CN104730129B - A kind of electrochemical method based on ethyl carbamate content in double enzyme modified electrode quick detection solution - Google Patents

Abstract

The invention discloses an electrochemical method for rapidly detecting the content of ethyl carbamate in a solution based on double-enzyme modified electrodes, belonging to the technical field of food safety detection. In the invention, the ethyl carbamate degrading enzyme and glutamate dehydrogenase are co-modified on the electrode surface to construct the ethyl carbamate electrochemical sensor. Urethane degrading enzymes hydrolyze ethyl carbamate to ethanol, CO ₂ and ammonia, and under the catalysis of glutamate dehydrogenase, ammonia reacts with the co-substrate α-ketoglutarate to generate glutamate, accompanied by reduced The coenzyme nicotinamide adenine dinucleotide (NADH) is oxidized. Therefore, the system converts the content of urethane into the change of NADH content, and utilizes the change of the current signal generated by the latter on the electrode surface to realize the detection of the content of urethane in the liquid system. The method can quickly detect ethyl carbamate in solution and simulated rice wine system, and the detection limit is as low as 5.26 nmol·L ^‑1 , which provides an effective means for the detection of ethyl carbamate in fermented food and beverages.

Description

A method for rapid detection of ethyl carbamate content in solution based on dual-enzyme modified electrodes Electrochemical method

technical field

The invention relates to an electrochemical method for quickly detecting the content of ethyl carbamate in a solution by using double-enzyme modified electrodes, and belongs to the technical fields of analytical chemistry and food safety detection.

Background technique

Ethyl carbamate (C ₂ H ₅ OCONH ₂ , CAS#51-79-6, referred to as EC) is a by-product widely present in fermented food and alcoholic beverages. Experimental studies on monkeys, mice, hamsters, and mice have proved that EC is a genotoxic and carcinogenic substance that can be rapidly absorbed by the gastrointestinal tract and skin, and can cause lung cancer, lymphoma, liver cancer, and skin cancer. and other diseases. It is mainly ingested and accumulated in the human body through alcoholic beverages such as rice wine, sake, wine, cider, brandy or whiskey, and has a carcinogenic effect on the human body through the metabolism of cytochrome P450. Based on the above epidemiological and experimental data, EC was defined as a possible human carcinogen (2B) by the International Cancer Organization and the U.S. Environmental Protection Agency. By 2007, it was reclassified as a 2A carcinogen with carcinogenic effects on humans. In view of this, many countries in the world have successively set limits on the content of urethane in drinking alcohol.

my country has a large population base, and produces and consumes a large amount of fermented wine and food every year, such as rice wine, white wine, beer, soy sauce, vinegar, fermented bean curd, etc. This means that our country will accumulate excess EC due to the intake of a large amount of alcoholic beverages and fermented foods. Therefore, it is urgent to propose methods and strategies to control and reduce the EC content in fermented foods and alcoholic beverages in my country. At the same time, it is imperative to develop accurate, simple, cheap and time-saving detection methods.

At present, the commonly used detection methods for ethyl carbamate at home and abroad are based on gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), etc. combined with other sensitive detector methods, such as liquid chromatography - Electrospray tandem mass spectrometry (HPLC-EIS-MS/MS), high performance liquid chromatography-fluorescence detector (HPLC-FLD), forward liquid chromatography-pressure chemical ionization tandem mass spectrometry (NP-LC-APCI-MS/ MS), multidimensional headspace solid-phase microextraction combined with gas chromatography and flame ionization detector (HS-SPME/GC/FID), one-dimensional gas chromatography-mass spectrometry (MEPS/GC-MS), high performance liquid chromatography combined with Q Exactive Hybrid quadrupole-orbitrap mass spectrometry (UHPLC-MS/MS), etc. Although these methods can detect the content of ethyl carbamate in alcoholic beverages to a certain extent, most of them require pretreatment of samples, complicated operations, time-consuming and labor-consuming, poor reproducibility, large operating system errors, and two Extraction and elution with organic solvents such as methyl chloride pose potential safety hazards to the health of operators. In addition, a large amount of organic solvent will be used in the extraction process, which needs to be evaporated to remove the organic solvent, resulting in poor separation effect and affecting the recovery rate of the sample and the accuracy of the test result. Most importantly, the various monitoring and analysis systems required by these methods need to be supported by large precision instruments and professional operators. At the same time, it is also accompanied by high requirements for testing instruments, high maintenance and care costs for the instruments, testing can only be carried out in professional laboratories with large and expensive instruments, and it is difficult to popularize and use them.

With the development of biosensors, dual-enzyme and multi-enzyme biosensors have gradually attracted people's attention and have been widely used. At present, many enzyme sensors have been used for the detection of ammonia, urea, heavy metal ions, hydrogen peroxide, phenol, organophosphate pesticides, ascorbic acid, glutamic acid and other amino acids in water. However, no enzymatic sensor for detecting EC content has been reported so far. On the basis of screening the bacterial strains producing urethane degrading enzyme in the early stage, the invention constructs a dual enzyme modified electrode sensor for urethane degrading enzyme and glutamic acid dehydrogenase to realize rapid detection of EC.

Contents of the invention

The purpose of the present invention is to provide an electrochemical method for rapidly detecting the content of ethyl carbamate in a solution, and the technical problem to be solved is to construct a dual-enzyme modified electrode sensor required for detecting ethyl carbamate. The urethane degrading enzyme (Urethanase, Ure) immobilized on the electrode surface can hydrolyze urethane to produce ethanol, CO ₂ and NH ₄ ⁺ , under the catalysis of glutamate dehydrogenase (GLDH) ammonia and co- The substrate α-ketoglutarate reacts to generate glutamate, accompanied by oxidation of the reduced coenzyme nicotinamide adenine dinucleotide (NADH). In this way, the system converts the content of urethane into the change of NADH content, and uses NADH to produce a change in the current response value on the electrode surface after a certain voltage is applied to realize the detection of the content of urethane in the liquid system.

Technical scheme of the present invention: an electrochemical method for quickly detecting the content of ethyl carbamate in a solution, characterized in that the ethyl carbamate EC The signal is converted into the reduced coenzyme nicotinamide adenine dinucleotide NADH signal for electrochemical detection, the ethyl carbamate degrading enzyme Ure hydrolyzes ethyl carbamate to generate ethanol, CO ₂ and ammonia, and the glutamate dehydrogenase GLDH Under the catalysis, ammonia reacts with the co-substrate α-ketoglutarate to generate glutamic acid, and at the same time, NADH is oxidized. The content of ethyl carbamate is electrochemically detected by using the change of current signal generated by the oxidation of NADH on the electrode surface under a specific voltage. ; the steps are:

(1) Construction of dual-enzyme modified electrodes

CS/silane sol-gel sol- Gel system, adding urethane degrading enzyme Ure40 U·mL ^-1 and glutamate dehydrogenase GLDH 16 U·mL ^-1 to this organic-inorganic hybrid material to prepare CS/gelatin/GPTMS/Ure/GLDH biocomposite The material is added dropwise on the surface of the graphite electrode, and dried in a refrigerator at 4°C to form a film to form a double-enzyme modified electrode;

The optimal reaction system of dual enzyme-modified electrode sensor for EC detection is 25 mmol L ^-1 PBS buffer, the optimal pH is 6.0, and the optimal concentration of α ^- ketoglutarate in the reaction system is 10 mmol L -1 ¹ , NADH concentration is 0.5 mmol·L ^-1 ;

(2) Establish a standard curve

Place the double-enzyme modified electrode in a solution containing 0-500 μmol L ^-1 EC, 300 mmol L ^-1 α-ketoglutarate 200 μL, and 7.5 mmol L ^-1 NADH 400 μL, with pH6.0, 25 mmol·L ^-1 PBS buffer solution was adjusted to 6 mL, and the resulting solution was mixed evenly, and the double-enzyme modified electrode was inserted immediately, and a voltage of 0.55 V was applied, and the current-time scanning was performed, and the reaction was carried out for 5 min, and NADH was observed on the electrode surface. The current response change value of the generated current response curve at 120 s is corresponding to the change value of the EC concentration and the current response value of the current response curve at 120 s within 5 min to make a standard curve;

(3) Calculate the EC content in the sample

After the double-enzyme modified electrode was placed in the rice wine sample to react for 5 min, the current response change value of the current response curve generated by NADH on the electrode surface at 120 s was detected, and the ethyl carbamate in the rice wine sample to be tested was calculated by comparing with the standard curve. ester content.

The established method can quickly detect and accurately quantify the content of ethyl carbamate in the solution sample. The detection limit in the simulated rice wine sample is 5.26 nmol·L ^-1 , the linear correlation coefficient is 0.9989, and the EC recovery rate is 95.17%-103.79%. , the relative standard deviation is 2.86%-8%.

The repeatability, reproducibility, anti-interference performance and storage stability of the double-enzyme modified electrode sensor are good.

Beneficial effects of the present invention: the present invention has successfully constructed a dual-enzyme sensor by optimizing the construction conditions of the dual-enzyme modified electrode sensor, the optimal reaction buffer system, and the concentration of α-ketoglutarate in the detection solution. An electrochemical method for the rapid detection of ethyl carbamate was established. This method overcomes many deficiencies in traditional methods.

Description of drawings

Figure 1 Determination of the optimal reaction buffer system for the dual-enzyme modified electrode sensor.

Fig. 2 Standard curve for detection of ethyl carbamate concentration by electrochemical method of dual-enzyme modified electrode sensor.

detailed description

Example 1. Optimum addition of chitosan (CS), gelatin (gelatin) and γ-(2,3-epoxypropoxy)propyltrimethoxysilane (GPTMS) in the CS/silane sol-gel system solution The amount is determined.

Before constructing the dual-enzyme modified electrode sensor, the conditions were optimized for the concentrations of chitosan, gelatin and GPTMS in the CS/silane sol-gel system solution.

First, 0, 0.025, 0.05, 0.075, 0.10 and 0.125 g of chitosan powder were dissolved in 25 mL of 1% acetic acid solution, and stirred evenly to prepare chitosan (mass volume percent, the same below) with concentrations of 0, 0.1%, 0.2%, 0.3%, 0.4% and 0.5% solutions. After the chitosan was completely dissolved, 1 mol·L ^-1 NaOH solution was added to the solution to adjust the pH to 6.0. Add 1% glycerol as a protective agent. Take 50 μL of chitosan solution, 25 μL of 16 U mL ^-1 urethane degrading enzyme and 25 μL of 16 U mL- ¹ glutamate dehydrogenase, and mix well. 15 μL of the above CS/silane biological mixture solution was added dropwise on the surface of the pyrolytic graphite electrode, and placed in a refrigerator at 4 °C to dry to form a film to prepare the CS/Ure/GLDH dual enzyme electrode sensor. The double-enzyme modified electrode sensors prepared under the above conditions containing 0, 0.1%, 0.2%, 0.3%, 0.4% and 0.5% chitosan were placed in a solution containing 0.5 mmol·L ^-1 EC, 0.5 mmol·L ^-1 Current-time sweep was carried out in the detection solution of α-ketoglutarate and 0.5 mmol·L ^-1 NADH. After observing the reaction time for 5 minutes, the current response value of NADH in the solution on the electrode surface was detected. The results showed that when the concentration of chitosan was 0.2%, the current response value of NADH produced on the electrode surface of the prepared dual-enzyme modified electrode sensor was the smallest after 5 min of reaction. So the optimal concentration of CS in CS/Ure/GLDH solution is 0.2%.

On the basis of determining the concentration of chitosan, add 0, 0.025, 0.05, 0.075, 0.10 and 0.125 g of gelatin to the 0.2% chitosan solution respectively, that is, the gelatin concentration is 0, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, stir. After the gelatin was completely dissolved, 1 mol·L ^-1 NaOH solution was added to the solution to adjust the pH to 6.0. Add 1% glycerol as a protective agent. Take 50 μL of CS/gelatin solution, 25 μL of 16 U mL ^-1 ethyl carbamate degrading enzyme and 25 μL of 16 U mL ^-1 glutamate dehydrogenase, and mix well. 15 μL of the CS/silane biological mixture solution was added dropwise on the surface of the pyrolytic graphite electrode, and dried in a refrigerator at 4 °C to form a film to prepare a CS/gelatin/Ure/GLDH dual-enzyme modified electrode sensor. The dual-enzyme-modified electrode sensor obtained under the above conditions was respectively placed in a detection solution containing 0.5 mmol L ^-1 EC, 0.5 mmol L ^-1 α-ketoglutarate and 0.5 mmol L ^-1 NADH for current- time scan. Observe the size of the NADH current response value at 5 min reaction time respectively. When the gelatin concentration was 0.3%, the current response value of NADH was the smallest after 5 min of reaction. So the optimal concentration of gelatin in CS/gelatin/Ure/GLDH solution is 0.3%.

Add 0, 250, 500, 750, 1000 and 1250 μLGPTMS dropwise to the solution containing 0.2% chitosan and 0.3% gelatin, that is, the concentration of GPTMS is 0, 1%, 2%, 3%, 4% and 5% respectively. %, stirred evenly, hydrolyzed and cross-linked at room temperature for 12 h. Then 1 mol·L ^-1 NaOH solution was added to the solution to adjust the pH to 6.0. Add 1% glycerol as a protective agent. Take 50 μL of CS/gelatin/GPTMS solution, 25 μL of 16 U mL ^-1 ethyl carbamate degrading enzyme and 25 μL of 16 U mL ^-1 glutamate dehydrogenase, and mix well. 15 μL of the biological mixture solution was added dropwise on the surface of the pyrolytic graphite electrode, and dried in a refrigerator at 4 °C to form a film to prepare a CS/gelatin/GPTMS/Ure/GLDH dual-enzyme modified electrode sensor. The dual-enzyme-modified electrode sensor obtained under the above conditions was respectively placed in the detection solution containing 0.5 mmol L ^-1 EC, 0.5 mmol L ^-1 α ^- ketoglutarate and 0.5 mmol L ^-1 NADH for current- time scan. After 5 min reaction time, the NADH current response change value was observed respectively. The results showed that when the concentration of GPTMS was 3%, the prepared dual-enzyme electrode sensor had the largest change in the current response to NADH after 5 min of reaction. So the optimal concentration of GPTMS in CS/gelatin/GPTMS/Ure/GLDH solution is 3%.

Example 2. Determination of the optimal reaction buffer system for dual-enzyme modified electrode sensors

First prepare the double-enzyme modified electrode, prepare 25 mmol L ^-1 citrate buffer solution with pH 4.0, 4.5 and 5.0, and 25 mmol L ^-1 phosphate buffer solution with pH 5.5, 6.0 and 6.5, and prepare with corresponding pH buffer solution 300 mmol·L ^-1 α-ketoglutarate, 0.1 mmol·L ^-1 EC and 7.5 mmol·L ^-1 NADH solution. Take 5100 μL buffer solution with different pH, add 200 μL 300 mmol L ^-1 α-ketoglutaric acid, 300 μL 0.1 mmol L ^-1 EC and 400 μL 7.5 mmol L ^-1 NADH solution and mix evenly to prepare different pH detection system. The prepared dual-enzyme-modified electrode sensors were respectively placed in the above-mentioned detection systems with different pH for current-time scanning. After reacting for 5 min, observe the magnitude of the current response value generated by NADH on the electrode surface. The results are shown in Figure 1. In the phosphate buffer at pH 6.0, after 5 min of reaction, the current response value of the remaining NADH in the solution was the smallest.

Example 3 Construction of double enzyme modified electrode

a. Enzyme source: urethane-degrading enzyme, the urethane-degrading enzyme-producing bacterial strain CGMCC No.5763 (the strain was screened from the intestinal tract of mice, and it was determined that the bacterium was Penicillium variabile , named as P. variabile JN-A525. It has been disclosed in the patent 201410533042.1 "Zhou Nandi, Lu Xiaoxia and Tian Yaping: A Spectrophotometric Method for Rapid Detection of Ethyl Carbamate Content". Gained after filtration and concentration; glutamate dehydrogenase is a commercially available enzyme;

b. Preparation of enzyme solution and other solutions: Dissolve urethane degrading enzyme and glutamate dehydrogenase in 25mmol·L ^-1 PBS buffer, so that the enzyme activities of the two are 40 U·mL ^-1 and 16U· mL ^-1 ; prepare 300mmol·L ^-1 α-ketoglutaric acid solution; 7.5mmol·L ^-1 NADH and different concentrations of urethane standard solutions;

c. Preparation of CS/silane sol-gel system: Accurately weigh 0.05 g of chitosan and 0.075 g of gelatin and dissolve in 25 mL of 1% acetic acid solution. After complete dissolution, add 1M NaOH dropwise to adjust the pH of the solution to 6.0. 1% glycerol was added as a protective agent, and then 750 μL of GPTMS (γ-(2,3-glycidoxy)propyltrimethoxysilane) was slowly added dropwise to the above solution, and stirred evenly at room temperature for 12 h, the prepared CS/silane sol-gel system;

d. Preparation of CS/gelatin/GPTMS/Ure/GLDH biocomposite: Take 50 μL of the CS/silane sol-gel system prepared above, add 40 U·mL ^-1 urethane degrading enzyme 25 μL and 16 U · mL ^-1 glutamate dehydrogenase 25 μL, mix well, that is the prepared CS/gelatin/GPTMS/Ure/GLDH biocomposite material;

e. Construction of dual-enzyme-modified electrode sensors: Add 15 μL of the CS/gelatin/GPTMS/Ure/GLDH biocomposite material prepared above to the surface of the cleaned pyrolytic graphite electrode, and place it in a refrigerator at 4°C to dry to form a film. That is, the prepared dual-enzyme modified electrode.

Example 4 Establishment of Standard Curve for Detection of Ethyl Carbamate Concentration by Dual Enzyme Modified Electrode Sensor

Prepare mother liquors with concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1 mmol L ^-1 with urethane with a purity greater than 99%;

Add appropriate volume and concentration of urethane mother liquor to the detection system, so that the concentration of urethane in the system is 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 , 100, 200, 300, 400, 500 μmol·L ^-1 ; then add 300 mmol·L ^-1 α-ketoglutaric acid 200 μL, 7.5 mmol·L ^-1 NADH 400 μL and a certain volume of pH6.0, 25 mmol L ^-1 PBS buffer, so that the total volume of the detection system is 6 mL (i.e. 10 mmol L ^-1 α-ketoglutaric acid, 0.5 mmol L ^-1 NADH solution); after mixing the obtained solution , and immediately insert the double-enzyme-modified electrode, and perform current-time sweep at a voltage of 0.55 V. After reacting for 5 min, ^NADH The current response change value ΔI _{120 s} is 0, 0.0345, 0.04055, 0.06935, 0.09685, 0.10505, 0.12675, 0.1706, 0.2528, 0.348075, 0.435475, 0.537675, 0.54675, 5, 0.68.08, 0.6367 Taking the concentration of ethyl carbamate (μmol L ^-1 ) as the abscissa and the corresponding ΔI _{120 s} as the ordinate, the standard curve of the concentration of ethyl carbamate detected by the dual-enzyme modified electrode sensor was plotted, as shown in Figure 2 .

Example 5 Rapid detection of ethyl carbamate by dual enzyme modified electrode sensor

Add a certain amount of urethane mother liquor to the simulated rice wine sample (the simulated rice wine was prepared by adding absolute ethanol to the ^pH6 . After being placed in the simulated rice wine sample for 5 min, the current response change value of the current response curve generated by NADH on the electrode surface at 120 s was detected, and the content of ethyl carbamate in the simulated rice wine sample was calculated by comparing with the standard curve.

Claims (2)

1. the electrochemical method of ethyl carbamate content in a kind of quick detection solution, it is characterised in that utilize urethane Urethanes signal is converted into reduced coenzyme niacinamide by ester digestive enzyme and the cascade reaction of glutamate dehydrogenase enzymatic Adenine-dinucleotide signal carries out Electrochemical Detection, and urethane ester hydrolysis is generated second by urethanes digestive enzyme Alcohol, CO₂And ammonia, ammonia and cosubstrate α-ketoglutaric acid reaction generation glutamic acid, simultaneous under glutamate dehydrogenase enzymatic Reduced coenzyme NADH is oxidized, and utilizes the core of reduced coenzyme nicotinamide adenine two under specific voltage Thuja acid aoxidizes the change for producing current signal in electrode surface, and Electrochemical Detection is carried out to ethyl carbamate content；Step is：

（1）The double enzyme modified electrodes of structure

0.2% chitosan, 0.3% gelatin and 3% γ-(oxygen of 2,3- epoxies third) propyl trimethoxy silicane are added into the vinegar of 25 mL 1% Chitosan/silane collosol-gelatum systems are prepared in acid solution, by the UmL of urethanes digestive enzyme 40^-1And glutamic acid Dehydrogenase 16 UmL^-1Add in this organic-inorganic hybrid material and prepare chitosan/gelatin/γ-(oxygen of 2,3- epoxies third) propyl group Trimethoxy silane/urethanes digestive enzyme/glutamte dehydrogenase Biocomposite material, is added dropwise in graphite electrode surface, Drying and forming-film in 4 DEG C of refrigerators is placed in, builds enzyme modified electrode in pairs；

It is 25 mmolL that double enzyme modified electrodes, which are used for reaction system when urethanes detects,^-1PBS, pH are 6.0, the concentration of α-ketoglutaric acid is 10 mmolL in reaction system^-1；Reduced coenzyme NADH is dense Spend for 0.5 mmolL^-1；

（2）Establish standard curve

Double enzyme modified electrodes are placed in containing 0~500 μm of olL^-1Ethyl carbamate concentration, 300 mmolL^-1α -one penta The μ L of diacid 200 and 7.5 mmolL^-1The μ L of reduced coenzyme NADH 400, with pH6.0,25 mmol·L^-1PBS is settled to 6 mL, after the solution of gained is well mixed, is immediately inserted into double enzyme modified electrodes, applies 0.55 V voltages, current-vs-time scanning is carried out, react 5 min, observation reduced coenzyme NADH is in electricity Current-responsive changing value of the current-responsive curve caused by the surface of pole in 120 s, ethyl carbamate concentration and electric current are rung Answer changing value of current-responsive value of the curve at 120 s in 5 min corresponding, make standard curve；

（3）Calculate ethyl carbamate content in sample

Double enzyme modified electrodes are placed in yellow rice wine sample to be measured after reacting 5 min, detection reduced coenzyme nicotinamide adenine two Current-responsive changing value of the nucleotides in current-responsive curve caused by electrode surface at 120 s, passes through contrast standard curve Calculate ethyl carbamate content in yellow rice wine sample to be measured.

2. the electrochemical method of ethyl carbamate content, its feature exist in quick detection solution according to claim 1 5.26 nmolL are limited in urethanes lowest detection of this pair of enzyme modified electrode in solution and simulation yellow rice wine^-1, ammonia The base Ethyl formate rate of recovery is 95.17%~103.79%, and relative standard deviation is 2.86%~8%.