Fluorescent probe for detecting glutathione and preparation and application thereof

Abstract

This invention discloses a method for preparing a fluorescent probe for detecting glutathione. PCN-224, ZnCl2, and DMF are added to a reaction vessel and dissolved thoroughly to obtain a mixture. The mixture is then transferred to a hydrothermal reactor and heated at 100-150°C for 10-20 hours to obtain the fluorescent probe. This invention uses Zn... 2+ The fluorescent probe PCN-224(Zn) was synthesized by incorporating TCPP ligands into PCN-224. 2+ The alteration of the N-N ring structure in TCPP caused the fluorescence quenching of PCN-224. GSH, rich in thiol groups, can react with Zn. 2+ The reaction will Zn 2+ By stripping PCN-224, PCN-224(Zn) is converted back to PCN-224, achieving fluorescence recovery and thus enabling the detection of GSH. This invention can detect GSH in complex sample environments and exhibits high sensitivity and selectivity for glutathione.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of glutathione detection. More particularly, the present application relates to a fluorescent probe for detecting glutathione and its preparation and application. BACKGROUND

[0002] Glutathione (GSH), chemical name is N-(N-L-γ-glutamyl-L-cysteinyl) glycine, is a kind of tripeptide containing γ-amide bond and thiol, which is composed of glutamic acid, cysteine and glycine, and is the most abundant non-protein thiol in cells

[98] Glutathione exists in almost every cell of the body, and more than 90% of glutathione exists in the reduced state (GSH), and less than 10% of glutathione exists in the disulfide state (i.e. oxidized glutathione GSSG). GSH participates in various physiological activities in the body, maintains the biological function of cells, forms an important redox pair in cells with oxidized glutathione (GSSG), and maintains the GSH / GSSG ratio to maintain the redox balance in cells. Glutathione also exists universally in plants and is a rich and indispensable thiol in plants, which participates in various biological processes such as scavenging of reactive oxygen species, redox signaling, sulfur storage and transport, detoxification of harmful substances, and metabolism of some compounds. Therefore, it is crucial to detect the concentration level of GSH in the body and edible plants.

[0003] At present, GSH can be detected by GSH kit, chromatography, spectroscopy, electrochemical detection and other methods. Spectroscopy has developed rapidly in recent years and is divided into ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy. Fluorescence spectroscopy utilizes the fluorescence properties of materials to realize the detection of GSH, and has great space and potential for improvement in high selectivity and high sensitivity detection of GSH.

[0004] Shu's group designed a reversible fluorescent probe (GeP) based on the nucleophilic addition and dissociation of intracellular GSH to GeP for detecting GSH. The probe shows a fast response time to GSH and a 20-fold fluorescence change, and can exhibit excellent targeting to mitochondria and monitor GSH in mitochondria. Wang's group designed a ZnS bright dot off-fluorescent sensor. Co 2+The fluorescence of ZnS bright sub-particle can be quenched, and GSH can further quench the fluorescence of ZnS, so that GSH can be indirectly detected. The detection concentration range is 1-300 μM, and the detection limit can reach 0.48 μM. Yue's research group synthesized a spindle-shaped zinc metal-organic framework material (Zn-DTBA) with H2DTBA ligand water stability, which can be used for simultaneous detection of glutathione and cysteine. Cysteine can react with the disulfide bond in the H2DTBA ligand, thereby destroying the crystal structure of Zn-DTBA and quenching its fluorescence. And GSH can interact with the direct connection between the ligand and the metal node, causing the structure of Zn-DTBA to collapse, releasing Zn 2+ ions and ligands. Therefore, the sensor can simultaneously detect cysteine and glutathione, and has different fluorescence responses, which will not affect the selectivity of the sensor to one of the analytes. However, many fluorescence detection methods cannot simultaneously consider both selectivity and sensitivity, or have good selectivity but need to improve sensitivity, or have good sensitivity but need to improve selectivity. Therefore, it is necessary to construct a fluorescence sensor with high sensitivity and high selectivity. SUMMARY

[0005] An object of the present application is to solve at least the above problems and provide at least the advantages to be described later.

[0006] An object of the present application is to provide a fluorescence probe for detecting glutathione, which can detect GSH in a complex sample environment and has high sensitivity and high selectivity to glutathione.

[0007] In order to achieve these objects and other advantages of the present application, a preparation method of a fluorescence probe for detecting glutathione is provided, PCN-224, ZnCl2 and DMF are added to a reaction container, and a mixed solution is obtained by fully dissolving, the mixed solution is transferred to a hydrothermal reaction kettle, and heated at 100-150℃ for 10-20h to obtain the fluorescence probe PCN-224(Zn).

[0008] Preferably, the preparation method of PCN-224 is: 0.1-0.5mmol of TCPP (meso-tetrakis(4-carboxyphenyl) porphyrin), 1-5mmol of ZrOCl2·8H2O and 200-250mmol of BA (benzoic acid) are added to 10mL of DMF (N,N-dimethylformamide), and ultrasonic dissolution is performed for 1-10min, then the mixture is transferred to a hydrothermal reaction kettle with polytetrafluoroethylene as the inner liner, and placed in an oven, heated at 100-150℃ for 12-24h to obtain a dark purple mixture, the mixture is washed with DMF for 2-3 times, and dried to obtain a purple solid PCN-224.

[0009] Preferably, the concentration of PCN-224 in the mixed solution is 1.5-3.5 mg / mL, and the concentration of ZnCl2 in the mixed solution is 0.04-0.44 mM.

[0010] Preferably, the ultrasonic wave is used to dissolve for 1-10 min.

[0011] The fluorescent probe for detecting glutathione is prepared by the preparation method of the fluorescent probe for detecting glutathione.

[0012] The application of the fluorescent probe for detecting glutathione in the detection of glutathione.

[0013] The present application at least includes the following beneficial effects:

[0014] First, the present application modifies the TCPP ligand doped into PCN-224 to synthesize the fluorescent probe PCN-224(Zn). 2+ The TCPP ligand doped into PCN-224 is changed to synthesize the fluorescent probe PCN-224(Zn). 2 + The structure of the N-N ring in TCPP is changed to cause the fluorescence quenching of PCN-224, and GSH is rich in sulfhydryl groups, which can react with Zn 2+ The Zn 2+ is stripped from PCN-224, so that PCN-224(Zn) becomes PCN-224 to achieve the effect of fluorescence recovery, thereby realizing the detection of GSH.

[0015] Second, the present application has high sensitivity and high selectivity in the detection of glutathione by the fluorescent probe PCN-224(Zn) constructed by zinc ion modification of porphyrin-based metal-organic framework.

[0016] Other advantages, objects and features of the present application will be partly embodied by the following description, and will be partly understood by those skilled in the art through research and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The synthesis step diagram of PCN-224(Zn) of the present application;

[0018] Figure 2 The synthesis condition optimization results of PCN-224(Zn) of the present application; wherein (a) is the ZnCl2 concentration optimization; (b) is the PCN-224 concentration optimization;

[0019] Figure 3The characterization results of PCN-224(Zn) and PCN-224; wherein (a) is a fluorescence spectrum; (b) is an ultraviolet-visible absorption spectrum; (c) is a photo under a daylight lamp and an ultraviolet lamp box;

[0020] Figure 4 The fluorescence intensity column chart of PCN-224(Zn) reacting with GSH in HEPES, PBS, Tris-HCl and H2O;

[0021] Figure 5 The XPS spectrum of PCN-224(Zn);

[0022] Figure 6 The TEM graph and particle size statistical column chart of PCN-224(Zn); wherein (a) is a TEM graph; (b) is a particle size statistical column chart;

[0023] Figure 7 The XRD spectrum of PCN-224 and PCN-224(Zn);

[0024] Figure 8 The infrared spectrum of TCPP, PCN-224 and PCN-224(Zn);

[0025] Figure 9 The stability results of PCN-224(Zn) placed for different time; wherein (a) is fluorescence stability; (b) is hydration particle size stability;

[0026] Figure 10 The selectivity results of PCN-224(Zn) for GSH detection; wherein (a) is a fluorescence spectrum of PCN-224(Zn) reacting with different substances; (b) is a fluorescence intensity column chart of selectivity and anti-interference experiment of PCN-224(Zn) for GSH detection;

[0027] Figure 11 The sensitivity results of PCN-224(Zn) for GSH detection; wherein (a) is a titration curve; (b) is a fluorescence enhancement spectrum;

[0028] Figure 12 The XPS spectra of PCN-224(Zn) before and after reacting with GSH. DETAILED DESCRIPTION

[0029] The application will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement the application according to the description and drawings.

[0030] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] It should be noted that the experimental methods in the following embodiments are conventional methods, and the reagents and materials are commercially available unless otherwise specified.

[0032] The application provides a preparation method of a fluorescent probe for detecting glutathione.

[0033] In another technical solution, the concentration of PCN-224 in the mixed solution is 1.5-3.5 mg / mL, and the concentration of ZnCl2 in the mixed solution is 0.04-0.44 mM.

[0034] In another technical solution, the ultrasonic wave is used for fully dissolving for 1-10 min.

[0035] In another technical solution, the preparation method of PCN-224 is as follows: 0.1-0.5 mmol of TCPP, 1-5 mmol of ZrOCl2·8H2O and 200-250 mmol of BA are added into 10 mL of DMF, the ultrasonic wave is used for fully dissolving for 1-10 min, the mixture is transferred into a hydrothermal reaction kettle with polytetrafluoroethylene as an inner liner and then placed into an oven, and the mixture is heated at 100-150 ℃ for 12-24 h to obtain a dark purple mixture; the mixture is washed with DMF for 2-3 times, dried, and then a purple solid PCN-224 is obtained.

[0036] The fluorescent probe for detecting glutathione is prepared by the preparation method of the fluorescent probe for detecting glutathione.

[0037] The fluorescent probe for detecting glutathione is prepared by the preparation method of the fluorescent probe for detecting glutathione.

[0038] Example 1

[0039] The application provides a preparation method of a fluorescent probe for detecting glutathione.

[0040] 1) Synthesis of PCN-224: 0.1 mmol of TCPP, 1 mmol of ZrOCl2·8H2O and 200 mmol of BA are added into 10 mL of DMF, the ultrasonic wave is used for fully dissolving for 1 min, the mixture is transferred into a hydrothermal reaction kettle with polytetrafluoroethylene as an inner liner, and then placed into an oven, and the mixture is heated at 100 ℃ for 24 h to obtain a dark purple mixture; the mixture is washed with DMF for 2 times, dried, and then a purple solid PCN-224 is obtained.

[0041] 2) PCN-224, ZnCl2 were added into a reaction vessel, DMF was added to dissolve PCN-224 to a concentration of 1.5 mg / mL in DMF and ZnCl2 to a concentration of 0.04 mM in DMF, and ultrasonic was applied to dissolve the mixture for 1 min, the mixture was transferred into a hydrothermal reactor, and heated at 100 °C for 20 h, the obtained dark green solid was washed with DMF for 3 times, and dispersed in a HEPES buffer solution to obtain the fluorescent probe PCN-224(Zn).

[0042] Example 2

[0043] A preparation method of a fluorescent probe for detecting glutathione, comprising the following steps:

[0044] 1) Synthesis of PCN-224: 0.5 mmol of TCPP, 5 mmol of ZrOCl2·8H2O and 250 mmol of BA were added into 10 mL of DMF, and ultrasonic was applied to dissolve the mixture for 10 min, the mixture was transferred into a hydrothermal reactor with polytetrafluoroethylene as an inner liner, and placed into an oven, and heated at 150 °C for 12 h to obtain a dark purple mixture, the mixture was washed with DMF for 3 times, and dried to obtain a purple solid PCN-224;

[0045] 2) PCN-224, ZnCl2 were added into a reaction vessel, DMF was added to dissolve PCN-224 to a concentration of 3.5 mg / mL in DMF and ZnCl2 to a concentration of 0.44 mM in DMF, and ultrasonic was applied to dissolve the mixture for 10 min, the mixture was transferred into a hydrothermal reactor, and heated at 150 °C for 10 h, the obtained dark green solid was washed with DMF for 3 times, and dispersed in a HEPES buffer solution to obtain the fluorescent probe PCN-224(Zn).

[0046] Example 3

[0047] A preparation method of a fluorescent probe for detecting glutathione, comprising the following steps:

[0048] 1) Synthesis of PCN-224: 0.2 mmol of TCPP, 3.6 mmol of ZrOCl2·8H2O and 216 mmol of BA were added into 10 mL of DMF, and ultrasonic was applied to dissolve the mixture for 5 min, the mixture was transferred into a hydrothermal reactor with polytetrafluoroethylene as an inner liner, and placed into an oven, and heated at 120 °C for 18 h to obtain a dark purple mixture, the mixture was washed with DMF for 3 times, and dried to obtain a purple solid PCN-224;

[0049] 2) PCN-224, ZnCl2 were added into the reaction vessel, and DMF was added to dissolve PCN-224 to a concentration of 2.0 mg / mL in DMF and ZnCl2 to a concentration of 0.28 mM in DMF, and ultrasonic dissolution was performed for 5 min, and a mixed solution was obtained by sufficient dissolution, and the mixed solution was transferred to a hydrothermal reaction kettle, and heating was performed at 120°C for 12 h, and the obtained dark green solid was washed with DMF for 3 times, and dispersed in a HEPES buffer solution to obtain the fluorescent probe PCN-224(Zn).

[0050] Test 1, synthesis and characterization of PCN-224(Zn)

[0051] I. Synthesis of PCN-224(Zn)

[0052] As shown in Figure 1 , the present application synthesizes PCN-224 first, and then Zn 2+ is doped into the TCPP ligand to synthesize PCN-224(Zn). When Zn 2+ is successfully doped into TCPP, the N-H bond of the N-N ring in TCPP is destroyed, resulting in fluorescence weakening, so that the fluorescence quenching efficiency of PCN-224(Zn) relative to PCN-224 can be used as a reference basis for the amount of Zn 2+ that is successfully doped, and the greater the fluorescence intensity quenching efficiency, the more Zn 2+ ions are doped. Based on this, the amount of ZnCl2 and PCN-224 is optimized.

[0053] First, the amount of ZnCl2 is optimized, 6 beakers are taken, the amount of PCN-224 is fixed, 25 mg of PCN-224 is added, and different amounts of ZnCl2 (0.04, 0.12, 0.2, 0.28, 0.36, 0.44 mM) are added, 10 mL of DMF is added to each of the 7 beakers, and ultrasonic dissolution is performed for 5 min. Transfer to a hydrothermal reaction kettle, heat at 120°C for 12 h. The obtained dark green solid is washed with DMF for 3 times, dispersed in a HEPES buffer solution, and the fluorescence spectrum is measured.

[0054] Then, the amount of PCN-224 is optimized, 6 beakers are taken, the amount of ZnCl2 is fixed, 0.28 mM is added to each beaker, and different amounts of PCN-224 (10, 15, 20, 25, 30, 35 mg) are added, 10 mL of DMF is added to each of the 7 beakers, and ultrasonic dissolution is performed for 5 min. Transfer to a hydrothermal reaction kettle, heat at 120°C for 12 h. The obtained dark green solid is washed with DMF for 3 times, dispersed in a HEPES buffer solution, and the fluorescence spectrum is measured.

[0055] Figure 2(a) To optimize the results of ZnCl2, with the increase of the amount of Zn 2+ , the fluorescence quenching efficiency of PCN-224(Zn) showed an upward trend. When the amount of Zn 2+ was 0.28 mM, the fluorescence quenching efficiency of PCN-224(Zn) was the highest. With the continuous increase of the amount of Zn 2+ , the fluorescence quenching efficiency of PCN-224(Zn) gradually decreased, so 0.28 mM was the optimal amount of ZnCl2.

[0056] Figure 2 (b) To optimize the results of PCN-224, the concentration of PCN-224 was increased while the amount of ZnCl2 was fixed at 0.28 mM. The fluorescence quenching efficiency of PCN-224(Zn) showed an increasing trend. When the concentration of PCN-224 was 2.0 mg / mL, the fluorescence quenching efficiency of PCN-224(Zn) was the highest. Since the amount of ZnCl2 that can combine with PCN-224 is fixed and has been fully combined with PCN-224, increasing the concentration of PCN-224 will not form more PCN-224(Zn), and the fluorescence quenching efficiency of the solution will gradually decrease. Therefore, the optimal concentration of PCN-224 is 2.0 mg / mL.

[0057] II. Characterization of PCN-224(Zn)

[0058] PCN-224(Zn) was characterized. As shown in Figure 3 (a), the dispersion of PCN-224 showed purple under daylight and emitted red fluorescence under UV lamp box irradiation. The dispersion of PCN-224(Zn) showed light yellow-green under daylight and no fluorescence could be observed by naked eye under UV lamp box irradiation. PCN-224(Zn) was characterized by fluorescence spectrum, UV-Vis absorption spectrum, and XPS. As shown in Figure 3 (b), the Q-band UV absorption peak of PCN-224 at 500-700 nm belongs to the characteristic peak of N-N ring in TCPP, with four small peaks. Compared with PCN-224, the Q-band UV absorption peak of PCN-224(Zn) at 500-700 nm decreased to two, which is because the incorporation of Zn 2+ replaced the H in the middle of N-H bond, changed the N-H bond to N-Zn bond, and changed the structure of TCPP ligand. As shown in Fig. 3(c), the strong UV absorption peak at about 410 nm showed red shift, which is because the porphyrin ring deformed, and the degree of red shift of the absorption peak is related to the degree of deformation of the porphyrin ring. Figure 3(b), compared with the fluorescence peak of PCN-224, the fluorescence peak of PCN-224(Zn) is blue-shifted from 664 nm to 610 nm due to the change of TCPP ligand, and the fluorescence intensity is greatly weakened due to the influence of metalation on the luminescent group of porphyrin.

[0059] (I) Effect of buffer solution on the performance of fluorescent probe

[0060] Four portions of PCN-224(Zn) were dissolved in HEPES, Tris-HCl, PBS and H2O buffer solution respectively, so that the concentration of PCN-224(Zn) was 20.0 μg / mL, 4 μM of GSH was added to each mixed solution respectively, and the fluorescence spectra before and after the addition of GSH were measured.

[0061] The results are shown in Figure 4 , PCN-224(Zn) has the strongest fluorescence enhancement response to GSH in HEPES, while PBS is mainly composed of phosphate, which can interact with ZrO in PCN-224(Zn) to form Zr-OP and break the electronic transfer between the metal node and the organic ligand, so that the fluorescence of the material is greatly enhanced. When PCN-224(Zn) is dispersed in Tris-HCl and H2O, the fluorescence enhancement response to GSH is not sensitive, so HEPES is selected as the buffer solvent.

[0062] (II) Measurement of XPS of PCN-224(Zn)

[0063] The XPS measurement of PCN-224(Zn) was carried out, Figure 5 (a) is the XPS full spectrum of PCN-224(Zn), and the peak at 1022.8 eV corresponds to the 2p of Zn element 1 / 2 . As Figure 5 (d) In the high-resolution 2p spectrum of Zn, Zn2p spin splitting is divided into two peaks, Zn2p 1 / 2 and Zn2p 3 / 2 , indicating that Zn 2+ is successfully doped into PCN-224. As Figure 5 (b) is the high-resolution spectrum of C1s, which is fitted to show that it contains three types of C atoms, C=C, C=N and C=O. Figure 5 (c) is the high-resolution Zr3d spectrum, which is the characteristic peak of zirconium cluster in PCN-224.

[0064] (III) TEM characterization and particle size statistics of PCN-224(Zn)

[0065] TEM characterization and particle size statistics of PCN-224(Zn) were carried out. As Figure 6As shown, PCN-224(Zn) is a spindle-shaped crystal with an average grain size of 122 nm, exhibiting good morphology and dispersibility. Compared to PCN-224, the edges of PCN-224(Zn) become more irregular, which may be due to the unevenness of Zn. 2+ The incorporation of [something] caused the structure of some TCPP to be damaged, resulting in the detachment of some framework structures.

[0066] (iv) XRD pattern of PCN-224(Zn)

[0067] Compare the XRD patterns of PCN-224(Zn) and PCN-224, such as Figure 7 As shown, PCN-224 has good phase purity and possesses the characteristic crystal planes of PCN-224, but the synthesized PCN-224(Zn) has some issues with Zn content. 2+ The incorporation of Zn leads to an incomplete crystal structure and decreased crystallinity, which may be due to the presence of Zn. 2+ The incorporation of [a substance] causes a certain degree of deformation in the porphyrin structure.

[0068] (V) FTIR Analysis of PCN-224(Zn)

[0069] To verify Zn 2+ PCN-224 was successfully incorporated, further illustrating the elemental binding mechanism, which was then analyzed using FTIR. Figure 8 The infrared spectra of TCPP, PCN-224, and PCN-224(Zn) are shown below. Figure 8 As shown in (a), TCPP at 960cm -1 The characteristic NH peak of the porphyrin N-N ring is present at the same location, and PCN-224 also has this NH peak at the same position, indicating that the porphyrin ring structure in PCN-224 remains intact. Furthermore, NH bonds (960 cm⁻¹) were observed in the infrared spectrum of PCN-224(Zn). -1 The disappearance of ) and N-Zn bonds (1006cm) -1 The formation of Zn. 2+ PCN-224 was successfully incorporated by substituting the H ion on the NH bond in TCPP. The C-OH bond (1246 cm⁻¹) of PCN-224 and PCN-224(Zn) was also incorporated. -1 ) and C=O bond (1696cm) -1 The asymmetric vibration intensity of Zr6 is much lower than that of TCPP, which is attributed to the carboxyl coordination provided by Zr6 and benzoic acid.

[0070] (vi) Stability analysis of PCN-224(Zn)

[0071] To explore its stability, PCN-224(Zn) was dispersed in HEPES buffer solvent, and its fluorescence spectrum and DLS hydration particle size spectrum within seven days were measured. As shown in Figure 9 (a), PCN-224(Zn) remained essentially unchanged in fluorescence intensity within seven days, and its fluorescence intensity was maintained at about 5089.2. DLS results show that Figure 9 (b)), PCN-224(Zn) remained essentially unchanged in average particle size within seven days, and maintained at about 224.3 nm. The two results prove that PCN-224(Zn) has good stability and will not change its properties after placement, which is convenient for storage.

[0072] Experiment two, selectivity of PCN-224(Zn) for GSH detection

[0073] Take several portions of 2 mL of PCN-224(Zn) HEPES buffer solution containing 20.0 μg / mL, and add GSH (4 μM), histidine (His), cysteine (Cys), ascorbic acid (AA), lysine (Lys), glutamic acid (Glu(E)), glycine (Gly), isoleucine (Iso), serine (Ser), proline (Pro), valine (Val), glucose (Glu(G)), oxidized glutathione (GSSG), human serum albumin (HSA), bovine serum albumin (BSA), Ba 2+ , Na + , Ca 2+ , NO2 - , wherein the concentration ratio of GSH to Glu(E), Cys, Gly is 1:25 (4 μM:100 μM), and the concentration ratio of other ions is 1:5 (4 μM:20 μM), and the fluorescence spectra before and after adding GSH and interfering substances are measured.

[0074] Take another several portions of 2 mL of PCN-224(Zn) HEPES buffer solution containing 20.0 μg / mL, and add equal amount of GSH (4 μM) in each portion, and then add GSH (4 μM), histidine (His), cysteine (Cys), ascorbic acid (AA), lysine (Lys), glutamic acid (Glu(E)), glycine (Gly), isoleucine (Iso), serine (Ser), proline (Pro), valine (Val), glucose (Glu(G)), oxidized glutathione (GSSG), human serum albumin (HSA), bovine serum albumin (BSA), Ba 2+ , Na + , Ca 2+ , NO2 -, still set the concentration ratio of GSH to Glu(E), Cys, Gly as 1:25 (4 μM: 100 μM), and the concentration ratio of GSH to other substances as 1:5. As shown in Fig. 2(a), PCN-224(Zn) only has a strong fluorescence response to GSH, and has no obvious fluorescence response to high concentrations of Glu(E), Gly, and Cys, and has no strong fluorescence response to other substances. To ensure that PCN-224(Zn) can detect GSH in a more complex environment, GSH and other interfering substances are simultaneously added to the PCN-224(Zn) dispersion, and the concentration ratio of GSH to Glu(E), Cys, and Gly is still set as 1:25 (4 μM: 100 μM), and the concentration ratio of GSH to other interfering substances is still set as 1:5. As shown in Fig. 2(b), in the presence of GSH, high concentrations of Glu(E), Gly, Cys, and other interfering substances cannot affect the fluorescence response of PCN-224(Zn) to GSH, indicating that PCN-224(Zn) can well detect GSH in complex biological samples or food samples.

[0075] The selectivity of PCN-224(Zn) for detecting GSH was tested under the optimal detection conditions. GSH (4 μM), histidine (His), cysteine (Cys), ascorbic acid (AA), lysine (Lys), glutamic acid (Glu(E)), glycine (Gly), isoleucine (Iso), serine (Ser), proline (Pro), valine (Val), glucose (Glu(G)), oxidized glutathione (GSSG), human serum albumin (HSA), bovine serum albumin (BSA), Ba 2+ , Na + , Ca 2+ , NO2 - , and K were added to the PCN-224(Zn) dispersion, and the concentration ratio of GSH to Glu(E), Cys, and Gly was set as 1:25 (4 μM: 100 μM), and the concentration ratio of GSH to other substances was set as 1:5. As shown in Fig. 2(a), PCN-224(Zn) only has a strong fluorescence response to GSH, and has no obvious fluorescence response to high concentrations of Glu(E), Gly, and Cys, and has no strong fluorescence response to other substances. To ensure that PCN-224(Zn) can detect GSH in a more complex environment, GSH and other interfering substances are simultaneously added to the PCN-224(Zn) dispersion, and the concentration ratio of GSH to Glu(E), Cys, and Gly is still set as 1:25 (4 μM: 100 μM), and the concentration ratio of GSH to other interfering substances is still set as 1:5. As shown in Fig. 2(b), in the presence of GSH, high concentrations of Glu(E), Gly, Cys, and other interfering substances cannot affect the fluorescence response of PCN-224(Zn) to GSH, indicating that PCN-224(Zn) can well detect GSH in complex biological samples or food samples. Figure 10 (a) shows that PCN-224(Zn) only has a strong fluorescence response to GSH, and has no obvious fluorescence response to high concentrations of Glu(E), Gly, and Cys, and has no strong fluorescence response to other substances. To ensure that PCN-224(Zn) can detect GSH in a more complex environment, GSH and other interfering substances are simultaneously added to the PCN-224(Zn) dispersion, and the concentration ratio of GSH to Glu(E), Cys, and Gly is still set as 1:25 (4 μM: 100 μM), and the concentration ratio of GSH to other interfering substances is still set as 1:5. As shown in Fig. 2(b), in the presence of GSH, high concentrations of Glu(E), Gly, Cys, and other interfering substances cannot affect the fluorescence response of PCN-224(Zn) to GSH, indicating that PCN-224(Zn) can well detect GSH in complex biological samples or food samples. Figure 10 (b) shows that in the presence of GSH, high concentrations of Glu(E), Gly, Cys, and other interfering substances cannot affect the fluorescence response of PCN-224(Zn) to GSH, indicating that PCN-224(Zn) can well detect GSH in complex biological samples or food samples.

[0076] Test three, sensitivity of PCN-224(Zn) for detecting GSH

[0077] A number of portions of PCN-224(Zn) HEPES buffer solution 2 mL containing 20.0 μg / mL were taken, and different concentrations of GSH (0.01, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 12, 14 μM) were added, and the fluorescence spectra before and after adding GSH were measured.

[0078] In 20 μg / mL PCN-224(Zn) dispersion liquid, different concentrations of GSH were added respectively, and the reaction was carried out for 4 min, Figure 11 (b) is the fluorescence enhancement spectrum of PCN-224(Zn), and the fluorescence intensity of PCN-224(Zn) gradually increases with the increase of the concentration of GSH. As shown in Figure 11 (a), the fluorescence intensity difference of PCN-224(Zn) before and after adding GSH is taken as the vertical coordinate, and the fluorescence intensity difference of PCN-224(Zn) and the concentration of GSH show a good linear relationship, and the fluorescence intensity difference of PCN-224(Zn) increases with the increase of the concentration of GSH. Taking the concentration of GSH as the horizontal coordinate and the fluorescence intensity difference before and after adding GSH as the vertical coordinate, the standard curve is drawn, and the linear relationship is fitted as Y = 3017.4X + 110.6. Within the linear relationship range of 0.01 μM to 6.00 μM of GSH, high sensitivity detection of GSH can be realized, and the correlation coefficient R 2 = 0.999, and the lowest detection limit (LOD) is as low as 1.5 nM.

[0079] Test four, verification of the detection mechanism of PCN-224(Zn) for GSH

[0080] After the reaction of GSH and PCN-224(Zn), the Zn quencher in PCN-224(Zn) can be eliminated, and after the elimination of Zn, it can be characterized by XPS, and the characteristic peak of Zn 2p will disappear or decrease. Therefore, by measuring the XPS of PCN-224(Zn) before and after the reaction with GSH, the change of the peak intensity of the Zn 2p characteristic peak in the spectrum is observed to reflect the change of the element, so as to verify the reaction mechanism of PCN-224(Zn) for detecting GSH.

[0081] As shown in Figure 12 , PCN-224(Zn) has a characteristic peak of Zn 2p in addition to Zr, C, O and N, but with the addition of GSH, GSH reacts with Zn 2+ and takes away Zn 2+ . The Zn 2p characteristic peak disappears, proving that Zn 2+ has been successfully taken away from PCN-224(Zn).

[0082] Test five, effect test

[0083] The standard addition recovery method was used for actual sample experiment. The fetal bovine serum was diluted ten times in 2 mL HEPES buffer solution, and the solution was prepared in triplicate. PCN-224(Zn) was added to each solution to make the concentration of PCN-224(Zn) 20.0 μg / mL, different concentrations of GSH (0.1, 0.5, 1.0 μM) were added respectively, and the fluorescence spectra before and after adding GSH were measured.

[0084] The vegetable juice was filtered and diluted ten times in 2 mL HEPES buffer solution, and the solution was configured in three parts, 2.0 mg / mL PCN-224(Zn) was added to each part, and different concentrations of GSH (0.1, 0.5) were added respectively, and the fluorescence spectra before and after the addition of GSH were measured.

[0085] The results of PCN-224(Zn) detecting GSH in fetal bovine serum and spinach juice actual samples are shown in Table 1.

[0086] Table 1 PCN-224(Zn) detecting GSH in fetal bovine serum

[0087]

[0088] Under the optimal detection conditions, the GSH in fetal bovine serum was detected by the standard addition recovery method, and 0.1, 0.5, 1 μM GSH was added respectively, and the average recovery of the detection results was between 99.5% and 100.4%. Similarly, under the optimal detection conditions, the GSH in spinach juice was detected by the standard addition recovery method, and 0.1, 0.5 μM GSH was added respectively, and the average recovery of the detection results was between 99.4% and 100.2%. The above results show that the fluorescence probe PCN-224(Zn) for detecting GSH has good reliability, and PCN-224(Zn) can be used for detection in actual samples.

[0089] Although the embodiments of the present application have been disclosed as above, it is not limited to the application listed in the specification and the embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily realized by those skilled in the art, and therefore the present application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. Use of a fluorescent probe in the detection of glutathione for non-disease diagnostic and therapeutic purposes, characterized in that, PCN-224, ZnCl2 and DMF were added into a reaction vessel, and a mixed solution was obtained by fully dissolving PCN-224, ZnCl2 and DMF, and the mixed solution was transferred to a hydrothermal reaction kettle, and heated at 100-150 ℃ for 10-20 h to obtain a dark green solid, which was washed with DMF and dispersed in a HEPES buffer solution to obtain a fluorescent probe PCN-224(Zn) having a fluorescence quenching property, wherein the fluorescent probe PCN-224(Zn) realizes fluorescence recovery by stripping Zn 2+ Changing the structure of the N-N ring in TCPP causes PCN-224 to quench fluorescence, and GSH is rich in sulfhydryl groups, which can bind with Zn 2+ The reaction strips Zn 2+ from PCN-224, so that PCN-224(Zn) becomes PCN-224 to achieve the effect of fluorescence recovery; The concentration of PCN-224 in the mixed solution is 1.5-3.5 mg / mL, and the concentration of ZnCl2 in the mixed solution is 0.04-0.44 mM; The ultrasonic wave is used for fully dissolving for 1-10 min; The preparation method of PCN-224 is as follows: meso-tetra (4-carboxyphenyl) porphyrin TCPP, ZrOCl2*8H2O and benzoic acid BA are added into DMF, the ultrasonic wave is used for fully dissolving for 1-10 min to make the concentration of TCPP 0.01-0.05 mmol / mL, the concentration of ZrOCl2*8H2O 0.1-0.5 mmol / mL and the concentration of BA 20-25 mmol / mL, the mixture is transferred into a hydrothermal reactor with polytetrafluoroethylene as an inner liner, is put into an oven, is heated at 100-150 DEG C for 12-24 h, a dark purple mixture is obtained, the mixture is washed with DMF for 2-3 times, and is dried to obtain a purple solid PCN-224; In the formula, DMF is N,N-dimethylformamide; TCPP is meso-tetra (4-carboxyphenyl) porphyrin; and BA is benzoic acid.

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