The present invention relates to a simple method for preparing an ethanol biosenser, applicable to NADH or ethanol detection in the fermentation field, clinical medicine and food engineering.
In recent years, with the rapid development of economy, the concept of sustainability, energy, environment and other issues are getting more prominent. Promoting the use of fuel ethanol is an important strategic initiative to alleviate energy and environmental problems. Fuel ethanol as the most successful biomass alternative in the world has formed a new energy industry in the United States, Brazil, the European Union and other countries and regions. China started the fuel ethanol industry during the “Tenth Five-Year Plan” period. After more than a decade, China has grown into the third largest producer and user of biofuel ethanol in the world, following the United States and Brazil. As the most important source of ethanol, ethanol fermentation has received a lot of attention. In the process of ethanol fermentation, the concentration of ethanol is one of the main parameters of fermentation, which on the one hand influences the growth of yeast and on the other hand influences the catalytic performance of various enzymes involved in the fermentation process. Generally, the fermentation process will cease when the concentration of ethanol reaches 14%. Therefore, the detection of ethanol concentration is particularly important in the field of fermentation. The conventional ethanol detection methods include spectrophotometry, chromatography and colorimetry. These methods usually require pre-treatment, take a longtime in detection and get results later, so they are unable to provide real-time concentration values.
Electrochemical sensors have gained much attention due to the advantages of easy operation, low cost, stable performance and high accuracy. The core of electrochemical sensors lies in the sensing electrodes, including the development of high-performance sensing materials and the preparation of sensing chips. No research results have been reported in the literature on the use of biosensors for real-time detection of ethanol and NADH, and there is still a research gap in the technology for online real-time monitoring of ethanol and NADH.
An objective of the present invention is to prepare an ethanol biosenser, which is used for accurate detection of ethanol concentration during fermentation. The preparation process of the biosensor is simple and has a low cost and a good application value. The technical solution of the present invention: A biosenser, with the following preparation steps:
Synthetic solution A is an anionic acid solution. Synthetic solution B is a cationic acid solution. In order to form uniform cubic particles, the two synthetic solutions shall have the same pH value and ion concentration. Synthetic solutions A and B are dropwise added simultaneously to a beaker at the same addition rate by micro syringe pump. After the addition, it is stirred for a certain time. Then a certain amount of solution B is dropwise added at the same addition rate again. After the addition, the synthetic solution is centrifugally cleaned several times and then transferred to a beaker. Deionized water is added to obtain a nickel hexacyanoferrate suspension.
A chloroauric acid solution is dropwise added to the nickel hexacyanoferrate suspension by micro syringe pump. After the addition, a reducing solution is dropwise added to the suspension. After the addition, centrifugal cleaning and drying are conducted to obtain gold nanoparticles/nickel hexacyanoferrate mixed powder. The gold nanoparticles/nickel hexacyanoferrate mixed powder is evenly mixed with carbon ink at a certain mass ratio to obtain gold nanoparticles/nickel hexacyanoferrate/carbon mixed ink.
The gold nanoparticles/nickel hexacyanoferrate/carbon mixed ink is fixed on a support by the screen-printing technique to form a working electrode. An ethanol dehydrogenase mixed solution containing a certain amount of glutaraldehyde is prepared. A certain amount of the mixed enzyme solution is taken and evenly applied on the working electrode. The working electrode is dried at low temperature in a refrigerator to obtain a biosensing chip for ethanol detection.
Preferably, in step 1, the ion concentrations of synthetic solution A and synthetic solution B are both in the range of 0.001-0.1M, and the pH values are both in the range of 1-6; the temperature of crystallization reaction is 10-60° C.; the injection rates of synthetic solution A and synthetic solution B are both in the range of 100-1,00001 min.
Preferably, in step 1, the anion donor is one of K3[Fe(CN)6] and K4[Fe(CN)6], and the cation donor is one of NiCl2, NiSO4 and Ni(NO3)2; the acid solution is one of hydrochloric acid, sulfuric acid and nitric acid.
Preferably, in step 1, the stirring time is 10 min-1 h, and the volume of added solution B is 30-90 ml.
Preferably, in step 1, the centrifugal rate is 5,000 r/min-10,000 r/min, the centrifugal time is 3 min-15 min, the centrifugal times are 2 to 5 times, and the volume of deionized water is 10-100 mL.
Preferably, in step 2, the reducing solution is one of sodium citrate, ascorbic acid and glucose.
Preferably, in step 2, the molar ratio of chloroauric acid to the reducing substance in the reducing solution is 1:5-1:15.
Preferably, in step 2, the mass ratio of gold nanoparticles/nickel hexacyanoferrate powder to carbon ink is 1:5-1:20; in step 3, the support is one of PVC, PET and alumina.
Preferably, in step 3, the concentration of ethanol dehydrogenase solution is 0.1-1 U/μL, the volume percent of glutaraldehyde in the mixed enzyme solution is 0.5%-2%, the volume of the mixed enzyme solution applied on the working electrode is 1-5 μl, and the ethanol biosensing chip is dried at 0-10° C.
The present invention provides an ethanol and/or NADH detection method. The biosensing chip prepared by the foregoing method is used for the detection.
The present invention provides an application of the biosensing chip obtained by the foregoing preparation method in ethanol and/or NADH detection.
The detection of ethanol by electrochemical method is mainly based on the catalytic oxidation of NADH by electrode materials at a certain voltage and the generation of an electric current, which reflects the content of ethanol. Nickel hexacyanoferrate has good catalytic performance and can effectively catalyze the oxidation of NADH. The present patent uses nickel hexacyanoferrate as a sensing material and introduces gold nanoparticles to improve the electrical conductivity of the material. By controlling the nanostructure of the material, gold nanoparticles/nickel hexacyanoferrate, a nanocomposite material with high catalytic selectivity for NADH, is obtained, and an ethanol biosenser is prepared in combination with the screen-printing technique. Thanks to the excellent electrocatalytic properties, stability and biocompatibility of the material, the prepared biosenser has a broader detection range than existing sensors do and enables dilution-free detection of ethanol in fermentation broth.
The present invention will be further described below with reference to embodiments from which the foregoing objectives, features and advantages of the present invention will be more evident. It should be noted that if without conflict, the embodiments of the present invention and the features in the embodiments can be combined.
The description below illustrates many details to fully understand the present invention, but the present invention can also be implemented in other ways different from this description, so the present invention is not limited to the embodiments disclosed below.
Below the technical solution of the present invention is described in detail.
An ethanol biosensing chip, with the following preparation process:
The sensing chip detects ethanol by the following steps: Connect the contacts of the reference electrode, counter electrode and working electrode of the sensing chip to an electrochemical workstation, do a timed amperometric current test on ethanol in a phosphate buffer solution (PBS) with a pH value of 7.0 and containing 0.1 mM coenzyme NAD+, and draw a working curve of ethanol concentration and response current.
The calculation shows that the ethanol detection sensitivity of the biosensing chip obtained in this embodiment is 1.69 μA·mM−1·cm−2, and the detection limit is as low as 0.01 mM. After the test, the modified electrode was kept in a pH 7.0 PBS at 0° C. for one week and its response signal was 94% of the initial signal; after a month, its response signal was 86% of the initial signal, suggesting that this chip has very good stability.
An ethanol biosensing chip, with the following preparation process:
The detection method is the same as that in Embodiment 1.
The calculation shows that the ethanol detection sensitivity of the biosensing chip obtained in this embodiment is 1.89 μA·mM−1·cm−2, and the detection limit is as low as 0.01 mM. After the test, the modified electrode was kept in a pH7.0 PBS at 0° C. for one week and its response signal was 89% of the initial signal; after a month, its response signal was 82% of the initial signal, suggesting that this chip has very good stability.
An ethanol biosensing chip, with the following preparation process:
The detection method is the same as that in Embodiment 1.
The calculation shows that the ethanol detection sensitivity of the biosensing chip obtained in this embodiment is 2.06 μA·mM−1·cm−2, and the detection limit is as low as 0.01 mM. After the test, the modified electrode was kept in a pH7.0 PBS at 0° C. for one week and its response signal was 88% of the initial signal; after a month, its response signal was 83% of the initial signal, suggesting that this chip has very good stability.
An ethanol biosensing chip, with the following preparation process:
The calculation shows that the ethanol detection sensitivity of the biosensing chip obtained in this embodiment is 1.36 μA·mM−1·cm−2, and the detection limit is as low as 0.01 mM. After the test, the modified electrode was kept in a pH7.0 PBS at 0° C. for one week and its response signal was 86% of the initial signal; after a month, its response signal was 79% of the initial signal, suggesting that this chip has very good stability.
The following method is used to detect the concentration of ethanol in fermentation broth by the ethanol biosensing chips prepared in Embodiments 1 to 4:
Based on the foregoing examples, the four kinds of ethanol biosensing chips prepared are used to detect the concentration of ethanol in fermentation broth by the timed amperometric current method. Firstly, ethanol with a known concentration is used as a standard sample. The content of ethanol in the fermentation broth is calculated by calculating the ratio of the current responded by ethanol to the current responded by the fermentation broth. The results are as shown in Table 1:
As shown in Table 1, the obtained ethanol biosensing chips can be used to detect the concentration of ethanol in fermentation broth effectively. The gold nanoparticles/nickel hexacyanoferrate obtained in Embodiment 1 was shot under an electron microscope. The results are as shown in
This embodiment provides an application case for NADH detection by biosensing chip.
The sensing chip obtained in Embodiment 1 is used for the detection.
The application method comprises the following steps:
Connecting the contacts of the reference electrode, counter electrode and working electrode of the sensing chip to an electrochemical workstation, doing a timed amperometric current test on ethanol in a phosphate buffer solution (PBS) with a pH value of 7.0, and drawing a working curve of NADH concentration and response current.
The calculation shows that the NADH detection sensitivity of the biosensing chip obtained in this embodiment is 96.86 μA·mM−1·cm−2, and the detection limit is as low as 0.05 mM. After the test, the modified electrode was kept in a pH7.0 PBS at 0° C. for one week and its response signal was 96% of the initial signal; after a month, its response signal was 91% of the initial signal, proving that this chip has very excellent long-term stability.
Number | Date | Country | Kind |
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202010979555.0 | Sep 2020 | CN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/CN2020/121110 | 10/15/2020 | WO |