CN106908504A - A kind of chloroalkene determines device and its detection method - Google Patents

Abstract

The invention relates to a chlorinated olefin measuring device and a detecting method thereof, belonging to the technical field of environmental monitoring. The assay device includes a water bath device, a biosensor device, a biofilm device, an electromagnetic stirring device and a detection device. The water bath device provides a constant temperature environment; the biosensor device includes AgCl/Ag ₂ S film working electrode and Ag/AgCl reference electrode; the biofilm device is fixed below the working electrode, including 2 porous nitrocellulose membranes and fixed between the two membranes. The degrading bacteria between them; the electromagnetic stirring device ensures the uniform mixing of the buffer solution; the detection device is used to measure the chloride ion concentration after the bacteria degrade the chlorinated olefin, and finally convert it into the measured chlorinated olefin concentration. The invention uses microbial degradation to measure the content of chlorinated olefins, without secondary pollution, simple device, reusable, extremely short reaction time, low detection limit, high reproducibility, and can resist the interference of a certain range of temperature and pH fluctuations , to ensure efficient and accurate detection of chlorinated olefins in groundwater.

Description

A kind of chloroalkene measuring device and its detection method

technical field

The invention belongs to the technical field of environmental monitoring, and relates to a chlorinated olefin measuring device and a detecting method thereof.

Background technique

Insufficient water sources and water source pollution are the most immediate problems facing China's drinking water sources, and the problem of drinking water source pollution is particularly prominent. At present, about 97% of China's cities have groundwater pollution, and the degree is divided into serious pollution, moderate pollution and light pollution, and the basic clean groundwater is only 3%. Groundwater pollution is a phenomenon in which the quality of groundwater decreases due to changes in the chemical composition, physical properties, and biological characteristics of groundwater caused by human activities. The main causes of groundwater pollution are: direct discharge of industrial wastewater to the ground, intrusion of polluted surface water into the underground aquifer, infiltration of human and animal feces or contaminated water due to excessive use of pesticides, etc. The result of pollution is to increase the content of harmful components, bacteria, organic matter, etc. in groundwater. Polluted groundwater has a serious impact on human health and industrial and agricultural production.

Trichlorethylene (TCE), tetrachlorethylene (PCE) and dichlorethylene (DCE) are three chlorinated olefins widely used in industry, and are often used as metal surface treatment agents, electroplating cleaners, degreasing agents and extractants , It can also be used in organic synthesis and pesticide production. Chlorinated olefins have become one of the most common types of refractory pollutants in groundwater due to improper handling and management, as well as their leakage during widespread use. Chlorinated olefins are biologically toxic, and can be continuously enriched in the environment through the food chain. When they enter the human body, they will damage the central nervous system, cause damage to the liver, kidney, heart, and trigeminal nerve, and are carcinogenic, which is a serious hazard to human health. The use of microbial remediation technology to treat chlorinated olefins in groundwater has attracted extensive attention. Chlorinated olefins can be degraded by the co-metabolism of aerobic and anaerobic bacteria in the presence of co-substrates such as methane, ammonia, and aromatic compounds. Among them, methanotrophs OB3b is a commonly used degrading bacteria.

Biosensors are sensitive to biological substances and can convert their concentrations into electrical signals for detection. The identification element is an immobilized biologically sensitive material, and is equipped with a physical and chemical transducer and a signal amplifier. Biosensors are often used in the field of water environment and atmospheric environment monitoring. Since chlorinated alkenes are volatile chlorine-containing aliphatic compounds and are difficult to be directly degraded by microorganisms, there are very few studies on the determination of chlorinated alkenes using biosensors. The traditional determination method is gas chromatography, which takes a long time to analyze, requires pretreatment, and the steps of solvent extraction and purge and trap are relatively complicated, and the experimental equipment is relatively expensive. Therefore, it is necessary to propose a simple and rapid chlorinated olefin determination device.

Contents of the invention

The problem to be solved by the present invention is to overcome the defects of the prior art, provide a relatively simple device that can quickly detect chlorinated olefins in groundwater, and a detection method based on the device, which is dedicated to the detection of chlorinated olefins in groundwater. effective monitoring.

In order to achieve the above object, the present invention provides a chlorinated olefin assay device, including a water bath device, a biosensor device, a biofilm device, an electromagnetic stirring device and a detection device; wherein, the biosensor device includes a reaction tank and a chloride ion electrode, The reaction tank is placed in a water bath device, and the chloride ion electrode includes an AgCl/Ag ₂ S film working electrode and an Ag/AgCl reference electrode, and both electrodes are immersed in the reaction tank; the biofilm device includes two parallel arranged porous Nitrocellulose membrane and degradative bacteria fixed between two layers of porous nitrocellulose membrane, the biofilm device is fixed on the bottom of the AgCl/Ag ₂ S membrane working electrode; the electromagnetic stirring device is divided into two parts and placed in the water bath device respectively Below and inside the biosensor device, the detection device is connected to the biosensor device.

As a further improvement of the present invention, the water bath device includes: a thermometer, a temperature controller, an electric heating unit, a water outlet, and a shell with an upper end open.

As a further improvement of the present invention, the electromagnetic stirring device includes an electromagnetic stirring chamber and an electromagnetic stirrer; wherein, the electromagnetic stirring chamber is placed under the water bath device, and the electromagnetic stirrer is placed at the bottom of the reaction tank in the biosensor device.

As a further improvement of the present invention, the detection device includes an amplifier, an ion detector and an electronic recorder; wherein the amplifier is connected to two chlorine ion electrodes, the ion detector is connected to the amplifier, and the electronic recorder is connected to the ion detector.

As a further improvement of the present invention, the diameter of the porous nitrocellulose membrane is 20 mm, and the pore size is 0.45 μm; the degrading bacteria are methane-oxidizing bacteria OB3b.

The detection method based on the chlorinated olefin measuring device may further comprise the steps:

(1) Add the solution to be tested in the reaction pool containing phosphate buffer solvent, the chlorinated olefins in the solution to be tested contact the fixed degrading bacteria through the lower porous nitrocellulose membrane, and the methanotrophic bacteria OB3b degrades the chlorinated olefins , the released chloride ions are received by the AgCl/Ag ₂ S membrane working electrode that selectively detects chloride ions through the upper porous nitrocellulose membrane;

(2) The ion signal is monitored by the amplifier, ion detector and electronic recorder through the working electrode, and the obtained chloride ion concentration is converted into the concentration of chlorinated olefins. The response of the biosensor device is evaluated by the steady-state method, and the chlorine The corresponding relationship diagram of olefins and chloride ion concentration, and then obtain the concentration of chlorinated olefins in the solution to be tested.

Compared with prior art, the beneficial effect that the present invention reaches is:

(1) Microbial degradation is used to measure the content of chlorinated olefins, avoiding the use of harmful chemical reagents, safe and non-toxic, no secondary pollution, more environmentally friendly, and reusable.

(2) Compared with the traditional detection method of chlorinated olefins, the more complex steps such as solvent extraction and purge and trap are omitted, the operation is more convenient, the equipment is simple, the cost is low, and the reaction time is greatly shortened. And lower than general biosensors.

(3) Using a sensitive biosensor to measure chlorinated olefins, the detection limit is lower than that of the traditional method, and the reproducibility is high, which provides a guarantee for the accuracy of the detection results.

(4) The degrading bacterium methanotroph OB3b has certain resistance to pH value and temperature changes, and the measuring device can achieve high accuracy and sensitivity within a certain range.

(5) The electromagnetic stirring device keeps the water body in the reactor in a flowing state, ensures the high mixing and high disturbance of the buffer in the reactor, ensures the complete degradation of the chlorinated olefins by the degrading bacteria, and improves the accuracy of the detection results.

Description of drawings

Fig. 1 is a structural schematic diagram of a chlorinated olefin measuring device.

Fig. 2 is the corresponding relationship figure of trichlorethylene concentration and chloride ion concentration in embodiment 1.

Fig. 3 is the corresponding relationship diagram of temperature and chloride ion concentration in embodiment 1.

Fig. 4 is the corresponding relationship figure of pH value and chloride ion concentration in embodiment 1.

In Fig. 1: 1. Electromagnetic stirring chamber, 2. Water bath device, 3. Thermometer, 4. Reaction cell, 5. AgCl/Ag ₂ S film working electrode, 6. Ag/AgCl reference electrode, 7. Electromagnetic stirrer, 8. Amplifier, 9. Ion detector, 10. Electronic recorder, 11. Porous nitrocellulose membrane, 12. Degrading bacteria.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings.

As shown in Figure 1, an efficient and sensitive chlorinated olefin determination device includes a water bath device 2, a biosensor device, a biofilm device, an electromagnetic stirring device, and a detection device.

Further, the water bath device includes a thermometer 3, a temperature controller, an electric heating unit, a water outlet, and a shell with an upper end opening; the water bath device can be heated by the electric heating unit, and the temperature is detected by the thermometer 3 and the temperature sensor , and maintain a constant temperature by the temperature controller.

Further, the biosensor device includes a reaction cell 4, an AgCl/Ag ₂ S film working electrode 5 and an Ag/AgCl reference electrode 6 for selectively detecting chloride ions; the reaction cell 4 is placed in the water bath device, The reaction pool 4 contains 10 mM phosphate buffer, and the AgCl/Ag ₂ S film working electrode 5 and Ag/AgCl reference electrode 6 for selectively detecting chloride ions are soaked in the reaction pool 4. in the phosphate buffered saline.

Further, the biofilm device comprises two porous nitrocellulose membranes 11 arranged in parallel and degrading bacteria 12 fixed between two layers of the porous nitrocellulose membranes; the diameter of the porous nitrocellulose membranes 11 is 20mm , with a pore size of 0.45 μm; the degrading bacteria 12 are methanotrophs OB3b; the biofilm device is fixed on the bottom of the AgCl/Ag ₂ S membrane working electrode 5 that selectively detects chloride ions with nylon and an O-ring.

Further, the electromagnetic stirring device includes an electromagnetic stirring chamber 1 and an electromagnetic stirrer 7; the electromagnetic stirring chamber 1 is placed under the water bath device 2, and the electromagnetic stirrer 7 is placed in the reaction chamber of the biosensor device. The middle part of the bottom of the pool 4; the electromagnetic stirring chamber 1 can control the electromagnetic stirrer 7 by magnetic force, thereby controlling different rotation speeds, so that the buffer solution in the reaction pool 4 is fully mixed.

Further, the detection device includes an amplifier 8, an ion detector 9 and an electronic recorder 10; the amplifier 8 is connected to the chloride ion electrode, the ion detector 9 is connected to the amplifier 8, and the electronic recorder The instrument 10 is connected with the ion detector 9 again.

The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the technical solutions of the present invention more clearly, but not to limit the protection scope of the present invention.

Example 1

The content of trichlorethylene was determined by the degradation of trichlorethylene by methanotrophic bacteria OB3b. The medium components used for the growth of methanotrophic bacteria OB3b were: 1.0g MgSO ₄ 7H ₂ O; 0.2g CaCl ₂ ; 0.004g ethylenediamine Iron tetraacetate; 1.0 g KNO ₃ ; 0.05% trace element solution (2.86 g H ₃ BO ₃ , 1.81 g MnCl ₂ 4H ₂ O, 0.22 g ZnSO ₄ 7H ₂ O, 0.08 g CuSO ₄ 5H ₂ O, 0.021g Na ₂ MoO ₄ , add sterile water to 1L, finally add 1 drop of concentrated sulfuric acid), dissolve in 1L distilled water, adjust the pH of the medium to about 7.0, and finally add 2 mL of sterile phosphate Buffer (pH 7.0). Under the condition of 30°C, the methanotroph OB3b was inoculated into the above-mentioned medium, and cultured in a gas environment of methane:air (1:2) at a rotation speed of 250rpm until the stationary phase.

The methanotrophs OB3b cultivated to the stationary phase were centrifuged at 4°C and 8000rpm for 5min, and washed twice with 10mM phosphate buffer (pH 7.0); the cells in the supernatant were aspirated into the Immobilized on a porous nitrocellulose membrane (diameter 20 mm, pore size 0.45 μm). Afterwards, another porous nitrocellulose membrane was covered on the fixed microbial membrane, so that the methanotrophic bacteria OB3b was sandwiched between the two membranes, and the preparation of the biofilm device was completed.

The working electrode and the reference electrode were first soaked in the reaction tank filled with 10mM phosphate buffer (pH 7.0), the temperature of the reaction tank was controlled by a constant temperature water bath to maintain at 30°C, and the buffer solution was continuously stirred by an electromagnetic stirring device. When the output signal is stable, trichlorethylene is injected into the reaction pool containing phosphate buffer solvent, and trichlorethylene contacts with fixed degrading bacteria through the lower porous nitrocellulose membrane, and the methanotrophic bacteria OB3b degrades trichlorethylene and releases Chloride ions are received by the AgCl/Ag ₂ S membrane working electrode that selectively detects chloride ions through the upper porous nitrocellulose membrane, and the ion signal is monitored through amplifiers, ion detectors and electronic recorders, and the obtained chloride ion concentration Converted to the concentration of trichlorethylene; the response of the sensor is evaluated by the steady-state method, and its response characteristics are shown in Figure 2, and the concentration of trichlorethylene is measured in the range of 0.05-4.5mg/L, and the concentration of trichlorethylene and The concentration of ^chloride ions is linear, and R is 0.9984; the detection limit of this method for detecting trichlorethylene is 0.05mg/L, and the relative standard deviation is 6.65%, and the reproducibility is high; The reaction time is 6 minutes, which is lower than the 10 minutes of general biosensors.

Set different temperature and pH value gradients to test the performance of the biosensor device for determining the concentration of trichlorethylene. The change of chloride ion concentration with temperature is shown in Figure 3, and the change of chloride ion concentration with pH value is shown in Figure 4. The device can accurately measure trichlorethylene in the temperature range of 20-40°C and the pH value range of 6.0-8.5, the optimum temperature is 30°C, and the optimum pH value is 7.0.

Claims (7)

1. a kind of chloroalkene determines device, it is characterised in that the measure device includes that water bath device, biology sensor are filled Put, Biological membrane device, electromagnetic mixing apparatus and detection means；Wherein, the biosensor arrangement include reaction tank and chlorine from Sub-electrode, reaction tank is placed in water bath device, and chlorine ion electrode includes AgCl/Ag₂S films working electrode and Ag/AgCl references electricity Pole, two kinds of electrodes are immersed in reaction tank；The Biological membrane device include 2 parallel arrangement porous nitrocellulose films and The bacterium for degrading between two-layer porous nitrocellulose film is fixed on, the Biological membrane device is fixed on AgCl/Ag₂S films work Electrode base；Electromagnetic mixing apparatus are divided into two parts and are respectively placed in inside water bath device lower section and biosensor arrangement, inspection Device is surveyed to be connected with biosensor arrangement.

2. a kind of chloroalkene according to claim 1 determines device, it is characterised in that the water bath device includes temperature The shell of meter, temperature controller, electric heating unit, delivery port and upper end open.

3. a kind of chloroalkene according to claim 1 determines device, it is characterised in that the electromagnetic mixing apparatus include Electromagnetic agitation chamber and electromagnetic agitation；Wherein, electromagnetic agitation chamber is placed in water bath device lower section, and electromagnetic agitation is placed in bio-sensing The bottom of pond of reaction tank in device device.

4. a kind of chloroalkene according to claim 1 determines device, it is characterised in that the detection means includes amplifying Device, ion detection instrument and electronic recorder；Wherein, amplifier is connected with two kinds of chlorine ion electrodes, ion detection instrument and amplifier It is connected, electronic recorder is connected with ion detection instrument again.

5. a kind of chloroalkene according to claim 1 determines device, it is characterised in that the porous nitrocellulose film A diameter of 20mm, aperture is 0.45 μm.

6. a kind of chloroalkene according to claim 1 determines device, it is characterised in that the bacterium for degrading is methane oxygen Change bacterium OB3b.

7. the detection method of device is determined based on the chloroalkene described in claim 1, is comprised the following steps：

(1) under to adding solution to be measured, the chloroalkene in solution to be measured to pass through in the reaction tank containing phosphate-buffered solvent Layer porous nitrocellulose film is contacted with fixed bacterium for degrading, and methane-oxidizing bacteria OB3b degrades to chloroalkene, release Go out the AgCl/Ag that chlorion is selectively detected chlorion by upper strata porous nitrocellulose film₂S films working electrode is received；

(2) ion signal is monitored by amplifier, ion detection instrument and electronic recorder by working electrode, by what is obtained Chlorine ion concentration is converted to the concentration of chloroalkene, and the response of biosensor arrangement is estimated by steady state method, makes chlorine For alkene and the corresponding relation figure of chlorine ion concentration, and then obtain concentration of the Chlorine in Solution to be measured for alkene.