CN116559102A - Rapid detection method of glutathione and cysteine - Google Patents

Abstract

A rapid detection method for glutathione and cysteine of the present invention is based on the activity of Fe-N-C@Hemin nano-enzymes, and utilizes the principle of inhibition of the oxidase-like activity of Fe-N-C@Hemin by small molecules of sulfhydryl groups. The detection ability of Fe‑N‑C@Hemin nanozyme to glutathione (GSH) and cysteine (Cys) was verified; with Fe‑N‑C@Hemin as the main material of the sensor, a nanozyme based on the A biosensor for the rapid and sensitive detection of glutathione and cysteine.

Description

A kind of rapid detection method of glutathione and cysteine

technical field

The invention belongs to the technical field of food safety detection, and in particular relates to a rapid detection method for glutathione and cysteine.

Background technique

Cysteine is a common amino acid in organisms, and it is also a representative substance of small sulfhydryl molecules. It plays an important role in various physiological activities, including signal transmission, protein biosynthesis, phospholipid metabolism, etc. The imbalance of cysteine level will lead to Damage to various tissues. Glutathione is an endogenous active peptide, which is the most common biothiol in the living system. It is composed of glutamic acid, cysteine and glycine, and exists in every cell in the human body. It acts as An antioxidant that plays an important role in regulating redox balance, maintaining the immune system, cell signal transduction, maintaining protein structure, gene transcription regulation and other processes. Currently, glutathione levels have been shown to be a key indicator of diseases such as skin, blood, cancer, liver damage, diabetes and Alzheimer's disease. Glutathione can be formed endogenously from cysteine, glutamic acid and glycine in the human body, but the content of glutathione formed in the human body is far from enough to meet the needs of normal physiological activities of the human body. In addition, glutathione will accelerate oxidation when aging, stress and disease lead to increased active oxygen, and glutathione in the body will be consumed by metabolism and excretion. When the level of endogenous antioxidants such as glutathione is low, the human body is very susceptible to disease, so it is necessary to obtain glutathione from food or supplements. Accurate quantification of glutathione levels in food provides an important reference for daily glutathione supplementation.

At present, the detection of glutathione is still mainly concentrated in the medical field, and there are few detection methods and related researches in the food field. Studies have shown that sulfhydryl molecules can bind Fe-NC single-atom nanozymes and inhibit nano-enzymes. Enzyme activity, therefore, the present invention explores the inhibitory ability of cysteine and glutathione to the OXD activity of Fe-NC@Hemin nanomaterials with heme (Hemin) as the source of organic iron, and the effect on glutathione in serum The research on the performance of detection in two types of matrices, food and food, can not only provide a detection method for the diagnosis of human glutathione-related diseases, but also provide a certain guiding value for functional foods containing glutathione. In addition, the use of oxidase activity can also avoid the defect of _insufficient stability caused by the easy decomposition of _H2O2 required for POD activity in previous studies.

Contents of the invention

In order to realize the rapid detection of glutathione and cysteine, the oxidase-like activity of Fe-N-C@Hemin and the inhibition of its oxidase-like activity by sulfhydryl groups were used to realize the detection of glutathione and cysteine. rapid detection.

First, the present invention provides a method for detecting the activity mechanism of Fe-N-C@Hemin oxidases:

The detection method includes: (1) verification of aerobicity; (2) verification of electron paramagnetic energy spectroscopy; (3) detection of specific fluorescent probes for reaction intermediates; (4) verification of active oxygen indicator combined with free radical scavenger The main reaction intermediate.

The verification of aerobicity is as follows: firstly add 10 uL TMB to the 0.1 mg/mL Fe-N-C@Hemin solution, pass air and nitrogen respectively, measure the difference in absorbance at 652 nm with a UV spectrophotometer, if the difference is large , indicating that the reaction requires the participation of oxygen;

The electron paramagnetic spectroscopy method is verified as follows: using electron paramagnetic spectroscopy, using 5,5-dimethyl-1-pyrroline oxide (DMPO) as a specific probe to capture the reaction intermediate in the process of catalytic TMB color development, Measure the resonance spectrum and analyze its characteristic peaks to judge the existence of reaction intermediates;

The specific fluorescent probe detection of the reaction intermediate is as follows: three kinds of fluorescent probes (TA, SOGC, DHE) are selected as free radical indicators for the oxidation of TMB catalyzed by Fe-NC@Hemin, which are used to detect OH, ¹ O ₂ . ;

The methods for verifying the main reaction intermediates of the active oxygen indicator combined with the free radical scavenger include (1) fluorescence method; (2) colorimetric method. The fluorescence method is to use the active oxygen fluorescent probe DCFH-DA as the labeling signal, add OH, ¹ O ₂ , The corresponding three free radical scavengers: isopropanol, sodium azide and superoxide dismutase, analyze the weakening degree of fluorescence after adding the corresponding scavengers; Add Fe-NC@Hemin and TMB to the solution, and analyze the weakening degree of the absorbance at 652 nm after adding the corresponding scavenger.

Compared with Fe-N-C inorganic nanozyme, the Fe-N-C@Hemin nanozyme provided by the present invention has more excellent OXD activity:

The Km value of Fe-N-C was 0.09022 mM, and the Km value of Fe-N-C@Hemin was 0.05905 mM compared with the oxidase-like kinetic parameters of the OXD activity.

The present invention provides a speculative mechanism for the oxidase-like activity of Fe-N-C@Hemin.

The mechanism is based on the aforementioned experimental verification, and it is speculated that the catalytic mechanism of Fe-NC@Hemin oxidase is as follows: Fe-NC@Hemin can catalyze the dissolved oxygen adsorbed on its surface to produce , /> Extremely unstable in aqueous solution, it will react quickly to form H ₂ O ₂ and ¹ O ₂ , and H ₂ O ₂ and /> The reaction generates OH. In addition, Fe-NC@Hemin can also directly catalyze the generation of H ₂ O ₂ by Fenton-like reaction/> and •OH.

Based on the above mechanism discovery, the present invention develops and adopts the application of Fe-N-C@Hemin in the rapid detection of cysteine or glutathione, specifically:

The first aspect of the present invention provides the application of Fe-N-C@Hemin as an oxidoreductase.

In a specific embodiment, the method is used to detect the content of glutathione or cysteine.

A second aspect of the present invention provides a kit for specifically detecting glutathione or cysteine, said kit comprising:

Fe-N-C@Hemin, TMB, sodium acetate buffer.

In a specific embodiment, according to the kit, the working concentration of Fe-N-C@Hemin is 20~50 μg/mL; the working concentration of TMB is 0.2~0.5 mM; the pH of the sodium acetate buffer is 3~50 μg/mL; 5.

The third aspect of the present invention is the use of the kit described in the second aspect in the detection of glutathione or cysteine content.

In a specific embodiment, wherein the specific application method is:

1) Mix the sample to be tested with sodium acetate buffer, the volume ratio of the two is 1:8~10;

2) Add Fe-N-C@Hemin single-atom nanozyme, and then add TMB reaction

3) Measure the absorbance at 652 nm, calculate the inhibition rate, construct a linear regression equation, and calculate the concentration.

Preferably, in step 2), the working concentration of Fe-N-C@Hemin is 20-50 μg/mL; the working concentration of TMB is 0.2-0.5 mM; the pH of the sodium acetate buffer is 3-5.

More preferably, the reaction time of step 2) is 10-20 min; the reaction pH is 3-5.

Yet another specific embodiment, wherein the formula for calculating the inhibition rate is (A ₀ -A)/A ₀ ×100, wherein A ₀ is a group without adding the sample to be tested.

In another specific embodiment, the sample is food or serum, and when the sample is serum, the application is for non-diagnostic purposes.

Fe-N-C@Hemin With the above technical solution, the present invention has at least the following advantages and beneficial effects:

The invention establishes a colorimetric detection method for glutathione and cysteine through Fe-N-C@Hemin nanozyme. First, the oxidase-like activity and mechanism of Fe-N-C@Hemin were verified. Secondly, based on the excellent OXD activity of Fe-N-C@Hemin, a colorimetric biosensor was constructed for the detection of Cys and GSH by utilizing the inhibition of sulfhydryl groups on the activity of Fe-N-C@Hemin, and it has good activity in both serum and food matrices. The recovery rate indicates the feasibility of the sensor in actual sample detection.

Compared with existing methods, the present invention can achieve at least one of the following beneficial effects:

(1) The detection method of the present invention is based on the excellent oxidase-like activity of Fe-N-C@Hemin nanozyme, and the reaction is rapid. The entire reaction can be completed within 20 minutes, and the detection result can be obtained. Compared with the traditional detection method, the detection time is shorter Faster to meet the needs of rapid detection. It can be preliminarily qualified by naked eye observation, and the detection of absorbance at 652 nm can accurately and quickly reflect the quantitative results.

(2) The detection specificity of the present invention is good, and the metal ions, protein components and sugars that often appear in food and serum are used to analyze the specific detection ability of the detection method for cysteine and glutathione, and the results It shows that the method can specifically recognize cysteine and glutathione, meeting the specific detection requirements.

(3) Easy operation: the detection can be completed only by using nanozyme materials and TMB, the operation steps are simple, avoiding cumbersome steps, and the detection results can be obtained with the least operation steps.

Description of drawings

Figure 1 shows the oxidase-like properties of Fe-N-C@Hemin: Figure 1A is the schematic diagram of single-atom nanozyme simulating oxidase; Figure 1B is the UV-Vis absorption spectrum under different systems.

Figure 2 shows the kinetic parameters of Fe-N-C and Fe-N-C@Hemin nanozyme oxidase. Figure 2A is the Michaelis-Menten equation of Fe-N-C; Figure 2B is the Michaelis-Menten equation of Fe-N-C@Hemin (substrate is TMB), the inset is the Lineweaver-Burk curve; Figure 2C is the enzyme activity SA.

Figure 3 is a study on the catalytic mechanism of Fe-N-C@Hemin oxidase activity. Figure 3A UV-Vis spectra under air and nitrogen atmosphere; Figure 3B Absorbance at 652 nm under air and nitrogen atmosphere.

Fig. 4 is the fluorescence spectrum diagram of the detection reaction intermediate of three kinds of fluorescent probes. Figure 4A terephthalic acid; Figure 4B singlet oxygen green fluorescent probe; Figure 4C dihydroethidium.

Figure 5 is the electron paramagnetic resonance spectrum, Figure 5A: the characteristic peak of OH; Figure 5B: the characteristic peak of ¹ O ₂ ; Figure 5C: characteristic peaks.

Fig. 6 is the fluorescence ratio Fig. 6A and the absorbance diagram Fig. 6B under the treatment of three radical scavengers; F ₀ is the fluorescence intensity of DCFH-DA and Fe-NC@Hemin at 525 nm without adding radical scavengers.

Figure 7 shows the oxidase-like mechanism of Fe-N-C@Hemin.

Figure 8 shows the optimization results of the sensing system, Figure 8A: Fe-N-C@Hemin concentration; Figure 8B: TMB concentration; Figure 8C: reaction pH value; Figure 8D: reaction time.

Figure 9 shows the specificity of the sensing system to glutathione and cysteine.

Figure 10 shows the detection sensitivity of glutathione (Figure 10A) and cysteine (Figure 10B).

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

Example 1 Verification of the activity mechanism of Fe-N-C@Hemin-like oxidases

1. Experimental materials

3,3',5,5'-tetramethylbenzidine (TMB), terephthalic acid, dihydroethidium, singlet oxygen green fluorescent probe, 2',7'-dichlorodihydrofluorescein di Acetate was purchased from commercially available products.

2. Verification of Fe-N-C@Hemin-like oxidase properties

In order to study the oxidase activity of nanomaterials, TMB was added to the two materials to explore their absorbance at 652 nm. As shown in Figure 1, when only Fe-N-C and Fe-N-C@Hemin exist, there is no color development, and there is no obvious peak at 652 nm, indicating that no oxidation reaction occurs in this system. When TMB was added to the system, both nanozymes had obvious peaks, the absorbance values of Fe-N-C and Fe-N-C@Hemin at 652 nm were 1.088 and 1.626, respectively, and Fe-N-C@Hemin was at 652 nm The absorbance value of Fe-N-C@Hemin is higher than that of Fe-N-C at 652 nm, indicating that Fe-N-C@Hemin has oxidase activity, which can oxidize TMB to oxTMB, and the oxidation ability is higher than that of Fe-N-C nanozyme. The above results prove that Fe-N-C@Hemin nanozyme has excellent oxidase-like activity.

The enzyme kinetic parameters of the two nanozymes were fitted by the Michaelis-Menten equation. The results are shown in Figure 2. The Michaelis constant Km of Fe-N-C is 0.09022 mM, the maximum reaction rate is 8.584 μmol/min, and the SA is 1.335 U/min. mg (Figure 2A, Figure 2C), the Michaelis constant Km of Fe-N-C@Hemin is 0.05905 mM, the maximum reaction rate Vmax is 9.315 μmol/min, and SA is 8.829 U/mg (Figure 2B, Figure 2C), which shows that Fe The oxidase activity of -N-C@Hemin was better than that of Fe-N-C.

3. Verification of Fe-N-C@Hemin-like oxidase properties

The mechanism of TMB color development catalyzed by Fe-N-C@Hemin was preliminarily explored. As shown in Figure 3A-B, TMB color development experiments were carried out under air and nitrogen atmosphere respectively. It can be seen from the figure that when detected under nitrogen atmosphere, , the absorbance value at 652 nm was greatly weakened, which was significantly different from that detected in air, which indicated that the Fe-N-C@Hemin nanozyme catalyzed TMB color development requires the participation of oxygen, which further explained the oxidase-like activity of Fe-N-C@Hemin.

In order to explore what kind of free radicals are produced during the color development of TMB by Fe-NC@Hemin, three fluorescent probes TA (Fig. 4A), SOGC (Fig. 4B) and DHE (Fig. 4C) were selected as the catalysts of Fe-NC@Hemin. The free radical indicator of TMB oxidation is used to detect • OH, ¹ O ₂ , . As shown in Figure 4A-Figure 4C, compared with the control group, the Fe-NC@Hemin group showed peaks corresponding to the wavelength of the fluorescent probe, indicating that Fe-NC@Hemin can catalyze dissolved oxygen to generate • OH, ¹ O ₂ , /> .

Electron paramagnetic resonance spectroscopy was used for detection, and 5,5-dimethyl-1-pyrroline oxide (DMPO) was used as a specific probe to capture the reaction intermediates in the chromogenic process of catalytic TMB. As shown in Figure 5A, it corresponds to the characteristic peak of OH 1:2:2:1; Figure 5B corresponds to the characteristic peak of ¹ O ₂ 1:1:1; Figure 5C shows six peaks of four big and two small, corresponding to The characteristic peaks further confirmed the existence of three free radicals.

In order to explore which free radical plays a major role in the process of Fe-NC@Hemin catalyzed TMB reaction, two verification methods were adopted. Firstly, use the fluorescence method, use the active oxygen fluorescent probe DCFH-DA as the labeling signal, add OH, ¹ O ₂ , The corresponding three free radical scavengers: isopropanol, sodium azide and superoxide dismutase, as shown in Figure 6, when the three active oxygen scavengers are added, the fluorescence is weakened. Among them, adding isopropanol Fluorescence decreased the most when compared with the control group by 85.24%, indicating that • OH was the main reaction intermediate. At the same time, the colorimetric method was used to verify that Fe-NC@Hemin and TMB were added to the solution, and the absorbance value decreased significantly after adding free radical scavenger. Among them, the absorbance decreased most obviously when adding isopropanol, which was lower than that of the control group. 0.21, which also shows that the oxidation ability of Fe-NC@Hemin is mainly derived from • OH (Fig. 6A-B).

Based on the above verification results, it is speculated that the oxidase-like catalytic mechanism of Fe-NC@Hemin is as follows (Figure 7): Fe-NC@Hemin can catalyze the dissolved oxygen adsorbed on its surface to produce , /> Extremely unstable in aqueous solution, it will react quickly to form H ₂ O ₂ and ¹ O ₂ , and H ₂ O ₂ and /> The reaction generates OH. In addition, Fe-NC@Hemin can also directly catalyze the generation of H ₂ O ₂ by Fenton-like reaction/> and •OH.

Example 2 Detection condition optimization

1. Experimental materials

3,3',5,5'-Tetramethylbenzidine, glutathione, and cysteine were all purchased from commercially available products.

2. Condition optimization of a rapid detection method for glutathione and cysteine

To detect glutathione and cysteine, first add 10 μL of the test substance to 84 μL sodium acetate buffer, and then add 4 μL of Fe-N-C@Hemin single-atom nanozyme with a concentration of 1 mg/mL, Finally, add TMB solution, measure its absorbance at 652 nm after reacting for a certain period of time, and calculate the inhibition rate. The nanozyme concentration, TMB concentration, pH and reaction time during the reaction were optimized respectively.

The results are shown in Figure 8A. Firstly, the concentration of nanozyme was optimized. When the final concentration of nanozyme was in the range of 10, 20, 30, 40, and 50 μg/mL, the color development trend gradually deepened with the increase of nanozyme concentration. , when the final concentration of Fe-N-C@Hemin nanozyme is 40 μg/mL, the color development is the best, so the optimal final concentration of nanozyme is 40 μg/mL;

At this concentration, the effect of TMB concentration on the OXD activity of Fe-N-C@Hemin was explored. TMB solutions with final concentrations of 0, 0.2, 0.4, 1, 2, 4, and 10 mM were selected and reacted at 37°C for 15 min. The results were As shown in Figure 8B, when the final concentration of TMB is 0.4 mM, the OD652 nm is the highest, so when the final concentration of TMB is 0.4 mM, it is the best color development concentration;

The pH of the reaction system will affect the catalytic ability of the nanozyme, so 2.6, 3.6, 4.6, 5.6, 7.6, and 8.6 were selected as the optimal pH to explore the optimal pH of the sensor. The effect of pH on the OXD activity of TMB catalyzed by Fe-N-C@Hemin is shown in Figure 8C. When the pH is 3.6, the OD652 nm absorbance is as high as 0.60. As the pH increases, the OD652 nm gradually decreases, indicating that Fe-N-C@Hemin The color rendering effect under acidic conditions is better than that under neutral and alkaline conditions.

Finally, optimize the color development time, as shown in Figure 8D. As the time increases, the color development gradually increases, and there is a big difference between the color development time of 15 minutes and 5 minutes. Considering the needs of rapid detection and the experimental optimization results, we choose 15 min is the best response time.

Example 3 Verification of the specific detection of glutathione and cysteine

Use common ions, proteins, etc. to explore the substances that affect the activity of Fe-N-C@Hemin-like OXD, as shown in Figure 9, it can be seen from the figure that cysteine and glutathione can significantly inhibit the activity of Fe-N-C@Hemin-like OXD The OXD activity shows that the constructed sensor system has specificity for the detection of glutathione and cysteine.

The sensitivity determination of embodiment 4 detection method

A sensor based on Fe-NC@Hemin single-atom nanozyme was constructed for the detection of Cys and GSH. Take 10 μL of Cys/GSH solutions with concentrations of 0, 0.1, 0.25, 0.5, 0.75, and 1 mM, and add them to 84 μL of sodium acetate buffer (pH=3.6). Then 4 μL of Fe-NC@Hemin single-atom nanozyme with a concentration of 1 mg/mL was added, and after incubation for 5 min, 2 μL of TMB with a concentration of 20 mM was added. Continue to react at 37°C for 15 min. The absorbance at 652 nm was measured with a microplate reader, and the inhibition rate was calculated as (A ₀ -A)/A ₀ ×100, where A ₀ was a group without adding the sample to be tested.

As shown in Figure 10, cysteine and glutathione were detected, and the detection limit was calculated by LOD=3.3σ/s, and the linear regression equation of Cys between 0.025 and 0.2 mM was y=276.5x+23.48, The detection limit is 0.513 μM, the linear regression equation of GSH in the range of 0.05~0.8 mM is y=60.53x+32.78, and the detection limit LOD is 3.1 μM.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. Use of fe-N-c@hemin as oxidoreductase.
2. 2. Use according to claim 1, for detecting the glutathione or cysteine content.
3. 3. A kit for specifically detecting glutathione or cysteine, said kit comprising:

   Fe-N-C@Hemin, TMB and sodium acetate buffer solution.
4. 4. The kit according to claim 3, wherein the working concentration of Fe-N-C@Hemin is 20-50 μg/mL; the working concentration of TMB is 0.2-0.5 mM; the pH of the sodium acetate buffer solution is 3-5.
5. 5. Use of the kit according to claim 3 or 4 for detecting glutathione or cysteine content.
6. 6. The application according to claim 5, wherein the specific application method is:

   1) Mixing a sample to be tested with a sodium acetate buffer solution, wherein the volume ratio of the sample to be tested to the sodium acetate buffer solution is 1:8-10;

   2) Adding Fe-N-C@Hemin monoatomic nano enzyme, and then adding TMB for reaction;

   3) The absorbance at 652 and nm was measured, the inhibition was calculated, a linear regression equation was constructed, and the concentration was calculated.
7. 7. The use according to claim 6, wherein in step 2), the working concentration of Fe-N-c@hemin is 20-50 μg/mL; TMB has a working concentration of 0.2 to 0.5 mM.
8. 8. The use according to claim 7, wherein the reaction time of step 2) is 10-20 min; the pH value of the reaction is 3-5.
9. 9. The use according to claim 6, wherein the inhibition ratio calculation formula is (a ₀ -A)/A ₀ X 100, wherein A ₀ A group to which no sample to be tested was added.
10. 10. Use according to any one of claims 6 to 9, wherein the sample is food or serum, and wherein when the sample is serum, the use is for non-diagnostic purposes.