阅读说明：本技术 一种微生物bod传感器及其制备方法和应用 (Microbial BOD sensor and preparation method and application thereof ) 是由 张俣乐 吴晨硕 唐宇轩 于 2020-12-18 设计创作，主要内容包括：本发明提供了一种微生物BOD传感器,包括工作电极、对电极和参比电极,所述工作电极包括基础电极以及设于基础电极表面的修饰石墨烯和细胞糊；所述基础电极表面设有透析膜,所述透析膜包覆基础电极表面、修饰石墨烯及细胞糊。本发明基于石墨烯修饰电极的BOD传感器相较于传统BOD传感器具有更低的检测极限和更高的灵敏度,同时也提高了电极稳定性,能够更灵敏、更稳定地检测水体中溶解氧的含量。本发明还提供了一种微生物BOD传感器的制备方法和应用。(The invention provides a microbial BOD sensor which comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode comprises a basic electrode, and modified graphene and cell paste which are arranged on the surface of the basic electrode; and a dialysis membrane is arranged on the surface of the basic electrode, and the dialysis membrane covers the surface of the basic electrode, the modified graphene and the cell paste. Compared with the traditional BOD sensor, the BOD sensor based on the graphene modified electrode has lower detection limit and higher sensitivity, improves the stability of the electrode, and can detect the content of dissolved oxygen in a water body more sensitively and more stably. The invention also provides a preparation method and application of the microbial BOD sensor.)

1. A microbial BOD sensor is characterized by comprising a working electrode, a counter electrode and a reference electrode, wherein the working electrode comprises a basic electrode, and modified graphene and cell paste which are arranged on the surface of the basic electrode;

and a dialysis membrane is arranged on the surface of the basic electrode, and the dialysis membrane covers the surface of the basic electrode, the modified graphene and the cell paste.

2. The microbial BOD sensor of claim 1, wherein the cell paste is a saccharomyces cerevisiae cell paste.

3. The microbial BOD sensor of claim 1, wherein the counter electrode is a Pt electrode, the reference electrode is an AgCl reference electrode, and the base electrode is a glassy carbon electrode.

4. A method for preparing a microbial BOD sensor is characterized by comprising the following steps:

providing a base electrode, polishing the surface of the base electrode by adopting aluminum oxide powder, ultrasonically cleaning the base electrode, transferring the base electrode into a PBS (phosphate buffer solution) of graphene oxide, deoxidizing the PBS of the graphene oxide, and scanning the base electrode by adopting a cyclic voltammetry method to prepare a graphene modified electrode;

coating cell paste on the surface of the graphene modified electrode, drying, and covering the surface of the graphene modified electrode with a dialysis membrane to obtain a working electrode;

and providing a counter electrode and a reference electrode, and assembling the microbial BOD sensor by combining the working electrode.

5. The method for preparing a BOD sensor according to claim 4, wherein the concentration of graphene oxide in the PBS solution of graphene oxide is 0.5-2 mg/mL, the concentration of PBS is 0.01-0.15 mol/L, and the pH value of the PBS solution of graphene oxide is 9-10.

6. The method for preparing a BOD sensor of claim 4, wherein the range of the cyclic voltammetry scan is-1.5-0.5V, and the range of the cyclic voltammetry scan is 10-30 mV/s.

7. The method for preparing a BOD sensor of microorganisms according to claim 4, wherein the specific operation of performing ultrasonic cleaning on the base electrode is as follows: and (3) putting the basic electrode into absolute ethyl alcohol and secondary deionized water in sequence for ultrasonic cleaning, wherein the ultrasonic power is 100-300W, and the ultrasonic time is 0.5-1 h.

8. Use of a microbial BOD sensor according to any of claims 1 to 3 for the biochemical oxygen demand determination of water quality.

Technical Field

The invention relates to the technical field of sensors, in particular to a microbial BOD sensor, and also relates to a preparation method of the microbial BOD sensor and application of the microbial BOD sensor in water body detection.

Background

Biochemical Oxygen Demand (BOD) refers to the amount of dissolved Oxygen consumed by microorganisms present in water during reactions that biochemically degrade organic matter. Usually expressed in mg/l or percentage, ppm. A large number of microorganisms exist in wastewater and sewage, and the microorganisms need dissolved oxygen for biochemical reaction, so BOD is a comprehensive index reflecting the content of organic pollutants in water. The higher the BOD value, the more organic pollutants in the water body and the more serious the water quality pollution.

The methods commonly used at present for detecting BOD are mainly electrode method and spectroscopic method. In the electrode method, the oxygen electrode used is easily affected by the environment, and has a high detection limit and a limited sensitivity. In an actual water body test environment, sewage and wastewater are often accompanied by larger pH value fluctuation and larger metal ion content, and a common electrode is easily influenced by a water quality environment to cause large test result deviation.

The standard for measuring BOD at present is HJ 505 + 2009 Water quality five-day Biochemical Oxygen Demand (BOD)₅) The measurement dilution and inoculation method of (1) and the HJ/T86-2002 'quick determination method of a microbial sensor for measuring Biochemical Oxygen Demand (BOD) of water quality', wherein the most common BOD online detection method in the market is an electrode method. Including the use of microbial fuel cells (patent No. CN 101620201 a) and the use of microbial membrane modified electrodes (patent No. CN 103843184 a), and the use of more recently applied titanium dioxide modified electrodes (patent No. CN1774627B), of which the use of microbial membrane electrode sensors is the most common.

The principle of the microbial film electrode method is that when a water sample contacts with a microbial film, dissolved oxygen in the water sample is consumed by the contact of biochemically degradable organic matters and the microbial film, and the oxygen content diffused to the surface of the oxygen electrode is reduced. When the diffusion speed of the biodegradable organic matter in the water sample is constant like the microbial film, the oxygen content diffused to the oxygen electrode is also constant, and constant electric connection is generated between the electrodes. Because the current magnitude and the oxygen content decrease have a quantitative relation, the biochemical oxygen demand in the water sample can be estimated through experiment and formula fitting calculation.

The existing microbial BOD sensor is greatly influenced by the environment of water (pH value, metal ion content and the like), the detection sensitivity is poor, and the allowable relative deviation of a standard method is +/-15%.

Disclosure of Invention

In view of the above, the invention provides a microbial BOD sensor, and also provides a preparation method of the microbial BOD sensor and an application of the microbial BOD sensor in water quality biochemical oxygen demand determination, so as to solve the problems of great influence by the water environment, poor detection sensitivity and the like of the existing microbial BOD sensor.

In a first aspect, the invention provides a microbial BOD sensor, which comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode comprises a base electrode, and modified graphene and cell paste which are arranged on the surface of the base electrode;

and a dialysis membrane is arranged on the surface of the basic electrode, and the dialysis membrane covers the surface of the basic electrode, the modified graphene and the cell paste.

The electrode is modified by the graphene material, and due to the specific inert response and excellent conductivity of the graphene, the electrode can be better suitable for multi-factor water body testing. The high conductivity of graphene is used as an electrode material, and the catalytic capability of graphene provides the capability of mediating electron transfer reaction. Compared with the traditional BOD sensor, the BOD sensor based on the graphene modified electrode has lower detection limit and higher sensitivity, improves the stability of the electrode, and can detect the content of dissolved oxygen in a water body more sensitively and more stably.

The graphene-modified electrode is an oxygen electrode because the value of BOD is estimated by the oxygen consumption of microorganisms in the reaction. And after the electrode is modified by graphene materials, a layer of fixed microbial film is coated, and when the microbes consume and analyze dissolved oxygen in water, the content of the dissolved oxygen consumed by the microbes in the water is reflected through current change due to potential change generated by ion migration. The graphene electrode has higher sensitivity and lower detection limit, and the output signal is in a linear relationship with the value of the biological oxygen demand BOD in the detected water sample, so that the value of the biological oxygen demand BOD in the water sample is rapidly detected.

The graphene modified electrode is used, so that the stability of the electrode in different environments is improved, the test result is less influenced by the pH value and metal ions, and meanwhile, due to the high conductivity of the graphene, the graphene modified electrode is more sensitive to the change of the concentration of a trace of object to be tested, can more sensitively capture the change of the concentration of organic matters in a water body, and can detect the content of the organic matters with lower concentration (the detection limit is about 10 times improved).

Preferably, the cell paste is a saccharomyces cerevisiae cell paste.

Preferably, the counter electrode is a Pt electrode, the reference electrode is an AgCl reference electrode, and the base electrode is a glassy carbon electrode.

In a second aspect, the present invention also provides a method for preparing a microbial BOD sensor, comprising the following steps:

providing a base electrode, polishing the surface of the base electrode by adopting aluminum oxide powder, ultrasonically cleaning the base electrode, transferring the base electrode into a PBS (phosphate buffer solution) of graphene oxide, deoxidizing the PBS of the graphene oxide, and scanning the base electrode by adopting a cyclic voltammetry method to prepare a graphene modified electrode;

coating cell paste on the surface of the graphene modified electrode, drying, and covering the surface of the graphene modified electrode with a dialysis membrane to obtain a working electrode;

and providing a counter electrode and a reference electrode, and assembling the microbial BOD sensor by combining the working electrode.

The preparation method of the microbial BOD sensor in the second aspect of the invention has the advantages of simple steps, low cost and the like, and can be used for large-scale industrial production. The preparation method of the microbial BOD sensor is improved by utilizing the existing working electrode, the graphene modified electrode can be prepared by simple electro-adsorption, and the working electrode is prepared by coating cell paste and covering the cell paste with a semipermeable membrane.

Preferably, in the graphene oxide PBS solution, the concentration of the graphene oxide is 0.5-2 mg/mL, the concentration of the PBS is 0.01-0.15 mol/L, and the pH value of the graphene oxide PBS solution is 9-10.

Preferably, the scanning range of the cyclic voltammetry is-1.5-0.5V, and the scanning range of the cyclic voltammetry is 10-30 mV/s.

Preferably, the specific operation of performing ultrasonic cleaning on the base electrode is as follows: and (3) putting the basic electrode into absolute ethyl alcohol and secondary deionized water in sequence for ultrasonic cleaning, wherein the ultrasonic power is 100-300W, and the ultrasonic time is 0.5-1 h.

In a third aspect, the present invention also provides a use of a microbial BOD sensor according to the first aspect of the present invention for the determination of biochemical oxygen demand in water.

The microbial BOD sensor is applied to the water quality biochemical oxygen demand measurement, has faster detection rate, higher sensitivity and lower detection limit, and the output signal is in a linear relation with the value of the biological oxygen demand BOD in a detected water sample.

Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

In order to more clearly illustrate the contents of the present invention, a detailed description thereof will be given below with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic structural diagram of a BOD sensor of a microorganism provided in one embodiment of the present invention;

FIG. 2 is a graph of a calibration curve of BOD concentration versus response current of a microbial BOD sensor provided by another embodiment of the present invention.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

In a first aspect, the invention provides a microbial BOD sensor, which comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode comprises a base electrode, and modified graphene and cell paste which are arranged on the surface of the base electrode;

and a dialysis membrane is arranged on the surface of the basic electrode, and the dialysis membrane covers the surface of the basic electrode, the modified graphene and the cell paste.

Preferably, the cell paste is a saccharomyces cerevisiae cell paste.

Preferably, the counter electrode is a Pt electrode, the reference electrode is an AgCl reference electrode, and the base electrode is a glassy carbon electrode.

In a second aspect, the present invention also provides a method for preparing a microbial BOD sensor, comprising the following steps:

providing a base electrode, polishing the surface of the base electrode by adopting aluminum oxide powder, ultrasonically cleaning the base electrode, transferring the base electrode into a PBS (phosphate buffer solution) of graphene oxide, deoxidizing the PBS of the graphene oxide, and scanning the base electrode by adopting a cyclic voltammetry method to prepare a graphene modified electrode;

coating cell paste on the surface of the graphene modified electrode, drying, and covering the surface of the graphene modified electrode with a dialysis membrane to obtain a working electrode;

and providing a counter electrode and a reference electrode, and assembling the microbial BOD sensor by combining the working electrode.

Preferably, in the graphene oxide PBS solution, the concentration of the graphene oxide is 0.5-2 mg/mL, the concentration of the PBS is 0.01-0.15 mol/L, and the pH value of the graphene oxide PBS solution is 9-10.

Preferably, the scanning range of the cyclic voltammetry is-1.5-0.5V, and the scanning range of the cyclic voltammetry is 10-30 mV/s.

Preferably, the specific operation of performing ultrasonic cleaning on the base electrode is as follows: and (3) putting the basic electrode into absolute ethyl alcohol and secondary deionized water in sequence for ultrasonic cleaning, wherein the ultrasonic power is 100-300W, and the ultrasonic time is 0.5-1 h.

In a third aspect, the present invention also provides a use of a microbial BOD sensor according to the first aspect of the present invention for the determination of biochemical oxygen demand in water.

See example 1 for a method of making a microbial BOD sensor and the resulting microbial BOD sensor.

Example 1

A method for preparing a microbial BOD sensor comprises the following steps:

providing a glassy carbon electrode as a basic electrode, polishing the surface of the basic electrode by adopting alumina powder with the average fineness of 0.05 mu m, then sequentially carrying out ultrasonic cleaning in absolute ethyl alcohol and secondary deionized water, wherein the ultrasonic power is 150W, the ultrasonic time is 0.5h, and transferring to room temperature for drying after the ultrasonic cleaning is finished. And transferring the base electrode into a PBS (phosphate buffer solution) solution of graphene oxide, wherein the concentration of the graphene oxide is 1mg/mL, the concentration of the PBS is 0.05mol/L, and the pH value of the PBS solution of the graphene oxide is 9.18. And introducing nitrogen to remove oxygen in the oxidized graphene PBS, and scanning for a plurality of cycles at a scanning speed of 20mV/s within a potential range of-1.5-0.5V by adopting a cyclic voltammetry method to prepare the graphene modified electrode. The unique two-dimensional plane conjugated structure of the graphene can generate pi-pi interaction with biomolecules with aromatic groups such as DA (dimethyl DA) and the like to generate electron transfer, so that the electrochemical detection of the biomolecules is facilitated.

And (2) coating distilled water on the surface of the graphene modified electrode (working electrode), uniformly coating saccharomyces cerevisiae cell paste on the surface of the graphene modified electrode, drying, covering a dialysis membrane on the surface of the working electrode, immersing in water, and fixing by using a rubber ring to obtain the working electrode. A microbial BOD sensor is built by a three-electrode system which takes a working electrode modified by graphene, Ag/AgCl as a reference electrode and a platinum wire as a counter electrode.

As shown in fig. 1, the microbial BOD sensor includes a working electrode 1 (specifically the working electrode prepared in example 1), a counter electrode (Pt counter electrode), and a reference electrode (Ag/AgCl reference electrode). The working electrode 1 comprises a basic glassy carbon electrode, and modified graphene and cell paste which are arranged on the surface of the glassy carbon electrode, and a dialysis membrane is further arranged on the surface of the glassy carbon electrode and covers the whole surface of the glassy carbon electrode, the modified graphene and the cell paste.

Effect embodiment:

the microbial BOD sensor prepared in example 1 was used to measure the biochemical oxygen demand of water. The microbial BOD sensor prepared in example 1 was placed in 1L of distilled water and stabilized for 10 minutesThe rows are aligned. The resulting current curve was stabilized and set as the baseline. Introducing oxygen into water at a speed of 1L/min by using an air duct, circulating for 10 minutes by using a water pump, maintaining the dissolved oxygen content in the water at 10mg/L, and obtaining electrochemical detection current A when the dissolved oxygen is consumed by the microorganisms in biochemical reaction₀. Then the microbial BOD sensor prepared in the example 1 is placed in a sample solution of BOD with the concentration of 10mg/L to be measured, a water pump is utilized to circulate for 10 minutes, the content of dissolved oxygen in water is maintained at 10mg/L, and when the microbes perform biochemical reaction and consume the dissolved oxygen, electrochemical detection current A is obtained₁. By A₂-A₁And removing the influence of blank background to obtain the current magnitude of the microbial BOD sensor during working. Under the same experimental conditions, the electrochemical detection current A of the microbial BOD sensor during working under different concentrations of BOD sample solutions (20mg/L, 40mg/L, 80mg/L and 160mg/L) is respectively tested_n. By using the difference A of the current in the microbial BOD sensor between the solution to be measured and the distilled water_n-A₀As an ordinate, BOD concentration is plotted as an abscissa and data are fitted, and the electrochemical detection current and BOD standard concentration value have good linear relation (figure 2), which shows that the microbial BOD sensor prepared by the method can be used for electrochemical detection of BOD in water quality.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.