Materials, reagents and applications for the detection of vitamin B2 and vitamin B6

Abstract

The present invention relates to the technical field of vitamin B2 and vitamin B6 detection, and specifically to materials, reagents, and applications for detecting vitamin B2 and vitamin B6. The reagent is a novel quantum dot material prepared by grafting polyether ester imide with butylamine lead iodine and diphenylthiourea. The material is prepared by coating the quantum dot material on the surface of a glassy carbon electrode. This material can not only detect vitamin B2 or vitamin B6, respectively, but also exhibits significantly higher detection sensitivity than known quantum dot fluorescent probes.

Description

Technical Field

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

[0002] Vitamins are a type of micronutrient necessary to maintain the body's normal physiological functions and promote its health. Although the body's vitamin content is small, they play a very important role in the body's material and energy metabolism. Vitamins can be divided into fat-soluble vitamins and water-soluble vitamins based on their solubility. B vitamins are an important type of water-soluble vitamin, 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 development and function maintenance of the body's systems. 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 primarily in two biologically active forms: flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Its water solubility is relatively low and it is primarily absorbed in the upper gastrointestinal tract. Vitamin B2, primarily as a coenzyme, participates in biological oxidation reactions and energy metabolism in the body and is involved in the metabolism of multiple vitamins, including folate, vitamin B12, and vitamin B6. Vitamin B2 deficiency interferes with iron absorption, leading to a decrease in iron storage and impairing growth and development. Vitamin B6 exists in three naturally occurring forms: pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM). These forms cannot be synthesized in the human body and must be obtained through the diet. In the intestine, vitamin B6 is absorbed in its dephosphorylated form. PN, PL, and PM are transported via the blood to the liver, where they are phosphorylated by pyridoxal kinase to exert their biological effects. Metabolism also occurs in the liver, where they are converted to pyridoxic acid (PA) for excretion through the kidneys. Vitamin B6 is one of the important water-soluble vitamins that helps maintain the function of the body's central nervous system and also plays an important auxiliary role in many metabolic reactions.

[0004] To meet consumer demand for nutritious and healthy foods, the market has introduced fortified foods. B vitamins, as a common nutritional fortifier, are found in a wide variety of foods. The levels of these fortifiers in these foods are a key aspect of food regulation. Therefore, qualitative and quantitative testing of B vitamin levels in these foods is crucial for monitoring their nutritional and safety profiles.

[0005] Therefore, quantitative detection of B vitamins in blood, urine, and tissue fluid is crucial for evaluating nutritional status, assessing the harmful effects of nutrition-related diseases, and assessing the effectiveness of nutritional interventions. Qualitative and quantitative detection of vitamin levels in nutritional supplements is also crucial for monitoring their nutritional and safety. Therefore, establishing specific, rapid, and sensitive detection methods for B vitamins holds significant potential. Summary of the Invention

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

[0007] One of the objectives 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, and injecting n-butylamine under stirring; continuing stirring, adding toluene, centrifuging, collecting the precipitate, and drying.

[0008] Furthermore, the molar ratio of butylamine lead iodide and diphenylthiourea is 5:1, and the weight of polyether ester imide is no 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 further comprises the steps of washing the obtained precipitate with DMF, centrifuging, and then taking the precipitate for purification.

[0010] Furthermore, in this method, polyether ester imide was purchased from Sigma, product model 700207.

[0011] Specifically, in the method, the centrifugation is performed at 10,000 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 objectives 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 using the above method; preparing an aqueous solution containing 1.0% chitosan and 0.5% acetic acid by mass, and ultrasonically mixing the solution with a DMF solution containing 5% of the reagent at a volume ratio of 3:1 to obtain a coating solution; polishing a glassy carbon electrode (diameter Φ = 3 mm) with 0.5 μm alumina until smooth, then rinsing with distilled water, then ultrasonically treating the electrode with a 1:1 volume ratio of ethanol and water for 1 minute, rinsing with distilled water, and air-drying the electrode. Cyclic voltammetry is then performed from -0.2 to 0.6 V using 1 mM K3[Fe(CN)6] as an electrolyte solution to ensure a redox peak potential difference of less than 80 mV; and applying a 6.0 μL droplet of the coating solution to the surface of the treated glassy carbon electrode, and air-drying the electrode 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 present invention prepares a novel quantum dot material by grafting polyether ester imide with butylamine lead iodide and diphenylthiourea. The quantum dot material is coated on the surface of a glassy carbon electrode to produce a sensor capable of simultaneously detecting vitamin B2 and vitamin B6.

[0018] After 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 UV 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 spectrum of polyether ester imide (I) and quantum dots (II) prepared by the present invention ( 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 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 curves (A) of the quantum dot sensor prepared in the present invention for simultaneous detection of B2 and B6, and the fluorescence curves of the fluorescent probes provided in the control group for detection of B2 and B6, respectively. The red line is B2, and the black line is B6.

[0023] Figure 4 Stability evaluation results of the sensor provided to the experimental group and the fluorescent probe provided to the control group. Figure 4 A is the ECL response curve of the sensor in the experimental group before and after UV 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 in 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 objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely illustrative of the present invention and are not intended to limit the present invention. Reagents not described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and are known in the 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 sonicated for 1 h to fully dissolve them. 0.747 g of polyetheresterimide (700207, Sigma) was then added. 1 mL of n-butylamine was injected at 80 rpm and stirred for 5 min. 25 mL of toluene was added and mixed, and the mixture was centrifuged at 10,000 rpm for 5 min. The precipitate was vacuum-dried in a glove box and washed twice with DMF. The washed precipitate was centrifuged at 10,000 rpm for 5 min and vacuum-dried to obtain quantum dots.

[0027] Figure 1 Curve I in A is the infrared graph 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 3240 cm -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 1575 cm is due to the stretching vibration of C=O. -1 The absorption peak at 1206 cm is attributed to the deformation vibration of NH.-1 The absorption peak at 2967 cm is due to the stretching vibration of C=N. Compared with curve I, curve II has a peak at 2967 cm -1 and 2920cm -1 The absorption peak 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°, respectively, corresponding to the (111), (200), (220) and (222) crystal planes, which show obvious diffraction peak intensities. This shows that the addition of polyetherimide 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 minutes; the clarified solution was then transferred to a 50 mL polytetrafluoroethylene-lined stainless steel autoclave, placed in a constant temperature forced air drying oven at 180°C for 10 hours, and finally cooled to room temperature; the resulting brown solution was centrifuged at 9000 rpm for 15 minutes; 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 hours; finally, the obtained pure quantum dot solution was placed in a vacuum drying oven and dried at 60°C for 72 hours 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, 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] A glassy carbon electrode (diameter Φ = 3 mm) was polished to smoothness with 0.5 μm alumina, 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. After rinsing with distilled water and naturally drying, cyclic voltammetry (CV, -0.2 to 0.6 V) was performed using 1 mM K3[Fe(CN)6] as the electrolyte solution to ensure that the redox peak potential difference was less than 80 mV.

[0033] A 6.0 μL droplet of the coating liquid was applied to the surface of the glassy carbon electrode after the above treatment and allowed to dry naturally to obtain a sensor.

[0034] Example 3: Characterization of Sensors

[0035] Test conditions: A three-electrode electrochemical detection system was used, with a GCE electrode or a quantum dot-coated GCE electrode (i.e., serving as the sensor) as the working electrode, a platinum (Pt) wire electrode as the counter electrode, and a silver chloride (Ag / AgCl) electrode as the reference electrode. The detection substrate solution consisted of 0.14 M K₂S₂O₂ in 0.1 M phosphate buffer (PBS) (pH 7.4). The ECL signal was recorded over a range of 0 to -2.0 V (photomultiplier tube voltage 800 V, negative scan direction, starting potential 0 V) ​​at a scan rate of 100 mV·s⁻¹. The sensor was characterized using cyclic voltammetry (CV) and ECL response curves.

[0036] Figure 2 (A) is the CV graph of the sensor in a 5 mM potassium ferricyanide solution containing 0.1 M potassium chloride. The redox peak current of the quantum dot-coated GCE electrode 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 ferricyanide to the electrode surface to participate in the reaction, indicating that the quantum dots have been successfully modified onto 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 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 a three-electrode electrochemical detection system was constructed as described above. -10 ~10 -4 M) of the base solution 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 of B2 is I-I0=1035.2lgC+10584.5, and the linear equation of B6 is I-I0=1109.5lgC+13548.7. Among them, I0 represents the ECL intensity of the base solution without vitamins, I represents the ECL intensity of 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 by the electrode coated with quantum dots provided by the present invention is 5.64×10-10 The detection limit of vitamin B6 was 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 decreased, the fluorescence intensity increased. A standard curve was prepared with the concentration of the standard solution as the horizontal axis and the fluorescence quenching efficiency of the system as the vertical axis. The obtained standard curve of vitamin B2 was (F0-F) / F=0.0103C+0.0536, and the obtained standard curve of vitamin B6 was (F0-F) / F=0.0089C+0.0983, wherein the fluorescence quenching efficiency of the system was (F0-F) / F, F0 and F were the fluorescence intensities of the solution without vitamins and the solution containing vitamins after reacting with quantum dots, respectively, and C represented the concentration of the vitamin. The detection limit was 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 was calculated that the detection limit of vitamin B2 was 1.25×10 -7 The detection limit of microorganism B6 was 9.085×10 -6 M.

[0041] In addition, the formulation contains 10 -5 M's 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 based on their different response times, and simultaneously detect vitamin B2 and vitamin B6.

[0042] In addition, the formulation contains 10 -5 M's 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: 50 mL of milk was heated to 75°C in a water bath, and then 0.5 mol / L HCl solution was slowly added dropwise while stirring until the pH was 5.0. After 30 min of reaction, heating was stopped and the solution was cooled to room temperature. The treated milk was centrifuged at 9000 rpm for 15 min, the supernatant was filtered through a 0.22 μm microporous membrane, and the filtrate was collected and a final concentration of 10 -5 M vitamin B2 solution, containing a final concentration of 10 -5 M vitamin B6 solution, containing 10 -5 M's 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 were compared with each other. -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 solution were high. -5 M's 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's 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 quantum dot-coated electrode provided by the present invention.

[0052] 1. Time stability

[0053] The quantum dot-coated electrode provided by the present invention was stored in a 4°C refrigerator away 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 quantum dot-coated electrode provided by the present invention was irradiated under UV light (UVA-340, 0.76W @ 340nm, 8 hours of illumination, BPT: 60℃, 4 hours of condensation, BPT: 50℃), specifically referring to GB / T 16422.3. The ECL signal was detected by continuous cyclic scanning in the range of 0 to -2.0V. The results are shown in Figure 2. Figure 4 As shown in Figure 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 UV light (UVA-340, 0.76W@340nm, 8h illumination, BPT: 60℃, 4h condensation, BPT: 50℃), according to GB / T 16422.3. The fluorescence intensity at 430nm was measured. Figure 4 As shown in B, the fluorescence intensity of the fluorescent probe provided by the control group at 430nm decreased significantly after being irradiated under ultraviolet light, indicating that it is sensitive to ultraviolet rays and is less stable than the electrode coated with quantum dots provided in this application.

[0057] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection 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; 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 10,000 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: The reagent prepared by any one of claims 1 to 5; An aqueous solution containing 1.0% chitosan and 0.5% acetic acid was prepared, and the solution was ultrasonically mixed with a DMF solution containing 5% of the reagent at a volume ratio of 3:1 to obtain a coating solution; A glassy carbon electrode with a diameter of 3 mm was polished with 0.5 μm alumina until smooth, then rinsed with distilled water, ultrasonically treated with a 1:1 volume ratio of ethanol and water for 1 min, rinsed with distilled water, and naturally dried. Cyclic voltammetry was then performed from -0.2 to 0.6 V using 1 mM K3[Fe(CN)6] as the electrolyte solution to ensure that the redox peak potential difference was less than 80 mV. 6.0 μL of the coating droplet was applied to the surface of the glassy carbon electrode after the above treatment, and dried naturally to obtain the material.

8. A material for detecting vitamin B2 and vitamin B6, prepared by the method according to 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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