CN111189812B

CN111189812B - Method for detecting GSH (glutathione) by taking luminol-aluminum as fluorescent nano material

Info

Publication number: CN111189812B
Application number: CN202010036714.3A
Authority: CN
Inventors: 祁文静; 吴狄; 何洪昆
Original assignee: Chongqing Normal University
Current assignee: Chongqing Normal University
Priority date: 2020-01-14
Filing date: 2020-01-14
Publication date: 2022-07-15
Anticipated expiration: 2040-01-14
Also published as: CN111189812A

Abstract

The invention belongs to the technical field of fluorescence detection, and particularly discloses a method for detecting GSH by using luminol-aluminum as a fluorescent nano material. The invention utilizes the aggregation-induced fluorescence principle, and uses aluminum ions as coordination center ions to aggregate luminol together to form luminol-aluminum fluorescent nano materials (luminol-Al NPs). Based on Fluorescence Resonance Energy Transfer (FRET) principle and MnO₂The oxidation-reduction reaction of the nanosheets and glutathione, the luminol-Al NPs can be used for detecting the glutathione, the detection range is 0.2-20 mu M, the minimum detection limit is 45nM, other metal ions and small biological molecules have no interference on the detection of the glutathione, and the method has high sensitivity, selectivity and accuracy when used for detecting the glutathione.

Description

Method for detecting GSH (glutathione) by taking luminol-aluminum as fluorescent nano material

Technical Field

The invention relates to the technical field of fluorescence detection, in particular to a method for detecting GSH by using luminol-aluminum as a fluorescence nano material.

Background

Glutathione is a main nonprotein thiol in human cells and has a reducing property, so that it has various functions in cells, such as oxidation resistance, reaction with free radicals, and the like, as a reducing substance. In clinical medicine, glutathione can be used to treat a variety of diseases, including alzheimer's disease, alcoholic liver disease, hepatitis, and the like. Therefore, it is of great interest to search for a method for rapidly and sensitively detecting glutathione. To date, there are many methods reported in the literature for detecting glutathione, such as colorimetric methods, raman scattering detection methods, electrochemical methods, electrochemiluminescence methods, and fluorescence detection methods. In contrast, fluorescence detection methods are popular and used more often because of their advantages such as simplicity, rapidity, sensitivity, low cost, etc. For detecting glutathione by a fluorescence detection method, it is particularly important to find a suitable fluorescence probe, for example, carbon quantum dots, nitrogen-doped carbon nanosheets and near-infrared graphene quantum dots are all fluorescence probes for detecting glutathione reported by literatures, most of the fluorescence probes are quantum dots, and most of the synthesis process needs high temperature, high pressure or long reaction time. Therefore, it is valuable to find a fluorescent probe with short synthesis time and simple synthesis conditions for practical application.

Disclosure of Invention

In view of the above disadvantages of the prior art, an object of the present invention is to provide a method for detecting GSH using luminol-aluminum as a fluorescent nanomaterial, which is used to solve the problems of long synthesis time and complex synthesis conditions of fluorescent probes in the prior art.

To achieve the above and other related objects, the present invention provides a method for detecting GSH using luminol-aluminum as fluorescent nanomaterial, wherein luminol-aluminum is used as fluorescent nanomaterial (luminol-Al NPs), and MnO is used as the fluorescent nanomaterial₂The nano-sheet is a fluorescence quencher for detecting Glutathione (GSH).

Furthermore, the luminol-aluminum is a fluorescent nano material and is prepared from luminol solution and Al-containing Al³⁺The ionic solution is prepared by one-step reaction after mixing.

Optionally luminol and Al³⁺The molar ratio of the ions is equal.

Optionally, the concentration of the luminol solution is 5-20 mM, and the Al is contained³⁺The concentration of the ionic solution is 5-20 mM; preferably, the concentration of the luminol solution is 10mM, and the Al is contained³⁺The concentration of the ionic solution was 10 mM.

Optionally, the Al is contained³⁺The solution of the ions is one of aluminum nitrate and aluminum chloride solution, and preferably aluminum nitrate solution.

Further, the MnO is₂The preparation method of the nano sheet comprises the following steps: mixing MnCl₂·4H₂O aqueous solution was added to tetramethylammonium hydroxide (TMA. OH) and H₂O₂Stirring and reacting for 6-12 h at room temperature, and centrifuging to obtain MnO₂Nanosheets.

Alternatively, the MnCl₂·4H₂The concentration of the O aqueous solution is 0.2-0.4M, and MnCl is added₂·4H₂The dosage of the O aqueous solution is 7.5-15 mL; the concentration of tetramethylammonium hydroxide (TMA. OH) in the mixed solution is 0.5-0.8M, and H in the mixed solution₂O₂The concentration of (2) is 3 wt%, and the dosage of the mixed solution is 15-30 mL.

Further, when GSH is detected, the concentration of the luminol-aluminum solution is 10-50 mu M; preferably, the concentration of the luminol-aluminum solution is 50 μ M.

Further, the method has the detection range of 0.2-20 mu M for GSH, and the lowest detection limit is 45 nM.

Further, when GSH is detected, the MnO is₂The concentration of the nano-sheets is 20-70 mug/mL, preferably 60 mug/mL.

Further, when GSH is detected, the reaction temperature is 25-50 ℃, and 25 ℃ is preferred.

Further, the reaction time is 0-25 min, preferably 15min when GSH is detected.

The second aspect of the present invention provides a method for preparing a fluorescent nanomaterial for detecting GSH, wherein the fluorescent nanomaterial is luminol-aluminum fluorescent nanomaterial (luminol-Al NPs), and the preparation method comprises: the luminol-aluminum is a fluorescent nano material prepared from luminol solution and Al³⁺The ionic solution is prepared by one-step reaction after mixing.

Alternatively, luminol and Al³⁺The molar ratio of the ions is equal.

Optionally, the Al is contained³⁺The ionic solution is one of aluminum nitrate and aluminum chloride solution, and preferably aluminum nitrate solution.

The third aspect of the invention provides a fluorescent nano material for detecting GSH, which is prepared by the preparation method.

Further, the fluorescent nano material is in MnO₂GSH is selectively detected in the presence of the nanosheets.

The fourth aspect of the present invention provides the use of the above fluorescent nanomaterial in the detection of GSH.

Further, the detection range of the fluorescent nano material on GSH is 0.2-20 mu M, and the lowest limit of detection (LOD) is 45 nM.

As mentioned above, the method for detecting GSH by using luminol-aluminum as fluorescent nano material has the following beneficial effects:

the invention utilizes an aggregation induced fluorescence principle (AIE) and takes aluminum ions as coordination center ions at room temperature to aggregate luminol together to form luminol-aluminum fluorescent nano material (luminol-Al NPs), and has simple synthesis mode and short synthesis time. Due to the emission spectrum of the luminol-Al NPs and manganese dioxide (MnO)₂) The absorption spectra of the nanoplates overlap, thus MnO₂The nanosheets can quench the fluorescence intensity of the luminol-Al NPs through the Fluorescence Resonance Energy Transfer (FRET) principle. MnO in the presence of reduced glutathione₂The nanosheets are reduced to Mn²⁺Such that the intensity of the quenched fluorescence increases with increasing glutathione concentration. Therefore, based on FRET and redox reaction, the luminol-Al NPs can be used for detecting glutathione, the detection range is from 0.2 mu M to 20 mu M, the lowest limit of detection (LOD) is 45nM, other metal ions and biological small molecules (such as L-lysine, L-threonine and L-valine) have no interference on the detection of the glutathione, and the method has higher sensitivity and selectivity on the detection of the glutathione. The method is used for detecting the glutathione in the serum sample, the recovery rate is between 101.48% and 103.23%, and the fluorescence enhancement method has better accuracy in the biological analysis.

Drawings

FIG. 1 shows the synthesis of Luminol-Al NPs and its principle diagram of fluorescence enhancement method for detecting GSH based on FRET.

FIG. 2 shows a TEM image (A) and a fluorescence spectrum (B) of Luminol-Al NPs and MnO in example 1 of the present invention₂TEM (C) and SEM (D) images of the nanoplatelets.

FIG. 3 shows a fluorescence spectrum (upper panel) and a sample photograph (lower panel) of the Luminol-Al NPs system in example 2 of the present invention.

FIG. 4 shows MnO in example 3 of the present invention₂And (3) a result chart of the influence of the concentration of the nanosheets on the detection of glutathione by the luminol-Al NPs.

FIG. 5 shows glutathione and MnO in example 3 of the present invention₂Reaction temperature of nanosheetAnd (3) a result graph of the influence of the glutathione concentration on the detection of glutathione.

FIG. 6 shows glutathione and MnO in example 3 of the present invention₂And (3) a result graph of the influence of the reaction time of the nanosheets on glutathione detection.

FIG. 7 shows the fluorescence spectrum (A) and the linear operation curve (B) of the system in example 4 of the present invention under different concentrations of glutathione.

FIG. 8 is a graph showing the results of selectivity of fluorescence detection of glutathione using luminol-Al NPs in example 4 of the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

As shown in FIG. 1, the present invention utilizes luminol-aluminum fluorescent nanomaterials (luminol-Al NPs) and MnO₂Nanosheets, glutathione detected by Fluorescence Resonance Energy Transfer (FRET). The luminol-aluminum fluorescent nano material (luminol-Al NPs) applied by the invention is based on an Aggregation Induced Emission (AIE) principle, and can be used for obtaining the luminol-Al NPs with stable fluorescence within two minutes at room temperature.

The AIE material is widely applied to the fields of biological imaging, environmental monitoring, organic light emitting diodes and the like, compared with other materials, the AIE material has the characteristics of low background and high light stability, and is very suitable for being used as a fluorescent probe. The luminol-Al NPs synthesized by the invention take aluminum ions as coordination center ions, and the luminol is often used as a luminescent substrate.

MnO₂The nano-sheet is a two-dimensional nano-material, which is often used as a photoelectric material and energyAs a material having an oxide nanomaterial, MnO, a material for storing and converting quantity, a catalytic material, etc₂The nano-sheet can oxidize 3, 3, 5, 5-Tetramethylbenzidine (TMB) and can also react with reduced glutathione to generate Mn²⁺So that MnO can be utilized₂Nanosheets for detection of glutathione. MnO (MnO)₂The rice flake has absorption in the wavelength range of 250-600 nm, the emission wavelength of luminol-Al NPs is about 430nm, and MnO₂The ultraviolet absorption spectrum of the nano-sheet is overlapped with the emission spectrum of luminol-Al NPs, MnO₂The nano-sheet can form fluorescence resonance energy transfer with luminol-AlNPs, and the fluorescence intensity of the luminol-Al NPs is quenched. Glutathione can convert MnO into MnO₂The nanosheets are decomposed into divalent manganese ions, such that the quenched fluorescence intensity is responsive to MnO₂The nanosheets are gradually restored by being decomposed by glutathione.

The materials and reagent sources used in the following examples are as follows:

luminol, manganese chloride, hydrogen peroxide (30%), tetramethylammonium hydroxide pentahydrate (TMA. OH) and glutathione (reduced form), bovine serum samples were purchased from Sigma-Aldrich (Beijing, China). L-valine, L-threonine, L-lysine were purchased from national chemical of drug stock control (Shanghai, China). Glacial acetic acid, sodium acetate, calcium chloride, ferric chloride, potassium chloride, and aluminum nitrate were purchased from chengdong chemical reagent ltd (chengdu, china). Water and 0.2M HAc-NaHAc buffer solution (pH 7.0) were used for dilution during the experiment. Ultrapure water was used in all experiments.

The apparatus used in the following examples is as follows:

fluorescence spectra were obtained on a Perkin Elmer LS55 luminescence spectrometer (Perkin Elmer Instruments, u.k). Ultraviolet-visible (UV-vis) absorption spectra were obtained on a UV2550 ultraviolet-visible spectrophotometer (Shimadzu, Japan). Transmission Electron Microscope (TEM) images were obtained on a JEM-2100F transmission electron microscope (JEOL, Japan). Fluorescence photographs were taken at 365nm uv using a ZF-20D uv analysis device from rainflower instruments ltd, consortium, china. An S-25 digital pH meter (Shanghai Proc. Industriaceae industries, Shanghai, China) was used to detect the pH of the solution.

The specific implementation process is as follows:

example 1

1. Preparation of luminol-Al NPs

At room temperature, 1mL of 10mM luminol solution and 1mL of 10mM aluminum nitrate were mixed, and after two minutes, luminol-Al NPs were synthesized directly in one step. Diluting the luminol-Al NPs solution by 100 times, and placing the diluted solution in a refrigerator at 4 ℃ for glutathione detection.

2、MnO₂Preparation of nanosheets

First, 10mL of 0.3M MnCl was added over 15s₂·4H₂20mL of 0.6M TMA. OH and 3 wt% H were added to the aqueous O solution₂O₂In the mixture of (a). In the second step, the reaction solution was stirred at room temperature overnight, and then the precipitate was removed by centrifugation with ultrapure water and methanol, washed three times, and centrifuged at 10000rpm for 10 minutes. Finally, the precipitate was dried in a vacuum oven at 50 ℃. Before use, MnO is added₂Dissolving the nano sheet in water to obtain MnO of 1mg/mL₂Nanosheet solution for subsequent glutathione detection.

3. Step of detecting glutathione

First, 70. mu.L of 1mg/mL MnO₂Reacting the nanosheets with glutathione of different concentrations in 20 mu L of HAc-NaAc buffer solution with pH of 5.8 for 15 minutes; then 40. mu.L of a 50. mu.M solution of luminol-AlNPs was added; 200 μ L of 0.2M (pH 7.0) HAc-NaAc buffer and appropriate amount of water were added to maintain the final volume of the whole solution at 1mL, and the emission spectrum was detected at an excitation wavelength of 290 nm.

4. Detection step of glutathione in serum sample

Bovine serum samples were purchased from Sigma-Aldrich (Beijing, China) and were glutathione free. The accuracy of this method for detecting glutathione was investigated using standard additive recovery methods. To obtain recovery, three different concentrations of glutathione (5mM, 10mM and 30mM) were added to 100mL of 10-fold serum samples, respectively. Similar to the glutathione detection step above, the other steps are completed.

5. Luminol-Al NPs and MnO₂Synthesis and characterization of nanoplatelets

FIG. 2 shows TEM image (A) and fluorescence spectrum (B) of Luminol-Al NPs, MnO₂TEM image (C) and sem (d) of nanosheets. Wherein, c (Al)³⁺μ M): 0. 40, 60, 80, 100 and 200; c (luminol, mM): 0.8; HAc-NaAc buffer solution pH 7.0; temperature (. degree. C.): 25 (room temperature); excitation wavelength: 290 nm; emission wavelength: 430 nm; reaction time (min): 2.

as can be seen from FIG. 2, Al is added to the luminol solution³⁺Thereafter, luminol-Al NPs were formed, which have a network structure of aggregated small particles (FIG. 2A). At the same time, the fluorescence of the luminol-Al NPs follows that of Al³⁺The increase in concentration increased (FIG. 2B), confirming the synthetic principle of AIE. The synthesized luminol-Al NPs solution can be stored in a refrigerator at 4 ℃ for two weeks without great change of fluorescence intensity by measuring the fluorescence intensity. Synthetic MnO₂The nano-sheet is in a sheet structure (shown in figures 2C-D), has a wider absorption range at the position of 300-650nm, and is overlapped with the fluorescence of the emission spectrum of the luminol-Al NPs at the position of 400-500 nm.

Example 2

Luminol-Al NPs and MnO₂Feasibility characterization of nanosheet for measuring glutathione

FIG. 3 shows the fluorescence spectrum (upper panel) and the sample photo (lower panel) of the Luminol-Al NPs system. The red line represents the fluorescence spectrum of the luminol-Al NPs; purple line represents MnO₂Fluorescence spectrum of luminol-Al NPs in the presence of nanosheets; the green line represents the fluorescence spectrum of the luminol-Al NPs in the presence of glutathione; blue line indicates in MnO₂Fluorescence spectrum of luminol-Al NPs under the condition that the nano-sheets and the glutathione coexist. The test conditions were: c (luminol-Al NPs, μ M): 2; c (glutathione, μ M): 20; c (MnO)₂Nanoplatelets, μ g/mL): 60, adding a solvent to the mixture; 40mM HAc-NaAc buffer solution: pH7.0; temperature (. degree. C.): 25 (room temperature); glutathione and MnO₂The reaction time of the nanosheet is as follows: 10 minutes; excitation wavelength: 290 nm; emission wavelength: 430 nm. The photograph of the sample is taken under the irradiation of a 365nm ultraviolet lamp, wherein the photograph of the luminol-Al NPs solution is (a), the photograph of the mixture of the luminol-Al NPs solution and glutathione is (b), and the photograph of the luminol-Al NPs solution and MnO are₂Photograph of nanosheet(c) luminol-Al NPs solution and MnO₂A photograph of the nanoplatelets and glutathione mixture (d). The test conditions were: c (luminol-Al NPs, mM): 0.2; c (MnO)₂Nanosheet, mg/mL): 0.33; c (glutathione, mM): 2.

FIG. 3 shows fluorescence spectra with MnO added₂After the nano-plate is prepared, the fluorescence intensity of luminol-Al NPs at 430nm is reduced by 77%, which indicates that the luminol-Al NPs and MnO are reduced₂Feasibility of FRET between nanosheets. MnO due to glutathione₂Reduction of nanosheets to Mn²⁺The fluorescence spectra in FIG. 3 show that luminol-AlNPs and MnO were present in the presence of glutathione (20. mu.M)₂The fluorescence of the nanoplatelet mixture is enhanced. As can be seen from the fluorescence quenching and enhancement results, the luminol-Al NPs can be used as a fluorescent probe for detecting glutathione.

Example 3

Optimization of conditions for detecting glutathione by Luminol-Al NPs

First, this example explores MnO₂The influence of the concentration of the nanosheets on detection of glutathione by luminol-Al NPs. The results are shown in FIG. 4. The red bars represent the fluorescence intensity of the luminol-Al NPs; green bars for luminol-AlNPs and MnO₂Fluorescence intensity of the nanosheet mixed solution; blue bars represent luminol-Al NPs and MnO₂Fluorescence of the nanosheets and glutathione. The test conditions were: c (MnO)₂Nanoplatelets, μ g/mL): 20. 30, 40, 50, 60 and 70. c (luminol-Al NPs, μ M): 2; glutathione and MnO₂The reaction time of the nanosheet is as follows: 10 minutes; c (GSH, μ M) 20; temperature (. degree. C.): 25 deg.C (room temperature). 40mM HAc-NaAc buffer: the pH was 7.0. Excitation wavelength: 290 nm; emission spectrum: 430 nm. All data were from three determinations.

As can be seen from FIG. 4, MnO₂The concentration of the nano-sheets is from 20 mu g/mL to 70 mu g/mL, the fluorescence quenching efficiency of the luminol-Al NPs is gradually increased, 20 mu M glutathione is added, the fluorescence intensity is enhanced, and the optimal signal-to-noise ratio, MnO, is achieved in consideration of the fluorescence quenching and increasing effects₂The optimum concentration of nanoplatelets is chosen to be 60 μ g/mL.

Next, this example explores glutathione andMnO₂influence of different reaction temperatures of the nanosheets on glutathione detection. The results are shown in FIG. 5. The test conditions were: temperature (. degree. C.): 25 (room temperature), 37, 50. c (luminol-Al NPs, μ M): 2; c (glutathione, μ M): 20; c (MnO)₂Nanoplatelets, μ g/mL): 60; glutathione and MnO₂The reaction time of the nanosheet is as follows: 10 minutes; 40mM HAc-NaAc buffer: the pH was 7.0. Excitation wavelength: 290 nm; emission wavelength: 430 nm; all data were from three determinations.

As shown in fig. 5, this example explores three different reaction temperatures: 25 deg.C, 37 deg.C, 50 deg.C, considering fluorescence quenching and increasing effect and achieving larger signal-to-noise ratio, selecting 25 deg.C (room temperature) as glutathione and MnO₂The nanosheet has an optimal reaction temperature.

Finally, this example explores glutathione and MnO₂Influence of reaction time of the nanosheets on glutathione detection. The results are shown in FIG. 6. The test conditions were: glutathione and MnO₂Nanosheet reaction time was (min): momentary, 2, 5, 10, 15, 20, 25; temperature (. degree. C.): 25 (room temperature); c (luminol-Al NPs, μ M): 2; c (glutathione, μ M): 20; c (MnO)₂Nanoplatelets, μ g/ml): 60; 40mM HAc-NaAc buffer: the pH was 7.0. Excitation wavelength: 290 nm; emission wavelength: 430 nm; all data were from three determinations. As shown in FIG. 6, the present example searched for the effects of the reaction times of instant, 2, 5, 10, 15, 20, and 25 minutes, respectively, and the experimental data showed that after 15 minutes, glutathione and MnO were present₂The reaction of the nano-sheets is complete, and the fluorescence recovery of the luminol-Al NPs reaches the maximum. The reaction was chosen as glutathione and MnO for 15 minutes considering the effect of fluorescence quenching and increase and achieving a larger signal to noise ratio₂The nanosheets have an optimal reaction time.

Example 4

Sensitivity and selectivity of Luminol-Al NPs for detecting glutathione

Under the optimized experimental conditions, the present example studies the glutathione pairs at different concentrations for luminol-Al NPs and MnO₂Influence of fluorescence intensity of the nanosheet mixed solution.

FIG. 6 shows the fluorescence spectrum (A) and the linear working curve (B) of the system under the condition of different concentrations of glutathione. The test conditions were: c (glutathione, μ M): 0. 0.2, 0.5, 2, 5, 8, 15, 20; temperature (. degree. C.): 25 (room temperature); glutathione and MnO₂The reaction time of the nanosheet is as follows: 15 minutes; c (luminol-Al NPs, μ M): 2; c (glutathione, μ M): 20; c (MnO)₂Nanoplatelets, μ g/ml): 60, adding a solvent to the mixture; 40mM HAc-NaAc buffer: the pH was 7.0. Excitation wavelength: 290 nm; emission wavelength: 430 nm; all data were from three determinations.

As shown in FIG. 7A, as the glutathione concentration increased from 0 to 20. mu.M, luminol-Al NPs and MnO₂The fluorescence intensity of the nanosheet mixed solution gradually increases. FIG. 7B shows a linear plot between fluorescence intensity at 430nm and glutathione concentration. As can be seen from fig. 7B, the linear relationship between glutathione detected by luminol-Al NPs was good, I ═ 124.34+5.89c (μ M), and the correlation coefficient (r) was 0.9957.

This example compares the method for detecting glutathione of the present invention with other methods for detecting glutathione (Table 1), and finds that the limit of detection (LOD, 45nM) of the present method is lower than that reported in most other documents. Glutathione is one of the most abundant antioxidant small molecular weight thiols in vivo, mainly exists in cells, the concentration is 0.5 mM-10 mM, and LOD (45nM) can meet the clinical analysis requirement of glutathione. Therefore, the method for detecting the glutathione by using the luminol-Al NPs has high sensitivity.

TABLE 1 comparison with other methods for glutathione detection

Methods 1-10 in table 1 are from the following references:

1.Liu,Y.；Zhou,M.；Cao,W.；Wang,X.；Wang,Q.；Li,S.；Wei,H.,Light-responsive metal-organic framework as an oxidase mimic for cellular glutathione detection.Anal.Chem.2019,91(13),8170-8175.

2.Peng,C.；Xing,H.；Fan,X.；Xue,Y.；Li,J.；Wang,E.,Glutathione Regulated Inner Filter Effect of MnO2 Nanosheets on Boron Nitride Quantum Dots for Sensitive Assay.Anal.Chem.2019,91(9),5762-5767.

3.Jiao,Y.；Gao,Y.；Meng,Y.；Lu,W.；Liu,Y.；Han,H.；Shuang,S.；Li,L.；Dong,C.,One-step synthesis of label-free ratiometric fluorescence carbon dots for the detection of silver ions and glutathione and cellular imaging applications.ACS.Appl.Mater.Inter.2019,11(18),16822-16829.

4.Meng,H.-M.；Zhao,D.；Li,N.；Chang,J.,A graphene quantum dot-based multifunctional two-photon nanoprobe for the detection and imaging of intracellular glutathione and enhanced photodynamic therapy.Analyst 2018,143(20),4967-4973.

5.Yan,X.；Song,Y.；Zhu,C.；Song,J.；Du,D.；Su,X.；Lin,Y.,Graphene quantum dot-MnO2 nanosheet based optical sensing platform:a sensitive fluorescence“turn off–on”nanosensor for glutathione detection and intracellular imaging.ACS.Appl.Mater.Inter.2016,8(34),21990-21996.

dong, z. -z.; lu, l.; ko, c. -n.; yang, C.; li, S.; lee, m. -y.; leung, C. -H.; ma, D. -L., A MnO2 nano sheet-assisted glutathione detection platform using an iridium (iii) complex as a switch-on luminescence probe, nanoscale 2017,9(14), 4677-.

7.Liu,Z.；Cai,X.；Lin,X.；Zheng,Y.；Wu,Y.；Chen,P.；Weng,S.；Lin,L.；Lin,X.,Signal-on fluorescent sensor based on GQDs-MnO2 composite for glutathione.Anal.Methods 2016,8(11),2366-2374.

8.Zhang,R.；Zhong,X.；Chen,A.-Y.；Liu,J.-L.；Li,S.-K.；Chai,Y.-Q.；Zhuo,Y.；Yuan,R.,Novel Ru(bpy)2(cpaphen)2+/TPrA/TiO2 Ternary ECL System:An Efficient Platform for the Detection of Glutathione with Mn2+as Substitute Target.Anal.Chem.2019,91(5),3681-3686.

9.Gao,W.；Liu,Z.；Qi,L.；Lai,J.；Kitte,S.A.；Xu,G.,Ultrasensitive Glutathione Detection Based on Lucigenin Cathodic Electrochemiluminescence in the Presence of MnO2 Nanosheets.Anal.Chem.2016,88(15),7654-7659.

10.Venkateswara Raju,C.；Senthil Kumar,S.,Highly sensitive novel cathodic electrochemiluminescence of tris(2,2′-bipyridine)ruthenium(ii)using glutathione as a co-reactant.Chem.Commun.2017,53(49),6593-6596.

To analyze the selectivity of fluorescence detection of glutathione using luminol-Al NPs, the present example simultaneously applied the method to detect other common ions and small biological molecules, such as CaCl₂、FeCl₃NaCl, KCl, glucose (Glu), L-valine, L-threonine, L-lysine and the like. The results are shown in FIG. 8, in which glutathione and CaCl were added₂、FeCl₃NaCl, KCl, glucose (Glu), L-valine, L-threonine and L-lysine at a concentration of 20 μ M; temperature (. degree. C.): 25 (room temperature); glutathione and MnO₂The reaction time of the nanosheet is as follows: 15 minutes; c (luminol-Al NPs, μ M): 2; c (MnO)₂Nanoplatelets, μ g/ml): 60, adding a solvent to the mixture; 40mM HAc-NaAc buffer: the pH was 7.0. Excitation wavelength: 290 nm; emission wavelength: 430 nm; all data were from three determinations.

As can be seen from FIG. 8, CaCl₂、FeCl₃NaCl, KCl, glucose, L-valine, L-threonine and L-lysine do not affect luminol-Al NPs and MnO₂Fluorescence of the nanoplatelets. It was not shown to react to luminol-Al NPs and MnO similar to glutathione₂Enhancement of fluorescence of the nanosheets. This fluorescence enhancement is attributed to glutathione and MnO₂Reaction between nanosheets. Therefore, the use of luminol-Al NPs shows high selectivity for detecting glutathione to other substances.

In order to evaluate the accuracy of the luminol-Al NPs for detecting glutathione, the following experiment is carried out by adding glutathione into a serum sample by adopting a method for measuring the standard recovery rate. The average recovery of serum samples was shown in Table 2 by adding 5. mu.M, 10. mu.M and 15. mu.M glutathione to the serum, respectively. All Relative Standard Deviations (RSD) were below 5%. It can be seen that the assay of glutathione using luminol-Al NPs is accurate and reliable.

Table 2 results of measuring recovery of glutathione in serum with standard addition

In conclusion, the luminol-Al NPs synthesized by the method are obtained at low cost under the mild reaction condition at room temperature. The invention utilizes MnO₂The principle that the ultraviolet absorption of the nanosheets is overlapped with the fluorescence emission spectra of the luminol-Al NPs develops MnO by using the luminol-Al NPs as an energy donor₂The nano-sheet is a FRET system of an acceptor. Based on MnO₂The oxidation-reduction reaction of the nanosheet and glutathione develops a fluorescence enhancement method for detecting glutathione. The method has high detection sensitivity on glutathione, the linear range is 0.2-20 mu M, and the detection limit is 45 nM; has high selectivity, and other metal ions, L-valine, L-threonine, L-lysine and the like are not interfered; the accuracy is high, and the detection recovery rate of glutathione in the serum sample is 97.90-101.69%. Therefore, the invention has important significance for promoting the research of the AIE luminescent nano material and the biological analysis and clinical analysis of the glutathione.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A method for detecting GSH by using luminol-aluminum as a fluorescent nano material is characterized in that luminol-aluminum is used as the fluorescent nano material, and MnO is used₂The nano-sheet is a fluorescence quencher for detecting glutathione GSH; the luminol-aluminum fluorescent nano material is prepared from luminolThe solution of Al and Al³⁺The solution is prepared by one-step reaction after mixing.

2. The method of claim 1, wherein:

the MnO₂The preparation method of the nano sheet comprises the following steps: mixing MnCl₂·4H₂O aqueous solution was added to tetramethylammonium hydroxide TMA. OH and H₂O₂Stirring and reacting for 6-12 h at room temperature, and centrifuging to obtain MnO₂Nanosheets.

3. The method of claim 2, wherein: luminol and Al³⁺The molar ratio of the components is equal;

and/or, said MnCl₂·4H₂The concentration of the O aqueous solution is 0.2-0.4M, and MnCl is added₂·4H₂The dosage of the O aqueous solution is 7.5-15 mL; the concentration of TMA & OH in the mixed solution is 0.5-0.8M, and the concentration of H in the mixed solution is₂O₂The concentration of (2) is 3 wt%, and the dosage of the mixed solution is 15-30 mL.

4. The method of claim 1, wherein: the method has the detection range of 0.2-20 mu M for GSH, and the lowest detection limit is 45 nM;

and/or, when GSH is detected, the MnO₂The concentration of the nano sheets is 20-70 mu g/mL;

and/or when GSH is detected, the reaction temperature is 25-50 ℃;

and/or when GSH is detected, the reaction time is 0-25 min.

5. The method of claim 4, wherein: when GSH is detected, the MnO₂The concentration of the nano-sheets is 60 mug/mL, the reaction temperature is 25 ℃, and the reaction time is 15 min.

6. The application of luminol-aluminum as a fluorescent nano material in detecting GSH is characterized in that: the fluorescent nano material is in MnO₂Selectively detecting GSH in the presence of a nanosheet, wherein the luminol-aluminum fluorescent nanomaterial is prepared from a luminol solution and Al-containing Al³ ⁺The solution is prepared by one-step reaction after mixing.

7. Use according to claim 6, characterized in that: the detection range of the fluorescent nano material on GSH is 0.2-20 mu M, and the lowest detection limit is 45 nM.