CN111189783B - A kind of detection method and application of D-glucose - Google Patents

Abstract

The invention provides a detection method and application of D-glucose. The detection method provided by the invention has the advantages that the molybdenum dioxide at the end sealed by the chiral cysteine is matched with the glucose oxidase (Gox), so that the detection of D-glucose can be realized, and the detection method has no response to other sugar molecule samples other than D-glucose and has high selectivity; meanwhile, the detection sensitivity is high, and the D-glucose detection Limit (LOD) is low.

Description

A kind of detection method and application of D-glucose

technical field

The invention belongs to the technical field of chiral signal detection, and relates to a detection method and application of D-glucose.

Background technique

Chiral recognition of chiral molecules in enantiomers is very important, especially in the fields of stereoselective synthesis, biosensing, bioimaging, and medicine. Thus, the development of efficient methods for the efficient identification and quantification of enantiopure molecules has become a major motivation for the design of modern nanomaterials.

So far, surface-modified metal nanoparticles, semiconductors, and carbon-based nanomaterials have been intensively studied for chiral recognition of biomolecules. However, current methods mainly rely on tuning the interactions between nanomaterial surfaces and chiral ligands through surface modifications such as chemical bonding and electrostatic attraction. Although great progress has been made, the stability and sensitivity of chiral recognition of nanomaterials are still unsatisfactory.

Ligand-induced chiral transition metal oxides with tunable absorption wavelengths have attracted widespread interest due to their wide range of potential applications, including biosensing, hyperspectral imaging, and photodetection. For example, Kotov et al. disclosed that chiral WO3 _-x and _Co3O4 nanoparticles modified with chiral ligands such as proline, aspartic _acid and cysteine exhibited strong optical chirality , even enhanced when paramagnetism is present. In this case, this chirality is caused by the metal-ligand charge transfer (MLCT) effect, which results from the transition of unpaired electrons from metal delta orbitals to ligand-based π and π* orbitals, show that the valence state of metal atoms is very important for such atoms. There are still few studies on the origin of chirality in MLCT.

The detection of glucose is generally based on ultraviolet or fluorescence spectral analysis. Due to the universality of ultraviolet and fluorescence, that is, a large number of substances have ultraviolet absorption or fluorescence emission spectra, the detection specificity is low, and chiral glucose (D-glucose and L-glucose) cannot be directly identified. -glucose).

CN110231486A discloses a glucose detection method, including a glucose detection kit, including glucose oxidase solution, alkaline aqueous solution, MnO ₂ NPs and luminol working solution; a glucose detection method, which includes: 1) The sample to be tested is mixed with glucose oxidase solution with a concentration of 0.8-1.5 mg/mL, and reacted at 30-37 °C for 6-10 min; 2) Take 100 μL of the reaction solution and 100 μL of NaOH aqueous solution, and 10 μL with a concentration of 0.01-1.0 mg/mL mL MnO ₂ NPs and luminol working solution were mixed; 3) CL signal was measured, and the glucose concentration was calculated according to the regression equation. Although this patent can detect the concentration of glucose, it cannot distinguish the concentration of chiral glucose.

Therefore, there is a need to provide a detection method that can identify and detect chiral glucose.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a detection method and application of D-glucose. The detection method provided by the present invention utilizes chiral cysteine-terminated molybdenum dioxide and glucose oxidase (Gox) in combination to detect D-glucose, but has no response to other non-D-glucose sugar molecule samples. It has high selectivity; at the same time, it has high detection sensitivity and low D-glucose detection limit (LOD).

In order to achieve this object of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a method for detecting D-glucose, which comprises detecting by using chiral cysteine-terminated molybdenum dioxide.

In the present invention, since hydrogen peroxide can change the chemical valence of molybdenum dioxide, thereby changing the chiral signal of chiral cysteine-terminated molybdenum dioxide, glucose oxidase can only specifically catalyze D-glucose is oxidized to generate hydrogen peroxide. Therefore, D-glucose can be specifically identified and detected by using chiral cysteine-terminated molybdenum dioxide.

In the present invention, the detection method comprises the following steps:

(1) draw a standard curve;

(2) mixing the sample to be tested with chiral cysteine-terminated molybdenum dioxide, and detecting the circular dichroism spectrum of the mixed solution;

(3) According to the CD peak absolute value of the circular dichroism spectrum obtained in step (2), corresponding to the standard curve, determine the concentration of D-glucose in the sample to be tested.

In the present invention, the standard curve takes the concentration of D-glucose as the abscissa, and the CD peak absolute value of the circular dichroism spectrum of chiral cysteine-terminated molybdenum dioxide as the ordinate.

Because the CD peak change value of the circular dichroism spectrum of chiral cysteine-terminated molybdenum dioxide has a linear relationship with the hydrogen peroxide concentration change value, and the glucose oxidase has a linear relationship with the catalysis of D-glucose to generate hydrogen peroxide , therefore, the standard curve is a linear relationship, and each concentration of D-glucose corresponds to a unique CD peak absolute value.

That is, by testing the CD peak of the circular dichroism spectrum of the chiral cysteine-terminated molybdenum dioxide in the sample to be tested, the concentration of D-glucose in the sample to be tested can be obtained.

Preferably, the method for drawing the standard curve comprises the following steps:

(A) D-glucose solutions of different concentrations are respectively configured, and the D-glucose solution includes glucose oxidase;

(B) D-glucose solution is mixed with chiral cysteine-terminated molybdenum dioxide, and the CD peak absolute value of chiral cysteine-terminated molybdenum dioxide is detected by circular dichroism spectroscopy;

(C) Draw the standard curve with the concentration of D-glucose as the abscissa and the absolute value of the CD peak as the ordinate.

Preferably, the concentration of D-glucose in the D-glucose solution in step (A) is 0-100000 μM, such as 1 μM, 50 μM, 100 μM, 600 μM, 800 μM, 1000 μM, 5000 μM, 10000 μM, 50000 μM, 80000 μM, etc., preferably 200-1000 μM , such as 400 μM, 600 μM, 800 μM, etc.

Preferably, in step (A), at least 3 or more (eg 4, 5, 6, 7, 8, etc.) D-glucose solutions with different concentrations are prepared, more preferably 5 or more.

Preferably, in the D-glucose solution, the concentration of the glucose oxidase is 50-400U/mL, such as 100U/mL, 150U/mL, 200U/mL, 250U/mL, 300U/mL, 350U/mL, etc. , more preferably 100U/mL.

Preferably, in the mixed solution in step (B), the concentration of the chiral cysteine-terminated molybdenum dioxide is 0.1-100mmol/L, such as 0.2mmol/L, 0.5mmol/L, 1mmol/L, 2mmol/L, 5mmol/L, 10mmol/L, 12mmol/L, 20mmol/L, 50mmol/L, 80mmol/L, 90mmol/L, etc., more preferably 10mmol/L.

Preferably, the mixing time in step (B) is 10-120 min, such as 20 min, 30 min, 50 min, 100 min, 110 min, etc., more preferably 30 min.

Preferably, the chiral cysteine-terminated molybdenum dioxide comprises L-cysteine-terminated molybdenum dioxide and/or D-cysteine-terminated molybdenum dioxide.

Preferably, the preparation method of the chiral cysteine-terminated molybdenum dioxide is as follows:

The chiral cysteine-terminated molybdenum dioxide is obtained by reacting chiral cysteine with molybdenum trioxide.

Preferably, the molar ratio of the chiral cysteine and the molybdenum trioxide is (50-200):3, such as 60:3, 70:3, 80:3, 85:3, 90:3, 95:3, 100:3, 120:3, 150:3, 180:3, etc., more preferably 100:3.

Preferably, the reaction includes sonication for 5 min followed by incubation in the dark for 1 week.

Preferably, the molybdenum trioxide participates in the reaction in the form of a solution.

Preferably, the preparation method of the molybdenum trioxide comprises the steps:

The molybdenum disulfide and hydrogen peroxide are mixed and reacted to obtain the molybdenum trioxide.

Preferably, the chiral cysteine includes L-cysteine and/or D-cysteine;

Preferably, the chiral cysteine is L-cysteine, and the chiral cysteine-terminated molybdenum dioxide is L-cysteine-terminated molybdenum dioxide (L-Cys-MoO _2NP ).

Preferably, the chiral cysteine is D-cysteine, and the chiral cysteine-terminated molybdenum dioxide is D-cysteine-terminated molybdenum dioxide (D-Cys-MoO ₂ ).

To ensure the Cys-MoO ₂ NPs were well dispersed in the solution, the samples in the aqueous solution were purified by centrifugation for 3 min, the precipitate (if any) was removed at 8000 rpm, and kept at 20 °C before use.

In the second aspect, the present invention provides the application of the D-glucose detection method according to the first aspect in the detection of reactive oxygen radicals or dopamine in vivo.

Since cysteine-terminated molybdenum dioxide has a sensitive response to oxidants in the external environment, oxygen anions, hydroxyl radicals, dopamine, hypochlorite, hydrogen peroxide and other oxidants generated in the metabolism of biological cells are Both sensing and detection can be achieved by this method.

Compared with the prior art, the present invention has the following beneficial effects:

(1) In the present invention, because hydrogen peroxide can change the chemical valence of molybdenum dioxide, and then change the chiral signal of chiral cysteine-terminated molybdenum dioxide, and glucose oxidase can only be specific Therefore, the use of chiral cysteine-terminated molybdenum dioxide can specifically recognize and detect D-glucose.

(2) Because the CD peak change value of the circular dichroism spectrum of chiral cysteine-terminated molybdenum dioxide has a linear relationship with the change value of hydrogen peroxide concentration, and glucose oxidase catalyzes D-glucose to generate hydrogen peroxide. Therefore, the concentration of D-glucose has a linear relationship with the absolute value of the CD peak, that is, the CD peak of the circular dichroism spectrum of the chiral cysteine-terminated molybdenum dioxide in the sample to be tested can be used to obtain the desired value. Measure the concentration of D-glucose in the sample.

Description of drawings

FIG. 1 is an HRTEM image of D-Cys-MoO ₂ NP synthesized in Preparation Example 1. FIG.

FIG. 2 is a particle size distribution diagram of D-Cys-MoO ₂ NPs synthesized in Preparation Example 1 based on HRTEM.

3 is a circular dichroism spectrum diagram of Preparation Example 1. FIG.

4 is the XPS spectrum of the sample provided in Preparation Example 1.

FIG. 5 is a high-resolution XPS spectrum and fitting result of the Mo 3d region of the sample provided in Preparation Example 1. FIG.

FIG. 6 is a graph of the standard curve fitted in Example 1. FIG.

FIG. 7 is a graph of the standard curve fitted in Example 2. FIG.

Figure 8 is a graph showing the results of a study on the selectivity of D-Cys-MoO ₂ NPs with various sugars.

Figure 9 is a fitted graph of detection limits for D-glucose in a performance test.

Detailed ways

The technical solutions of the present invention are further described below through specific embodiments. It should be understood by those skilled in the art that the embodiments are only for helping the understanding of the present invention, and should not be regarded as a specific limitation of the present invention.

Preparation Example 1

A D-cysteine-terminated molybdenum dioxide nanoparticle (D-Cys-MoO ₂ NP), the preparation method is as follows:

( ₁ ) Preparation of MoO3 solution:

Under vigorous stirring, 0.8 _g of pristine black MoS2 powder ( ₅ mmol) was dissolved in 462.5 mL of deionized water, after which 37.5 mL of 30 wt% H2O2 was added to the prepared solution _; then, the black mixture became Clear yellow, the solution was heated to 80°C to remove _{excess H2O2} . The dispersion liquid to be mixed becomes transparent, and the heat treatment is stopped.

(2) Preparation of D-Cys-MoO ₂ NPs:

60 mg D-cysteine (D-Cys) was added to 1.5 mL (10 mM) of the MoO solution obtained in step ( ₁ ), the mixture was sonicated for 5 min, and then incubated in the dark for 1 week to obtain D-Cys- MoO ₂ NPs.

Preparation Example 2

An L-cysteine-terminated molybdenum dioxide nanoparticle (L-Cys-MoO ₂ NP), the preparation method refers to Preparation Example 1, the difference is that D-Cys is replaced by L-Cys.

performance test 1

The performance test of the chiral cysteine-terminated molybdenum dioxide nanoparticles provided in Preparation Example 1-2 is as follows:

(1) Particle size distribution: observe the particle size by high-resolution transmission electron microscope (HRTEM);

Fig. 1 is the HRTEM image of D-Cys-MoO ₂ NP synthesized in Preparation Example 1, and Fig. 2 is the particle size distribution diagram of D-Cys-MoO ₂ NP synthesized in Preparation Example 1 based on HRTEM, which was observed and counted 100 It can be seen from Figures 1 and 2 that the size of the chiral cysteine-terminated molybdenum dioxide nanoparticles is in the range of 24.9±0.8mm.

(2) Circular dichroism spectrum test: use JASCO J-1500 circular dichroism instrument to conduct circular dichroism spectrum test;

FIG. 3 is a circular dichroism spectrum diagram of Preparation Example 1. As can be seen from the diagram, a strong CD signal can be observed. Two CD bands were recorded at 376 nm and 580 nm, hereinafter referred to as peak (I) and peak (II), the chiral enantiomer of the ligand showed opposite linearity, while pure L- or D- Cysteine has a CD band at 220 nm, indicating that the chiral cysteine-terminated molybdenum dioxide nanoparticles are prepared by the present invention.

(3) Elemental analysis of samples: analysis by X-ray photoelectron spectroscopy (XPS);

Figure 4 is the XPS spectrum of the sample provided in Preparation Example 1, and Figure 5 is the high-resolution XPS spectrum of the Mo 3d region and the fitting result. Figure 4 confirms the existence of cysteine, molybdenum and oxygen. When fitting the high-resolution XPS spectrum of Mo 3d in 5, the splitting energy of the Mo 3d5/2-Mo 3d3/2 doublet is about 3.15 eV, and the fixed area ratio is 3:2. Two positions are used on the 3d5/2 peak, one for Mo(IV) and one for Mo(VI), at 229.2 eV and 232.5 eV, respectively, which is consistent with literature reports; this comparison of Mo(Mo VI) The high oxidation state contribution (< ₅ %) may be due to partial surface oxidation of MoO2 nanoparticles. The Mo IV ratio from the XPS results is about 94.8%. Both demonstrate the presence of molybdenum dioxide; the shoulder at 226.9 eV may be due to the S 2s contribution of the cysteine ligand grafted on the NP, which is also confirmed by the S 2p peak at 161.8 eV.

Example 1

A kind of detection method of D-glucose is as follows:

(1) Draw the standard curve:

a. Prepare 0.5mL aqueous solution of Gox and D-glucose, which contains 80μL of Gox (5U/μL) and D-glucose of different concentrations, and incubate the enzymatic reaction system for 30min;

Among them, the D-glucose concentrations were 200 μM, 400 μM, 600 μM, 800 μM and 1000 μM, respectively.

b. 1.5 mL of D-Cys-MoO ₂ solution (400 μL, 10 mM) was added to the above solution, the reaction system was continuously stirred for 30 min, and the CD peak of chiral cysteine-terminated molybdenum dioxide was detected by circular dichroism spectroscopy absolute value.

c. Draw a standard curve with the concentration of D-glucose as the abscissa and the absolute value of the CD peak as the ordinate.

(2) mixing the sample to be tested, D-Cys-MoO ₂ and the solvent, and detecting the circular dichroism spectrum of the mixed solution;

(3) According to the CD peak absolute value of the circular dichroism spectrum obtained in step (2), corresponding to the standard curve, determine the concentration of D-glucose in the sample to be tested.

FIG. 6 is a graph of the standard curve fitted in Example 1. It can be seen from the graph that the correlation coefficient R ² =0.988>0.98, indicating a good linear relationship.

Example 2

The difference from Example 1 is that D-Cys-MoO ₂ is replaced by L-Cys-MoO ₂ .

FIG. 7 is the standard curve obtained in Example 2, and it can be seen from the figure that the correlation coefficient R ² =0.997>0.99, indicating a good linear relationship.

performance test 2

The detection method provided by the embodiment is verified, and the method is as follows:

(1) Accuracy verification:

Prepare the test solution of D-glucose, which contains 80 μL of Gox (5U/μL) and D-glucose of different concentrations, and incubate the enzymatic reaction system for 30 minutes;

Among them, the D-glucose concentrations were 385 μM, 560 μM and 937 μM, respectively,

Then utilize the detection method that embodiment provides to detect, and the detection results are shown in Table 1:

Table 1

Detection method Measured value (μM) Preparation value (μM) error/% Example 1 400.4 385 4.0 Example 1 546.9 560 2.3 Example 1 925.8 937 1.2 Example 2 406.9 385 5.7 Example 2 539.3 560 3.7 Example 2 932.3 937 0.5

It can be known from the examples and performance tests that the detection method provided by the present invention can accurately detect D-glucose, with high detection accuracy and small error, and the error is less than 6%.

(2) Selectivity study: The system was used to detect different sugars, fructose, maltose, xylose, lactose, sucrose, L-glucose and D-glucose. - specific recognition of glucose;

Figure 8 is a graph showing the results of studies on the selectivity of D-Cys-MoO ₂ NPs to various sugars. It can be seen from Fig. 8 that the system only responded to D-glucose, and did not respond to L-glucose and other carbohydrates.

(3) Limit of detection (LOD): Prepare mixed solutions with D-glucose concentrations of 0 μM, 1 μM, 3 μM, 5 μM, 10 μM, 30 μM and 50 μM, respectively, which contain 80 μL of Gox (5U/μL), referring to the method for drawing a standard curve carry out testing;

Fig. 9 is a fitting curve diagram of detection limit. It can be seen from the figure that the fitting curve is y=a+bx, a=0.455, b=0.218, R ² =0.992; the calculated limit of detection (LOD) is 0.446 μM.

It can be seen from the examples and performance tests that the detection method provided by the present invention can realize the specific detection of D-glucose, with high detection accuracy and sensitivity, and at the same time, the detection limit of D-glucose is lower than 0.446 μM.

The applicant declares that the present invention illustrates the D-glucose detection method and application of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims (23)

1. The detection method of the D-glucose is characterized by comprising the step of detecting by using molybdenum dioxide blocked by chiral cysteine.

2. The method for detecting D-glucose according to claim 1, wherein the method comprises the steps of:

(1) drawing a standard curve;

(2) mixing a sample to be detected with molybdenum dioxide at the end sealed by chiral cysteine, and detecting the circular dichroism spectrum of the mixed solution;

(3) and (3) determining the concentration of the D-glucose in the sample to be detected according to the CD peak absolute value of the circular dichroism spectrum obtained in the step (2) and corresponding to the standard curve.

3. The method for detecting D-glucose according to claim 2, wherein the standard curve has a D-glucose concentration as an abscissa and an absolute value of a CD peak of a circular dichroism spectrum of the molybdenum dioxide capped with the chiral cysteine as an ordinate.

4. The method for detecting D-glucose according to claim 2 or 3, wherein the standard curve is plotted by the method comprising the steps of:

(A) respectively preparing D-glucose solutions with different concentrations, wherein the D-glucose solutions comprise glucose oxidase;

(B) mixing the D-glucose solution with molybdenum dioxide at the chiral cysteine end, and detecting the CD peak absolute value of the molybdenum dioxide at the chiral cysteine end by using circular dichroism;

(C) and drawing a standard curve by taking the concentration of the D-glucose as an abscissa and taking the absolute value of a CD peak as an ordinate.

5. The method for detecting D-glucose according to claim 4, wherein the concentration of D-glucose in the D-glucose solution in the step (A) is 0 to 100000. mu.M.

6. The method for detecting D-glucose as claimed in claim 5, wherein the concentration of D-glucose in the D-glucose solution in step (A) is 200 μ M and 1000 μ M.

7. The method for detecting D-glucose according to claim 4, wherein 3 or more D-glucose solutions having different concentrations are prepared in the step (A).

8. The method for detecting D-glucose according to claim 7, wherein more than 5 kinds of D-glucose solutions having different concentrations are prepared in the step (A).

9. The method for detecting D-glucose according to claim 8, wherein the concentration of the glucose oxidase in the D-glucose solution is 50 to 400U/mL.

10. The method for detecting D-glucose as claimed in claim 4, wherein the concentration of the molybdenum dioxide at the end of the chiral cysteine in the mixed solution in the step (B) is 0.1-100 mmol/L.

11. The method for detecting D-glucose in claim 10, wherein the concentration of the molybdenum dioxide at the chiral cysteine end-capping in the mixed solution in the step (B) is 10 mmol/L.

12. The method for detecting D-glucose in accordance with claim 4, wherein the mixing time in step (B) is 10 to 120 min.

13. The method for detecting D-glucose according to claim 12, wherein the mixing time in the step (B) is 30 min.

14. The method for detecting D-glucose as claimed in claim 1, wherein the molybdenum dioxide at the end of the chiral cysteine comprises molybdenum dioxide at the end of the L-cysteine and/or molybdenum dioxide at the end of the D-cysteine.

15. The method for detecting D-glucose as claimed in claim 1, wherein the preparation method of the molybdenum dioxide capped with the chiral cysteine is as follows:

chiral cysteine is used for reacting with molybdenum trioxide to obtain the molybdenum dioxide terminated by the chiral cysteine.

16. The method for detecting D-glucose as claimed in claim 15, wherein the molar ratio of the chiral cysteine to the molybdenum trioxide is (50-200): 3.

17. The method for detecting D-glucose according to claim 15, wherein the reaction comprises incubating for 1 week after 5min with ultrasound under dark conditions.

18. The method for detecting D-glucose according to claim 15, wherein the molybdenum trioxide is reacted in the form of a solution.

19. The method for detecting D-glucose according to claim 18, wherein the method for preparing molybdenum trioxide comprises the steps of:

and mixing molybdenum disulfide and hydrogen peroxide for reaction to obtain the molybdenum trioxide.

20. The method for detecting D-glucose in accordance with claim 15, wherein the chiral cysteine comprises L-cysteine and/or D-cysteine.

21. The method for detecting D-glucose as claimed in claim 20, wherein the chiral cysteine is L-cysteine, and the molybdenum dioxide capped by the chiral cysteine is L-cysteine-capped molybdenum dioxide.

22. The method for detecting D-glucose as claimed in claim 20, wherein the chiral cysteine is D-cysteine, and the molybdenum dioxide capped by the chiral cysteine is D-cysteine-capped molybdenum dioxide.

23. Use of the method for the detection of D-glucose according to any one of claims 1-22 in the detection of reactive oxygen radicals or dopamine in vivo.

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