CN113358592B - Procyanidine detection method based on sodium alginate-platinum nanoparticle oxidase activity - Google Patents
Procyanidine detection method based on sodium alginate-platinum nanoparticle oxidase activity Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Abstract
The invention discloses a method for detecting procyanidine based on sodium alginate-platinum nanoparticle oxidase activity. The invention relates to an ultraviolet visual analysis method for rapidly determining procyanidine based on oxidase activity of sodium alginate-platinum nano particles. Under the optimal color development condition, the detection linear range is 4-32.5 mu mol/L, the lowest detection limit is 2.0 mu mol/L, and the detection sensitivity is high; the correlation coefficient is 0.999, and the detection linearity is good; the relative standard deviation RSD is 0.6%, and the precision of the detection result is high. In addition, the platinum nano-particles used in the invention are simple and convenient to prepare, and the substrate TMB is cheap and easy to obtain and has strong operability. Meanwhile, the invention has the advantages of high stability, good reproducibility, strong specificity and the like, opens up a new path for the measurement of procyanidine, and is expected to be applied to the fields of screening, identification, research and analysis of flavonoid substances and the like.
Description
Technical Field
The invention provides a simple and feasible colorimetric analysis method for detecting procyanidins, which is an ultraviolet visible analysis method for rapidly detecting procyanidins based on oxidase activity of sodium alginate-platinum nano particles, and belongs to the technical fields of analytical chemistry and nano.
Background
Procyanidins, also known as condensed tannins, are a general term for a large class of polyphenols widely existing in various plants such as grapes, blackcurrants, mulberries, blueberries, pine bark, ginkgo biloba and the like. In addition to plants, many food-derived procyanidins are also available in daily drinks such as apple juice and red wine, and the oligomer, i.e. the oligomeric procyanidins, is a currently internationally recognized natural antioxidant effective in scavenging free radicals in human bodies. Procyanidins have strong biological activity and have unique effects in preventing and treating diseases and maintaining beauty and keeping young. Furthermore, the procyanidine has the functions of reducing blood fat, lowering blood pressure, resisting cancer, resisting radiation and the like. Therefore, the procyanidine is expected to play an important role in the fields of clinical treatment, food health care, skin care, beauty and the like.
However, due to the very complex composition and structure of procyanidins, a unified method for determining procyanidin content has not been formed internationally. Currently, the quantitative analysis method of procyanidins mainly comprises spectrophotometry and high performance liquid chromatography, and specifically comprises the following steps: sulfuric acid-vanillin method, ammonium molybdate method, n-butanol-hydrochloric acid-HPLC method and thiolysis-HPLC method. The analysis purposes of these methods are different, the quantitative analysis principles are different, and the reference substances used are generally different, but according to the prior art, no systematic evaluation and comparison for different methods are found. Moreover, the detection process of the method is complex and time-consuming, the technical requirement is high, the required instrument is expensive, and the method cannot be widely applied to the detection of procyanidine products. Therefore, a simple and efficient detection and analysis method is established for evaluating the oxidation resistance of procyanidins, is beneficial to medicine quality control and consumer guidance, and has important significance.
The invention adopts sodium alginate-platinum nano particles with good oxidase activity. Because the sodium alginate-platinum nano particles have high-efficiency oxidase activity on the substrate 3, 3', 5, 5' -tetramethyl benzidine hydrochloride (TMB), the oxidation of TMB can be rapidly catalyzed to develop blue, and meanwhile, based on the oxidation resistance of procyanidine, the oxidation of TMB can be inhibited, so that the progress of a color reaction is inhibited, and therefore, a colorimetric detection method for measuring procyanidine is constructed. The invention has the advantages of good stability, high sensitivity, good reproducibility, low cost and the like, opens up a new path for measuring the procyanidine actual sample, and is expected to be further applied to the field of screening and researching of flavonoid substances.
Disclosure of Invention
The invention aims to construct a color development system for inhibiting oxidation of sodium alginate-platinum nanoparticles on 3, 3', 5, 5' -tetramethyl benzidine hydrochloride (TMB) by procyanidine based on good oxidase activity of sodium alginate-platinum nanoparticles and aiming at oxidation resistance of procyanidine. Along withThe absorbance of the color development system at the maximum ultraviolet absorption wavelength of 450 nm is continuously reduced due to the increase of the procyanidine content. Then according to the absorbance difference delta A of the color development system at the wavelength 450 nm 450 （ΔA 450 = A 0 - A t Wherein A is 0 And A t The absorbance of the reaction product at 450 nm) for the final concentration of procyanidins, respectively, when procyanidins are absent and corresponding concentrations of procyanidins are present, a standard working curve is drawn based on a 10-base logarithm of the final concentration of procyanidins, so that the actual sample of procyanidins can be quantitatively detected.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a procyanidine detection method based on sodium alginate-platinum nanoparticle oxidase activity is characterized in that sodium alginate with good biocompatibility and no toxicity is used as a protective agent of a nano material to prepare sodium alginate-platinum nanoparticle, and the synthesized material can rapidly catalyze dissolved oxygen to generate O under mild conditions 2 •− And oxidizing 3, 3', 5, 5' -tetramethyl benzidine hydrochloride (TMB) to generate color reaction, and detecting procyanidins by utilizing good intrinsic analog oxidase activity.
The sodium alginate-platinum nanoparticles used were prepared by the following method: weighing 0.1 g sodium alginate, dissolving in acetic acid with concentration of 1 v/v% of 50 mL, stirring for 15 min to completely dissolve sodium alginate to obtain 0.2 m/v% sodium alginate solution; and adding chloroplatinic acid with the concentration of 2 mL of 10 mmol/L into 47 mL of 0.2 m/v% sodium alginate solution, stirring in a vortex manner in a dark place, then dropwise adding 1 mL of newly prepared sodium borohydride solution with the concentration of 0.2 mol/L, and stirring in a dark place after finishing the addition within 5 min to obtain sodium alginate-platinum nano particles, wherein the mass concentration of platinum element is 78.03 mg/L.
In the presence of dissolved oxygen in a solvent and without the participation of hydrogen peroxide, the sodium alginate-platinum nanomaterial can catalyze O 2 Generated O 2 •− And the generated O 2 •− Can react with ascorbic acid to be eliminated, so that the ultraviolet characteristic absorption peak of the ascorbic acid at 250 nm is disappeared, which is oneNovel oxidation mimetic enzymes.
Cathode O in the presence of oxygen 2 The reduction peak appears at-0.34V; under the condition of introducing nitrogen, no peak appears after oxygen is removed; this shows that sodium alginate-platinum nanoparticle is a novel oxidation mimic enzyme capable of catalyzing oxygen to undergo oxidation-reduction reaction.
Several factors influencing the color reaction, including the pH value of the reaction system, the temperature of the reaction system, the substrate concentration of the reaction system, etc., are optimized one by utilizing a single-factor controlled variable method to determine the influence of the external conditions of the oxidation reaction; by measuring absorbance A at 450 nm 450 To determine optimal conditions for facilitating color reactions; for the constructed sodium alginate-platinum nanoparticle-TMB color development system, the optimal reaction conditions are as follows: 50 The pH of the mmol/L phosphoric acid buffer solution was 4.5, the reaction temperature of the reaction system was 37℃and the reaction time was 5 minutes, and the concentration of the substrate 3, 3', 5, 5' -tetramethylbenzidine hydrochloride (TMB) solution was 0.15 mmol/L.
The method for detecting the procyanidine based on the activity of the sodium alginate-platinum nanoparticle oxidase is characterized by comprising the step that the absorbance value of a color development system at 450 and nm is obviously reduced along with the increase of the concentration of the added procyanidine; when the concentration of procyanidine in the reaction system reaches 1 mmol/L, the color reaction is almost completely inhibited; therefore, the procyanidine can inhibit the oxidase catalytic activity of the sodium alginate-platinum nanoparticle solution to a certain extent, and the concentration of the procyanidine can be primarily judged by naked eyes according to the color depth of the solution of the color development system.
The procyanidine detection method based on the sodium alginate-platinum nanoparticle oxidase activity is characterized in that the procyanidine detection method is based on the absorbance difference delta A of a color development system at the wavelength of 450 nm 450 Drawing a standard working curve for the logarithmic value of the final concentration of the procyanidine based on 10, thus realizing quantitative detection of the procyanidine actual sample, wherein the delta A 450 = A 0 - A t Wherein A is 0 And A t Is prepared from the raw materials without procyanidine and in the presence of procyanidineAbsorbance of the reaction product at 450 nm; the standard working curve has good linearity and delta A within the range of 4-32.5 mu mol/L of procyanidine concentration 450 = 1.1791·lgC OPC + 3.1941, correlation coefficient of 0.999, relative standard deviation RSD of 0.6% at OPC concentration of 10 μmol/L, n=9, and lowest detection limit of 2.0 μmol/L.
The method for detecting procyanidine based on sodium alginate-platinum nanoparticle oxidase activity is characterized in that 1 mmol/L of ascorbic acid is used for defining the antioxidation capacity of a detection system as 1U, and finally the antioxidation capacity of procyanidine in a grape seed sample is 2.85U/mg; then 10 mu mol/L, 15 mu mol/L and 20 mu mol/L of procyanidine standard substance solutions with different concentrations are selected, respectively added into sample solutions, uniformly mixed and then added into a color development system, the measurement steps are repeated to respectively measure the absorbance values of the procyanidine standard substance solutions, and the obtained absorbance values are substituted into a standard curve to obtain the labeled recovery rate of procyanidine in grape seed samples; under the standard adding level of three different concentrations, the standard adding recovery rate of procyanidine in grape seeds is 97.0-98.6%, the standard range required by 95-105% and the relative standard deviation is 0.5-3.4%.
The invention adopts the following technical scheme:
preparation of sodium alginate-platinum nano particles:
(1) Accurately weighing sodium alginate 0.1 g, adding into acetic acid solution (prepared from 500 mL glacial acetic acid and 4500 mL double distilled water) with concentration of 1% (v/v) 50 mL under stirring, and stirring thoroughly until sodium alginate is completely dissolved (stirring for about 15 min).
(2) The mixed solution of 3 mL was removed by a pipette, and chloroplatinic acid (H) was removed at a concentration of 0.01 mol/L in an amount of 2 mL 2 PtCl 6 ) The aqueous solution was added to the sodium alginate solution having the above concentration of 0.2% (m/v) and stirred uniformly (about 0.5. 0.5 h stirred at room temperature), and the resulting solution was pale yellow.
(3) Then 1 mL sodium borohydride solution with the concentration of 0.07 mol/L is removed and is dropwise added into the yellowish solution (NaBH) under vigorous stirring 4 The solution was freshly prepared and added within 5 min). With NaBH 4 The color of the solution gradually becomes dark, the solution is finally changed into a tan solution from light yellow, and then the solution is continuously stirred at a light-proof place for 1.5 to h, so that the sodium alginate modified platinum nano-particles are prepared (all glassware is washed by aqua regia and washed by double distilled water and dried before being used), and the prepared product is stored at the temperature of 4 ℃ in the light-proof place).
(II) detection of procyanidins in actual samples
Accurately weighing a proper amount of procyanidine standard substance, accurately preparing procyanidine solutions with a series of concentrations (0, 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.65, 0.7 and 0.8 mmol/L) by taking water as a solvent (the operation steps are carried out in a dark place). Then 50. Mu.L of procyanidine solution with different concentrations is added into 870. Mu.L of phosphoric acid buffer solution with the concentration of 50 mmol/L and the pH of 4.5, and a reaction system of 1 mL is formed by 50. Mu.L of TMB solution with the concentration of 3 mmol/L and 30. Mu.L of sodium alginate-platinum nanoparticle solution (obtained by diluting the platinum nanomaterial solution prepared in the following technical scheme 1 by 5 times). Immediately placing in a constant temperature water bath at 37deg.C for reaction for 5 min, adding 200 μL sulfuric acid with concentration of 2 mol/L to terminate the reaction, measuring ultraviolet absorbance at 450 nm, and passing through color development system absorbance difference DeltaA 450 A standard curve can be obtained by fitting a linear curve to the logarithmic value of the final concentration of procyanidine based on 10.
Similarly, a proper amount of ascorbic acid reagent is weighed, a solution with a certain concentration is prepared, 50 mu L of the solution is removed and added into 870 mu L of phosphoric acid buffer solution (the concentration of the solution is 50 mmol/L, the pH value is 4.5), and then 50 mu L of TMB solution with a concentration of 3 mmol/L and 30 mu L of sodium alginate-platinum nanoparticle solution (the platinum nanomaterial solution prepared in the technical scheme 1 is diluted 5 times and obtained by-15.61 mg/L) are added to form a reaction system of 1 mL. And reacted at 37℃for 5 min. After the reaction was quenched with acid, its UV absorbance at 450 nm was measured. The absorbance of the reaction product obtained at this time was compared with the absorbance obtained after the addition of no procyanidine (blank absorbance value) and the unit of enzyme activity U was defined (ascorbic acid is an internationally recognized substance having antioxidant activity, and herein, 1 mmol/L of ascorbic acid was defined as 1U against the oxidation resistance of the detection system).
The invention relates to a method for measuring procyanidine in procyanidine reference substances (extracted sources: seeds of grape of Vitaceae). Firstly, a proper amount of a sample to be measured is removed, the accurate measurement is carried out, and three distilled water is used for dissolving to prepare a solution to be measured with a certain concentration. Then, 50. Mu.L of the solution to be tested was removed and added to 870. Mu.L of the phosphate buffer solution (concentration: 50 mmol/L, pH=4.5). Then 50. Mu.L of TMB (3 mmol/L) solution and 30. Mu.L of sodium alginate-platinum nanoparticle solution (obtained by diluting the platinum nanomaterial solution prepared in the technical scheme 1 by 5 times, the mass concentration of platinum is 15.61 mg/L) are added into the buffer system for reaction (the reaction solution is immediately placed in a water bath at 37 ℃ C. For 5 min after shaking). After the acid addition, the absorbance was measured at a wavelength of 450 nm. And then, according to the drawn standard curve, carrying the measured absorbance into a fitting equation to obtain the concentration of the actual sample.
Labeling recovery rate of procyanidine in grape seeds
Weighing a proper amount of actual sample to prepare a grape seed sample solution with a certain concentration. The method comprises the steps of selecting procyanidine standard substance solutions with different concentrations, respectively adding the procyanidine standard substance solutions into sample solutions, uniformly mixing the sample solutions, adding the sample solutions into a color development system, respectively measuring absorbance values of the sample solutions by repeating the measuring steps of the technical scheme 2, substituting the absorbance values into a standard curve to obtain the recovery concentration of procyanidine in grape seed samples, and calculating to obtain the labeled recovery rate, so that the feasibility of detecting procyanidine in grape seeds is further evaluated.
The invention has the advantages that:
the invention uses sodium alginate with good biocompatibility and no toxicity as a protective agent of nano materials to prepare sodium alginate-platinum nano particles, and the synthesized materials can rapidly catalyze dissolved oxygen to generate O under mild conditions 2 •− And oxidize TMB to produce color reaction with good intrinsic pseudo-oxidase activity. Based on this, according to the original flowerThe antioxidant capacity of the green element establishes a colorimetric analysis method for measuring the procyanidine. Difference delta A of absorbance of color development system 450 Linear relationship with the logarithmic value of the procyanidin concentration based on 10. Under the optimal experimental condition, the linear range is 4-32.5 mu mol/L, the correlation coefficient is 0.999, the RSD is 0.6% (OPC concentration is 10 mu mol/L, n=9), and the lowest detection limit is 2.0 mu mol/L. The sodium alginate-platinum nano-particles needed by each detection are small in dosage, the chromogenic substrate (3, 3', 5, 5' -tetramethyl biphenyl amine hydrochloride) is common and easy to obtain, and the detection cost is low. Meanwhile, the method is used for detecting the procyanidine, has good linearity, is simple and easy to operate, has high sensitivity and good stability, opens up a new path for measuring the procyanidine actual sample, and is expected to be further applied to the field of screening and researching of flavonoid substances.
Drawings
FIG. 1 is an ultraviolet-absorption spectrum of a sodium alginate-platinum nanoparticle-TMB color development system of the invention.
Fig. 2 is an ultraviolet absorbance spectrum of ascorbic acid in acetate buffer of the invention (ph=4) after 5 min incubation under different conditions.
FIG. 3 is a cyclic voltammogram of the sodium alginate-platinum nanoparticle modified electrode redox reaction of the present invention in an aerobic and nitrogen environment, respectively.
FIG. 4 shows the effect of pH of the reaction system of the present invention on the color development system of sodium alginate-platinum nanoparticle catalyzed oxidation 3, 3', 5, 5' -tetramethylbenzidine hydrochloride.
FIG. 5 is the effect of the temperature of the reaction system of the present invention on the color development system of sodium alginate-platinum nanoparticle catalyzed oxidation of 3, 3', 5, 5' -tetramethylbenzidine hydrochloride.
FIG. 6 is a graph showing the effect of chromogenic substrate concentration on sodium alginate-platinum nanoparticle catalyzed oxidation of 3, 3', 5, 5' -tetramethylbenzidine hydrochloride chromogenic system.
FIG. 7 shows the inhibition effect of procyanidins with different concentrations on a sodium alginate-platinum nanoparticle-TMB color development system.
FIG. 8 shows the present inventionAbsorbance difference Δa of color system 450 A graph of linear relationship with the base 10 log procyanidin concentration.
Detailed Description
The invention provides a rapid colorimetric analysis method for procyanidine, and relates to a sodium alginate-platinum nanoparticle-TMB color development system. Procyanidine can inhibit chromogenic system, or scavenge oxygen free radical O generated by sodium alginate-platinum nanoparticle catalyzed dissolved oxygen 2 •− Or compete with the substrate TMB for binding sites for the mimic enzyme, thereby inhibiting the color reaction of the chromogenic system and thereby altering the uv absorbance value of the solution at 450 nm. Adding a series of standard samples containing procyanidins with different concentrations into a system for reaction, and according to ultraviolet absorbance A of a solution of a color development system 0 (blank) and A t The difference value (containing procyanidine) can be used for drawing a standard curve, so that the procyanidine can be detected. The technical scheme of the detection method of the invention is further described below with reference to the accompanying drawings and a plurality of embodiments.
Example 1:
the sodium alginate-platinum nanoparticle is prepared by taking sodium alginate as a protective agent and reducing chloroplatinic acid under the action of sodium borohydride. Firstly, sodium alginate 0.1. 0.1 g was weighed accurately, added to an acetic acid solution (prepared from 500 mL glacial acetic acid and 4500. 4500 mL double distilled water) with a concentration of 1% (v/v) of 50 mL under stirring, and stirred well until sodium alginate was completely dissolved (stirring about 15 min). Then, the mixed solution of 3 mL was removed by a pipette, and chloroplatinic acid (H) having a concentration of 2 mL of 0.01 mol/L was removed 2 PtCl 6 ) The aqueous solution was added to the sodium alginate solution having the above concentration of 0.2% (m/v) and stirred uniformly (about 0.5. 0.5 h stirred at room temperature), and the resulting solution was pale yellow. Finally, 1. 1 mL sodium borohydride solution with a concentration of 0.07 mol/L was removed and dropped dropwise into the pale yellow solution (NaBH) with vigorous stirring 4 The solution was freshly prepared and added within 5 min). With NaBH 4 Continuously dripping, gradually darkening, and finally changing into brown solution from yellowish to darkStirring is continued for 1.5 and h, and then the platinum nano particles modified by sodium alginate are prepared (all glassware is washed by aqua regia and washed by double distilled water and dried before being used, and the prepared product is stored in a dark place at the temperature of 4 ℃).
Example 2:
the sodium alginate-platinum nanomaterial solution prepared in example 1 is taken to react with 3, 3', 5, 5' -tetramethylbenzidine hydrochloride (TMB) solution with a certain concentration in an EP tube containing phosphate buffer. Incubating the mixed solution in a constant-temperature water bath at 37 ℃ for 5 min, and then sweeping the spectrum of the reaction product to obtain an ultraviolet-absorption spectrum; reacting again under the conditions, stopping the reaction with 200 mu L of sulfuric acid with the concentration of 2 mol/L immediately after the mixed solution is subjected to constant-temperature water bath for 5 min, sweeping a spectrum, and determining the maximum absorption wavelength; in addition, control experiments were performed: the reaction conditions are unchanged, sodium alginate and chloroplatinic acid are respectively used for replacing sodium alginate-platinum nano particles to be added into a reaction system, and an ultraviolet-absorption spectrum is measured by an ultraviolet spectrophotometer. As shown in the inset of fig. 1, in the presence of sodium alginate-platinum nanosolvent, the solution changed from colorless to blue, and a characteristic absorption peak appeared at 652 nm wavelength (fig. 1), with no color development in the blank. Proves that the sodium alginate-platinum nano solution can oxidize chromogenic substrates 3, 3', 5, 5' -tetramethyl benzidine hydrochloride (TMB) without the participation of hydrogen peroxide, and is a novel simulated oxidase; the sulfuric acid-added reaction solution turns from blue to yellow (fig. 1, inset), and has a strong characteristic absorption peak at 450 nm wavelength (fig. 1); for the control group: sodium alginate and chloroplatinic acid are respectively used for replacing sodium alginate-platinum nano particles to be added into a reaction system, under the same condition, the sodium alginate and chloroplatinic acid do not cause a substrate to generate a color reaction, and a product sweep spectrum does not generate an obvious absorption peak, which indicates that the material components do not have catalytic activity when being singly present, and further proves that the oxidase-like activity of the material is derived from a nanocomposite.
Example 3:
the sodium alginate-platinum nanomaterial solution prepared in example 1 (platinum mass concentration 78.03/mg/L) was taken in an EP tube containing acetate buffer (pH=4)Adding 250 mu mol/L ascorbic acid solution, uniformly mixing, placing the EP tube in a constant-temperature water bath at 37 ℃ for incubation for 5 min, and measuring the ultraviolet-absorption spectrum by an ultraviolet spectrophotometer; then, the sodium alginate-platinum nanomaterial solution prepared in the technical scheme 1 is taken in an EP tube containing acetic acid buffer (ph=4), and nitrogen 2 h is introduced to remove oxygen. Then adding 250 mu mol/L ascorbic acid solution, uniformly mixing, placing the EP tube in a constant-temperature water bath at 37 ℃ for incubation for 5 min, and sweeping an ultraviolet visible absorption spectrum; in addition, the buffer solution is used for replacing sodium alginate-platinum nano material solution, and the ultraviolet visible-absorption spectrum of the ascorbic acid is scanned by the same method. As shown in fig. 2, ascorbic acid exhibits a characteristic absorption peak at 250 nm in the detection wavelength range; when nitrogen is introduced into the solution to remove oxygen, only sodium alginate-platinum nano solution and ascorbic acid coexist, and at the moment, the absorption peak of the ascorbic acid is slightly changed, namely, the ascorbic acid is slightly oxidized; when oxygen and sodium alginate-platinum nanomaterial solution exist in the solution, an absorption peak does not appear in the ultraviolet spectrogram, which indicates that the ascorbic acid is completely consumed, namely O generated by the catalysis of dissolved oxygen by the sodium alginate-platinum nanomaterial solution 2 •− The reaction with ascorbic acid and the elimination by ascorbic acid prove that sodium alginate-platinum nanoparticles in example 1 are a novel oxidation mimetic enzyme.
Example 4:
cyclic voltammetry (scanning rate 50 mV/s, KOH concentration 0.1 mol/L) in an aerobic and nitrogen-passing environment, respectively, by oxidation-reduction reaction on an electrode modified with sodium alginate-platinum nanoparticles. As shown in fig. 3, in the presence of oxygen, cathode O 2 The reduction peak appears at-0.34V. In the case of nitrogen, no peak appears after oxygen removal. These results demonstrate that the sodium alginate-platinum nanoparticle prepared in example 1 exhibits an inherent oxidase-like activity and is capable of catalyzing the oxidation-reduction reaction of oxygen, which further demonstrates the conclusion that the sodium alginate-platinum nanoparticle solution obtained in example 1 has a simulated oxidase activity.
Example 5:
the sodium alginate-platinum nanomaterial solution prepared in example 1 and the substrate TMB solution are respectively reacted in a series of phosphoric acid buffer solutions with different pH values (the pH values are respectively 2, 3, 4, 4.5, 5, 6, 7 and 8, the concentration is 50 mmol/L), the reaction temperature is 37 ℃, and the temperature is kept constant for 5 min. Thereafter, 200. Mu.L of H having a concentration of 2 mol/L was added 2 SO 4 To terminate the reaction, and then to measure the absorbance A at 450 nm 450 . The relative ratio of absorbance was plotted against pH to determine the optimum pH. (sodium alginate-platinum nanoparticle is diluted by 10 times with phosphate buffer solution with corresponding pH value in advance, and then 30 mu L of the sodium alginate-platinum nanoparticle, 920 mu L of 50 mmol/L of phosphate buffer solution and 50 mu L of TMB with 3 mmol/L of TMB form a reaction system of 1 mL) As can be seen from FIG. 4, the pH has a significant influence on a color development system, and the pH of the phosphate buffer solution is optimized for better detection of procyanidins. The enzymatic activity of the sodium alginate-platinum nanoparticle reaches the highest value at the pH value of 4.5, so that the optimal pH value of the color development system is 4.5.
Example 6:
30. Mu.L of sodium alginate-platinum nanomaterial solution (prepared by diluting the sodium alginate-platinum nanomaterial solution prepared in example 1 with a phosphate buffer solution having a pH of 4.5 and a concentration of 50 mmol/L by a factor of 10) was added to an EP tube containing 920. Mu.L of the phosphate buffer solution (50 mmol/L, pH=4.5) and reacted with 50. Mu.L of TMB solution having a concentration of 3 mmol/L at constant temperature in a water bath for 5 minutes. (A series of reaction temperatures of 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ respectively) then 200. Mu.L of H at a concentration of 2 mol/L was added 2 SO 4 To terminate the reaction and then measure the absorbance A at 450 nm 450 . The temperature is plotted as the relative ratio of absorbance to determine the optimum reaction temperature. As shown in FIG. 5, A is a reaction temperature increases 450 And also increases with it; the best color reaction effect is achieved when the temperature is 37 ℃; when the temperature continues to rise, A 450 And has a descending trend. Thus, the present invention employs 37 ℃ as the optimal reaction temperature.
Example 7:
mu.L of sodium alginate-platinum nanomaterial solution (prepared in example 1 by diluting the sodium alginate-platinum nanomaterial solution with a phosphate buffer solution having a pH of 4.5 and a concentration of 50 mmol/L by a factor of 10) was added to an EP tube containing 920. Mu.L of the phosphate buffer solution (50 mmol/L, pH=4.5). In the same manner, several portions were taken and incubated with 50. Mu.L of TMB solutions of different concentrations (0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mmol/L, respectively) at 37℃for 5 min. Then, H was added at a concentration of 200. Mu.L at 2 mol/L 2 SO 4 The reaction was stopped and its absorbance A at 450 nm was measured 450 . The optimal substrate concentration values in the series of concentrations were determined by plotting the relative ratio of absorbance versus TMB concentration. As can be seen from fig. 6, as the TMB concentration increases, so does a 450; when the TMB concentration is greater than 0.15 mmol/L, the A450 changes slowly. Thus, the present invention selects TMB at 0.15 mmol/L as the optimal concentration for the color system.
Example 8:
accurately weighing a proper amount of procyanidine standard substance, and accurately preparing procyanidine solutions with different concentrations (0, 0.08, 0.2, 0.5 and 0.7 mmol/L) by using triple distilled water as a solvent (light-shielding operation). Then 50. Mu.L of procyanidin solution with different concentrations is added into EP containing 870. Mu.L of phosphoric acid buffer solution (50 mmol/L, pH=4.5) respectively, and a reaction system of 1 mL is formed by 50. Mu.L of TMB solution (3 mmol/L) and 30. Mu.L of sodium alginate-platinum nanoparticle solution (prepared in example 1, the sodium alginate-platinum nanoparticle solution is diluted 5 times by phosphoric acid buffer solution with pH value of 4.5 and concentration of 50 mmol/L). Immediately after rapid and thorough mixing, the mixture was placed in a thermostatic water bath at 37 ℃ for 5 min of incubation. After the reaction is finished, 2 mmol/L sulfuric acid is added to stop the reaction, and then an ultraviolet visible-absorption spectrum is measured by an ultraviolet spectrophotometer. As shown in fig. 7, the absorbance value of the color development system at 450 nm decreased significantly with increasing concentrations of procyanidins added. When the concentration of procyanidine in the reaction system reached 1 mmol/L, the color reaction was almost completely suppressed. Therefore, procyanidins can inhibit the oxidase catalytic activity of sodium alginate-platinum nanoparticle solution to a certain extent.
Example 9:
accurately weighing a proper amount of procyanidine standard substance, and accurately preparing procyanidine solutions (operating in dark place) with a series of concentrations (0, 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.65, 0.7 and 0.8 mmol/L) by using triple distilled water as a solvent. Then 50 mu L of procyanidine solution with different concentrations are respectively added into each EP tube containing 870 mu L of phosphoric acid buffer solution (50 mmol/L, pH=4.5), and a reaction system of 1 mL is formed by 50 mu L of TMB solution with the concentration of 3 mmol/L and 30 mu L of sodium alginate-platinum nanoparticle solution diluted 5 times. After rapid and full mixing, the mixture is immediately placed in a constant-temperature water bath at 37 ℃ for reaction for 5 min, acid is added to stop the reaction after the completion, the absorbance at the position of 450 nm is measured, and the absorbance difference delta A450 (delta A 450 = A 0 - A t Wherein A is 0 And A t The absorbance of the reaction product at 450 nm) plotted against the final concentration of procyanidins, with 10 base log, was fitted to a standard working curve, without procyanidins and in the presence of procyanidins at the corresponding concentrations, respectively. As shown in FIG. 8, under the optimal color development condition, the standard working curve obtained by the method has good linearity (delta A) within the range of 4-32.5 mu mol/L of procyanidine concentration 450 = 1.1791·lgC OPC + 3.1941), the correlation coefficient was 0.999, the relative standard deviation RSD was 0.6% (OPC concentration 10 μmol/L, n=9), and the lowest detection limit was 2.0 μmol/L.
Example 10:
weighing a proper amount of actual sample, and dissolving with three distilled water to prepare grape seed sample solution with a certain concentration. Then, 50. Mu.L of the solution to be tested was removed and added to 870. Mu.L of the phosphate buffer solution (50 mmol/L, pH=4.5). Then, 50. Mu.L of TMB (3 mmol/L) solution and 30. Mu.L of sodium alginate-platinum nanoparticle solution (obtained by diluting the platinum nanomaterial solution prepared in example 15 times-platinum mass concentration: 15.61. 15.61 mg/L) were added to the buffer system to perform a reaction (the reaction solution was immediately placed in a 37 ℃ C. Environment for 5 minutes after shaking). After the acid addition, the absorbance was measured at a wavelength of 450 nm. And then, according to the drawn standard curve, carrying the measured absorbance into a fitting equation to obtain the concentration of the actual sample.
An appropriate amount of ascorbic acid reagent is weighed, a solution with a certain concentration is prepared, 50 mu L of the solution is removed and added into 870 mu L of phosphoric acid buffer solution (50 mmol/L, pH=4.5), and 50 mu L of TMB solution with a concentration of 3 mmol/L and 30 mu L of sodium alginate-platinum nanoparticle solution are added to form a reaction system of 1 mL. And reacted at 37℃for 5 min. After the reaction was quenched with acid, its absorbance at 450 nm was measured. The absorbance of the reaction product measured this time was compared with the absorbance (blank absorbance value) measured after no procyanidine was added, and the enzyme activity unit U was defined. (1 mmol/L of ascorbic acid was defined as 1U for the oxidation resistance of the test system for evaluation of procyanidins in grape seed samples.)
And selecting procyanidine standard substance solutions (10 mu mol/L, 15 mu mol/L and 20 mu mol/L) with different concentrations, respectively adding the procyanidine standard substance solutions into the sample solutions, uniformly mixing the procyanidine standard substance solutions, adding the procyanidine standard substance solutions into a color development system, repeatedly measuring the absorbance values of the procyanidine standard substance solutions, respectively measuring the absorbance values of the procyanidine standard substance solutions, substituting the absorbance values into a standard curve to obtain the procyanidine recovery concentration in grape seed samples, and calculating to obtain the labeled procyanidine recovery rate in grape seeds. Finally, the antioxidant capacity of procyanidine in the grape seed sample is 2.85U/mg. From Table 1, it can be seen that the standard recovery of procyanidins from grape seeds varies from 97.0% to 98.6% over the standard range required for 95-105% at three different concentration levels, with a relative standard deviation in the range of 0.5-3.4%.
Table 1: labeling recovery rate of procyanidine in grape seeds
Claims (5)
1. A procyanidine detection method based on sodium alginate-platinum nanoparticle oxidase activity is characterized by utilizing sea with good biocompatibility and no toxicitySodium alginate is used as a protective agent of nano materials to prepare sodium alginate-platinum nano particles, and the synthesized material can rapidly catalyze dissolved oxygen to generate O under mild conditions 2 •− Oxidizing 3, 3', 5, 5' -tetramethyl benzidine hydrochloride to generate color reaction, and detecting procyanidins by utilizing good intrinsic analog oxidase activity; the absorbance value of the color development system at 450 nm is obviously reduced along with the increase of the concentration of the added procyanidine; when the concentration of procyanidine in the reaction system reaches 1 mmol/L, the color reaction is almost completely inhibited; therefore, the procyanidine can inhibit the oxidase catalytic activity of the sodium alginate-platinum nanoparticle solution to a certain extent, and the concentration of the procyanidine can be primarily judged by naked eyes according to the color depth of the solution of a color development system;
according to the absorbance difference delta A of the color development system at the wavelength of 450 nm 450 Drawing a standard working curve for the logarithmic value of the final concentration of the procyanidine based on 10, thus realizing quantitative detection of the procyanidine actual sample, wherein the delta A 450 = A 0 - A t Wherein A is 0 And A t Absorbance of the reaction product at 450 nm in the absence of procyanidins and in the presence of procyanidins at the respective concentrations, respectively; the standard working curve has good linearity and delta A within the range of 4-32.5 mu mol/L of procyanidine concentration 450 = 1.1791·lgC OPC + 3.1941, correlation coefficient of 0.999, relative standard deviation RSD of 0.6% at OPC concentration of 10 μmol/L, n=9, minimum detection limit of 2.0 μmol/L;
the antioxidant capacity of 1 mmol/L ascorbic acid to the detection system is defined as 1U, and the antioxidant capacity of procyanidine in the grape seed sample is finally detected to be 2.85U/mg; then 10 mu mol/L, 15 mu mol/L and 20 mu mol/L of procyanidine standard substance solutions with different concentrations are selected, respectively added into sample solutions, uniformly mixed and then added into a color development system, the measurement steps are repeated to respectively measure the absorbance values of the procyanidine standard substance solutions, and the obtained absorbance values are substituted into a standard curve to obtain the labeled recovery rate of procyanidine in grape seed samples; under the standard adding level of three different concentrations, the standard adding recovery rate of procyanidine in grape seeds is 97.0-98.6%, the standard range required by 95-105% and the relative standard deviation is 0.5-3.4%.
2. The method for detecting procyanidine based on sodium alginate-platinum nanoparticle oxidase activity according to claim 1, wherein the sodium alginate-platinum nanoparticle used is prepared by the following method: weighing 0.1 g sodium alginate, dissolving in acetic acid with concentration of 1% (v/v) of 50 mL, stirring for 15 min to completely dissolve sodium alginate to obtain 0.2% (m/v) sodium alginate solution; and adding chloroplatinic acid with the concentration of 2 mL of 10 mmol/L into 47 mL of 0.2% (m/v) sodium alginate solution, stirring in a vortex manner in a dark place, then dropwise adding a newly prepared sodium borohydride solution with the concentration of 1 mL of 0.2 mol/L, and stirring in a dark place after finishing the addition within 5 min to obtain sodium alginate-platinum nano particles, wherein the mass concentration of platinum element is 78.03 mg/L.
3. The method for detecting procyanidine based on sodium alginate-platinum nanoparticle oxidase activity as claimed in claim 1 or 2, wherein sodium alginate-platinum nanomaterial can catalyze O in a solvent with dissolved oxygen and without hydrogen peroxide 2 Generated O 2 •− And the generated O 2 •− Can react with ascorbic acid to be cleared by the ascorbic acid, so that the ultraviolet characteristic absorption peak of the ascorbic acid at 250 nm disappears, and is a novel oxidation mimic enzyme.
4. The method for detecting procyanidine based on sodium alginate-platinum nanoparticle oxidase activity according to claim 1 or 2, wherein the method is characterized in that under the condition of oxygen, a cathode O 2 The reduction peak appears at-0.34V; under the condition of introducing nitrogen, no peak appears after oxygen is removed; this shows that sodium alginate-platinum nanoparticle is a novel oxidation mimic enzyme capable of catalyzing oxygen to undergo oxidation-reduction reaction.
5. Seaweed-based according to claim 1 or 2The method for detecting the procyanidine of the sodium-platinum nanoparticle oxidase activity is characterized in that several factors influencing the color reaction, including the pH value of a reaction system, the temperature of the reaction system, the substrate concentration of the reaction system and the like, are optimized one by utilizing a single-factor controlled variable method so as to determine the influence of external conditions of the oxidation reaction; by measuring absorbance A at 450 nm 450 To determine optimal conditions for facilitating color reactions; for the constructed sodium alginate-platinum nanoparticle-TMB color development system, the optimal reaction conditions are as follows: 50 The pH of the mmol/L phosphoric acid buffer solution was 4.5, the reaction temperature of the reaction system was 37℃and the reaction time was 5 minutes, and the concentration of the substrate 3, 3', 5, 5' -tetramethylbenzidine hydrochloride solution was 0.15 mmol/L.
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