Material and reagent for detecting vitamin B2 and vitamin B6 and application

Abstract

The invention relates to the technical field of detection of vitamin B2 and vitamin B6, in particular to a material and a reagent for detecting vitamin B2 and vitamin B6 and application. The reagent is a novel quantum dot substance prepared by grafting polyether ester imide with butylamine lead iodine and diphenylthiourea. The material is prepared by coating the quantum dot substance on the surface of a glassy carbon electrode. The material can be used for respectively detecting vitamin B2 or vitamin B6, and the detection sensitivity is obviously higher than that of a known quantum dot fluorescent probe.

Description

Technical Field

[0001] The present invention relates to the technical field of vitamin B2 and vitamin B6 detection, and in particular to materials, reagents and applications for detecting vitamin B2 and vitamin B6. Background Art

[0002] Vitamins are a type of micronutrients necessary to maintain the body's normal physiological functions and promote the body's health. Although the body's vitamin content is small, it plays a very important role in the body's material and energy metabolism. Vitamins can be divided into fat-soluble vitamins and water-soluble vitamins according to their solubility, and B vitamins are an important class of water-soluble vitamins, including vitamin B1, vitamin B2, vitamin PP, vitamin B6, folic acid, vitamin B12, pantothenic acid, biotin, etc., which play a very important role in the body's system development and function maintenance. B vitamins are widely present in the body's tissues and organs, but their distribution is uneven.

[0003] Vitamin B2 (also known as riboflavin) exists in the body in two biologically active forms: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). It has relatively low water solubility and is mainly absorbed in the upper gastrointestinal tract. Vitamin B2 mainly participates in biological oxidation reactions and energy metabolism in the body in the form of a coenzyme, and participates in the metabolism of multiple vitamins such as folic acid, vitamin B12, and vitamin B6. Vitamin B2 deficiency interferes with the absorption of iron in the body, resulting in a decrease in the amount of stored iron, and also affects the growth and development of the body. Vitamin B6 includes three naturally occurring forms: pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM). It cannot be synthesized in the human body and needs to be obtained through diet. In the intestine, vitamin B6 is absorbed in a dephosphorylated form. PN, PL, and PM are transported to the liver through the blood, phosphorylated by pyridoxal kinase, and then exert biological effects. Its metabolism also occurs in the liver and is converted into pyridoxic acid (PA) and excreted through the kidneys. Vitamin B6 is one of the important water-soluble vitamins. It helps maintain the function of the body's central nervous system and also plays an important auxiliary role in many metabolic reactions.

[0004] In order to meet people's demand for nutritious and healthy food, the market has launched related nutritional fortified foods. As a common nutritional fortifier, B vitamins have a large number of related nutritional fortified foods. The content level of nutritional fortifiers in related foods is an important aspect of food supervision. Therefore, qualitative and quantitative detection of B vitamin levels in related foods is of great significance for monitoring their nutrition and safety.

[0005] It can be seen that the quantitative detection of B vitamins in the body's blood, urine, tissue fluid, etc. is an important link in evaluating its nutritional level, judging the damage of nutrition-related diseases and the effectiveness of nutritional intervention measures; qualitative and quantitative detection of vitamin levels in nutritional supplements is also important for monitoring its nutrition and safety. Therefore, the establishment of a specific, rapid and sensitive detection method for related B vitamins has important application prospects. Summary of the invention

[0006] In view of this, the present invention provides materials, reagents and applications for detecting vitamin B2 and vitamin B6, so as to solve one of the above-mentioned technical problems to a certain extent.

[0007] One of the purposes of the present invention is to provide a method for preparing a reagent for detecting vitamin B2 and vitamin B6. The method comprises: dissolving butylamine lead iodide and diphenylthiourea in DMF under ultrasonic conditions; adding polyether ester imide, injecting n-butylamine under stirring conditions; adding toluene after continued stirring, centrifuging, taking the precipitate, and drying to obtain the reagent.

[0008] Furthermore, the molar ratio of butylamine lead iodide and diphenylthiourea is 5:1, and the weight of polyether ester imide is not more than twice that of diphenylthiourea, otherwise excessive precipitates are easily generated during the reaction, resulting in low purity of the final quantum dots.

[0009] Furthermore, the method also includes the steps of washing the obtained precipitate with DMF, centrifuging, and then taking the precipitate for purification.

[0010] Furthermore, in the method, polyether ester imide is purchased from Sigma, and the product model is 700207.

[0011] Specifically, in the method, the centrifugation is performed at 10000 rpm for 5 minutes.

[0012] One of the purposes of the present invention is to provide a reagent for detecting vitamin B2 and vitamin B6, which is prepared by the above method.

[0013] One of the purposes of the present invention is to provide a method for preparing a material for detecting vitamin B2 and vitamin B6. The method comprises: preparing a reagent by the above method; preparing an aqueous solution containing chitosan with a mass fraction of 1.0% and 0.5% acetic acid, and ultrasonically mixing it with a DMF solution containing 5% of the reagent at a volume ratio of 3:1 to obtain a coating liquid; polishing a glassy carbon electrode (diameter Φ=3mm) with 0.5μm alumina until smooth, then rinsing it with distilled water, and then ultrasonically treating it with an ethanol aqueous solution with a volume ratio of 1:1 for 1min, rinsing it with distilled water, and drying it naturally, using 1mM K3[Fe(CN)6] as an electrolyte solution, performing a -0.2 to 0.6V cyclic voltammetry scan to make the redox peak potential difference less than 80mV; taking 6.0μL of the coating droplet and applying it on the surface of the glassy carbon electrode after the above treatment, and drying it naturally to obtain a sensor.

[0014] One of the purposes of the present invention is to provide a material for detecting vitamin B2 and vitamin B6, which is prepared by the above method.

[0015] One of the purposes of the present invention is to provide the use of the above reagents or materials in detecting vitamin B2 and vitamin B6.

[0016] Compared with the prior art, the present invention at least discloses the following technical effects:

[0017] The invention obtains a novel quantum dot material by grafting polyether ester imide with butylamine lead iodine and diphenylthiourea. The quantum dot material is coated on the surface of a glassy carbon electrode to obtain a sensor capable of simultaneously detecting vitamin B2 and vitamin B6.

[0018] Through comparison, it was found that the sensor can not only detect vitamin B2 or vitamin B6 separately, but also has a detection sensitivity significantly higher than that of known quantum dot fluorescent probes. It can also overcome the defect that existing quantum dot fluorescent probes cannot detect vitamin B2 and vitamin B6 at the same time.

[0019] In addition, the sensor provided by the present invention has time stability and ultraviolet stability, and can also overcome the defect that the existing quantum dot fluorescent probe is sensitive to ultraviolet rays. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The infrared spectra of polyether ester imide (I) and quantum dots (II) prepared by the present invention are ( Figure 1 A), and the XPS spectrum of the quantum dots (II) prepared by the present invention ( Figure 1 B).

[0021] Figure 2 The characterization results of the quantum dot sensor (red line) and the GCE electrode (black line) prepared in the present invention are shown in FIG. Figure 2 A is the CV curve, Figure 2B is the ECL response curve.

[0022] Figure 3 The ECL response curve (A) of the quantum dot sensor prepared in the present invention for simultaneously detecting B2 and B6, and the fluorescence curves of the fluorescent probe provided in the control group for detecting B2 and B6 respectively. The red line is B2, and the black line is B6.

[0023] Figure 4 Stability evaluation results of the sensors provided to the experimental group and the fluorescent probes provided to the control group. Figure 4 A is the ECL response curve of the sensor in the experimental group before and after ultraviolet irradiation, with the black line before irradiation and the red line after irradiation. Figure 4 B is the fluorescence intensity curve of the fluorescent probe of the control group before and after ultraviolet irradiation, with the black line before irradiation and the red line after irradiation. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0025] Example 1 Preparation of quantum dots

[0026] Experimental group: In a glove box, 0.01 mol of butylamine lead iodide and 0.002 mol of diphenylthiourea were added to 9 mL of DMF, and ultrasonicated for 1 h to fully dissolve them, and then 0.747 g of polyether ester imide (700207, Sigma) was added. After 1 mL of n-butylamine was injected at 80 rpm, stirring was continued for 5 min, and 25 mL of toluene was added and mixed. The mixture was centrifuged at 10000 rpm for 5 min, and the precipitate was vacuum dried in the glove box. The precipitate was washed twice with DMF, centrifuged at 10000 rpm for 5 min, and vacuum dried to obtain quantum dots.

[0027] Figure 1 Curve I in A is the infrared image of polyether ester imide. Figure 1 Curve II in A is the infrared image of quantum dots. Figure 1 A shows that quantum dots appear at 3240cm -1 and 3410cm -1 The absorption peak is due to the stretching vibration of OH and NH at 1723 cm -1 The absorption peak at 1575cm is due to the stretching vibration of C=O. -1 The absorption peak at 1206 cm-1 is attributed to the deformation vibration of NH-1 The absorption peak at 2967cm is due to the stretching vibration of C=N. Compared with curve I, curve II has a peak at 2967cm -1 and 2920cm -1 The absorption peaks at 1464 cm -1 The absorption peak was enhanced, indicating that the quantum dots were successfully synthesized in the butylamine lead iodine grafted polymer.

[0028] Figure 1 B shows that the diffraction angles of the obtained quantum dots are 25°, 29°, 40° and 57°, corresponding to the (111), (200), (220) and (222) crystal planes, respectively, which show obvious diffraction peak intensities. This indicates that the addition of polyether ester imide causes changes in the butylamine lead iodine matrix, thereby causing lattice compression of the quantum dots provided by the experimental group.

[0029] Control group: 0.2 g of thiamine nitrate was added to 20 mL of ultrapure water, and then stirred in a constant temperature water bath at 45°C for 15 min; the clarified solution was then transferred to a 50 mL polytetrafluoroethylene-lined stainless steel high-pressure reactor, placed in a constant temperature forced air drying oven at 180°C for 10 h, and finally cooled to room temperature; the resulting brown solution was centrifuged at 9000 rpm for 15 min; the supernatant was filtered with a 0.22 μm microporous filter membrane to remove insoluble large particles; the filtrate was then dialyzed with a dialysis membrane with a molecular weight cutoff of 100 Da for 48 h; finally, the obtained pure quantum dot solution was placed in a vacuum drying oven and dried at 60°C for 72 h to obtain a dark brown solid, which was the fluorescent probe of the control group.

[0030] Example 2 Preparation of sensor

[0031] An aqueous solution of 1.0% chitosan (98% deacetylation degree, Sigma-Aldrich Chemical Company) and 0.5% acetic acid was prepared and ultrasonically mixed with a DMF solution containing 5% quantum dots provided by the above experimental group at a volume ratio of 3:1 to obtain a coating solution.

[0032] The glassy carbon electrode (diameter Φ = 3 mm) was polished with 0.5 μm alumina until smooth, then rinsed with distilled water, and then ultrasonically treated with a mixture of ethanol and distilled water (volume ratio of 1:1) for 1 min. It was rinsed with distilled water, dried naturally, and then cyclic voltammetry scans (CV, -0.2~0.6 V) were performed using 1 mM K3[Fe(CN)6] as the electrolyte solution to make the redox peak potential difference less than 80 mV.

[0033] Take 6.0 μL of the coating droplet and apply it on the surface of the glassy carbon electrode after the above treatment, dry it naturally, and obtain a sensor.

[0034] Example 3: Characterization of Sensors

[0035] Test conditions: A three-electrode electrochemical detection system was used, with the working electrode being a GCE electrode or a GCE electrode coated with quantum dots (i.e., as a sensor), the counter electrode being a platinum (Pt) wire electrode, and the reference electrode being a silver chloride electrode (Ag / AgCl) electrode. Detection base solution: 0.1M phosphate buffer solution (PBS) (pH=7.4) containing 0.14M K2S2O8. The ECL signal was recorded by scanning at a scan rate of 100mV·s-1 in the range of 0 to -2.0V (the photomultiplier tube voltage was 800V, the scanning direction was negative scanning, and the starting potential was 0V). The sensor was characterized by cyclic voltammetry (CV) and ECL response curves.

[0036] Figure 2 (A) is the CV graph of the sensor in a 5 mM potassium ferrocyanide solution containing 0.1 M potassium chloride. The redox peak current of the GCE electrode coated with quantum dots is significantly greater than the current intensity of the GCE. This is because N-CQDs themselves have good conductivity. In addition, chitosan and quantum dots can attract more negatively charged potassium ferrocyanide to the electrode surface to participate in the reaction, indicating that the quantum dots have been successfully modified on the electrode.

[0037] Figure 2 (B) is the ECL signal corresponding to the two sensors. Compared with GCE, the GCE electrode coated with quantum dots shows a strong ECL response, indicating that the quantum dots have good ECL activity and are successfully modified onto the GCE electrode.

[0038] Example 4: Detection of standard solution

[0039] Experimental group: Electrochemical detection was performed using the electrodes coated with quantum dots provided by the present invention, and the above three-electrode electrochemical detection system was constructed. -10 ~10 -4 M) to detect ECL signals. -10 ~10 -4 M) base solution to detect ECL signal. The increase in ECL signal is linearly related to the logarithmic value of the concentration of vitamin B2 or B6 in the base solution. The linear equation for B2 is I-I0=1035.2lgC+10584.5, and the linear equation for B6 is I-I0=1109.5lgC+13548.7. Wherein, I0 represents the ECL intensity detected in the base solution without vitamins, I represents the ECL intensity detected in the base solution containing vitamins, and C represents the concentration of vitamins. The detection limit is calculated according to 3σ / S, where σ is the standard deviation obtained from 10 parallel blank measurements, and S is the slope of the calibration curve. It can be calculated that the detection limit of vitamin B2 detected using the electrode coated with quantum dots provided by the present invention is 5.64×10-10 M, the detection limit of vitamin B6 is 1.25×10 -10 M.

[0040] Control group: Prepare pH=7.0 BR buffer solution containing 8.75 mg / mL control group quantum dots, add different concentrations of vitamin B2 or B6 standard solution to the solution, so that the final test concentration is (10 -10 ~10 -4 M), and recorded their fluorescence spectra respectively, with a scanning range of 340-600nm. As the concentration of the standard solution decreases, the fluorescence intensity increases. A standard curve is prepared with the concentration of the standard solution as the horizontal coordinate and the fluorescence quenching efficiency of the system as the vertical coordinate. The obtained standard curve of vitamin B2 is (F0-F) / F=0.0103C+0.0536, and the obtained standard curve of vitamin B6 is (F0-F) / F=0.0089C+0.0983, wherein the system fluorescence quenching efficiency is (F0-F) / F, F0 and F are the fluorescence intensities of the solution without vitamins and the solution containing vitamins after reacting with quantum dots, respectively, and C represents the concentration of the vitamin. The detection limit is calculated according to 3σ / S, where σ is the standard deviation obtained from 10 parallel blank measurements, and S is the slope of the calibration curve. It is calculated that the detection limit of vitamin B2 is 1.25×10 -7 M, the detection limit of microorganism B6 was 9.085×10 -6 M.

[0041] In addition, the formulation contains 10 -5 M B2 and contains 10 -5 The base solution of B6 of M was tested in the experimental group. Figure 3 As shown in A, vitamin B2 (about 51.6s) and vitamin B6 (about 57.1s) have different response times, and the response time of vitamin B6 is short. That is, electrochemical detection using the electrode coated with quantum dots provided by the present invention can distinguish vitamin B2 and vitamin B6 according to the different response times, and detect vitamin B2 and vitamin B6 at the same time.

[0042] In addition, the formulation contains 10 -5 M B2 and contains 10 -5 The B6 solution of M was tested as a control group. Figure 3 As shown in B, the peak values ​​and intensities of the fluorescence intensity curves of vitamin B2 (red line) and vitamin B6 (black line) are almost the same, and vitamin B2 and vitamin B6 cannot be effectively distinguished, and vitamin B2 and vitamin B6 cannot be detected at the same time.

[0043] Example 5: Detection of actual samples

[0044] Preparation of sample solution: Take 50mL of milk and heat it to 75℃ in a water bath, then slowly add 0.5mol / L HCl solution while stirring until pH=5.0, stop heating after 30min reaction and cool to room temperature; centrifuge the treated milk at 9000rpm for 15min, take the supernatant and filter it with a 0.22μm microporous membrane, collect the filtrate, add 10 -5 M vitamin B2 solution, containing a final concentration of 10 -5 M vitamin B6 solution containing 10 -5 M B2 and contains 10 -5 The B6 solutions of M were used as three sample solutions and stored at 2-8°C.

[0045] Experimental group: The three sample solutions were tested using the above experimental group method.

[0046] Control group: The three sample solutions were tested in the same manner as the control group above.

[0047] As shown in Table 1, the experimental group and the control group contained a final concentration of 10 -5 M vitamin B2 solution, containing a final concentration of 10 -5 The recovery rate and RSD of the solution containing 10 M vitamin B6 were similar, indicating that the repeatability and stability of the detection of vitamin B2 or vitamin B6 were high. -5 M B2 and contains 10 -5 The solution of B6 containing 10 M still maintained good repeatability and stability, while the control group could not pass the test -5 M B2 and contains 10 -5 M's B6 solution can only analyze vitamin B2 or vitamin B6, and the repeatability and stability are significantly improved, indicating that there is a competitive binding relationship between the vitamin B2 and vitamin B6 in the test solution and the fluorescent probe of the control group, which interferes with each other, resulting in a decrease in the detection repeatability and stability, which is not conducive to its long-term detection.

[0048] Table 1

[0049]

[0050] Example 6: Stability Evaluation

[0051] This example further studies the time stability and UV stability of the electrode coated with quantum dots provided by the present invention.

[0052] 1. Time stability

[0053] The electrode coated with quantum dots provided by the present invention was placed in a 4°C refrigerator and protected from light. After 30 days, the ECL signal was detected by continuous cyclic scanning in the range of 0 to -2.0 V. The ECL signal at -2.0 V remained above 90% of its original value.

[0054] 2. UV stability

[0055] The electrode coated with quantum dots provided by the present invention was irradiated under ultraviolet light, UVA-340, 0.76W@340nm, 8h illumination, BPT: 60℃, 4h condensation, BPT: 50℃, specifically referring to GB / T 16422.3. The ECL signal was detected by continuous cyclic scanning in the range of 0~-2.0V. The results are as follows Figure 4 As shown in A, the electrode coated with quantum dots provided by the present invention is subjected to ultraviolet irradiation, and its ECL signal at -2.0V is maintained at more than 90% of its original value.

[0056] The fluorescent probe provided in the control group was irradiated under ultraviolet light, UVA-340, 0.76W@340nm, 8h illumination, BPT: 60℃, 4h condensation, BPT: 50℃, refer to GB / T 16422.3 for details. The fluorescence intensity at 430nm was detected. Figure 4 As shown in B, the fluorescence intensity of the fluorescent probe provided by the control group at 430nm decreased significantly when irradiated with ultraviolet light, indicating that it is sensitive to ultraviolet rays and its stability is not as good as the electrode coated with quantum dots provided in the present application.

[0057] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a reagent for detecting vitamin B2 and vitamin B6, characterized in that: include: Dissolve butylamine lead iodide and diphenylthiourea in DMF under ultrasonic conditions; add polyether ester imide, and inject n-butylamine under stirring conditions; Continue stirring, add toluene, centrifuge, take the precipitate, and dry it.

2. The preparation method according to claim 1, characterized in that: The molar ratio of butylamine lead iodide to diphenylthiourea is 5:

1.

3. The preparation method according to claim 1, characterized in that: The weight of the polyetheresterimide is not more than twice that of the diphenylthiourea.

4. The preparation method according to claim 1, characterized in that: The method also includes a step of purifying the precipitate.

5. The preparation method according to claim 1, characterized in that: Specifically, the centrifugation is performed at 10000 rpm for 5 minutes.

6. A reagent for detecting vitamin B2 and vitamin B6, which is prepared by the method according to any one of claims 1 to 5.

7. A method for preparing a material for detecting vitamin B2 and vitamin B6, characterized in that: include: A reagent prepared according to any one of claims 1 to 5; An aqueous solution containing 1.0% chitosan and 0.5% acetic acid by mass fraction is prepared, and the solution is ultrasonically mixed with a DMF solution containing 5% of the reagent at a volume ratio of 3:1 to obtain a coating liquid; A glassy carbon electrode with a diameter of Φ = 3 mm was polished to be smooth with 0.5 μm alumina, then rinsed with distilled water, and then ultrasonically treated with an ethanol-water solution with a volume ratio of 1:1 for 1 min, rinsed with distilled water, and naturally dried. Then, a cyclic voltammetry scan from -0.2 to 0.6 V was performed using 1 mM K3[Fe(CN)6] as the electrolyte solution to make the redox peak potential difference less than 80 mV; Take 6.0 μL of the coating droplet and apply it on the surface of the glassy carbon electrode after the above treatment, dry it naturally, and obtain the material.

8. A material for detecting vitamin B2 and vitamin B6, prepared by the method of claim 7.

9. Use of the reagent according to claim 6 or the material according to claim 8 in detecting vitamin B2 and vitamin B6.

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