CN110006885B - A method for quantitative analysis of alcohol based on dual-enzyme-inorganic nanoflower composites - Google Patents
A method for quantitative analysis of alcohol based on dual-enzyme-inorganic nanoflower composites Download PDFInfo
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
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- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
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Abstract
本发明公开了一种基于双酶‑无机纳米花复合材料的酒精定量分析方法。所述方法包括将醇氧化酶(AOX)、辣根过氧化物酶(HRP)、CaCl2溶液加入磷酸盐缓冲溶液中，室温静置反应，得到双酶‑无机纳米花复合材料(AOHNF)。反应条件简单，得到的材料酶活性和稳定性均显著增强。再利用AOX催化氧化溶液中酒精发生反应生成H2O2，HRP催化TMB与H2O2反应使溶液呈现蓝色，测定吸收波长在650nm处的吸光度，从而得出溶液中酒精浓度的大小。分析方法操作简单，检测结果灵敏度高。本发明所述的基于双酶‑无机纳米花复合材料的酒精定量分析方法具有操作简单、灵敏度高、稳定性好等特点，发展前景良好。
The invention discloses a method for quantitative analysis of alcohol based on a dual-enzyme-inorganic nano-flower composite material. The method includes adding alcohol oxidase (AOX), horseradish peroxidase (HRP), and CaCl 2 solution into a phosphate buffer solution, and allowing the reaction to stand at room temperature to obtain a dual-enzyme-inorganic nanoflower composite material (AOHNF). The reaction conditions are simple, and the enzymatic activity and stability of the obtained material are significantly enhanced. Then use AOX to catalyze the reaction of alcohol in the oxidation solution to generate H 2 O 2 , and HRP catalyzes the reaction of TMB and H 2 O 2 to make the solution appear blue, and measure the absorbance at 650nm to obtain the alcohol concentration in the solution. The analysis method is simple to operate and the detection result is highly sensitive. The alcohol quantitative analysis method based on the dual-enzyme-inorganic nanoflower composite material of the present invention has the characteristics of simple operation, high sensitivity, good stability and the like, and has good development prospects.
Description
Technical Field
The invention belongs to the field of quantitative analysis, and particularly relates to an alcohol quantitative analysis method based on a double-enzyme-inorganic nanoflower composite material.
Background
With the development of the times, the detection of alcohol compounds with high sensitivity, high selectivity and high accuracy is widely applied in many different fields, and the rapid, sensitive, selective and quantitative detection of alcohol substances is also of great significance to industries such as law enforcement, chemical engineering, pharmacy, fermentation and the like, wherein the precise and rapid determination of ethanol content is very important in clinical and forensic analysis. In addition, simple, rapid and economical methods of alcohol analysis are also used in the pulp, food and beverage industries to control the fermentation process and the quality of its products. In the processes of traffic enforcement, forensics, clinical analysis and the like, the commonly used alcohol detection technology is expiration detection, saliva detection and urine blood detection. The expiration method is a non-invasive, convenient and rapid diagnosis method, generally an expiration type alcohol detector is used for determination, and generally, the alcohol content in the breath is unstable, so that the breath is easily influenced by the expiration amount of a person to be determined, unabsorbed alcohol remained in the oral cavity and the like, and the determination result is wrong; the saliva rule is mainly to use an enzyme test strip for determination, and the method is quick, cheap and easy to operate and is generally applied to emergency departments; the urine test method is to detect the alcohol concentration of urine by extracting urine of a person concerned, and the blood test method is mainly to extract ethanol in blood to detect the content of the ethanol, and is generally used when accidents are handled and taken as a basis. The method for detecting the concentration of the alcohol in blood and urine by using the enzyme method has good accuracy, repeatability and specificity, but cannot detect on site, and only can be handed to a professional institution for indoor test, and if the concentration is not timely checked, a large error can occur.
In recent years, many methods for alcohol determination have been reported, such as colorimetry, chromatography, redox titration, refractive index method, and the like, wherein colorimetry and chromatography are relatively more applied methods. The conventional colorimetric method mainly uses potassium dichromate for determination, but has a disadvantage of not conforming to the "beer's law", in which chromium ions, an oxidation product, are green and mixed with yellow dichromate ions being measured in various concentrations, causing errors in colorimetric results, resulting in loss of stability of the determination results. The chromatography is used for detecting alcohol in blood or body fluid, and two technologies of headspace sampling and direct sample injection are commonly used for replacingThe method replaces the prior solvent extraction or distillation method, has high automation and stable and reliable measurement results, but has complex measurement process, is easy to cause pollution to injection ports, columns and the like, has long time consumption, and cannot meet the requirement of rapid quantitative measurement of alcohol. To overcome these disadvantages, NAD-based has emerged+Alcohol is analyzed and tested by various alcohol biosensors depending on alcohol dehydrogenase or alcohol oxidase, the methods mainly utilize the catalytic activity of the bio-enzyme on alcohol substances, and the methods are also successfully applied to monitoring alcohol criminals in criminal judicial law, medical supervision and alcohol determination in the fermentation process. Such sensors have high selectivity, but their associated use costs are high and do not adequately address the disadvantages of alcohol detection, such as low sensitivity, low stability and narrow linear operating range in practical applications. The biological enzyme has low stability, is not easy to store for a long time, and is volatile, which brings certain limitation to the method for detecting the alcohol by the biological enzyme.
Enzymes have been extensively studied as multifunctional tools due to their unique properties such as high reaction specificity, high catalytic activity and low toxicity, but natural free enzymes suffer from inherent disadvantages such as high preparation and purification costs, low operational stability, sensitive catalytic activity to environmental conditions and difficult recovery. In order to overcome these disadvantages, a concept of immobilizing enzymes has been reported, and embedding enzymes into nanomaterials has become a hot spot of research in recent years. Research shows that the enzyme immobilized on the nanometer material has the characteristics of wider pH and temperature adaptation range and higher thermal stability than natural enzyme. Since the nano material with a three-dimensional structure is superior to other nano materials in terms of surface area, reactivity, stability and the like, the three-dimensional nano material becomes a hot spot for researching enzyme immobilization in recent years.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a quantitative alcohol analysis method based on a double-enzyme-inorganic nanoflower composite material. The method mainly utilizes enzyme and inorganic material to form a flower-shaped nano structure, and double-enzyme-inorganic nano flower is constructed to measure alcohol. The reaction system relates to two enzymes, namely alcohol oxidase and horseradish peroxidase, belongs to a multi-enzyme cascade reaction, and has the main advantages that the active sites of the enzymes are close to each other in a nano material, the distance between the enzymes and reactants is reduced, the diffusion of enzyme intermediates in the reaction is minimized, and the reactant concentration around the enzymes is increased, so that the overall reaction efficiency and specificity are enhanced. According to the invention, inorganic phosphate is selected as a carrier to fix alcohol oxidase AOX and horseradish peroxidase HRP, so that a multi-enzyme co-immobilization reaction system is realized. Compared with free enzyme, the double-enzyme inorganic nanoflower has obviously enhanced enzyme activity and stability. Thereby improving the sensitivity of alcohol detection.
The purpose of the invention is realized by the following technical scheme:
a quantitative alcohol analysis method based on a double-enzyme-inorganic nanoflower composite material comprises the following steps:
step 1) adding an alcohol oxidase solution and a horseradish peroxidase solution into a phosphate buffer solution, and then adding CaCl2Mixing the solution, standing, centrifuging after the reaction is finished, removing supernatant, adding phosphate buffer solution, and uniformly mixing to obtain the double-enzyme-inorganic nanoflower composite material;
step 2) mixing the double-enzyme-inorganic nanoflower composite material prepared in the step 1) with a series of sample solutions with different alcohol concentrations, adding a color developing agent, performing ultraviolet-visible spectral scanning, recording an absorbance value, and taking the alcohol concentration as an abscissa and the absorbance value as an ordinate to obtain a standard curve;
and 3) obtaining the alcohol concentration of the measured sample according to a standard curve method.
Preferably, the concentration of the phosphate buffer solution of step 1) is 10 mM.
Preferably, the CaCl of step 1)2The concentration of the solution was 100 mM.
Preferably, the temperature of the reaction in step 1) is 30 ℃ and the reaction time is 48 h.
Preferably, the color developer in step 2) is TMB solution.
According to the alcohol quantitative analysis method based on the double-enzyme-inorganic nanoflower composite material, the alcohol oxidase and the horseradish peroxidase are co-immobilized on the inorganic phosphate framework to form the nanoflower-shaped structure, so that the contact area of reaction is effectively increased, the loss of an intermediate product is prevented to the maximum extent, and meanwhile, the larger enzyme activity is maintained. More specifically, the invention has the following beneficial effects:
(1) the preparation of the inorganic nanoflower composite material with multi-enzyme combination does not need any organic reagent at normal temperature and normal pressure, is simple to operate, has special material appearance and high specific surface area, and greatly improves the activity and stability of the enzyme.
(2) The alcohol detection can be completed only by an ultraviolet-visible spectrophotometer, complex operation is not needed, a large-scale instrument is not needed, and the alcohol detection is more convenient and practical.
(3) The linear range of the quantitative analysis of the alcohol measured by the experiment is 0.1-2.5 mg/mL, and in China, drunk driving is judged when the concentration of the alcohol in the body is more than or equal to 0.2mg/mL, drunk driving is judged when the concentration of the alcohol in the body is more than or equal to 0.8mg/mL, and the method can be well applied to accurate quantitative judgment of drunk driving.
Drawings
FIG. 1 is a diagram of a method for preparing a double enzyme-inorganic nanoflower composite (AOHNF);
FIG. 2 is an SEM image of a dual enzyme-inorganic nanoflower composite;
FIG. 3 is a visible spectrum obtained by quantitative analysis and detection of alcohol;
FIG. 4 is a graph showing the change of absorbance at 650nm with alcohol concentration measured by the alcohol quantitative analysis method.
Detailed Description
The technical solutions of the present invention are further described in detail with reference to the following specific embodiments, but it should be noted that the following embodiments are only used for describing the content of the present invention and do not limit the scope of the present invention.
Example 1
A quantitative alcohol analysis method based on a double-enzyme-inorganic nanoflower composite material comprises the following steps:
(1) preparing a double-enzyme-inorganic nano-flower composite material: as shown in FIG. 1, 15. mu.L each of 1.0mg/mL Alcohol Oxidase (AOX) solution and 1.0mg/mL horseradish peroxidase (HRP) was added to 1.41mL Phosphate Buffered Saline (PBS), and 168. mu.L 100mM CaCl was added2Adding the solution into the solution, mixing uniformly, standing, incubating at 30 ℃ for 48h, taking out, centrifuging at 10000r/min for 15min, removing supernatant, adding 150 μ L PBS, and mixing uniformly to obtain the double-enzyme-inorganic nanoflower composite material (AOHNF) required by the experiment.
As shown in fig. 2, the double-enzyme-inorganic nanoflower composite material is characterized by SEM, and the double-enzyme-inorganic nanoflower composite material is uniform nanoflower-shaped spheres with an average diameter of about 6 μm, uniform distribution, uniform morphology, good dispersibility, and a multilayer three-dimensional structure.
(2) And (3) quantitatively detecting alcohol: firstly, preparing alcohol solutions with different concentrations (0, 0.1, 0.2, 0.3, 0.5, 0.7, 0.8, 1.0, 1.2, 1.5, 1.75, 2.0, 2.2 and 2.5, the unit is mg/mL), respectively and uniformly mixing 5 muL of AOHNF with 100 muL of alcohol solutions with different concentrations, placing the mixture in a constant temperature incubator, incubating at 37 ℃ for 5min, adding 100 muL of TMB solution, placing the incubator again in the constant temperature incubator, and incubating at 37 ℃ for 5 min. The absorption peak spectrogram of the reacted solution is determined by an ultraviolet-visible spectrophotometer by using a 350 mu L micro cuvette.
In the process of alcohol oxidase oxidative decomposition of alcohol, H is produced2O2HRP as another enzyme in the composite material of double enzyme-inorganic nanometer flower2O2And TMB is used as a substrate of the methanol gasoline to carry out catalytic reaction, so that a blue substance is finally generated, and the color of the solution is gradually deepened along with the increase of the alcohol concentration. And the absorbance of the alcoholic solution with different concentrations and AOHNF at the wavelength of 650nm is different, and fig. 3 shows the relationship between the alcohol concentration and the absorbance at the wavelength of 650nm, and the absorbance gradually increases with the increase of the alcohol concentration from 0mg/mL to 2.5mg/mL, and the increase range is most obvious when the alcohol concentration is 0.1-2.0 mg/mL. Therefore, the detection range of the detection method is 0.1-2.5 mg/mL。
The absorbance of the alcohol at 650nm after the reaction of the alcohol with different concentrations with AOHNF is proportional to the alcohol concentration, and as shown in fig. 4, the linear regression equation is that Y is 0.08974X +0.06762, X represents the alcohol concentration (mg/mL), and Y represents the corresponding absorbance. Therefore, when an alcohol solution with unknown concentration is obtained, the concentration of the original alcohol solution can be obtained only by measuring the absorbance of the alcohol solution after the reaction and then finding out the corresponding alcohol concentration from the linear relation. Compared with the headspace gas chromatography method specified by the national industry standard, the alcohol sample in the standard needs to be heated in a headspace sample injector or a constant temperature heater for 15min to be detected, the method only needs 10min to prepare the sample to be detected, the time required by the method is more time-saving compared with the time required by the standard method, in the standard method, the prepared sample to be detected also needs to enter a gas chromatograph, the final quantitative test is carried out on the sample to be detected by recording the peak area of ethanol and tert-butyl alcohol in a detection material and adding the detection material, the method only needs to measure the absorbance of the prepared sample to be detected to measure the alcohol concentration of the original solution, and the detection process is more convenient.
Claims (2)
1. A quantitative alcohol analysis method based on a double-enzyme-inorganic nanoflower composite material is characterized by comprising the following steps:
step 1) adding an alcohol oxidase solution and a horseradish peroxidase solution into a phosphate buffer solution, and then adding CaCl2Mixing the solution, standing, centrifuging after the reaction is finished, removing supernatant, adding phosphate buffer solution, and uniformly mixing to obtain the double-enzyme-inorganic nanoflower composite material;
the concentration of the phosphate buffer solution is 10 mM; the CaCl is2The concentration of the solution is 100 mM;
the reaction temperature is 30 ℃, and the reaction time is 48 hours;
step 2) mixing the double-enzyme-inorganic nanoflower composite material prepared in the step 1) with a series of sample solutions with different alcohol concentrations, adding a color developing agent, performing ultraviolet-visible spectral scanning, recording an absorbance value, and taking the alcohol concentration as an abscissa and the absorbance value as an ordinate to obtain a standard curve;
and 3) obtaining the alcohol concentration of the measured sample according to a standard curve method, wherein the linear range of the alcohol quantitative analysis is 0.1-2.5 mg/mL.
2. The method for quantitatively analyzing alcohol based on the double-enzyme-inorganic nanoflower composite material according to claim 1, wherein the color developing agent in step 2) is a TMB solution.
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| CN111504986A (en) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | Method for rapidly detecting diamine biogenic amine |
| CN111504987A (en) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | A method for rapid detection of diamine biogenic amines using inorganic hybrid nanoflower enzyme |
| CN112683890B (en) * | 2020-12-03 | 2024-09-24 | 南京师范大学 | Kit for rapidly detecting glucose based on double-enzyme-hydrogel composite material and detection method thereof |
| CN119757724A (en) * | 2024-12-26 | 2025-04-04 | 吉林如诺生物科技有限公司 | Preparation method of double-enzyme inorganic functional nanoflower and application of double-enzyme inorganic functional nanoflower in preparation of competitive ELISA kit for clonorchiasis sinensis detection |
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