Nanometer material for detecting tea polyphenol and ascorbic acid by spectrophotometry

Abstract

The application discloses a kind of nanometer materials for detecting tea polyphenols and ascorbic acid by spectrophotometry, and belongs to the field of chemical analysis.The nanometer material is prepared by the following method: first, using microbial matter as adsorbent to adsorb palladium ions in palladium ion solution, second, using filter membrane with pore size of 0.22 μm or 0.45 μm to separate the microbial matter adsorbing palladium ions, then adding reducing agent dropwise into deionized water suspending the microbial matter to reduce, and obtaining palladium nanometer material after cleaning.The prepared nanometer material is used for detecting the concentration of tea polyphenols and ascorbic acid by spectrophotometry, has good stability, is simple to operate, does not need to rely on complex instruments such as high performance liquid chromatography, and reduces the error of determining reaction end point by naked eye in titration method.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, and in particular to a nanomaterial for spectrophotometric detection of tea polyphenols and ascorbic acid. Background Technology

[0002] Tea polyphenols are secondary metabolites of tea leaves, composed of various flavonoid compounds. They prevent and treat diseases by scavenging free radicals and regulating the activity of different types of oxidases in the body. Positive effects of tea polyphenols on the treatment of diseases such as cancer, diabetes, Alzheimer's disease, Parkinson's disease, and cardiovascular diseases have been reported. Ascorbic acid (vitamin C, Vc) is an essential micronutrient in the human diet, and its levels are usually maintained through the intake of fresh fruits and vegetables. Vc enhances immunity, prevents chronic diseases, promotes skin health, and has anti-aging effects. Vc deficiency can lead to gingivitis, bone diseases, myocardial degeneration, scurvy, and other diseases, while excessive Vc may cause increased urate levels, uric acid stones, and rashes. Furthermore, as a natural antioxidant, Vc is widely used in beverages and dietary supplements. Therefore, the quantitative detection of tea polyphenol and Vc concentrations in samples is extremely important.

[0003] Currently, methods for measuring the content of tea polyphenols and vitamin C include high-performance liquid chromatography (HPLC), fluorescence analysis, titration, and spectrophotometry. While each of these methods has its own advantages, they still suffer from drawbacks such as expensive instruments, complex operation, poor reagent stability, slow detection speed, and unsatisfactory accuracy. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of existing technologies and provide a nanomaterial for spectrophotometric detection of tea polyphenols and ascorbic acid. This invention is achieved through the following means:

[0005] A method for preparing nanomaterials for spectrophotometric detection of tea polyphenols and ascorbic acid, comprising:

[0006] (1) Microbial material was used as an adsorbent for adsorption in palladium ion solution;

[0007] (2) Use a filter membrane with a pore size of 0.22 μm or 0.45 μm to separate the microbial material adsorbed with palladium ions into solid and liquid phases;

[0008] (3) The isolated microbial material was resuspended in deionized water and a reducing agent was added dropwise for reduction. The reduction reaction was carried out in a shaker. After washing, a nanomaterial for spectrophotometric detection of tea polyphenols and ascorbic acid was obtained.

[0009] Furthermore, the adsorbent in step (1) includes, but is not limited to: microbial cells, microbial residue, and modified microorganisms; the concentration of the palladium ion solution is 100–1000 mg / L, and the volume is 10–1000 mL; the amount of adsorbent used is 1–10 g / L; the adsorption conditions are: carried out in a shaker or stirred with a stirrer at a speed of 100–300 rpm, a temperature of 10–50 °C, and a time of 3–12 h.

[0010] Furthermore, the adsorbent has an adsorption capacity of >100 mg / g for palladium ions, the initial concentration of the palladium ion solution is 500 mg / L, and the volume is 100 mL; the amount of adsorbent used is 1 g / L; the adsorption conditions are: rotation speed 180 rpm, temperature 30℃, and time 6 h.

[0011] Furthermore, the reducing agent in step (3) includes, but is not limited to: sodium formate, sodium acetate, and sodium borohydride; the volume of the deionized water is 10 to 1000 mL; and the final concentration of the reducing agent is 1 to 100 mmol / L.

[0012] Furthermore, the volume of the deionized water is 100 mL; the reducing agent is sodium formate with a final concentration of 20 mmol / L.

[0013] The present invention also discloses a nanomaterial for spectrophotometric detection of tea polyphenols and ascorbic acid prepared according to any of the above preparation methods.

[0014] This invention also discloses an application of the above-mentioned nanomaterials in the detection of tea polyphenols and ascorbic acid, comprising:

[0015] (1) 1 mg of nanomaterial was added to a sodium acetate-acetic acid buffer solution with pH 3.5-5 containing 3,3',5,5'-tetramethylbenzidine (TMB). The reaction system volume was 2800 μL. After reacting in the dark for 0.1-1 h, 200 μL of tea polyphenol or ascorbic acid solution with known concentration gradients was added respectively. The absorbance at 652 nm was measured using a UV-Vis spectrophotometer. The absorbance values ​​of tea polyphenol or ascorbic acid concentrations were used to create a standard curve.

[0016] (2) Take a sample solution containing tea polyphenols or vitamin C and add it to a sodium acetate-acetic acid buffer solution containing TMB. After reacting in the dark for 0.1-1 h, use a UV-Vis spectrophotometer to test the absorbance at 652 nm. The TMB concentration, pH of the sodium acetate-acetic acid buffer solution and reaction time are the same as those in step (1) to establish a standard curve reaction system. The concentration of tea polyphenols or ascorbic acid in the sample to be tested can be obtained according to the above standard curve.

[0017] Furthermore, in step (1), the final concentration of TMB is 10 mmol / L and the volume is 30–2000 μL; the concentration range of the known concentration gradient of tea polyphenols and ascorbic acid is 0.1–300 μmol / L.

[0018] Furthermore, the TMB volume is 1500 μL.

[0019] Furthermore, the sodium acetate-acetic acid buffer solution in step (1) has a pH of 4.0 and a volume of 1300 μL;

[0020] The light-protected reaction time is 0.5 hours.

[0021] Compared with the prior art, the advantages of the present invention are as follows:

[0022] This invention utilizes palladium nanomaterials prepared from microbial substrates as a carrier, which serve as a detection catalyst for spectrophotometric determination of tea polyphenols and ascorbic acid (Vc) content in solutions. This method exhibits good stability, is simple to operate, and eliminates the need for complex instruments such as high-performance liquid chromatography, thus reducing the error associated with visually determining the reaction endpoint in titration methods. Attached Figure Description

[0023] Figure 1 These are TEM images of the palladium nanomaterials in Examples 1 and 2.

[0024] Figure 2 The images show the XRD patterns of palladium nanomaterials in Examples 1 and 2.

[0025] Figure 3 This is a standard curve for detecting tea polyphenols in palladium nanomaterials in Example 1.

[0026] Figure 4 This is a standard curve for ascorbic acid detection of palladium nanomaterials in Example 2. Detailed Implementation

[0027] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0029] Example 1

[0030] (1) Pichia pastoris was used as an adsorbent. It was added to a palladium chloride solution with a palladium ion concentration of 500 mg / L and a volume of 100 mL. The amount of adsorbent added was 1 g / L. The adsorbent was shaken on a shaker at 30 °C and a speed of 180 rpm for 6 h.

[0031] (2) Microbial material adsorbed with palladium ions was separated using a 0.45 μm pore size filter membrane.

[0032] (3) Sodium formate solution was added dropwise to 100 mL of deionized water for reduction, bringing the final sodium formate concentration to 20 mmol / L. The reduction reaction was carried out in a shaker. After washing, palladium nanomaterials were obtained. Figure 1 TEM images show that the surface of the microbial plasmid is loaded with nanoparticles of 2.0-4.0 nm, and through... Figure 2 Further XRD analysis revealed that the surface nanoparticles were palladium nanoparticles, confirming that the method was used to form palladium nanoparticles.

[0033] (4) Take 1 mg of palladium nanomaterial, add 1300 μL of sodium acetate-acetic acid buffer solution with pH 4.0, then add 1500 μL of TMB with a concentration of 10 mmol / L. After reacting in the dark for 0.5 h, add 200 μL of tea polyphenol solutions with concentrations of 10, 20, 40, 60, 80, 100, 120, 150, 180, and 200 μmol / L, respectively. Measure the absorbance at 652 nm using a UV-Vis spectrophotometer. Plot a standard curve using tea polyphenol concentration and absorbance values, such as... Figure 3 As shown, a standard curve was obtained: Y = 1.2829 - 0.00586X. Here, Y represents absorbance, and X represents the concentration of the tea polyphenol solution, in μmol / L.

[0034] (5) Take 200 μL of the solution sample to be tested for tea polyphenol concentration, add 1300 μL of sodium acetate-acetic acid buffer solution with pH 4.0, and then add 1500 μL of TMB with a concentration of 10 mmol / L. After reacting in the dark for 0.5 h, use a UV-Vis spectrophotometer to test the absorbance at 652 nm. The concentration of tea polyphenols in the sample to be tested can be obtained according to the above standard curve.

[0035] Example 2

[0036] (1) Pichia pastoris was used as an adsorbent and added to a palladium chloride solution with a palladium ion concentration of 500 mg / L. The amount of microbial adsorbent added was 1 g / L. The adsorbent was shaken on a shaker at 30°C and 180 rpm for 6 h.

[0037] (2) Microbial material adsorbed with palladium ions was separated using a 0.45 μm pore size filter membrane.

[0038] (3) Sodium formate solution was added dropwise to 100 mL of deionized water for reduction, bringing the final sodium formate concentration to 20 mmol / L. The reduction reaction was carried out in a shaker. After washing, palladium nanomaterials were obtained. Figure 1TEM images show that the surface of the microbial plasmid is loaded with nanoparticles of 2.0-4.0 nm, and through... Figure 2 Further XRD analysis revealed that the surface nanoparticles were palladium nanoparticles, confirming that the method was used to form palladium nanoparticles.

[0039] (4) Take 1 mg of palladium nanomaterial, add 1300 μL of sodium acetate-acetic acid buffer solution with pH 4.0, then add 1500 μL of TMB with a concentration of 10 mmol / L. After reacting in the dark for 0.5 h, add 200 μL of vitamin C solution with concentrations of 0.1, 0.5, 1, 5, 10, 15, 20, and 25 μmol / L respectively. Measure the absorbance at 652 nm using a UV-Vis spectrophotometer. Plot a standard curve using vitamin C solution concentration and absorbance values. Figure 4 As shown, a standard curve was obtained: Y = 1.19433 - 0.03414X. Here, Y is the absorbance, and X is the concentration of the Vc solution, in μmol / L.

[0040] (5) Take 200 μL of the solution sample with the Vc concentration to be tested, add 1300 μL of sodium acetate-acetic acid buffer solution with pH 4.0, and then add 1500 μL of TMB with a concentration of 10 mmol / L. After reacting in the dark for 0.5 h, use a UV-Vis spectrophotometer to test the absorbance at 652 nm. The Vc concentration in the sample to be tested can be obtained according to the above standard curve.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of nanomaterials in detection of tea polyphenols and ascorbic acid, comprising: (1) 1 mg of nanomaterials is added to a sodium acetate-acetic acid buffer solution containing 10 mmol / L of 3,3',5,5'-tetramethylbenzidine with a volume of 30-2000 μL, the volume of the reaction system is 2800 μL, after 0.1-1 h of dark reaction, 200 μL of 0.1-300 mmol / L tea polyphenols or ascorbic acid solution is added, and the absorbance at 652 nm is tested by using a UV-visible spectrophotometer, and the absorbance value of tea polyphenols or ascorbic acid concentration is used as a standard curve; (2) a sample solution containing tea polyphenols or ascorbic acid is added to a sodium acetate-acetic acid buffer solution containing 3,3',5,5'-tetramethylbenzidine, after 0.1-1 h of dark reaction, the absorbance at 652 nm is tested by using a UV-visible spectrophotometer, and the 3,3',5,5'-tetramethylbenzidine concentration, sodium acetate-acetic acid buffer solution pH and reaction time are synchronous steps (1) to establish a standard curve reaction system; (3) the tea polyphenols or ascorbic acid concentration in the sample to be tested can be obtained according to the above standard curve; wherein: the nanomaterials in step (1) are prepared by the following method: ① 1-10 g / L of Pichia pastoris is used as an adsorbent to adsorb in a palladium ion solution with a concentration of 100-1000 mg / L and a volume of 10-1000 mL, the adsorption rotation speed is 100-300 rpm, the temperature is 10-50 °C, and the time is 3-12 h; ② the microorganism mass adsorbed with palladium ions is solid-liquid separated by using a filter membrane with a pore size of 0.22 μm or 0.45 μm; ③ the separated microorganism mass is resuspended in 10-1000 mL of deionized water, and sodium formate is added dropwise for reduction, the reduction reaction is carried out in a shaking bed, the final concentration of sodium formate is 1-100 mmol / L, and after washing, a nanomaterial for detecting tea polyphenols and ascorbic acid by spectrophotometry is obtained, and the microorganism mass is loaded with 2.0-4.0 nm nanoparticles on the surface.

2. The application according to claim 1, wherein: the volume of 3,3',5,5'-tetramethylbenzidine in step (1) is 1500 μL.

3. The application according to claim 1, wherein: the pH of the sodium acetate-acetic acid buffer solution in step (1) is 4.0, and the volume is 1300 μL; the dark reaction time is 0.5 h.

4. The application according to claim 1, wherein: the concentration of the palladium ion solution in step ① is 500 mg / L, and the volume is 100 mL; the amount of the adsorbent used is 1 g / L; the adsorption conditions are: rotation speed 180 rpm, temperature 30 °C, and time 6 h.

5. The application according to claim 1, wherein: the volume of deionized water in step ③ is 100 mL; the final concentration of sodium formate is 20 mmol / L.

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