CN101892046B - Colorimetric fluorescence probe for high selectivity multiple biological thiol and preparation method thereof - Google Patents
Colorimetric fluorescence probe for high selectivity multiple biological thiol and preparation method thereof Download PDFInfo
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- CN101892046B CN101892046B CN 201010209443 CN201010209443A CN101892046B CN 101892046 B CN101892046 B CN 101892046B CN 201010209443 CN201010209443 CN 201010209443 CN 201010209443 A CN201010209443 A CN 201010209443A CN 101892046 B CN101892046 B CN 101892046B
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- 0 *c(c1n[s]nc11)c(*)c(C(c2c(*)c(*)c(*)c(*)c22)=O)c1C2=O Chemical compound *c(c1n[s]nc11)c(*)c(C(c2c(*)c(*)c(*)c(*)c22)=O)c1C2=O 0.000 description 2
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Abstract
The invention relates to a colorimetric fluorescence probe for high selectivity multiple biological thiol and a preparation method thereof, particularly to the colorimetric fluorescence probe which uses selenazole ring as an identification receptor, takes an anthraquinone derivant as an information report functional group, can high selectively distinguish and identify cysteine, homocysteine and glutathione and the preparation method thereof, belonging to the technical field of life science. The colorimetric fluorescence probe has the following structure formula as shown in the specification. The colorimetric fluorescence probe is prepared by grinding and reacting a diamine compound corresponding to the colorimetric fluorescence probe for the high selectivity multiple biological thiol and selenium dioxide in an agate mortar for 15-90 min at the grinding temperature of 10-35 DEG C, the mole ratio of the corresponding diamine compound to the selenium dioxide is 1:1-5, and the reaction formula is shown in the specification. The invention can directly distinguish and identify the cysteine, the homocysteine and the glutathione by eyes, and carry out imaging analysis on biological thiol in living cells. The invention has the advantages of simple method, environmental protection and industrial production.
Description
Technical field
The present invention relates to colorimetric fluorescence probe for high selectivity multiple biological thiol and preparation method thereof, be specifically related to a class take the selenazoles ring as identification receptor, take anthraquinone derivative as the report information functional group, and can highly selective Division identification halfcystine, the colorimetric fluorescence probe of homocysteine and gsh and preparation method thereof, belong to the Life Sci-Tech field.
Background technology
Mercaptan is a very important material of class in life system and chemical science.Small molecules mercaptan extensively is distributed in cell, blood plasma and tissue, plays an important role in redox equilibrium with keeping in organism at the important physiological activity of participation.Gsh (glutathione, GSH) be the abundantest small molecules sulfur alcohol compound (1～10mM) that exists in cell, can combine with the toxic compounds that enters body, heavy metal ion or carcinogenic substance etc., short its excretes, in therefore playing and detoxification.In addition; gsh is a kind of antioxidant of the sulfydryl of protective enzyme and other protein; it is the chief component of non-albumen sulfydryl in cell; participate in intracellular redox reaction; reduced form (GSH) plays keying action with the ratio of Sleep-promoting factor B (GSSG) content in keeping vivo oxidation reduction balance, the ANOMALOUS VARIATIONS of this ratio will cause the generation of the diseases such as heart trouble, tumour and multiple abalienation.Halfcystine (cysteine, Cys) and homocysteine (also referred to as homocysteine, homocysteine, Hcy) are also two important sulfur alcohol compounds that exist in organism.The reduction of cysteine content often can cause grow slowly, hair fades, oedema, listless, hepatic diseases, skin injury and the disease such as thin and weak.Homocysteine will be accompanied by the diseases such as myocardial infarction, exhausting, venous thromboembolism in the rising of Plasma.The content of homocysteine also may cause senile dementia (Alzheimer ' s disease), nervous center defective, conceived complication, enteritis and osteoporosis diseases higher than normal value.In addition, the isocyatic rising of oxidation state Cys and Hcy in blood sample, perhaps the reduction of reduced form GSH concentration is all the distinctive characteristic index of oxidative stress.Due to contain mercaptan amino acid can well reflect cellular oxidation stress degree, so in blood sample, the amino acid whose change in concentration of thio-alcohol has begun to serve as the clinical indices of the various metabolic disturbance diseases of diagnosis and detection.
Given this, developed the detection of various analysis for biological thiol, especially colorimetric and fluorescent spectrometry have obtained abundant development and have obtained larger progress, but only are confined to: improve the sensitivity of probe and selectivity, shortening time of response with can realize detecting in real time and the broadening useful range to satisfy requirement in practical application etc.Recently, the people such as de Silva have elaborated the superiority of multivariate analysis probe on Nature, and wherein more topmost is that it can overcome and analyzes the plurality of target thing and need to inject simultaneously the deficiency that multiple probe brings.Though polynary probe has certain development, almost all for analysis hydrogen ion and metallic cation, and the probe of these biological thiols of Division identification still has no report effectively.Therefore, the polynary probe that the synthetic class of design can the multiple biological thiol of Division identification becomes the major objective that the present invention will solve.
Summary of the invention
The objective of the invention is can not the Division identification halfcystine in order to solve prior art, homocysteine and gsh be in the problem of active somatic cell intensive amount, proposes colorimetric fluorescence probe for high selectivity multiple biological thiol and preparation method thereof.
The objective of the invention is to be achieved through the following technical solutions.
Colorimetric fluorescence probe for high selectivity multiple biological thiol of the present invention, a class are take the selenazoles ring as identification receptor, with the multiple biological thiol colorimetric fluorescence probe of anthraquinone derivative as the report information functional group, and its structural formula is as follows:
In formula: R
1, R
2, R
3, R
4, R
5, R
6A kind of in hydrogen atom, alkyl, alkoxyl group, sulfonic group or ester group; R
1, R
2, R
3, R
4, R
5, R
6Can be identical or different.
The preparation method of colorimetric fluorescence probe for high selectivity multiple biological thiol of the present invention is: carry out griding reaction with the corresponding diamino compounds of colorimetric fluorescence probe for high selectivity multiple biological thiol and tin anhydride in agate mortar and make, grinding temperature is 10～35 ℃, milling time is 15～90min, and the mol ratio of corresponding diamino compounds and tin anhydride is 1: 1～5; Its reaction structure formula is:
The colorimetric fluorescence probe for high selectivity multiple biological thiol that aforesaid method obtains can produce different absorption spectrum simultaneous distinct colors from halfcystine, homocysteine and gsh effect respectively to be changed and fluorescence emission spectrum, thereby realizes the selectivity identification to halfcystine, homocysteine and gsh; And all can not cause the obvious change of absorption spectrum and fluorescence emission spectrum with other non-mercaptan amino acid effects, and these occurrences of amino acid do not disturb to the quantitative assay of biological thiol; But hatch after 10～60min just transfered cell with cell, thereby realize the mensuration to biological thiol content in cell.
Mentality of designing: anthraquinone and its derivative Chang Zuowei chromophoric group and fluorophore are widely used in the design of negatively charged ion and metal ion probe, and these probes often are accompanied by the variation of solution colour and fluorescence spectrum in the process of identification target compound.In addition, the conclusion of having reported and the result of study that we obtain show that the selenazoles ring can be used as the acceptor of highly selective identification mercaptan.On this basis, we design and have synthesized with two amido anthraquinone derivatives as report information functional group and the organic selenazoles ring mercaptan probe as acceptor.We guess that in a single day the diamines in two amido anthraquinone derivatives forms the selenazoles loop type, and absorption spectrum has blue shift largely.When add biological thiol in probe solution after, the dismemberent of selenazoles will cause the red shift of absorption spectrum.Importantly, other functional groups in biological thiol (as amino, carboxyl etc.) may with anthraquinone in carbonyl the effect of multi-form or degree occurs, thereby cause the appearance of red shift Absorption and fluorescence spectrum in various degree, and then they are distinguished.
Beneficial effect
The enough eyes of the present invention's energy are Division identification halfcystine, homocysteine and gsh directly, and can carry out imaging analysis to biological thiol in viable cell, and this preparation method is simple, environmental protection, and cost is low, but suitability for industrialized production.
Description of drawings
Fig. 1 is that different analytes are on the impact of target compound (10 μ M) absorption spectrum;
Fig. 2 is that different analytes are on the impact of target compound (10 μ M) fluorescence spectrum;
Fig. 3 is the variation that there is lower target compound solution colour in different analytes;
Fig. 4 is that the GSH of different concns (0～900 μ M) is on the impact of target compound (10 μ M) absorption spectrum;
Fig. 5 is that the GSH of different concns (0～900 μ M) is on the impact of target compound (10 μ M) absorption spectrum;
Fig. 6 is that the GSH of different concns (20～120 μ M) is on the impact of target compound (10 μ M) absorption spectrum;
Fig. 7 is that the GSH of different concns (0～900 μ M) is on the impact of target compound (10 μ M) fluorescence spectrum;
Fig. 8 is that the GSH of different concns (0～900 μ M) is on the impact of target compound (10 μ M) fluorescence spectrum;
Fig. 9 is that the GSH of different concns (0～100 μ M) is on the impact of target compound (10 μ M) fluorescence spectrum;
Figure 10 is that different analytes are on the impact of target compound (10 μ M) absorption spectrum quantitative analysis GSH (300 μ M) result;
Figure 11 is that different analytes are on the impact of target compound (10 μ M) fluorescence spectrum quantitative analysis GSH (300 μ M) result;
Figure 12 is the co-focusing imaging figure of target compound mark live body HeLa cell;
The co-focusing imaging figure of Figure 13 live body HeLa cell that to be the target compound mark processed through NEM.
Embodiment
The present invention will be further described below in conjunction with embodiment.
Embodiment
With 238.2mg (1mmol) 1,2-two amido anthraquinones and 221.9mg (2mmol) tin anhydride be porphyrize in agate mortar respectively, then with two kinds of compound after porphyrize, continue to grind 1h, then the mixture after grinding is dissolved in trichloromethane, filters, and boils off solvent under decompression, resistates separates through column chromatography, trichloromethane is done eluent, then leacheate is removed the trichloromethane eluent, gets target compound sterling 261.8mg, yield 84%, reaction formula is as follows:
The nucleus magnetic hydrogen spectrum of the target compound that obtains and high resolution mass spectrum characterization data are as follows:
1H-NMR?(400MHz，DMSO-d
6)δ(*10
-6)：7.95(t，J＝7.0Hz，2H)，8.20(d，J＝7.6Hz，2H)，8.34(d，J＝2.8Hz，2H)；
HRMS(ESI?positive)：[M+H]
+calcd?for?C
14H
7N
2O
2Se?314.96676，found314.96644。
The target compound that obtains is carried out discriminance analysis to assay, assay is halfcystine Cys, homocysteine Hcy, gsh GSH, arginine Arg, L-Ala Ala, tyrosine Tyr, Methionin Lys, histidine, aspartic acid Asp, α-amino-isovaleric acid Val, leucine Leu, tryptophane Try, methionine(Met) Met, proline(Pro) Pro, phenylalanine Phep, Serine Ser, Threonine Thr, L-glutamic acid Glu and glycine Gly, the concentration of assay is 300 μ M, and its concrete discriminance analysis step is:
Under 25 ℃, target compound and the assay of 10 μ M is dissolved in the mixed system of phosphate buffer soln and ethanol, mix after 1h test UV spectrum and fluorescence spectrum and observe simultaneously its colour-change; The phosphate buffer soln that adds is 20mM, and its pH is 7.4, and the volume ratio of ethanol and water is 1: 1; Excitation wavelength used is 490nm, exciting and launching slit width is all 5nm, the UV spectrum spectrogram that test obtains as shown in Figure 1, the fluorescence spectrum spectrogram that obtains of test as shown in Figure 2, its colour-change as shown in Figure 3, in its figure, 1 represents target compound and Cys, 2 represent target compound and Hcy, 3 represent target compound and GSH, and 4 represent target compound, and 5 represent target compound and other naturally amino acid whose mixtures;
The target compound that obtains is carried out quantitative analysis to GSH, and its concrete steps are:
Under 25 ℃, the GSH that the target compound of 10 μ M and target compound and concentration is respectively 10 μ M, 20 μ M, 30 μ M, 40 μ M, 50 μ M, 60 μ M, 70 μ M, 80 μ M, 90 μ M, 100 μ M, 120 μ M, 140 μ M, 160 μ M, 200 μ M, 300 μ M, 400 μ M, 500 μ M, 600 μ M, 700 μ M, 800 μ M, 900 μ M is dissolved in the mixed system of phosphate buffer soln and ethanol, mixes test UV spectrum and fluorescence spectrum after 1h; The phosphate buffer soln that adds is 20mM, and its pH is 7.4, and the volume ratio of ethanol and water is 1: 1; Excitation wavelength used is 490nm, and exciting and launching slit width is all 5nm, the UV spectrum spectrogram that obtains of test as shown in Figure 4 and Figure 5, the absorbancy at target compound 490nm place and the linear relationship of GSH concentration are as shown in Figure 6; The fluorescence spectrum spectrogram that obtains of test as shown in Figure 7 and Figure 8, the fluorescence intensity at target compound 594nm place and the linear relationship of GSH concentration are as shown in Figure 9;
The impact of disturbance thing on target compound quantitative test GSH, its concrete steps are:
under 25 ℃, 10 μ M target compounds, 300 μ MGSH and 300 μ M chaff interferences are dissolved in the mixed system of phosphate buffer soln and ethanol test UV spectrum and fluorescence spectrum after mixing 1h, the phosphate buffer soln that adds is 20mM, and its pH is 7.4, and the volume ratio of ethanol and water is 1: 1, excitation wavelength used is 490nm, and exciting and launching slit width is all 5nm, chaff interference is respectively Arg, Ala, Tyr, Lys, His, Asp, Val, Leu, Try, Met, Pro, Phe, Ser, Thr, Glu and Gly, the UV spectrum spectrogram that obtains of test as shown in figure 10, in figure, histogram is the absorbancy at the 490nm place, the fluorescence spectrum spectrogram that obtains of test as shown in figure 11, in figure, histogram is the fluorescence intensity at the 594nm place, in Figure 10 and Figure 11, histogram represents respectively target compound (1), target compound+GSH+Arg (2), target compound+GSH+Ala (3), target compound+GSH+Tyr (4), target compound+GSH+Lys (5), target compound+GSH+His (6), target compound+GSH+Asp (7), target compound+GSH+Val (8), target compound+GSH+Leu (9), target compound+GSH+Try (10), target compound+GSH+Met (11), target compound+GSH+Pro (12), target compound+GSH+Phe (13), target compound+GSH+Ser (14), target compound+GSH+Thr (15), target compound+GSH+Glu (16), target compound+GSH+Gly (17) and target compound+GSH (18).
The cell imaging experiment:
Measure the ability of biological thiol in active somatic cell in order to investigate target compound, we are applied to target compound the imaging analysis experiment of live body HeLa cell; After target compound (10 μ M) is hatched 30min, present strong red fluorescence as shown in figure 12 in the tenuigenin of HeLa cell, this result shows that target compound can permeates cell membranes and can targeting ground mark tenuigenin; In addition, we first carry out pre-treatment with the concentration of biological thiol in the reduction cell with 50 μ M NEM to cell, and then hatch 30min with target compound (10 μ M), and result shows that red fluorescence obviously reduces as shown in figure 13; These results show being caused by the change of biological thiol content in cell really of change in fluorescence of target compound, and concrete steps are:
HeLa cell (Beijing consonance medical university cell centre) is 10 with DMEM/F12 nutrient solution (containing 10% foetal calf serum, 50U/mL penicillin, 50 μ g/mL Streptomycin sulphates) adjustment cell density
-6Individual cell/mL is placed in aseptic culture dish, cultivates in 37 ℃ of 5% CO2gas incubator; First clean 3 times with the DMEM of serum-free, remove not adherent cell, and then be placed in respectively two 6 orifice plates with the nutrient solution dilution and cultivate for experiment used; A part is directly hatched 30min with 10 μ M target compound solution; A part is first hatched 2h with the concentration of biological thiol in the reduction cell with 50 μ MNEM, and then hatches 30min with 10 μ M target compound solution.Take the fluorescence micrograph of above-mentioned cell with laser confocal microscope, excitation light source: green glow; Before Laser scanning confocal microscopy, remove nutrient solution, and then rinse twice with PBS buffered soln.
Claims (2)
1. colorimetric fluorescence probe for high selectivity multiple biological thiol, it is characterized in that: take the selenazoles ring as identification receptor, with the multiple biological thiol colorimetric fluorescence probe of anthraquinone derivative as the report information functional group, its structural formula is as follows:
In formula: R
1, R
2, R
3, R
4, R
5, R
6A kind of in hydrogen atom, alkyl, alkoxyl group, sulfonic group or ester group; R
1, R
2, R
3, R
4, R
5, R
6Identical or different.
2. the preparation method of a colorimetric fluorescence probe for high selectivity multiple biological thiol claimed in claim 1, it is characterized in that: carry out griding reaction with the corresponding diamino compounds of colorimetric fluorescence probe for high selectivity multiple biological thiol and tin anhydride in agate mortar and make, grinding temperature is 10～35 ℃, milling time is 15～90min, and the mol ratio of corresponding diamino compounds and tin anhydride is 1: 1～5; Its reaction structure formula is:
The colorimetric fluorescence probe for high selectivity multiple biological thiol that aforesaid method obtains can produce different absorption spectrum simultaneous distinct colors from halfcystine, homocysteine and gsh effect respectively to be changed and fluorescence emission spectrum, thereby realizes the selectivity identification to halfcystine, homocysteine and gsh; And all can not cause the obvious change of absorption spectrum and fluorescence emission spectrum with other non-mercaptan amino acid effects, and these occurrences of amino acid do not disturb to the quantitative assay of biological thiol; But hatch after 10～60min just transfered cell with cell, thereby realize the mensuration to biological thiol content in cell.
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| CN103088554A (en) * | 2011-10-31 | 2013-05-08 | 中国科学院合肥物质科学研究院 | Porous membrane mixed by 1,4-dihydroxy anthraquinone and cellulose, preparation method and usage |
| CN103084073B (en) * | 2011-10-31 | 2015-06-03 | 中国科学院合肥物质科学研究院 | Porous membrane composed of cellulose doped with 1,4-dihydroxy anthraquinone and bivalent copper ion and preparation method and application thereof |
| CN102585802A (en) * | 2012-01-31 | 2012-07-18 | 天津理工大学 | Novel water-soluble sulfydryl fluorescent probe, and preparation method and application thereof |
| CN108101867B (en) * | 2017-12-15 | 2020-05-19 | 湖北大学 | Preparation method and application technology of fluorescent probe for detecting glutathione |
| CN108947994A (en) * | 2018-07-13 | 2018-12-07 | 济南大学 | A kind of biological thiol fluorescence probe and its application |
| CN112209942B (en) * | 2020-10-14 | 2023-10-31 | 中南大学 | Fluorescent probe for distinguishing and detecting cysteine, homocysteine and glutathione |
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