CN113281318B - Fluorescent-colorimetric dual-signal detection of Fe by MoSe2@ Fe nano composite material3+And GSH - Google Patents

Abstract

The invention discloses a method based on MoSe₂Detection of Fe by adopting fluorescence-colorimetric dual-signal sensor of @ Fe nano composite material³⁺And GSH by adding Fe³⁺，Fe³⁺Inhibit MoSe₂The fluorescence intensity of @ Fe and the fluorescence intensity of the material after GSH is added are recovered to construct MoSe₂The fluorescence sensor of @ Fe nano composite material detects Fe by establishing a standard curve through fluorescence intensity measured by a fluorescence photometer³⁺And GSH concentration; using TMB as a colorimetric redox indicator, adding Fe³⁺Then, Fe³⁺Enhancing the enzyme activity of the nano composite material to deepen blue; then GSH is added, and can directly reduce oxTMB to lighten blue color, thereby visually detecting Fe³⁺And the concentration of GSH. The invention has the advantages of time saving, labor saving, low detection limit, good accuracy and simple and rapid detection.

Description

MoSe2Fluorescence-colorimetric dual-signal detection of Fe by @ Fe nano composite material3+And GSH

Technical Field

The invention belongs to the technical field of biological analysis and detection, and particularly relates to a method for detecting a biological sample based on MoSe₂Detection of Fe by adopting fluorescence-colorimetric dual-signal sensor of @ Fe nano composite material³⁺And methods of GSH.

Background

Iron ion (Fe)³⁺) Is an indispensable trace element of human body, is an important component of heme and iron-sulfur protein, and has good effects on enzyme catalysis, oxygen delivery, metabolism, transcriptional regulation and the likeMultiple physiological activities play an important role. Studies have shown that iron deficiency or excess in humans can lead to problems such as anemia, heart failure and tissue damage. Efficient and sensitive detection of Fe³⁺The method can more rapidly discover Fe in organisms³⁺Abnormal changes in the content, thereby effectively diagnosing and treating diseases, are also very important in the aspects of environment and food detection. To date, Fe³⁺The detection method is mature and mainly comprises an inductively coupled plasma mass spectrometry method, an atomic absorption spectrometry method and an electrochemical method. These detection methods have high precision and accuracy, but generally require complicated procedures, cumbersome sample handling and expensive equipment, and are difficult to adapt to rapid detection.

Glutathione (GSH), which consists of glutamic acid, cysteine (Cys) and glycine, is an important intracellular tripeptide widely found in plants, mammals, fungi and some prokaryotes. GSH, a low molecular weight biological thiol capable of scavenging reactive oxygen species, antitoxins and antimutagens, plays a critical role in maintaining the normal redox processes of the cellular system, and is also a very important mediator in many cellular functions. Meanwhile, GSH, as the most abundant small molecule sulfhydryl in cells, plays a very important role in the protection and detoxification functions of cells and also plays a very important regulatory role in many biological processes. More importantly, as one of endogenous antioxidants, the abnormal level of the antioxidant is closely related to cardiovascular diseases, liver injuries and the like. At present, various analysis methods such as high performance liquid chromatography, electrochemical method and the like are applied to the determination of GSH. However, these proposed analysis methods all require complicated and expensive instruments, long sample handling and complicated reaction procedures, and are disadvantageous for practical applications. A number of single chemical signal sensors have been reported for detecting Fe³⁺And GSH, but the detection result of the chemical sensor model combining two signals is more reliable and efficient than that of a single-signal chemical sensor, and chemical signal dual-mode sensors such as fluorescence-colorimetry, fluorescence-magnetic resonance, fluorescence-electrochemistry, colorimetry-photothermal and the like are reported in many ways. Thus, develop aSimple, economical and time-saving double-signal detection Fe³⁺And the method of GSH content are of great significance.

Disclosure of Invention

To solve the above problems, and to construct a fluorescence-colorimetric dual-signal sensor for detecting Fe in food³⁺And GSH. The invention provides a method based on MoSe₂Detection of Fe by adopting fluorescence-colorimetric dual-signal sensor of @ Fe nano composite material³⁺And methods of GSH.

MoSe of the invention₂The @ Fe nanocomposite is prepared by the following method:

step 1: mixing Fe (NO)₃)₃·9H₂Dissolving O, polyethylene glycol 6000, ammonium molybdate, sodium selenite and glutathione in the aqueous solution to form a uniform solution; transferring the mixture into a stainless steel autoclave according to a one-pot hydrothermal method and heating at 180 ℃ for 12 h; after cooling to room temperature, it was filtered through a 0.22 μm membrane to remove large particles.

Step 2: dialyzing the filtrate for 48 hours by using a 500MWCO dialysis membrane; finally, MoSe was obtained by freeze-drying₂@ Fe nanocomposite brown powder and stored in a refrigerator.

The MoSe-based film of the invention₂Detection of Fe by adopting fluorescence-colorimetric dual-signal sensor of @ Fe nano composite material³⁺And GSH, comprising the steps of:

step 1: verification of MoSe₂The activity of the @ Fe nanocomposite peroxidase mimic was determined by the characteristic absorption peak of TMB oxidation using TMB as the substrate.

Presence of only H₂O₂at-TMB, the solution was clear with no distinct absorption peak; in the presence of H alone₂O₂Or TMB as a substrate, MoSe is added₂@ Fe nanocomposite, the solution color was not significantly changed; description of MoSe₂@ Fe nanocomposite and H₂O₂Does not produce color change and has the mimic activity of oxidase.

With simultaneous addition of H₂O₂And TMB as a reaction substrate, MoSe₂@ Fe nanocompositeThe material enables the solution to generate macroscopic blue change, and a strong ultraviolet absorption peak is generated at 652 nm; it can be seen that MoSe₂The emission wavelength of @ Fe nanocomposite was 370nm, the absorption wavelength was 450nm, and the fluorescence intensity was very high.

Step 2: MoSe₂Enzyme kinetic analysis of @ Fe nanocomposites.

100 μ L of TMB solution, 100 μ L H₂O₂Solutions and 20. mu. LMoSE₂@ Fe homogeneous solution was added to 2.78mL of acetic acid buffer, and after 10 minutes of reaction, the effect of temperature and pH on enzyme activity was investigated by recording the absorbance at 652nm with a UV-Vis spectrophotometer; the enzyme activity was found to be optimal at pH 3.2 and temperature 45 ℃.

MoSe was calculated by measuring the stabilization time of TMB at various concentrations in a pH 3.2 environment at a temperature of 45 deg.C₂Kinetic parameters of @ Fe; 100 μ L of TMB and 20 μ L of MoSe₂@ Fe was added to 2.78mL of acetic acid buffer and MoSe was estimated by recording the absorbance of the mixed reaction solution at 652nm every 5 minutes₂Kinetics of @ Fe; kinetic parameters were calculated by the Michaelis-Menten equation: 1/V ═ by (KM/V)_max)(1/[S]+1/KM) in which [ S ] is present]Represents the substrate concentration; v_maxIs the maximum reaction rate; KM is the Michaelis constant.

And step 3: construction of MoSe₂The fluorescence sensor of @ Fe nano composite material detects Fe by establishing a standard curve through fluorescence intensity measured by a fluorescence photometer³⁺And GSH concentration.

At normal temperature, Fe with different concentrations is added³⁺Solution and optimum concentration of MoSe₂Mixing and shaking up @ Fe homogeneous solution in equal proportion, reacting for 10min, detecting fluorescence spectrum under 370nm excitation by using a fluorescence spectrophotometer, and obtaining emission spectrum at 445 nm; a certain concentration of Fe³⁺Solution and optimum concentration of MoSe₂Mixing and shaking up @ Fe solution in equal proportion, after reacting for 10min, detecting fluorescence spectrum under 370nm excitation by using a fluorescence spectrophotometer, obtaining emission spectrum at 445nm, adding glutathione solutions with different concentrations, continuing to react for 10min, and detecting fluorescence under 370nm excitation by using the fluorescence spectrophotometerSpectrum, emission spectrum was obtained at 445 nm.

Drawing and detecting Fe according to the measured spectrum³⁺And fluorescence spectrum of GSH concentration, and with Fe³⁺And a standard curve with GSH concentration on the abscissa and fluorescence intensity on the ordinate.

Measuring the change of the fluorescence intensity of the sample to be tested under the same experimental environment to quantify Fe³⁺And the actual concentration of GSH.

And 4, step 4: construction of MoSe₂And measuring an ultraviolet absorption spectrogram and a standard curve of the colorimetric sensor by using the @ Fe nanocomposite colorimetric sensor.

Mixing 100 μ L TMB solution and 100 μ L LH₂O₂Solution and 20. mu.L of MoSe₂@ Fe solution was added to 2.78mL of acetic acid buffer solution, and after a reaction time, Fe of various concentrations was added³⁺Continuing the reaction for 5min, recording the absorbance at 652nm by an ultraviolet-visible spectrophotometer, adding glutathione solutions with different concentrations, continuing the reaction for 5min, and recording the absorbance at 652nm by the ultraviolet-visible spectrophotometer; with Fe³⁺The concentration is abscissa, the absorbance Delta A is ordinate, and Fe is obtained³⁺A standard curve; and taking the concentration of the GSH as an abscissa and the absorbance delta A as an ordinate to obtain a GSH standard curve.

Under the same experimental environment, the change of the absorbance of a sample to be measured at 652nm is measured to realize the Fe control³⁺And quantification of GSH concentration.

The beneficial technical effects of the invention are as follows:

the MoSe provided by the invention for the first time₂The synthesis method of the @ Fe nano composite material adopts a one-step hydrothermal method, and the reaction condition is mild, green and simple; MoSe of the invention₂The @ Fe nano composite material has stable optical performance and good water solubility; has the dual properties of fluorescence emission peak and peroxide mimic enzyme, and can be used as a fluorescence and colorimetric dual-signal probe for directly detecting Fe3+ and GSH.

Compared with the traditional detection method, the method for detecting Fe3+ and GSH by using MoSe2@ Fe fluorescent and colorimetric method double signals provided by the invention can be used for detecting without expensive instruments and professionals for operation, and has the advantages of time saving, labor saving, low detection limit, good accuracy, simplicity and rapidness in detection.

Drawings

FIG. 1 shows MoSe₂SEM image of @ Fe nanocomposite.

FIG. 2 shows MoSe₂EDS energy spectrum of @ Fe nanocomposite.

FIG. 3 shows MoSe₂@Fe-TMB-H₂O₂、MoSe₂@Fe-TMB、MoSe₂@Fe-H₂O₂、TMB-H₂O₂Ultraviolet versus absorption graph of (a).

FIG. 4 shows MoSe₂@ Fe fluorescence excitation and emission spectra.

FIG. 5 is Fe³⁺And GSH vs MoSe₂Influence of the fluorescence intensity of @ Fe nanocomposite (in the figure, B is MoSe)₂@Fe)。

FIG. 6 is Fe³⁺And GSH vs MoSe₂@ Fe nanocomposite Oxidation of TMB, the Effect of absorbance was measured (in the figure, A is MoSe)₂@Fe+TMB+H₂O₂)。

FIG. 7 is temperature vs. MoSe₂Effect of the enzyme kinetics of @ Fe composite.

FIG. 8 shows pH vs. MoSe₂Effect of the enzyme kinetics of @ Fe composite.

FIG. 9 shows different concentrations H₂O₂The rate of enzyme reaction in the case of (2).

FIG. 10 shows the enzyme reaction rates at different concentrations of TMB.

FIG. 11 shows different concentrations H₂O₂The double reciprocal curve of the enzyme reaction rate in the case of (2).

FIG. 12 is a double reciprocal curve of the enzyme reaction rate at different concentrations of TMB.

FIG. 13 shows MoSe₂Detection of Fe by @ Fe fluorescent sensor³⁺Fluorescence spectrum and standard curve.

FIG. 14 shows MoSe₂The @ Fe fluorescence sensor detects the fluorescence spectrogram and standard curve of GSH.

FIG. 15 shows MoSe₂Detection of Fe by @ Fe colorimetric sensor³⁺Ultraviolet absorption spectrum and standard curve.

FIG. 16 shows MoSe₂The @ Fe colorimetric sensor detects the ultraviolet absorption spectrogram and the standard curve of the GSH.

FIG. 17 shows the fluorescence measurement of Fe by MoSe2@ Fe nanocomposite³⁺Selectivity of (2).

FIG. 18 is the selectivity of MoSe2@ Fe nanocomposites for fluorescence determination of GSH.

FIG. 19 is comparative color determination of Fe for MoSe2@ Fe nanocomposite³⁺Selectivity of (2).

FIG. 20 shows the selectivity of the MoSe2@ Fe nanocomposite for measuring GSH versus color.

Detailed Description

The invention is described in further detail below with reference to the figures and the detailed description.

MoSe of the invention₂The @ Fe nanocomposite is prepared by the following method:

mixing Fe (NO)₃)₃·9H₂Dissolving O, polyethylene glycol 6000, ammonium molybdate, sodium selenite and glutathione in the aqueous solution to form a uniform solution; transferring the mixture into a stainless steel autoclave according to a one-pot hydrothermal method and heating at 180 ℃ for 12 h; after cooling to room temperature, it was filtered through a 0.22 μm membrane to remove large particles. Dialyzing the filtrate for 48 hours by using a 500MWCO dialysis membrane; finally, MoSe was obtained by freeze-drying₂@ Fe nanocomposite brown powder and stored in a refrigerator.

The prepared MoSe2@ Fe nano composite material has stable optical property and good water solubility, and a Scanning Electron Microscope (SEM) can show that a sample is spherical particles with different sizes, and a large number of irregular particles are uniformly distributed on the spherical surface. The spherical particle structure provides a larger specific surface area for the peroxidase mimic activity, and increases the contact area with the subsequent reaction substrate (as shown in FIG. 1). As can be seen from the EDS (fig. 2), the doped sample was prepared to contain 1.81% of Fe element in addition to 77.13% of Mo element and 21.06% of Se element. In fig. 2, the unlabeled peak is an Au element peak occurring in the gold spraying operation during the scanning electron microscope, and in the detection sensitivity range, except for peaks corresponding to Fe, Mo and Se, no other impurity peaks exist on the sample, which indicates that the Fe element is successfully doped into the synthesized composite material.

The MoSe-based film of the invention₂Detection of Fe by adopting fluorescence-colorimetric dual-signal sensor of @ Fe nano composite material³⁺And GSH, comprising the steps of:

step 1: verification of MoSe₂The activity of the @ Fe nanocomposite peroxidase mimic was determined by the characteristic absorption peak of TMB oxidation using TMB (3,3',5,5' -tetramethylbenzidine) as the substrate.

As is evident from FIG. 3, in H alone₂O₂Or TMB as a substrate, MoSe is added₂@ Fe nanocomposite, the solution color was not significantly changed. This shows that MoSe₂@ Fe nanocomposite and H₂O₂Does not produce color change and has the mimic activity of oxidase. While adding H at the same time₂O₂And TMB as a reaction substrate, MoSe₂The @ Fe nano composite material can cause the macroscopic blue color change of the solution, and a strong ultraviolet absorption peak is generated at 652 nm. As shown in FIG. 4, it can be seen that MoSe₂The emission wavelength of @ Fe nanocomposite was 370nm, the absorption wavelength was 450nm, and the fluorescence intensity was very high.

By utilizing the fluorescence characteristic of the composite material, Fe is added under certain reaction conditions³⁺，Fe³⁺Inhibit MoSe₂The fluorescence intensity of @ Fe is recovered after GSH is added, and Fe is quantified by detecting the change amount (delta A1, delta A2) of the fluorescence intensity of the reaction solution under the optimum excitation wavelength³⁺And the purpose of GSH. We utilized 3,3',5,5' -Tetramethylbenzidine (TMB) as a widely used redox indicator in colorimetric assay systems. MoSe₂@ Fe nanocomposite as peroxide mimic enzyme, the substrate being coated with H₂O₂Upon oxidation, a blue oxidation state TMB (ox-TMB) is produced. After adding Fe³⁺Then, Fe³⁺Enhancing the enzyme activity of the nano composite material to deepen blue; then GSH is added, the GSH can directly reduce oxTMB to lighten blue color, thereby achieving visual effectChemical detection of Fe³⁺And the purpose of GSH (as shown in fig. 5, 6).

Step 2: MoSe₂Enzyme kinetic analysis of @ Fe nanocomposites.

Found through experiments, MoSe is found₂The peroxidase-like catalytic activity of @ Fe depends on pH, temperature, H₂O₂And TMB concentration. The effect of temperature (30-60 ℃) and pH (2.0-8.0) on enzyme activity was investigated.

100 μ L of TMB solution, 100 μ L H₂O₂Solutions and 20. mu. LMoSE₂@ Fe homogeneous solution was added to 2.78mL of acetic acid buffer, and after 10 minutes of reaction, the effect of temperature (30-60 ℃) and pH (2.0-8.0) on enzyme activity was investigated by recording the absorbance at 652nm by a UV-Vis spectrophotometer; the temperature effect curve is shown in FIG. 7 and the pH effect curve is shown in FIG. 8, and it can be seen that the enzyme activity is optimum at pH 3.2 and at a temperature of 45 ℃.

MoSe was calculated by measuring the stabilization time of TMB at various concentrations in a pH 3.2 environment at a temperature of 45 deg.C₂Kinetic parameters of @ Fe; 100 μ L of TMB and 20 μ L of MoSe₂@ Fe was added to 2.78mL of acetic acid buffer, and MoSe was evaluated by recording the absorbance value of the mixed reaction solution at 652nm every 5 minutes₂Kinetics of @ Fe; kinetic parameters were calculated by the Michaelis-Menten equation: 1/V ═ by (KM/V)_max)(1/[S]+1/KM) in which [ S ] is present]Represents the substrate concentration; v_maxIs the maximum reaction rate; KM is the Michaelis constant. Different concentrations of H₂O₂The reaction rates in the case of (2) are shown in FIG. 9, the reaction rates in the case of different concentrations of TMB are shown in FIG. 10, H₂O₂And the double reciprocal curves of TMB concentration versus reaction rate are shown in fig. 11 and 12.

The initial rate of the catalytic reaction and the concentration of the substrate are in a Mie's curve model within a certain concentration range. In the lower concentration range, the initial rate of reaction is linear with the concentration of substrate. At higher concentrations, the rate of reaction increases slowly with increasing substrate concentration until the rate of reaction no longer increases. H is calculated by using Lambert beer's law and the equation of double reciprocal curve of Mie's curve₂O₂And K of TMB_mValue respectivelyThe concentration was 0.209. mu.M and 1.182. mu.M.

And step 3: construction of MoSe₂The fluorescence sensor of @ Fe nano composite material detects Fe by establishing a standard curve through fluorescence intensity measured by a fluorescence photometer³⁺And GSH concentration.

At normal temperature, Fe with different concentrations is added³⁺Solution and optimum concentration of MoSe₂Mixing and shaking up @ Fe homogeneous solution in equal proportion, reacting for 10min, detecting fluorescence spectrum under 370nm excitation by using a fluorescence spectrophotometer, and obtaining emission spectrum at 445 nm; a certain concentration of Fe³⁺Solution and optimum concentration of MoSe₂Mixing and shaking the @ Fe solution in equal proportion, after reacting for 10min, detecting a fluorescence spectrum at the position of 445nm by using a fluorescence spectrophotometer under the excitation of 370nm, adding glutathione solutions with different concentrations, continuing the reaction for 10min, detecting the fluorescence spectrum at the position of 445nm by using the fluorescence spectrophotometer under the excitation of 370nm, and obtaining the emission spectrum at the position of 445 nm.

Drawing and detecting Fe according to the measured spectrum³⁺Fluorescence spectrum of (1), and with Fe³⁺The standard curve (FIG. 13) with the concentration on the abscissa and the fluorescence intensity on the ordinate shows the change in fluorescence intensity with Fe³⁺The concentration is linear between 40 and 300. mu.M. From the measured spectrum, a fluorescence spectrum of the concentration of the detected GSH was plotted, and a standard curve (as shown in fig. 14) with the concentration of the GSH as the abscissa and the fluorescence intensity as the ordinate was plotted, and it can be seen that the addition of GSH at different concentrations restored the fluorescence at 445nm, enhancing the fluorescence intensity. The change in fluorescence intensity is linear with GSH concentration between 15 and 50 μ M.

Measuring the change of the fluorescence intensity of the sample to be tested under the same experimental environment to quantify Fe³⁺And the actual concentration of GSH.

And 4, step 4: construction of MoSe₂And measuring an ultraviolet absorption spectrogram and a standard curve of the colorimetric sensor by using the @ Fe nanocomposite colorimetric sensor.

Mixing 100 μ L TMB solution and 100 μ L LH₂O₂Solution and 20. mu.L of MoSe₂@ Fe solution was added to 2.78mL of acetic acid buffer solution, reacted for a while, and addedDifferent concentrations of Fe³⁺Continuing the reaction for 5min, recording the absorbance at 652nm by an ultraviolet-visible spectrophotometer, adding glutathione solutions with different concentrations, continuing the reaction for 5min, and recording the absorbance at 652nm by the ultraviolet-visible spectrophotometer; with Fe³⁺The concentration is abscissa, the absorbance Delta A is ordinate, and Fe is obtained³⁺Standard curve (as shown in fig. 15); as can be seen from the figure, the change in absorbance is related to Fe³⁺The concentration is linear between 0 and 13.3. mu.M. The GSH standard curve was obtained using the GSH concentration as the abscissa and the absorbance Δ a as the ordinate (as shown in fig. 16). With the increase of the concentration of the GSH, the color of the solution changes from deep blue to colorless, which shows that the GSH can obviously inhibit the catalytic activity of the GSH. The change in absorbance (. DELTA.A 652nm) was linear with the GSH concentration between 0 and 33.3. mu.M.

Under the same experimental environment, the change of the absorbance of a sample to be measured at 652nm is measured to realize the Fe control³⁺And quantification of GSH concentration.

Selectivity is one of the important parameters that measure whether a new detection method actually detects an application. To assess selectivity, the method was tested for Fe by monitoring changes in absorbance and fluorescence intensity³⁺When using Cu²⁺、Sn²⁺、Zn²⁺、K⁺、Mg²⁺、 Mn²⁺、Ca²⁺、Na⁺、Pb²⁺、Ag⁺And the like, common amino acids such as Arg, Met, Cys, Glu, Asn, Ser, Gln, His, Lys and the like are selected when GSH is detected, and the response of different interferents is researched.

Under the optimum reaction conditions, Fe was measured³⁺While providing other metal cations (including Cu)²⁺，Sn²⁺，Zn²⁺，K⁺，Mg²⁺，Mn²⁺，Ca²⁺，Na⁺，Pb²⁺，Ag⁺Etc.), the measurement results are shown in fig. 17 and fig. 19.

Several familiar amino acids were added as controls (including Arg, Met, Cys, Glu, Asn, Ser, gin, His, Lys, etc.) to determine changes in the same parameters under the same conditions, as well as to determine changes in absorbance and fluorescence intensity of glutathione. The measurement results are shown in fig. 18 and 20.

Claims (1)

1. Based on MoSe₂Detection of Fe by adopting fluorescence-colorimetric dual-signal sensor of @ Fe nano composite material³⁺And GSH, characterized in that it comprises the following steps:

step 1: MoSe based on one-pot hydrothermal synthesis₂@ Fe nanocomposite;

s11: mixing Fe (NO)₃)₃·9H₂Dissolving O, polyethylene glycol 6000, ammonium molybdate, sodium selenite and glutathione in the aqueous solution to form a uniform solution; transferring the mixture into a stainless steel autoclave according to a one-pot hydrothermal method and heating at 180 ℃ for 12 h; after cooling to room temperature, it was filtered through a 0.22 μm membrane to remove large particles;

s12: dialyzing the filtrate for 48 hours by using a 500MWCO dialysis membrane; finally, MoSe was obtained by freeze-drying₂@ Fe nanocomposite brown powder, and stored in a refrigerator;

step 2: verification of MoSe₂The activity of the @ Fe nanocomposite peroxidase mimic is determined by using TMB as a substrate and a characteristic absorption peak of TMB oxidation;

presence of only H₂O₂at-TMB, the solution was clear with no distinct absorption peak; in the presence of H alone₂O₂Or TMB as a substrate, MoSe is added₂@ Fe nanocomposite, the solution color was not significantly changed; description of MoSe₂@ Fe nanocomposite and H₂O₂The color can not be changed, and the mimic activity of oxidase can not be generated;

with simultaneous addition of H₂O₂And TMB as a reaction substrate, MoSe₂The @ Fe nano composite material enables the solution to generate macroscopic blue change and generates a strong ultraviolet absorption peak at 652 nm; it can be seen that MoSe₂The emission wavelength of @ Fe nanocomposite was 370nm, the absorption wavelength was 450nm, and the fluorescence intensity was very high;

step (ii) of3：MoSe₂Enzyme kinetic analysis of @ Fe nanocomposites:

100 μ L of TMB solution, 100 μ L H₂O₂Solutions and 20. mu. LMoSE₂@ Fe homogeneous solution was added to 2.78mL of acetic acid buffer, and after 10 minutes of reaction, the effect of temperature and pH on enzyme activity was investigated by recording the absorbance at 652nm with a UV-Vis spectrophotometer; the enzyme activity is optimal when the pH is 3.2 and the temperature is 45 ℃ is measured;

MoSe was calculated by measuring the stabilization time of TMB at various concentrations in a pH 3.2 environment at a temperature of 45 deg.C₂Kinetic parameters of @ Fe; 100 μ L of TMB and 20 μ L of MoSe₂@ Fe was added to 2.78mL of acetic acid buffer and MoSe was estimated by recording the absorbance of the mixed reaction solution at 652nm every 5 minutes₂Kinetics of @ Fe; kinetic parameters were calculated by the Michaelis-Menten equation: 1/V ═ by (KM/V)_max)(1/[S]+1/KM) in which [ S ] is present]Represents the substrate concentration; v_maxIs the maximum reaction rate; KM is a Michaelis constant;

and 4, step 4: construction of MoSe₂The fluorescence sensor of @ Fe nano composite material detects Fe by establishing a standard curve through fluorescence intensity measured by a fluorescence photometer³⁺And GSH concentration:

at normal temperature, Fe with different concentrations is added³⁺Solution and optimum concentration of MoSe₂Mixing and shaking up @ Fe homogeneous solution in equal proportion, reacting for 10min, detecting fluorescence spectrum under 370nm excitation by using a fluorescence spectrophotometer, and obtaining emission spectrum at 445 nm; a certain concentration of Fe³⁺Solution and optimum concentration of MoSe₂Mixing and shaking up the @ Fe solution in equal proportion, after reacting for 10min, detecting a fluorescence spectrum by using a fluorescence spectrophotometer under the excitation of 370nm to obtain an emission spectrum at 445nm, adding glutathione solutions with different concentrations, continuing to react for 10min, detecting the fluorescence spectrum by using the fluorescence spectrophotometer under the excitation of 370nm to obtain the emission spectrum at 445 nm;

detecting Fe by drawing the measured spectrum³⁺And the fluorescence spectrum of GSH concentration, and as Fe³⁺And a standard curve with GSH concentration as abscissa and fluorescence intensity as ordinate;

identity of same entityMeasuring the change of the fluorescence intensity of the sample to be tested to quantify Fe under the test environment³⁺And the actual concentration of GSH;

and 5: construction of MoSe₂The @ Fe nano composite colorimetric sensor is used for measuring an ultraviolet absorption spectrogram and a standard curve of the colorimetric sensor:

mixing 100 μ L TMB solution and 100 μ L LH₂O₂Solution and 20. mu.L of MoSe₂@ Fe solution was added to 2.78mL of acetic acid buffer solution, and after a reaction time, Fe of various concentrations was added³⁺Continuing the reaction for 5min, recording the absorbance at 652nm by an ultraviolet-visible spectrophotometer, adding glutathione solutions with different concentrations, continuing the reaction for 5min, and recording the absorbance at 652nm by the ultraviolet-visible spectrophotometer; with Fe³⁺The concentration is abscissa, the absorbance Delta A is ordinate, and Fe is obtained³⁺A standard curve; taking the GSH concentration as an abscissa and the absorbance delta A as an ordinate to obtain a GSH standard curve;

under the same experimental environment, the change of the absorbance of a sample to be measured at 652nm is measured to realize the Fe control³⁺And quantification of GSH concentration.

Images