Preparation method and application of GSH fluorescence sensor based on ZIF-8 (at) MnO2

Abstract

The invention belongs to the field of biochemistry, and particularly relates to a preparation method and application of a GSH fluorescence sensor based on ZIF-8 (at) MnO2. The preparation method is relatively simple and convenient, and the required fluorescence sensor can be obtained through simple chemical reaction and physical treatment. The detection range of the GSH fluorescence sensor based on ZIF-8 (at) MnO2 is 2-20 [mu] M, the lower detection limit is about 0.41 [mu] M (3 sigma), in addition, the GSH recovery rate result of the method in a human serum sample is also satisfactory, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and particularly relates to a preparation method and application of a GSH fluorescence sensor based on ZIF-8@MnO 2 . Background Art

[0002] Glutathione (GSH), as the most common and abundant biothiol in vivo, plays a crucial role in maintaining the intracellular redox activity. Abnormal GSH levels are closely related to many diseases in humans, such as Parkinson's disease, liver cancer, Alzheimer's disease, and other cardiovascular diseases. Therefore, establishing a rapid and effective method to detect GSH in vivo is of great significance for understanding the functional process of GSH in cells and disease diagnosis.

[0003] In past reports, researchers have developed various methods for detecting GSH, such as colorimetry, electrochemistry analysis, mass spectrometry, high performance liquid chromatography, surface enhanced Raman scattering (SERS), and inductively coupled plasma mass spectrometry (ICP-MS). Although these methods can achieve the purpose of detecting GSH, they all have problems such as time-consuming, high cost, complex operation, and cumbersome sample preparation. Fluorescence method has attracted much attention of scientific researchers due to its advantages such as rapid response, high sensitivity, and simple equipment operation. However, most of the methods for detecting GSH based on fluorescence utilize the change of fluorescence intensity at a single wavelength. This traditional fluorescence detection method is easily affected by external factors such as environment and excitation light source. In order to overcome the shortcomings of traditional fluorescence method, some researchers have proposed the ratio fluorescence method, which uses the ratio of fluorescence intensities at two different wavelengths as the sensing signal. It can greatly weaken the influence of external factors and has a certain self-calibration function.

[0004] In recent years, metal-organic framework materials (MOFs) have attracted extensive attention of researchers due to their unique framework structure and excellent physical and chemical properties. Among them, zeolitic imidazolate framework material ZIF-8, as the most typical, common and most studied MOF material, has characteristics such as good crystallinity, high porosity, rich structural diversity, excellent chemical and thermal stability, etc., and thus is widely used in the fields of adsorption and separation, catalysis, biomedical science, and chemical sensing. For example, Lin et al. encapsulated amino-functionalized fluorescent carbon quantum dots into ZIF-8 material to construct a composite nanoprobe for detecting Cu 2+ ions. The research results show that the cumulative effect of ZIF-8 successfully amplifies the sensing signal of carbon quantum dots. Liu et al. established a new method for detecting Cu 2+The ratiometric fluorescence sensor (ZIF-8@rhodamine B), based on π-π interactions and hydrogen bonds between the N atoms of ZIF-8 and -COOH of rhodamine B, thus realizing the self-assembly of rhodamine B and ZIF-8. Sun et al. successfully used it for the detection of glucose by co-encapsulating glucose oxidase and gold nanoclusters into ZIF-8. It has been proven that in most cases, constructing hybrid nanocomposites using ZIF-8 as a template has shown superior performance.

[0005] As is well known, manganese dioxide nanomaterials (MnO 2 ) have attracted increasing attention due to their excellent properties such as simple synthesis, low toxicity, good stability, broad ultraviolet absorption, and peroxidase-like activity. Research shows that MnO 2 can quench the fluorescence of some substances through fluorescence resonance energy transfer (FRET) or inner filter effect (IFE), and can also be reduced to Mn 2+ ions, thereby restoring fluorescence. For example, Hu et al. constructed a novel fluorescence sensing platform for ultrasensitive detection of S1 nuclease activity based on MnO 2 nanosheets and FAM-labeled single-stranded DNA (FAM-ssDNA); Hu et al. established a novel ratiometric fluorescence probe (RF-probe) for sensitive and selective detection of proanthocyanidins (PAs) by synthesizing MnO 2 nanosheets and simultaneously regulating the photoluminescence of alloy Au / Ag nanoclusters (NCs) and thiamine (VB1).

[0006] Currently, there has been no report on the detection of GSH by a GSH fluorescence sensor based on ZIF-8@MnO 2 Therefore, we propose a new method for ratiometric fluorescence sensing of GSH based on ZIF-8@MnO 2 composite materials. Summary of the Invention

[0007] The purpose of the present invention is to provide a preparation method and application of a GSH fluorescence sensor based on ZIF-8@MnO 2 The preparation method is relatively simple, and the required fluorescence sensor can be obtained through simple chemical reactions and physical treatments. The detection range of the GSH fluorescence sensor based on ZIF-8@MnO 2 is 2 - 20 μM, and the lower detection limit is about 0.41 μM (3σ). In addition, the GSH recovery results in human serum samples by this method are also satisfactory.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] A GSH fluorescence sensor based on ZIF-8@MnO 2Preparation method of GSH fluorescence sensor, comprising the following steps:

[0010] 1) Synthesis of ZIF-8: Dissolve zinc nitrate hexahydrate in water to obtain a zinc nitrate solution; dissolve 2-methylimidazole in water to obtain a 2-methylimidazole solution; then add the 2-methylimidazole solution to the zinc nitrate solution, stir to obtain a milky white solution, then centrifuge the milky white solution, and then collect the white precipitate, wash it with deionized water, and dry it to obtain ZIF-8 powder;

[0011] 2) Synthesis of ZIF-8@MnO 2 : First, take the ZIF-8 obtained in step 1), disperse it in water, and continuously ultrasonicate it in an ice bath to obtain a ZIF-8 solution, and then drop the KMnO 4 solution into the ZIF-8 solution, stir to obtain a dark brown solution, then collect the product by centrifugation, wash it with water, and dry it to obtain the GSH fluorescence sensor.

[0012] Further, in step 1), the mass ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1:10.

[0013] Further, in step 1), the stirring is continuous stirring at 37 °C for 1 h.

[0014] Further, in step 2), the mass ratio of the ZIF-8 to the KMnO 4 is 2.5:1.

[0015] An application of the above GSH fluorescence sensor in detecting glutathione.

[0016] Further, the application comprises the following steps: Pipette the ZIF-8@MnO 2 solution into a centrifuge tube, then add GSH standard solutions with various concentrations, oscillate on a vortex oscillator for several seconds, then react at 45 °C, finally, add the OPD solution, oscillate on a vortex oscillator for several seconds to mix it evenly, then transfer it to a microcuvette, measure the fluorescence intensity under 340 nm excitation with a fluorescence spectrophotometer and record the experimental data, and then use F 565 / F 390 as the ordinate and the logarithm of the GSH concentration as the abscissa to make a linear relationship graph, and further quantitatively detect the concentration of the target substance GSH according to the fluorescence intensity ratio of F 565 / F 390 . The experimental principle of this application is as Figure 14 shown.

[0017] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

[0018] The preparation method of the GSH fluorescence sensor based on ZIF-8@MnO 2 is relatively simple. The required fluorescence sensor can be obtained through simple chemical reactions and physical treatments. The raw materials used in the preparation process are relatively easy to obtain, and the reaction conditions are mild, which is conducive to reducing production costs and improving production efficiency.

[0019] The present invention proposes a new application of a GSH ratio-type fluorescence sensing based on a ZIF-8@MnO 2 composite material. First, ZIF-8@MnO 2 composite material was prepared by in-situ synthesizing MnO 2 nanosheets on the surface of ZIF-8; at this time, the MnO 2 nanosheets would quench the light blue fluorescence centered at 390 nm generated by ZIF-8 under the excitation at 340 nm, which is mainly attributed to the broad ultraviolet absorption of the MnO 2 nanosheets. At the same time, by utilizing the peroxidase-like activity of the MnO 2 nanosheets, in the absence of the target GSH, OPD can be oxidized by the MnO 2 nanosheets into the fluorescent product 2,3-diaminophenazine (DAP), and a yellow fluorescence signal is generated at 565 nm; however, when the target GSH is present, GSH can reduce the MnO 2 nanosheets to Mn 2+ , thus significantly inhibiting the formation of DAP, resulting in the quenching of the fluorescence at 565 nm, and at the same time enhancing the light blue fluorescence centered at 390 nm of ZIF-8@MnO 2 .

[0020] In the present invention, the inventors optimized the main experimental conditions, including the concentration of ZIF-8@MnO 2 , the concentration of OPD, the reaction time, the reaction temperature, and the system pH. In addition, the selectivity of this method and the linear concentration range and detection limit for detecting GSH by ZIF-8@MnO 2 were also investigated. The experimental results show that the detection range of the developed GSH fluorescence sensor based on ZIF-8@MnO 2 is 2 - 20 μM, and the detection limit is about 0.41 μM (3σ). In addition, the GSH recovery results in human serum samples of this method are also satisfactory. Description of the Drawings

[0021] Figure 1 are the morphology diagrams of ZIF-8 and ZIF-8@MnO 2 in the present invention. Among them, (A) is the SEM image (left) of ZIF-8 and the EDS image (right) of ZIF-8; (B) is ZIF-8@MnO2 SEM image of (left), ZIF-8@MnO 2 EDS image of (right);

[0022] Figure 2 In the present invention, ZIF-8 and ZIF-8@MnO 2 Infrared spectrum;

[0023] Figure 3 In the present invention, ZIF-8 and ZIF-8@MnO 2 Elemental composition and chemical state diagram;

[0024] Figure 4 In the present invention, ZIF-8 and ZIF-8@MnO 2 XRD pattern;

[0025] Figure 5 In the present invention, ZIF-8, MnO 2 and ZIF-8@MnO 2 Potential change diagram;

[0026] Figure 6 In the present invention, ZIF-8@MnO 2 And feasibility exploration spectrum of detecting GSH with simple substrate OPD. Among them, (A) is the fluorescence spectrum of ZIF-8 (a), ZIF-8@MnO 2 (b) and ZIF-8@MnO 2 after adding OPD (c) (the inset is their photos under 365 nm ultraviolet light); (B) is the feasibility diagram of GSH fluorescence detection based on ZIF-8@MnO 2 (a: without GSH; b: with GSH, the inset is their photos under 365 nm ultraviolet light); (C) is the excitation (Ex) and emission (Em) spectra of ZIF-8 (the inset: the excitation (Ex) and emission (Em) spectra of the oxidation product DAP of OPD); (D) is the ultraviolet spectra of ZIF-8 and ZIF-8@MnO 2 ;

[0027] Figure 7 In the present invention, ZIF-8@MnO 2 Concentration optimization diagram;

[0028] Figure 8 In the present invention, concentration optimization diagram of OPD;

[0029] Figure 9 In the present invention, reaction time optimization diagram;

[0030] Figure 10 In the present invention, reaction temperature optimization diagram;

[0031] Figure 11 It is the pH optimization diagram of the system in the present invention;

[0032] Figure 12 It is ZIF-8@MnO in the present invention 2 Detection performance diagram of GSH, where (A) is the fluorescence spectrum diagram of ZIF-8@MnO in the presence of different concentrations of GSH (0, 1, 2, 3, 5, 8, 10, 20, 30, and 40 μM); 2 (B) is the calibration curve of the relationship between the fluorescence intensity ratio (F 565 / F 390 ) and the logarithm of the GSH concentration;

[0033] Figure 13 It is the fluorescence intensity ratio (F 565 / F 390 ) after adding different amino acids in the present invention;

[0034] Figure 14 It is the schematic diagram of the GSH ratio fluorescence detection principle based on ZIF-8@MnO in the present invention 2 . Specific embodiments

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] Embodiment

[0037] 1.1 Experimental reagents

[0038] Zinc nitrate hexahydrate (Zn(NO 3 ) 2 ·6H 2 O), 2-methylimidazole (C 4 H 6 N 2 ), potassium permanganate (KMnO 4 ), o-phenylenediamine (OPD), disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 ·12H 2 O), sodium dihydrogen phosphate dihydrate (NaH 2 PO 4 ·2H 2O) and sodium hydroxide (NaOH) were purchased from Xilong Chemical Co., Ltd. (Guangzhou, China). Glutathione (GSH), cysteine ​​(Cys), alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), proline (Pro), serine (Ser), threonine (Thr), histidine (His), arginine (Arg), glutamic acid (Glu), and tyrosine (Tyr) were from Sangon Biotechnology Co., Ltd. (Shanghai, China). All the above chemical reagents were of analytical grade (99%) and were used directly without further purification. In addition, deionized water (18.25 MΩ cm -1 ) were from an ultrapure water device (UPS-II-20L) from Yuechun Technology Co., Ltd. (Chengdu, China).

[0039] 1.2 Experimental instruments

[0040] The optical characterization of the materials was measured by UV-visible spectrometer (Beijing Puxi General Instrument Co., Ltd., China). Their morphological characterization was measured on a scanning electron microscope (SEM, SU-5000, Hi-Tech Corporation, Japan). Fourier transform infrared spectroscopy (FT-IR) was measured by Nicoleti S10 spectrometer (Thermo, USA). X-ray photoelectron spectroscopy (XPS) was measured by KRATOS AXIS His model (Krato, UK). Fluorescence excitation and emission spectra were measured using a Hitachi F-7000 fluorescence spectrophotometer (HITACHI, Japan). The analytical balance was purchased from Shanghai Tianmei Scientific Instrument Co., Ltd. (Shanghai, China). The pH meter (PHS-3C) was purchased from Precision Scientific Instruments Co., Ltd. (Shanghai, China). The vortex oscillator used was Vortex-BE1 from Qilin Bell Instrument Manufacturing Co., Ltd. (Haimen, China). The water bath was purchased from Beijing Kewei Yongxing Instrument Co., Ltd. (Beijing, China). Malvern Zeta potential analyzer (Zetasizer Nano ZS90) was from Malvern, UK.

[0041] 1.3 Synthesis of ZIF-8

[0042] 0.20 g Zn(NO 3 ) 2 6H 2O was dissolved in 4.0 mL of deionized water. 2.0 g of 2-methylimidazole was dissolved in 8.0 mL of deionized water (due to the large content of 2-methylimidazole, continuous ultrasonic assistance was required until dissolution). Then the prepared 2-methylimidazole solution was added to the zinc nitrate solution, and continuously stirred at 37 °C for 1 h to obtain a milky white solution. The milky white solution was then centrifuged at 10000 rpm for 15 min on a centrifuge, and then the white precipitate was collected and washed with deionized water. After vacuum drying at 80 °C for 24 h in an electrothermal vacuum drying oven, a ZIF-8 powder sample was obtained.

[0043] 1.4 ZIF-8@MnO 2 Synthesis

[0044] First, 0.05 g of the previously synthesized ZIF-8 was weighed and dispersed in 10 mL of deionized water, and continuously ultrasonicated in an ice bath. Then 10 mL of KMnO 4 (0.02 g) solution was dropped into the above solution. After continuously stirring for 60 min, a dark brown solution was obtained. The product was collected by centrifugation (10000 rpm, 5 min) and washed with deionized water. Then after vacuum drying at 80 °C for 24 h in an electrothermal vacuum drying oven, a brown powder sample was obtained. Finally, it was stored in a refrigerator at 4 °C for further use.

[0045] 1.5 Analysis procedure

[0046] Under the optimal experimental conditions, 0.5 mL of a 0.5 mg / mL ZIF-8@MnO 2 solution was accurately pipetted into a centrifuge tube, and then 0.1 mL of GSH standard solution with each concentration was added and oscillated on a vortex oscillator for several seconds. Then it was reacted at 45 °C for 20 min. Finally, 0.5 mL of a 12 mg / mL OPD was added to it, and the mixed solution was oscillated on a vortex oscillator for several seconds to fully mix it. Subsequently, it was transferred to a microcuvette, and the fluorescence intensity under 340 nm excitation was measured with a fluorescence spectrophotometer and the experimental data were recorded. Then, with F 565 / F 390 as the ordinate and the logarithm of the GSH concentration as the abscissa, a linear relationship graph was made. Furthermore, based on the fluorescence intensity ratio of F 565 / F 390 , the concentration of the target GSH was quantitatively detected.

[0047] 1.6 Pretreatment of actual samples

[0048] First, human serum samples (from volunteers at Guilin University of Technology Hospital) were centrifuged at 10,000 rpm for 10 min and then filtered through a 0.22 μm microporous membrane. After dilution, 100 μL of serum samples with different concentrations of GSH standard solution were added to ZIF-8@MnO 2 After reacting the solution at 45°C for 20 min, 0.5 mL of 12 mg / mL OPD was added to the solution, and the final volume of the mixed solution was 1.1 mL. The mixture was then vortexed on a vortex oscillator for several seconds to allow for full reaction. The analytical procedure was as described above.

[0049] 2 Results and discussion

[0050] 2.1 ZIF-8@MnO 2 Characterization

[0051] 2.1.1 ZIF-8@MnO 2 Morphological characterization

[0052] In order to study the ZIF-8 and ZIF-8@MnO 2 To better understand the morphology of the samples, a scanning electron microscope (SEM) was used to measure them. Figure 1 As shown in A, ZIF-8 is well dispersed and presents a uniform nanoscale regular octahedron shape; the EDS elemental analysis results on the right also show that the four elements C, N, O, and Zn are evenly distributed in ZIF-8. 2 After that, ZIF-8@MnO 2 The appearance of Figure 1 In B we can see that due to MnO 2 The ZIF-8@MnO 2 The composite material is in the shape of a flower cluster. At the same time, the EDS element analysis results on the right also show that the five elements C, N, O, Zn and Mn are evenly distributed in it. This proves that ZIF-8@MnO 2 The synthesis was successful.

[0053] 2.1.2 ZIF-8@MnO 2 FT-IR characterization

[0054] Next, we investigated ZIF-8 and ZIF-8@MnO 2 FT-IR characterization was performed. Figure 2 As shown, for ZIF-8, at 3442 cm -1 There is a characteristic peak near 2830cm -1There is a characteristic peak nearby, corresponding to the stretching vibration of C-H, at 1593 cm -1 The characteristic peak at the position corresponds to the stretching vibration of C=N, and the one at 1061 cm -1 corresponds to the stretching vibration of C-N, while the one at 1358 cm -1 , 1147 cm -1 and 776 cm -1 are the bending vibrations of the imidazole ring, and the characteristic absorption peak near 491 cm -1 is the stretching vibration of Zn-N. For MnO 2 @ZIF-8, in addition to the above typical ZIF-8 peaks, a peak band appears at ~530 cm -1 , which should come from the Mn-O vibration. The broad nature of this band indicates that the Mn in ZIF-8@MnO 2 can exist in different valence states.

[0055] 2.1.3 ZIF-8@MnO 2 XPS Characterization

[0056] Then, we also analyzed the elemental composition and chemical state of ZIF-8 and ZIF-8@MnO 2 by XPS characterization. As shown in Figure 3 A, compared with ZIF-8, in addition to the four elements C, N, O, and Zn, there is an additional peak of Mn in ZIF-8@MnO 2 , which undoubtedly indicates the formation of MnO 2 . As can be seen from Figure 3 B, the C1s high-resolution energy spectrum peaks of ZIF-8@MnO 2 correspond to C-C (283.9 eV), C-O (284.8 eV), and C=O (287.8 eV). The O1s high-resolution energy spectrum peaks ( Figure 3 C) are divided into four peaks at 529.2 eV, 530.8 eV, 531.6, and 532.5 eV, which correspond to Mn-O-Mn, Mn-O-H, H-OH, and C=O in the composite material respectively. Figure 3 As can be seen from Figure 3 D, the N1s high-resolution energy spectrum peaks can be divided into N-C (398.3 eV), N-H (399.1 eV), and N-Zn (400.7 eV). In the Zn2p high-resolution energy spectrum peaks ( 1 / 2 E), the two sharp peaks at 1215.2 eV and 1044.2 eV correspond to Zn2p 3 / 2 respectively. Figure 3 F is the high-resolution energy spectrum peak of Mn2p, and its two large sharp peaks are Mn2p 1 / 2 and Mn2p3 / 2 , and their corresponding binding energies are 653.3 eV and 641.4 eV. Among them, the Mn2p 3 / 2 peak can be divided into three peaks with binding energies of 640.6, 641.5, and 642.7 eV, respectively, which are attributed to Mn 4+ , Mn 3+ , and Mn 2+ . The spin energy separation between the two peaks is 11.9 eV, indicating that the Mn in ZIF-8@MnO 2 mainly exists in the form of tetravalent positive.

[0057] 2.1.4 ZIF-8@MnO 2 XRD Characterization

[0058] Then, we also carried out XRD characterization on ZIF-8 and ZIF-8@MnO 2 . As Figure 4 shown, ZIF-8 and ZIF-8@MnO 2 are well-crystallized. For ZIF-8 (black curve), we can observe a series of characteristic diffraction peaks, such as 2θ = 7.4° (011), 10.4° (002), 12.8° (112), 18.0° (222), etc. From the spectrum of ZIF-8@MnO 2 (red curve), it can be seen that the in-situ synthesis of MnO 2 will not affect the structure of ZIF-8.

[0059] 2.1.5 ZIF-8@MnO 2 Potential Characterization

[0060] Finally, we also carried out potential characterization on ZIF-8 and ZIF-8@MnO 2 . As Figure 5 shown, pure MnO 2 nanosheets have a negative potential, and its negative potential is -0.204 ± 0.021 mV. In contrast, ZIF-8 is positively charged, and its positive potential is +0.145 ± 0.046 mV. However, after the in-situ synthesis of MnO 2 , compared with ZIF-8, the potential of the ZIF-8@MnO 2 composite material is reduced to +0.098 ± 0.012 mV. The results of potential characterization once again verify the in-situ formation of MnO 2 nanosheets on ZIF-8.

[0061] 2.2 Feasibility Study

[0062] First, through the above analysis of ZIF-8 and ZIF-8@MnO 2After a series of characterizations, we have verified the successful synthesis of these materials. Then, the exploration of the feasibility of detecting GSH with ZIF-8@MnO 2 and the simple substrate OPD began. Figure 6 Figure A shows that under the excitation at 340 nm ( Figure 6 Figure C is the best excitation and emission spectrum of ZIF-8, and the inset is the best excitation and emission spectrum of the OPD catalytic product DAP), ZIF-8 itself produces a fluorescence emission peak centered at 390 nm (black curve). After in-situ synthesis of MnO 2 nanosheets on the surface of the ZIF-8 material, the fluorescence at 390 nm significantly decreases (red curve), because the relatively broad ultraviolet absorption (250 - 500 nm) of the MnO 2 nanosheets quenches the fluorescence of ZIF-8 at 390 nm through the FRET and IFE mechanisms ( Figure 6 The ultraviolet spectrum in Figure D also confirms this). At this time, when OPD is added, a strong fluorescence emission peak appears at 565 nm for ZIF-8@MnO 2 (blue curve), which is due to the peroxidase-like catalytic activity of the MnO 2 nanosheets, promoting the substrate OPD to produce a yellow fluorescent product DAP centered at 565 nm. At the same time, the emission bands of DAP and ZIF-8 are significantly distinguishable, which provides the possibility for the sensor based on ratio fluorescence proposed later.

[0063] Therefore, we began to construct a ratio fluorescence sensor for the detection of GSH. As Figure 6 shown in Figure B, compared with the blank group (black curve), when GSH is present (red curve), we can observe that the fluorescence intensity at 565 nm significantly weakens, while the fluorescence of ZIF-8 at 390 nm is restored (the inset is their picture under 365 nm ultraviolet light). This phenomenon can be explained as a special reaction occurring between GSH and the MnO 2 nanosheets, resulting in the MnO 2 nanosheets being directly reduced by GSH to Mn 2+ ions, thus reducing its performance in catalyzing OPD to generate DAP. The reaction mechanism between GSH and MnO 2 is as follows:

[0064] 2GSH + MnO 2 + 2H + = GSSG + Mn 2+ + 2H 2 O

[0065] 2.3 Optimization of experimental conditions

[0066] 2.3.1 ZIF-8@MnO 2 Concentration optimization

[0067] It was found that the intensity of the fluorescence signal is often highly related to the concentration of the sensor. Therefore, before performing GSH detection, we first optimized the concentration of ZIF-8@MnO 2 First, we prepared ZIF-8@MnO solutions with concentrations of 0.25, 0.5, 0.75, 1.0, and 1.25 mg / mL respectively. Then, 0.5 mL of the above ZIF-8@MnO solutions with different concentrations was taken, and 0.5 mL of OPD solution with a concentration of 12 mg / mL was added respectively. Each group of experiments was repeated three times in parallel. After shaking well, the fluorescence signal was measured on an F-7000 fluorescence spectrophotometer, and the fluorescence intensity was recorded. Then, the fluorescence intensity at the emission peak at 565 nm was denoted as F 2 2 565 390 565 390 ) was used as the ordinate, and the concentration of ZIF-8@MnO 2 was used as the abscissa. The results are as follows:

[0068] From Figure 7 we can see that the ability of ZIF-8@MnO with different concentrations to catalyze OPD is different. As the concentration of ZIF-8@MnO 2 increases, the ratio of the fluorescence intensity values (F 2 / F 565 / F 390 ) shows a trend of first increasing and then decreasing. When the concentration of Al-MOF is 1 mg / mL, F 565 / F 390 reaches the maximum value. In addition, the larger the value of F 565 / F 565 , the greater the degree of OPD catalyzed to produce DAP. Therefore, it can be concluded that the utilization efficiency of ZIF-8@MnO 2 in the system is the highest at this time. Therefore, the ZIF-8@MnO with a concentration of 1 mg / mL 2 was selected as the optimal condition for this experiment.

[0069] 2.3.2 OPD concentration optimization

[0070] ​​​​​Next, we optimized the concentration of OPD. First, OPD solutions with concentrations of 7, 8, 9, 10, 11, 12, and 13 mg / mL were prepared. Then, 0.5 mL of the above OPD solutions with different concentrations was taken, and 0.5 mL of ZIF-8@MnO solution with a concentration of 1 mg / mL was added to each. 2 For each group of experiments, three parallel operations were carried out. After shaking well, the fluorescence was measured on an F-7000 fluorescence spectrophotometer, and the fluorescence intensity was recorded. Then, the fluorescence intensity at the emission peak at 565 nm was denoted as F 565 , and the fluorescence intensity at 390 nm was denoted as F 390 . Then, with the ratio of the two fluorescence intensity values (F 565 / F 390 ) as the ordinate and the concentration of OPD as the abscissa, the results are as follows:

[0071] From Figure 8 it can be seen that as the concentration of OPD increases, the ratio of fluorescence intensity values (F 565 / F 390 ) shows a trend of first increasing and then decreasing. When the concentration of OPD is 12 mg / mL, F 565 / F 390 reaches the maximum value, indicating that the reaction between OPD and MnO 2 is the most sufficient at this time. Therefore, OPD with a concentration of 12 mg / mL was selected as the optimal concentration.

[0072] 2.3.3 Time Optimization

[0073] After exploring the optimal concentrations of ZIF-8@MnO 2 and OPD, we began to optimize the reaction conditions with the target substance GSH. First, we optimized the reaction time between ZIF-8@MnO 2 and GSH, and set 6 different reaction times of 0, 5, 10, 15, 20, and 25 min for investigation. Then, under the condition of the optimal concentration of ZIF-8@MnO 2 , 0.5 mL of ZIF-8@MnO solution with a concentration of 1 mg / mL was taken, and 0.1 mL of deionized water (blank group) and 0.1 mL of GSH with a concentration of 100 μM (sample group) were added respectively. After shaking well, 0.5 mL of OPD solution with a concentration of 12 mg / mL was added. Each group of experiments was carried out with three parallel operations, and then the fluorescence was measured on an F-7000 fluorescence spectrophotometer every five minutes, and the fluorescence intensity was recorded. Then, the fluorescence intensities of the two emission peaks of the experimental group where the ZIF-8@MnO 2 solution was mixed with GSH were denoted as F 2 and F 565 respectively.390 , and then we used the ratio of two fluorescence intensity values (F 565 / F 390 ) as the ordinate and the reaction time as the abscissa, and the results are as follows:

[0074] According to Figure 9 , we can find that at different reaction times, the reduction degree of ZIF-8@MnO 2 by GSH with a concentration of 100 μM is not very different. And as the reaction time prolongs, the ratio of fluorescence intensity values (F 565 / F 390 ) shows a trend of first decreasing and then leveling off. When the reaction time is 20 min, F 565 / F 390 reaches the minimum value and then remains relatively stable. And the smaller the value of F 565 / F 390 , the more Mn 2+ ions are reduced. That is to say, at this time, ZIF-8@MnO 2 reacts completely with the target substance GSH. Therefore, we choose the reaction time of 20 min as the optimal condition for this experiment.

[0075] 2.3.4 Temperature Optimization

[0076] Considering that the reaction temperature may have a certain impact on the system stability, for this reason, we optimized the reaction temperature. Based on the above optimization conditions, at the optimal reaction concentration (1.0 mg / mL) and the optimal reaction time (20 min) of ZIF-8@MnO 2 , we selected five temperature gradients of 4, 25, 37, 45, and 65 °C for research and investigation. First, take 0.5 mL of the ZIF-8@MnO 2 solution with a concentration of 1 mg / mL, and then add 0.1 mL of deionized water (blank group) and 0.1 mL of GSH with a concentration of 100 μM (sample group) respectively. After reacting for 20 min, add 0.5 mL of the OPD solution with a concentration of 12 mg / mL. Each group of experiments is repeated three times, shaken well, and then the fluorescence is measured on an F-7000 fluorescence spectrophotometer and the fluorescence intensity is recorded. Then, the fluorescence intensities of the two emission peaks of the experimental group where the ZIF-8@MnO 2 solution is mixed with GSH are respectively recorded as F 565 and F 390 , and then we use the ratio of two fluorescence intensity values (F 565 / F 390 ) as the ordinate and the reaction time as the abscissa, and the results are as follows:

[0077] From Figure 10As can be seen, different reaction temperatures have a significant impact on the fluorescence signal of this experiment. Moreover, as the reaction temperature increases, the ratio of fluorescence intensity values (F 565 / F 390 ) shows a trend of first decreasing and then increasing. When the reaction temperature is 45 °C, F 565 / F 390 reaches the minimum value. That is to say, the reaction between ZIF-8@MnO 2 and GSH is the most complete under the condition of 45 °C. Therefore, we choose 45 °C as the optimal reaction temperature for this experiment.

[0078] 2.3.5 Optimization of system pH

[0079] Considering that the pH value of the system may also have a certain impact on the chemical reaction process, we thus optimized the pH value. Based on the previous optimization conditions, we then investigated the influence of different pH values in the range of 3 - 9 on this experiment. First, ZIF-8@MnO 2 solutions were prepared using PBS buffer solutions with pH values of 3, 4, 5, 6, 7, 8, 9 respectively and deionized water. Then, at room temperature, 0.5 mL of a ZIF-8@MnO 2 solution with a concentration of 1 mg / mL was taken, and then 0.1 mL of deionized water (blank group) and 0.1 mL of GSH with a concentration of 100 μM (sample group) were added respectively. After reacting for 20 min, 0.5 mL of an OPD solution with a concentration of 12 mg / mL was added. Each group of experiments was repeated three times in parallel, and then the fluorescence was measured on an F-7000 fluorescence spectrophotometer and the fluorescence intensity was recorded. Next, the fluorescence intensities of the two emission peaks of the experimental group where the ZIF-8@MnO 2 solution was mixed with GSH were respectively denoted as F 565 and F 390 . Then, with the ratio of the two fluorescence intensity values (F 565 / F 390 ) as the ordinate and the pH value as the abscissa, the results are as follows:

[0080] As can be seen from Figure 11 , different pH values have a significant impact on the fluorescence signal of this experiment. This may be because strong acidity easily destroys the structure of ZIF-8@MnO 2 , thus affecting the sensing ability of GSH. The experimental results show that the experimental effect is the best when the pH is 8. Therefore, the optimal pH for this experiment is 8.

[0081] 2.4 GSH detection performance

[0082] After optimizing the key conditions of the experiment, we further investigated ZIF-8@MnO 2Detection performance for GSH. First, GSH solutions with different concentrations (0, 1, 2, 3, 5, 8, 10, 20, 30, 40 μM) were prepared separately, and then added to the ZIF-8@MnO solution with a concentration of 1 mg / mL as the sample group (three parallel samples in each group). Then, deionized water was added to the ZIF-8@MnO solution with a concentration of 1 mg / mL as the blank group (11 parallel samples). After reacting for 20 min, 0.5 mL of OPD solution with a concentration of 12 mg / mL was added, and then the fluorescence was measured on an F-7000 fluorescence spectrophotometer, and the fluorescence data were recorded. The fluorescence emission spectra at different GSH concentrations are shown in 2 Figure A. As the GSH concentration increases, the fluorescence intensity of DAP at 565 nm gradually decreases, while the fluorescence intensity of ZIF-8 gradually recovers. Then, the fluorescence intensities of the two emission peaks in the experimental group where the ZIF-8@MnO solution was mixed with GSH were denoted as F 2 and F Figure 12 respectively. Then, with the ratio of the two fluorescence intensity values (F 2 / F 565 ) as the ordinate and the logarithm of the GSH concentration (LogC 390 ) as the abscissa, the following results were obtained: 565 390 ) GSH As shown in Figure B, in the GSH concentration range of 2 - 20 μM, the ratio of the two fluorescence intensity values F

[0083] and the logarithm of the GSH concentration LogC Figure 12 showed a good linear relationship. The linear regression equation was F 565 / F 390 = -0.3857LogC GSH + 0.9876 (R 565 = 0.9982). Finally, according to the 3σ rule, the LOD of this linearity was approximately 0.41 μM. 390 GSH 2

[0084] 2.5 Selectivity investigation

[0085] To investigate the selectivity of this ZIF-8@MnO 2 for GSH, we selected 13 kinds of amino acids including GSH (GSH, Cys, Ala, Gly, Glu, Arg, Tyr, Leu, Pro, Ser, Thr, Val, His, and the concentration of each amino acid was 100 μM) and conducted fluorescence experiments with the ZIF-8@MnO 2 solution. First, take the ZIF-8@MnO solution with a concentration of 1 mg / mL 2 ​​​Add 0.5 mL of the solution, and then add 0.1 mL of deionized water (blank group) and 0.1 mL of various amino acids with a concentration of 100 μM (sample group) respectively. After reacting for 20 min, add 0.5 mL of OPD solution with a concentration of 12 mg / mL, then measure the fluorescence on an F-7000 fluorescence spectrophotometer and record the fluorescence data. The selective investigation results are as Figure 13 shown:

[0086] As can be seen from Figure 13 , when other amino acids are added to the sample group, the fluorescence intensity ratio F 565 / F 390 has a relatively high value and is similar to that of the blank group, indicating that these have no significant effect on the reaction; while when the target substance GSH is added, the fluorescence intensity ratio F 565 / F 390 has a significantly lowest value. Therefore, this method has good selectivity for GSH.

[0087] 2.6 Analysis of actual samples

[0088] To better prove the reliability and practicality of this method, we also carried out the detection of GSH in human serum. First, pretreat human serum, centrifuge it at a speed of 10000 rpm for 10 min, and then filter it with a 0.22 μm filter membrane to remove potential interferences. Then add diluted GSH standard solutions with different concentrations to the actual samples. Measure the fluorescence intensity in the same way as before. According to the results shown in Table 1, the spiked recovery rates of GSH in human serum are between 98.28 - 109.7%, and the relative standard deviations (RSD) are between 2.54 - 5.15% (n = 6), and the results are satisfactory. Therefore, the experimental results show that this method has good reliability and practicality.

[0089] Table 1 Determination of GSH content in human serum samples.

[0090] Table 1 Determination of GSH content in human serum samples.

[0091]

[0092] 3 Summary

[0093] The present invention uses ZIF-8@MnO 2 as a fluorescence probe, based on MnO 2Based on the peroxidase-like catalytic activity, broad ultraviolet absorption quenching of ZIF-8 fluorescence, and special reaction with GSH, a new ratiometric fluorescence sensing method for GSH was successfully established. This method has a fast response time, high sensitivity, and does not require specialized experimental personnel. Finally, the experimental results show that the detection linear range of this method for GSH is 2 - 20 μM, the LOD is about 0.41 μM (3σ rule), and it has good selectivity. In addition, the recovery results of actual samples are also satisfactory, indicating its good reliability and practicality.

[0094] The above description is a detailed description of the preferred feasible embodiment of the present invention, but the embodiment is not used to limit the patent application scope of the present invention. Any equivalent changes or modified changes completed under the technical spirit prompted by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing a GSH fluorescent sensor based on ZIF-8@MnO2, characterized in that: The following steps are involved: 1) Synthesis of ZIF-8: dissolving zinc nitrate hexahydrate in water to obtain a zinc nitrate solution; dissolving 2-methylimidazole in water to obtain a 2-methylimidazole solution; then adding the 2-methylimidazole solution to the zinc nitrate solution, stirring to obtain a milky white solution, then centrifuging the milky white solution, collecting a white precipitate, washing it with deionized water, and drying it to obtain ZIF-8 powder; 2) Synthesis of ZIF-8@MnO2: First, take the ZIF-8 from step 1), disperse it in water, and continuously sonicate it in an ice bath to obtain a ZIF-8 solution, then drop the KMnO4 solution into the ZIF-8 solution and stir to obtain a dark brown solution, then collect the product by centrifugation, wash it with water, and dry it to obtain the GSH fluorescent sensor.

2. The method for preparing the GSH fluorescent sensor based on ZIF-8@MnO2 according to claim 1, characterized in that: In step 1), the mass ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1:

10.

3. The method for preparing the GSH fluorescent sensor based on ZIF-8@MnO2 according to claim 1, characterized in that: In step 1), the stirring is continuous stirring at 37° C. for 1 h.

4. The method for preparing the GSH fluorescent sensor based on ZIF-8@MnO2 according to claim 1, characterized in that: In step 2), the mass ratio of the ZIF-8 to the KMnO4 is 2.5:

1.

5. Use of the GSH fluorescent sensor according to any one of claims 1 to 4 in detecting glutathione.

6. The use according to claim 5, characterized in that: The application comprises the following steps: transferring the ZIF-8@MnO2 solution into a centrifuge tube, adding various concentrations of GSH standard solutions, vortexing for several seconds, and reacting at 45°C, and finally, adding the OPD solution, vortexing for several seconds to fully shake it, and then transferring it into a micro-cuplet, measuring the fluorescence intensity under 340nm excitation by means of a fluorescence spectrophotometer and recording the experimental data, and then recording the fluorescence intensity by F 565 / F 390 With the logarithm of GSH concentration as the ordinate and the logarithm of GSH concentration as the abscissa, a linear relationship diagram is drawn. 565 / F 390 The concentration of the target GSH was quantitatively detected by the fluorescence intensity ratio of the two samples.

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