescent probe of the present invention compared with prior art, its positively effect is: 1) have good water-solubility and biocompatibility; 2) have higher sulfydryl and measure sensitivity; 3) change in fluorescence is rapid and before and after the GSH reaction, and fluorescence is stable, is suitable for the instant fluorometric assay of GSH in the system; 4) good stability can prolonged preservation use; 5) can get in the viable cell, selectivity and GSH effect cause probe molecule fluorescence significantly to strengthen.This fluorescent probe is the relevant biological signaling pathway of the biological sulfydryl of research, and meaning and the diagnosis of sulfydryl in some diseases or organism internal procedure provides research method preferably.
Description of drawings
Fig. 1 is the fluorescence spectrum variation diagram after water soluble fluorescence probe molecule 1-Cu (1 expression compound) cooperates.
Fig. 2 be the water soluble fluorescence probe with other metals ion ionization after fluorescence intensity change comparison diagram.
Fig. 3 is the fluorescence intensity change curve that water-soluble mercapto fluorescence probe detects sulfhydryl compound GSH.
Fig. 4 is the selectivity that metal ion match type fluorescent probe detects biological sulfhydryl compound.
Embodiment
Pass through the bright specifically the present invention of embodiment below, but the present invention does not receive the qualification of following embodiment.
Embodiment:
A kind of preparation method of said new type water-solubility mercapto fluorescence probe, step is following:
1) 4.66g aniline is dissolved in the 40mL water obtains aniline-water solution; Adding the 15mL mass percent concentration and be 37% concentrated hydrochloric acid dissolves aniline fully; Ice bath is cooled to 0 ℃, obtains anilinechloride solution, the 3.6g Sodium Nitrite is dissolved in the 12mL water obtain sodium nitrite in aqueous solution; Ice bath be cooled to after 0 ℃ with p.s. 2 speed be added drop-wise in the above-mentioned anilinechloride solution, make diazonium salt of aniline solution;
2) be that 1% NaOH aqueous solution 300mL adds in the 6.1g salicylic aldehyde and obtains salicylic aldehyde solution with mass percent concentration; Under 0 ℃ with diazonium salt of aniline solution with p.s. 3 speed be added drop-wise in the salicylic aldehyde alkali aqueous solution, drip mass percent concentration simultaneously and be 20% Na
2CO
3It is 8 that solution keeps pH, treat that diazonium salt of aniline solution dropwises after, throw out is used the absolute ethyl alcohol recrystallization, can make 2-hydroxyl-5-azobenzene salicylic aldehyde; The product fusing point is 127 ℃;
3) 2.1g 2-hydroxyl-5-azobenzene salicylic aldehyde, 0.96g sodium sulfite anhy 96 are dissolved in the 50mL absolute ethyl alcohol, stir down at 25 ℃ and obtain mixing solutions, the 1g O-Phenylene Diamine is dissolved in 25mLN; Obtain o-phenylenediamine solution in the dinethylformamide; Then o-phenylenediamine solution is added drop-wise in the above-mentioned mixing solutions, reacted 3 hours down, pour reactant into cold water at 80 ℃; Separate out yellow mercury oxide; After filtration under diminished pressure, the vacuum-drying, use acetone recrystallization twice again, make 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline;
4) with 1.5g 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline, 5.2ml mass percentage content be that 80% Hydrazine Hydrate 80,0.038g mass percentage content are that the 5%Pd/C catalyzer mixes with the 100mL absolute ethyl alcohol; Reacted 50 minutes down at 80 ℃; Filtered while hot then; Filtrating has been concentrated into crystal has separated out, filter cake is used ethyl alcohol recrystallization behind the suction filtration, make 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline;
5) with 1g 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline joins in the 50ml acetone and obtains acetone soln, under 60 ℃ of conditions, is stirred to dissolving, adds chloroacetyl chloride and triethylamine after being cooled to room temperature; Stirred 3 hours down at 25 ℃; This solution is poured in the water, faint yellow deposition occurred, left standstill 1 hour; The water flush cake is 3 times behind the suction filtration, make after the drying 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline;
6) with 0.453g 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline and 1.05mL mass percent concentration be that 33% Trimethylamine 99 ethanolic soln joins in the 50mL absolute ethyl alcohol; Under 80 ℃ of temperature, stirred 48 hours; Decompression steams 95% of ethanol volume; Be cooled to add after 25 ℃ the ether of surplus solution volume 250mL; Obtain white precipitate, suction filtration, drying, obtain white powder be title product-2-[5 '-(QAE alkali)-acetamido-2 '-hydroxy phenyl] benzoglyoxaline (TMACA-HPBI).
The master data of this new type water-solubility fluorescent probe:
The incarnadine powder, Mp:＞300 ℃;
1H?NMR(400MHz，DMSO-d
6)：4.48(s，2H，Ar-CH
2-)，7.06(d，1H，Ar-H)，7.28(dd，2H，Ar-H)，7.44(dd，1H，Ar-H)，7.66(dd，2H，Ar-H)，8.44(s，1H，Ar-H)，11.13(s，1H，-NH-)，13.19(br?s，2H，Ph-OH，-NH).Anal.Calcd?for?C
18H
21ClN
4O
2：C，59.91；H，5.87；N，15.53.Found：C，60.08；H，5.75；N，15.65。
The detection of this fluorescent probe and application:
1) fluorescence spectrum after water soluble fluorescence probe and bivalent cupric ion cooperate changes:
Probe molecule is soluble in water, to final concentration be 10 μ M.Get 3mL, drip the Cu that concentration is 500 μ M again
2+Solution, Cu in mixing solutions
2+Strength of solution is 13 μ M.The period detecting fluorescence intensity changes, and makes fluorescence intensity to Cu
2+The change curve of concentration, excitation wavelength is 320nm.Fig. 1 is the fluorescence spectrum variation diagram after water soluble fluorescence probe molecule 1-Cu cooperates.Along with the continuous increase of copper ion concentration, the fluorescence intensity of probe molecule in the aqueous solution reduces gradually.Work as Cu
2+Concentration is increased to 1.3 * 10
-3The time, probe molecule fluorescence with do not add Cu
2+Compare almost and disappear.As can be seen from Figure 1 after the reaction of this probe and bivalent cupric ion, fluorescence intensity decreases drastically, and this explanation has formed new title complex and made the ESIPT process be obstructed, the probe molecule fluorescent quenching along with the adding of cupric ion.
2) the water soluble fluorescence probe contrasts with other transition metal ion ionic reactions:
Probe molecule is soluble in water, to final concentration be 10 μ M.Get 3mL, dripping concentration again is the various metals ion aqueous solution of equal 500 μ M, makes that metal ion solution concentration is 13 μ M in the mixing solutions.The period detecting fluorescence intensity is with respect to the variation multiple of former probe, and excitation wavelength is the histogram that 320nm makes corresponding each transition metal ion of fluorescence intensification factor.Fig. 2 be the water soluble fluorescence probe with other metals ion ionization after fluorescence intensity change comparison diagram.In the phase concentration TMACA-HPBI aqueous solution, add the different metal ion of same concentrations, have only Cu
2+Change to the system fluorescence intensity is the most remarkable.As can be seen from Figure 2, this fluorescent probe is compared with other transition metal ions, to Cu
2+Have very high selectivity, can exclusive reaction.Fluorescence intensity has obvious variation before and after reaction, and other the common metals ions that exist in the biosystem can not cause tangible change to its fluorescence, can well get rid of the different metal ionic and disturb.
3) the water soluble fluorescence probe is to the fluorescence intensity-concentration curve of gsh:
Water intaking dissolubility fluorescent probe probe molecule, this compound is soluble in water, to final concentration be 10 μ M.Get 3mL, add Cu
2+Solution makes Cu in the mixing solutions
2+Strength of solution is that 13 μ M. drip the GSH solution that concentration is 500 μ M afterwards, and the GSH strength of solution is 39 μ M in mixing solutions.The period detecting fluorescence intensity changes, and excitation wavelength is 320nm, makes fluorescence intensity to the GSH concentration curve.Fig. 3 is the fluorescence intensity change curve that water-soluble mercapto fluorescence probe detects sulfhydryl compound GSH.In the 1-Cu aqueous solution, add GSH solution gradually, along with GSH concentration raises, the system fluorescence intensity strengthens gradually.From Fig. 3, can draw, fluorescence intensity has had significantly recovery behind the adding GSH solution, and this explanation GSH solution and bivalent cupric ion have formed new title complex, make probe compound dissociate out, and the ESIPT process is able to recover, and produces fluorescence.
4) probe selectivity that sulfhydryl compound is detected:
Water intaking dissolubility fluorescent probe probe molecule, it is soluble in water, to final concentration be 10 μ M.Get 3mL, add Cu
2+Solution makes Cu in the mixing solutions
2+Strength of solution is 13 μ M.Add concentration more respectively and be 500 μ M gsh (GSH), cysteine hydrochloride (L-Cys), mercaptoethylamine hydrochloride (MEA), Thiovanic acid (TGA) solution makes that the sulfhydryl compound strength of solution is 39 μ M in the mixing solutions.Fluorescence intensity is with respect to the variation multiple of former probe behind the mensuration adding sulfhydryl compound, and excitation wavelength is 320nm.Make the change curve of fluorescence intensification factor to sulfhydryl compound concentration.Fig. 4 is the selectivity that metal ion match type fluorescent probe detects biological sulfhydryl compound.The GSH detection sensitivity is superior to other three kinds of sulfhydryl compounds.As can be seen from Figure 4, compare with other sulfhydryl compound, this fluorescent probe has very high selectivity to reduced glutathion (GSH), can exclusive reaction.Fluorescence intensity has obvious variation before and after reaction, and other the common sulfhydryl compounds that exist in the biosystem can not cause tangible change to its fluorescence.Therefore, it can be applied to selectivity detection of biological sulfhydryl compound in the aqueous solution.
5) probe is distinguished normal cell and tumour cell fluorescence imaging:
Get the 1-Cu title complex and be dissolved in the deionized water, concentration is 0.1M, joins respectively in L929 and the Hela cell, and the final concentration of probe is 5 μ M.The L929 cell adds 10% foetal calf serum nutrient solution with Roswell Park Memorial Institute (RPMI1640) substratum to be cultivated, and the Hela cell adds 5% foetal calf serum nutrient solution with Dublecco ' Modified Eagle Media (DMEM) substratum and cultivates.After 12 hours, shine fluorescence photo.
Probe 1-Cu is very obvious to the detection effect of tumour cell.In normal cell L929, have only faint fluorescence to strengthen after adding probe, this possibly be that the GSH that produces of L929 cell background is relevant.And cell fluorescence significantly strengthens after in tumour cell Hela, adding probe, and this is to become big because a large amount of GSH of tumour cell metabolism generation makes in the cell with cell peripheral GSH partial concn.GSH and Cu
2+Form and make behind the new mixture 1 to dissociate out the ESIPT process takes place, probe molecule fluorescence recovers, and reaches the purpose that tumour cell is detected.
Claims (9)
1. new type water-solubility mercapto fluorescence probe is characterized in that: compound name be called 2-[5 '-(QAE alkali)-acetamido-2 '-hydroxy phenyl] benzoglyoxaline (TMACA-HPBI), its chemical molecular formula is C
18H
21N
4O
2Cl; This compound with 2-(2 '-hydroxy phenyl) benzoglyoxaline (HPBI) structure as fluorescent chromophore; Improve the water-soluble of molecule with trimethyl ammonium chloride as hydrophilic radical; Fusing point is higher than 300 ℃, and water-soluble back is to send blue-fluorescence under the ultra violet lamp of 365nm at wavelength, and the molecular structural formula of this fluorescent probe is:
2. preparation method of new type water-solubility mercapto fluorescence probe according to claim 1 is characterized in that step is following:
1) with the aniline aniline-water solution that obtains soluble in water; Adding concentrated hydrochloric acid dissolves aniline fully; Ice bath is cooled to 0-4 ℃, obtains anilinechloride solution, with the Sodium Nitrite sodium nitrite in aqueous solution that obtains soluble in water; Ice bath is cooled to be added drop-wise in the above-mentioned anilinechloride solution with the speed that p.s., 1-2 dripped after 0-4 ℃, makes diazonium salt of aniline solution;
2) the NaOH aqueous solution is added in the salicylic aldehyde obtain salicylic aldehyde solution, under 0 ℃ of temperature, diazonium salt of aniline solution is added drop-wise in the salicylic aldehyde alkali aqueous solution with the speed that p.s., 3-4 dripped, drip mass percent concentration simultaneously and be 20% Na
2CO
3It is 7-8 that solution keeps pH, treat that diazonium salt of aniline solution dropwises after, throw out is used the absolute ethyl alcohol recrystallization, can make 2-hydroxyl-5-azobenzene salicylic aldehyde;
3) 2-hydroxyl-5-azobenzene salicylic aldehyde, sodium sulfite anhy 96 are dissolved in the absolute ethyl alcohol, under 20-25 ℃ of temperature, stir and obtain mixing solutions, O-Phenylene Diamine is dissolved in N; Obtain o-phenylenediamine solution in the dinethylformamide; Then o-phenylenediamine solution is added drop-wise in the above-mentioned mixing solutions, reaction is 1-3 hour under 78-80 ℃ of temperature, pours reactant into cold water; Separate out yellow mercury oxide; After suction filtration, the vacuum-drying, use acetone recrystallization twice again, make 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline;
4) with 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline, mass percentage content be that 80% Hydrazine Hydrate 80, mass percentage content are that the 5%Pd/C catalyzer mixes with absolute ethyl alcohol; Reaction is 30-60 minute under 78-80 ℃ of temperature; Filtered while hot then; Filtrating has been concentrated into crystal has separated out, filter cake is used ethyl alcohol recrystallization behind the suction filtration, make 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline;
5) with 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline joins and obtains acetone soln in the acetone, under 60 ℃ of conditions, is stirred to dissolving, adds chloroacetyl chloride and triethylamine after being cooled to room temperature; Under 20-25 ℃ of temperature, stirred 3-5 hour; This solution is poured in the water, faint yellow deposition occurred, left standstill 1-2 hour; The water flush cake is 2-3 time behind the suction filtration, make after the drying 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline;
6) with 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline and mass percent concentration be that 33% Trimethylamine 99 ethanolic soln joins in the absolute ethyl alcohol; Under 70-80 ℃ of temperature, stirred 24-48 hour; Decompression steams 95% of ethanol volume; Be cooled to add after 20-25 ℃ the ether of 50 times of surplus solution volumes; Obtain white precipitate, suction filtration, drying, obtain white powder be title product-2-[5 '-(QAE alkali)-acetamido-2 '-hydroxy phenyl] benzoglyoxaline (TMACA-HPBI).
3. according to the preparation method of the said new type water-solubility mercapto fluorescence probe of claim 2; It is characterized in that: the mass percent concentration of said aniline-water solution is 12-14%; The mass percent concentration of concentrated hydrochloric acid is 37%, and aniline-water solution and concentrated hydrochloric acid volume ratio are 8: 3; The mass percent concentration of said sodium nitrite in aqueous solution is 30%, and the volume ratio of sodium nitrite in aqueous solution and anilinechloride solution is 1: 4.
4. according to the preparation method of the said new type water-solubility mercapto fluorescence probe of claim 2; It is characterized in that: the mass percent concentration of the said NaOH aqueous solution is 1%; Aqueous sodium hydroxide solution and salicylic aldehyde mass ratio are 50: 1, and salicylic aldehyde solution is 6: 1 with diazonium salt of aniline liquor capacity ratio.
5. according to the preparation method of the said new type water-solubility mercapto fluorescence probe of claim 2; It is characterized in that: the mass ratio of said 2-hydroxyl-5-azobenzene salicylic aldehyde, sodium sulfite anhy 96 and absolute ethyl alcohol is 2: 1: 150; O-Phenylene Diamine dissolving and N; The mass ratio of dinethylformamide is 1: 10, and o-phenylenediamine solution is 1: 12 with the mixed liquor volume ratio.
6. according to the preparation method of the said new type water-solubility mercapto fluorescence probe of claim 2, it is characterized in that: said 2-(5 '-azobenzene-2 '-hydroxy phenyl) benzoglyoxaline, mass percentage content be that 80% Hydrazine Hydrate 80, the mass ratio that mass percentage content is 5%Pd/C catalyzer and absolute ethyl alcohol are 13: 30: 1: 320.
7. according to the preparation method of the said new type water-solubility mercapto fluorescence probe of claim 2; It is characterized in that: said 2-(5 '-amino-2 '-hydroxy phenyl) benzoglyoxaline and acetone mass ratio be 1: 4, the volume ratio of acetone soln and chloroacetyl chloride and triethylamine is 20: 2: 3.
8. according to the preparation method of the said new type water-solubility mercapto fluorescence probe of claim 2, it is characterized in that: said 2-(5 '-chloro acetylamino-2 '-hydroxy phenyl) benzoglyoxaline, mass percent concentration be that 33% the Trimethylamine 99 aqueous ethanolic solution and the mass ratio of absolute ethyl alcohol are 1: 2: 90.
One kind according to claim 1 the new type water-solubility mercapto fluorescence probe application, it is characterized in that being used for the detection of biological sulfhydryl compound of the aqueous solution and vivo tumor cell, method is following:
1) said fluorescent probe is mixed with the aqueous solution of 10 μ M, then with Cu
2+Reaction forms the complex type fluorescent probe, adds the testing sample aqueous solution that contains different sulfhydryl compounds again, and reduced glutathion in the working sample (GSH) concentration is used for the external quick diagnosis of GSH concentration;
2) with above-mentioned title complex probe and active somatic cell co-cultivation; With no change before and after the normal cell L929 effect; After tumour cell Hela effect, can under the fluorescence electron microscope, observe in the cell and send blue-greenish colour fluorescence with cytolemma, can be used for detecting the vivo tumor cell.
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| CN111684277A (en) * | 2018-03-01 | 2020-09-18 | 埃科莱布美国股份有限公司 | Method for measuring benzimidazole compounds in water |
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| CN107037018B (en) * | 2016-10-28 | 2019-07-30 | 郑州大学 | A kind of application of the lysosome positioning fluorescence probe in near-infrared ratio test hydrazine |
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| CN111684277B (en) * | 2018-03-01 | 2023-10-20 | 埃科莱布美国股份有限公司 | Method for measuring benzimidazole compounds in water |
| CN108641710A (en) * | 2018-05-22 | 2018-10-12 | 中国人民解放军第二军医大学 | A kind of fluorescence probe and its preparation method and application of detection protein sulphur sulfhydrylation |
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| CN110746411A (en) * | 2019-11-15 | 2020-02-04 | 辽宁科技大学 | Fluorescent probe and preparation method thereof, fluorescent probe test paper and application thereof |
| CN110849856A (en) * | 2019-12-03 | 2020-02-28 | 浙江大学 | Application of salicylaldehyde hydrazone derivative with aggregation-induced emission performance in detection of nitrite ions |
| CN114460052A (en) * | 2022-01-11 | 2022-05-10 | 武汉理工大学 | Method for directly detecting concentration of sodium pyruvate based on fluorescent carbon quantum dots |
| CN114460052B (en) * | 2022-01-11 | 2023-06-20 | 武汉理工大学 | Method for directly detecting concentration of sodium pyruvate based on fluorescent carbon quantum dots |
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