CN113735796A - Design synthesis and property research based on phenothiazine reversible fluorescent probe - Google Patents
Design synthesis and property research based on phenothiazine reversible fluorescent probe Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 28
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 229950000688 phenothiazine Drugs 0.000 title claims abstract description 16
- 230000002441 reversible effect Effects 0.000 title claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 7
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 7
- 238000011160 research Methods 0.000 title claims description 3
- 238000013461 design Methods 0.000 title description 2
- 239000000523 sample Substances 0.000 claims abstract description 83
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- 230000004044 response Effects 0.000 claims abstract description 24
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- 239000007853 buffer solution Substances 0.000 claims abstract description 3
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- 230000008859 change Effects 0.000 claims description 13
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
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- 238000012360 testing method Methods 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- COLOSBBTKSNIMJ-UHFFFAOYSA-N 10-ethylphenothiazine-3-carbaldehyde Chemical compound O=CC1=CC=C2N(CC)C3=CC=CC=C3SC2=C1 COLOSBBTKSNIMJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000000862 absorption spectrum Methods 0.000 claims description 4
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- 230000007935 neutral effect Effects 0.000 claims description 4
- 238000004448 titration Methods 0.000 claims description 4
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 3
- IQZBMUCMEBSKSS-UHFFFAOYSA-N 10-ethylphenothiazine Chemical compound C1=CC=C2N(CC)C3=CC=CC=C3SC2=C1 IQZBMUCMEBSKSS-UHFFFAOYSA-N 0.000 claims description 3
- MATJPVGBSAQWAC-UHFFFAOYSA-N 2-cyano-n,n-dimethylacetamide Chemical compound CN(C)C(=O)CC#N MATJPVGBSAQWAC-UHFFFAOYSA-N 0.000 claims description 3
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N Vilsmeier-Haack reagent Natural products CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 3
- RDHPKYGYEGBMSE-UHFFFAOYSA-N bromoethane Chemical compound CCBr RDHPKYGYEGBMSE-UHFFFAOYSA-N 0.000 claims description 3
- 239000003068 molecular probe Substances 0.000 claims description 3
- 239000012312 sodium hydride Substances 0.000 claims description 3
- 229910000104 sodium hydride Inorganic materials 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims 1
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- 238000000605 extraction Methods 0.000 claims 1
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- 239000002994 raw material Substances 0.000 abstract 1
- 238000001308 synthesis method Methods 0.000 abstract 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 156
- 229960003180 glutathione Drugs 0.000 description 78
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 12
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 108010024636 Glutathione Proteins 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 150000003573 thiols Chemical class 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 150000001768 cations Chemical class 0.000 description 5
- 230000007423 decrease Effects 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- -1 thiol small molecules Chemical class 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 3
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
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- 238000002474 experimental method Methods 0.000 description 3
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- 239000007787 solid Substances 0.000 description 3
- 238000006845 Michael addition reaction Methods 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
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- 238000004611 spectroscopical analysis Methods 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
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- 239000007795 chemical reaction product Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
- C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
- C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
- C07D279/18—[b, e]-condensed with two six-membered rings
- C07D279/22—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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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- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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Abstract
本发明涉及的是荧光探针领域，本发明提供了一种能够对GSH有良好选择性的荧光探针的合成，具体涉及一种以吩噻嗪为发光团，以迈克尔反应受体‑活泼双键为反应位点，设计合成一种可逆的特异性识别GSH的荧光探针。结构式如下：。本发明的荧光探针对GSH具有高选择性和灵敏性，含有1%DMSO的PBS（pH=7.4）缓冲溶液中探针（10μM）对Cys(200μM)、Hcy(15μM)和GSH(5 mM)响应35 min后，通过紫外和荧光光谱仪检测结果显示，本探针能对GSH有良好选择性。荧光探针对GSH不同浓度的响应，随着GSH浓度的增加，探针在606 nm处荧光发射强度逐渐减弱，荧光发射强度与GSH的浓度在一定的范围内成线性关系，且荧光探针对GSH作用速度快、选择强、灵敏性高，且合成方法简单，原料易得，易于推广。The present invention relates to the field of fluorescent probes, and the invention provides the synthesis of a fluorescent probe with good selectivity for GSH, and specifically relates to a kind of fluorescent probe with phenothiazine as luminophore and Michael reaction acceptor-active double The bond is the reaction site, and a reversible fluorescent probe that specifically recognizes GSH is designed and synthesized. The structure is as follows: 
. The fluorescent probe of the present invention has high selectivity and sensitivity to GSH, and the probe (10 μM) in PBS (pH=7.4) buffer solution containing 1% DMSO is sensitive to Cys (200 μM), Hcy (15 μM) and GSH (5 mM) ) after 35 min of response, the detection results by UV and fluorescence spectrometers show that the probe has good selectivity to GSH. The response of fluorescent probes to different concentrations of GSH, with the increase of GSH concentration, the fluorescence emission intensity of the probe at 606 nm gradually weakened, and the fluorescence emission intensity was linearly related to the concentration of GSH within a certain range, and the fluorescent probe had a linear relationship with the concentration of GSH. GSH has the advantages of fast action speed, strong selection, high sensitivity, simple synthesis method, readily available raw materials and easy popularization.
Description
Technical Field
The invention relates to the field of fluorescent probes, in particular to a reversible fluorescent probe with good GSH selectivity, which is designed and synthesized by taking phenothiazine as a luminophore and taking Michael reaction acceptor-active double bonds as reaction sites.
Background
Glutathione is a small molecule mercaptan with the largest content in organisms, the content of other thiols in organisms such as cysteine is less than one percent of that of glutathione, and the content of homocysteine is less than one thousandth of that of glutathione. It is widely present in animals and plants, and plays important physiological roles in organisms, such as participating in intracellular redox reactions, intracellular signal transmission, and in vivo metabolism. Simultaneously, the glutathione also plays an important physiological role in a biochemical defense system in a human body, and the main physiological role of the glutathione is to remove free radicals in the human body, be used as an important antioxidant in the human body and protect molecules such as various proteins and enzymes.
In view of the fact that glutathione plays an important physiological role in organisms and the abnormal change of the content of glutathione in organisms is often related to certain diseases, the detection of glutathione attracts attention. There are many methods for detecting bio-thiol, such as electrochemical detection and high performance liquid chromatography, but these methods often have their own limitations, such as large sample size or long detection time. The detection of the fluorescence probe on the biological mercaptan has the advantages of low detection line, no damage, visualization, high speed and the like, and is increasingly viewed in the aspect of biological mercaptan detection.
In recent years, the development of biomolecular thiol fluorescent probes is long, and various fluorescent probes are generated, so that a new method is provided for detecting thiol in organisms. However, the selective detection of biomolecular thiols still faces a great challenge, and the physiological structures of different thiol small molecules are different, so that the selective fluorescent probe has great significance.
Disclosure of Invention
The invention provides synthesis of a fluorescent probe with good selectivity on GSH by taking phenothiazine as a fluorophore. In particular to a reversible fluorescent probe for recognizing GSH, which is designed and synthesized by taking phenothiazine as a luminophore and taking Michael reaction acceptor-active double bond as a reaction site.
The invention provides a fluorescent probe with good selectivity on GSH, wherein the name of the fluorescent probe is (E) -2-cyano-3- (10-ethyl-10H-phenothiazin-3-yl) -N, N-dimethylacrylamide, the name of the fluorescent probe is (E) -2-cyano-3- (10-ethyl-10H-phenothiazin-3-yl) -N, N-dimethylacrylamide, and the structural formula is as follows:
the preparation method of the fluorescent molecular probe comprises the following steps:
The application of the fluorescent probe comprises the following steps of distinguishing and detecting Cys, Hcy and GSH:
after 35 min of probe (10 μ M) in PBS (pH = 7.4) buffer containing 1% DMSO in response to Cys (200 μ M), Hcy (15 μ M) and GSH (5 mM), it can be seen from the UV absorption spectrum that the probe has almost no significant change in Cys and Hcy, but has significant response to GSH, and the peaks at 310 nm and 435 nm are significantly reduced. After the probe reacts with GSH, the fluorescence intensity of the probe is obviously reduced, and the probe has almost no obvious change to Cys and Hcy. The probe is considered to react with GSH, and the mercapto group and the active double bond have Michael addition reaction, thereby influencing the fluorescence of the probe. Cys and Hcy have limited interaction with the probe due to their low concentrations, and have high GSH content, with significant interaction with the probe. Thereby realizing the selection of the biological thiol GSH.
The application of the fluorescent probe, the titration of the probe and the GSH and the detection line measurement are as follows:
fluorescence intensity at 606 nm after reaction of probe (10 μ M) with GSH (1-10 mM) in PBS (pH =7.4, 1% DMSO) was monitored over a GSH concentration range of 0-10 mM. As the concentration of GSH increases, the fluorescence intensity of the emission peak at 606 nm also decreases. We performed tests with GSH at concentrations of 0-10 mM and fitted the probe linearly to the concentration of GSH (0-5 mM) based on the test results and found that at GSH concentrations less than 5mM, the fluorescence intensity is linearly related to the concentration of GSH with the equation of a straight line y = -147.11x + 764.89. When the GSH concentration reached 5mM, the fluorescence intensity remained within a relatively stable range with almost no change in fluorescence intensity, and the lowest detection limit for GSH was calculated to be 1.07. mu.M (0-10 mM for GSH in cells).
Drawings
FIG. 1 scheme for the synthesis of the probe.
FIG. 2 UV absorption and fluorescence emission spectra of probes (10 μ M) in PBS buffer containing 1% DMSO (pH = 7.4) in response to Cys (200 μ M), Hcy (15 μ M) and GSH (5 mM) for 35 min.
FIG. 3 is a graph of fluorescence emission spectra of probes (10. mu.M) in PBS buffer containing 1% DMSO (pH = 7.4) after 35 min response to 0-10 mM GSH, and a graph of fluorescence intensity at 606 nm and linear relationship in the range of 0-5 mM GSH.
Figure 4 graph of probe (10 μ M) versus GSH (5 mM) response in PBS buffer (pH = 7.4) containing 1% DMSO, fluorescence intensity at 606 nm as a function of time.
Figure 5 graph of probe (10 μ M) fluorescence intensity change at 606 nm in PBS buffer pH =4-10 (pH = 7.4) containing 1% DMSO; graph of fluorescence intensity at 606 nm after response 35 min for probe (10 μ M) and GSH (5 mM) in PBS buffer pH =4-10 containing 1% DMSO.
Figure 6 graph of fluorescence intensity at 606 nm of probes (10 μ M) after response to different amino acids and common cations in human in PBS buffer containing 1% DMSO (pH = 7.4).
Figure 7 graph of the change in fluorescence intensity at 606 nm of probe (10 μ M) and GSH (2 mM) in PBS buffer (pH = 7.4) containing 1% DMSO.
Detailed Description
EXAMPLE 1 preparation of fluorescent Probe
The preparation route of the fluorescent probe of the invention is shown in figure 1.
(1) Preparation of Compound a1
Phenothiazine (0.199 g, 1 mmol) was added to a reaction flask (100 mL), and 20 mL of DMSO was added to the reaction flask and dissolved by stirring. Sodium hydride (0.166 g, 1 mmol) was added to the reaction flask and the materials were mixed and stirred under nitrogen for 0.5 h. Measuring bromoethane (0.074 mL, 1 mmol), adding into a reaction bottle, reacting at room temperature under the protection of nitrogen for 24 hours, tracing by a TLC point plate, determining whether the reaction is finished, washing with distilled water after the reaction is finished, extracting with dichloromethane (40 mL, 3 times), drying with anhydrous magnesium sulfate, carrying out vacuum filtration, spinning off the excess solvent, selecting a proper eluent (PE: EA (v: v) = 10: 1), and purifying by column chromatography. Finally, 0.163 g of a white solid was obtained in a yield of 72%. 1H NMR (400 MHz, DMSO). delta.7.19 (t, J = 12.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 6.93 (t, J = 8.0 Hz, 2H), 3.92 (q, J = 8.0 Hz, 4H), 1.29 (t, J = 8.0 Hz, 3H).
(2) Preparation of intermediate compound a2
DMF (2 mL) was added to a two-necked flask (100 mL), and POCl3 (2 mL) was gradually added dropwise to the flask and refluxed at 50 ℃ under nitrogen for 30 min. Intermediate a1 (0.16 g, 0.7 mmol) was dissolved in 1, 2-dichloroethane (15 mL) and transferred to an isopiestic dropping funnel where it was added slowly to the two-necked flask. Stirring and refluxing for 12 hours at 90 ℃ under the protection of nitrogen, tracing by a TLC point plate, and determining whether the reaction is finished or not until the reaction is finished. Cooled to room temperature and quenched in an ice-water bath by slow addition of distilled water. The pH was adjusted to neutral with 20% NaOH solution, washed with distilled water, extracted with dichloromethane (40 mL, 3 times), dried over anhydrous magnesium sulfate, filtered under reduced pressure, the excess solvent was decanted off, and the appropriate eluent (PE: EA (v: v) = 10: 1) was selected and purified by column chromatography. Finally, 0.13g of a pale yellow solid was obtained in a yield of 75%. 1H NMR (400 MHz, DMSO). delta.9.79 (s, 1H) 7.73(d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 2H), 7.09(d, J = 8.0 Hz, 1H), 7.02 (t, J = 8.0 Hz, 1H), 4.00 (q, J = 8.0 Hz, 4H), 1.32 (t, J = 8.0 Hz, 3H).
(3) Preparation of fluorescent probes
Intermediate a2 (0.127 g, 0.5 mmol) was added to a reaction flask (100 mL), 20 mL of ethanol was measured and added to the flask and stirred to dissolve, 2-cyano-N, N-dimethylacetamide (0.084 g, 1.5 mmol) was added, 52. mu.L of piperidine was added, and the mixture was stirred and refluxed at 80 ℃ under nitrogen for 8 hours and followed by TLC spot plate to determine whether the reaction was complete. After the reaction was completed, the reaction mixture was washed with distilled water, extracted with dichloromethane (40 mL, 3 times), dried over anhydrous magnesium sulfate, suction-filtered under reduced pressure, and the excess solvent was removed by centrifugation, and an appropriate eluent (PE: EA (v: v) = 10: 1) was selected and purified by column chromatography. Finally, 0.078 g of a dark yellow solid is obtained, yield 45%. 1H NMR (400 MHz, CDCl 3): δ 7.77(d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.95(t, J = 8.0 Hz, 1H), 6.88 (t, J = 8.0 Hz, 2H), 3.97 (q, J = 8.0 Hz, 4H), 3.21(s, 3H), 3.06(s, 3H), 1.44 (t, J = 8.0 Hz, 3H), 13C NMR (100 MHz, CDCl3) δ 164.45, 150.86, 148.30, 143.12, 130.14, 128.90, 127.54, 127.46, 126.44, 124.39, 123.39, 123.10, 116.61, 115.36, 114.58, 3942, 3942.33.
Example 2 spectroscopic analysis of Probe and thiol response
To explore the response of the probe to different thiols, we performed a spectroscopic analysis of the reaction of the probe with the thiol. As shown in the figure, the spectral responses of the probe (10 [ mu ] M) and Cys (200 [ mu ] M), Hcy (15 [ mu ] M) and GSH (5 mM) are tested, and the probe has almost no obvious change on Cys and Hcy and has obvious response on GSH from the ultraviolet absorption spectrum, and the peaks at the wavelengths of 310 nm and 435 nm have obvious reduction. The same phenomenon can be seen from the fluorescence emission spectrum as shown in FIG. 2(b), and the fluorescence intensity of the probe is significantly reduced after the probe reacts with GSH, while the probe has almost no significant change for Cys and Hcy. The probe is considered to react with GSH, and the mercapto group and the active double bond have Michael addition reaction, thereby influencing the fluorescence of the probe. Cys and Hcy have limited interaction with the probe due to their low concentrations, and have high GSH content, with significant interaction with the probe.
Example 3 titration experiment of Probe with GSH
In order to investigate the response of the probe to GSH with different concentrations of 0-10 mM, we performed a titration experiment, and as can be seen from the fluorescence emission spectrum in fig. 3(a), as the GSH concentration increases, under the GSH condition of 0-5 mM, the fluorescence of the probe itself at 606 nm continuously decreases, which indicates that the probe has a very high sensitivity and can detect GSH with 0-5 mM, and that the fluorescence signal of the GSH with 6-10 mM is not clearly distinguished after the GSH and the probe stabilize, which may be due to the excessive concentration, so the probe cannot clearly distinguish GSH with 6-10 mM.
Example 4 Probe detection line assay
Fluorescence intensity at 606 nm after reaction of the probe (10 μ M) with GSH (1-10 mM) in PBS (pH 7.4, 1% DMSO) was monitored over a GSH concentration range of 0-10 mM. As the concentration of GSH increased, the fluorescence intensity of the emission peak at 606 nm also decreased, as shown in FIG. 3 (b). We performed tests with GSH at a concentration of 0-10 mM and fitted the probe linearly to the concentration of GSH (0-5 mM) according to the test results, and found that the fluorescence intensity at a concentration of GSH less than 5mM is linearly related to the concentration of GSH as shown in FIG. 3(c), and the equation for the straight line is y = -147.11x + 764.89. After the GSH concentration reached 5mM, the fluorescence intensity remained within a relatively stable range, with almost no fluorescence intensity. As shown in the figure, the lowest detection limit of GSH was calculated to be 1.07. mu.M (GSH in cells is 0-10 mM).
EXAMPLE 5 determination of Probe and GSH response time
The response time of probe 1 to GSH was measured, as shown in FIG. 4, using a probe concentration of 10. mu.M, and the fluorescence intensity at 606 nm after the addition of GSH (5 mM) was collected, from which it can be seen that the fluorescence intensity at 606 nm rapidly decreases after the addition of 5mM GSH and stabilizes within 35 min. The response of the probe and the GSH is very quick and is basically complete within 35 min, so the response time of the probe and the GSH is 35 min.
EXAMPLE 6 stability testing of probes
The stability of the probe was then tested and tested for its stability and ability to respond to GSH in PBS buffered solutions of different pH values, and the results are shown. As can be seen from FIG. 5(a), the fluorescence intensity of the probe itself hardly changes in the range of pH =4-10 in the PBS buffer solution, indicating that the probe itself has good pH stability and is not affected by pH to change the fluorescence intensity. Meanwhile, the response ability of the probe to GSH was also tested in the range of pH =4-10, and it can be seen from fig. 5(b) that the probe could not detect GSH well under acidic condition and could detect GSH well under neutral and alkaline condition in the range of pH = 7-10. The pH value under physiological conditions is 7.4, so the probe can be used for detecting GSH under physiological conditions.
EXAMPLE 7 Selective testing of probes
Next, a selectivity test is performed to determine whether the probe has good selectivity for GSH identification according to the test result, which is shown in fig. 6. The ability of the probe to respond to several common amino acids was tested, and it can be seen from the figure that the common amino acids were added to the 10 μ M probe added PBS (pH = 7.4), except that the fluorescence intensity of Hcy (15 μ M) and Cys (200 μ M) at 606 nm was slightly decreased, the fluorescence intensity of other amino acids at 606 nm did not change significantly, indicating that the probe hardly responded to other amino acids. Meanwhile, various cations exist in organisms, and then the response capability of the probe to the cations is tested, and the test result is shown in the figure, although partial cations and the probe respond, the responses of the cations and the probe are weak compared with the response of the probe to the GSH, and the influence of the responses on the amount of addition products in the organisms is negligible, so that the recognition of the probe to the GSH can be judged to have good selectivity.
Example 8 reversibility verification of Probe and GSH reaction
To confirm the reversibility of the reaction of the probe with GSH, a reversible mechanism verification experiment was performed to monitor the change in fluorescence intensity at 606 nm during the reaction, as shown in fig. 7. GSH (2 mM) is selected to react with a probe (10 mu M), the fluorescence intensity at 606 nm gradually decreases, the fluorescence intensity tends to be stable about 35 min, the reaction is indicated to reach an equilibrium state, and H is added into the system after the reaction is stable2O2(5 mM) was allowed to react with GSH, at which time the fluorescence intensity at 606 nm of the probe gradually recovered, which is an experimental phenomenon indicating that the reaction of the probe with GSH is a reversible reaction.
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| CN115232089B (en) * | 2022-07-04 | 2024-11-15 | 洛阳师范学院 | A vinyl phenothiazine fluorescent probe and its preparation method and application |
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