Heavy metal ion toxicity sensor as well as working electrode and preparation method thereof

Abstract

The invention belongs to the technical field of microbial electrochemical toxicity sensing, and particularly relates to a heavy metal ion toxicity sensor as well as a working electrode and a preparation method thereof. A working electrode of a heavy metal ion toxicity sensor comprises a base material; the biological membrane is attached to the surface of the base material; the protective layer is attached to the surface of the electroactive biological membrane, and the protective layer is polydopamine. The electroactive biological membrane of the anode is protected by using a polydopamine wrapping method, so that not only is the real-time online monitoring which is simple to operate, stable in result and rapid in feedback realized, but also the continuous detection frequency is effectively increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial electrochemical toxicity sensing, and in particular relates to a heavy metal ion toxicity sensor and a working electrode thereof and a preparation method thereof. Background Art

[0002] With the rapid development of industrialization and urbanization, heavy metal pollution has become increasingly prominent, posing a serious threat to the ecological environment and human health. Heavy metal ions are highly biologically toxic and enriched. Once they enter the environment, they are not only difficult to degrade naturally, but may also enter the human body through the food chain and cause diseases. Therefore, accurate and rapid detection of heavy metal ions in the environment is particularly important.

[0003] Traditional detection methods, such as atomic absorption spectroscopy and atomic fluorescence spectroscopy, have high accuracy and sensitivity, but are complex, costly, and time-consuming, making it difficult to meet the needs of rapid on-site detection. Therefore, the development of new heavy metal ion detection technologies has become a current research hotspot.

[0004] Microbial electrolytic cell toxicity sensor is a new technology that can monitor the toxicity of external poisons by monitoring the change of electric current to indirectly feedback the toxicity of poisons in the environment. This technology is easy to operate, low cost, and can achieve real-time online monitoring, so it has attracted widespread attention from scientific researchers. Among them, the electroactive biofilm on the working electrode is the most important part of the microbial electrolytic cell sensor. Most of the existing microbial electrolytic cell toxicity sensors will cause the death of the electroactive biofilm under the long-term impact of toxicity, which greatly limits the application of microbial electrolytic cell toxicity sensors. Therefore, how to reduce the death of biofilm on the working electrode and increase the number of uses of microbial electrolytic cell toxicity sensors is an urgent problem to be solved in this field. Summary of the invention

[0005] In view of the shortcomings of existing microbial electrolytic cell type toxicity sensors, such as poor stability, small detection range, and few reusable times, the present invention provides a heavy metal ion toxicity sensor, a working electrode thereof, and a preparation method thereof.

[0006] Specifically, the present invention is achieved through the following technical solutions:

[0007] A working electrode of a heavy metal ion toxicity sensor, comprising:

[0008] Base material;

[0009] a biofilm attached to the surface of the substrate;

[0010] A protective layer is attached to the surface of the electroactive biofilm.

[0011] The working electrode of the heavy metal ion toxicity sensor mentioned above, the substrate is a carbon brush.

[0012] In the working electrode of the heavy metal ion toxicity sensor, the electroactive microbial species on the biofilm are one or more of Shewanella, Geobacter sulfurreducens, Rhodopseudomonas palustris, and Geobacter anodoreducens.

[0013] In the working electrode of the heavy metal ion toxicity sensor, the protective layer is polydopamine.

[0014] On the other hand, the present invention provides a method for preparing the working electrode of the heavy metal ion toxicity sensor, comprising:

[0015] S1: immersing the substrate in a phosphate buffer and inoculating electroactive microbial strains to obtain a first intermediate working electrode;

[0016] S2: immersing the first intermediate working electrode in a dopamine hydrochloride solution, so that dopamine generates polydopamine on the surface of the first intermediate working electrode, thereby obtaining a second intermediate working electrode;

[0017] S3: placing the second intermediate working electrode into a heavy metal ion toxicity sensor, and subjecting the second intermediate working electrode to an extreme impact using a heavy metal ion solution to obtain a third intermediate working electrode;

[0018] S4: immersing the third intermediate working electrode in a dopamine hydrochloride solution, so that dopamine regenerates polydopamine on the surface of the third intermediate working electrode to obtain a working electrode.

[0019] In the above-mentioned method for preparing the working electrode of the heavy metal ion toxicity sensor, in step S1, the substrate is immersed in the phosphate buffer for 12-36 hours; the concentration of the phosphate buffer is 10-100 mmol / L.

[0020] In the above-mentioned method for preparing the working electrode of the heavy metal ion toxicity sensor, the preparation of the first intermediate working electrode in step S1 further comprises the following steps:

[0021] S11 uses the substrate as the anode of the heavy metal ion toxicity sensor, puts the culture solution in the main container, and uses nitrogen and carbon dioxide mixed gas aeration to deoxygenate;

[0022] S12 inoculates the culture solution with electroactive microbial strains and continues aeration and deoxygenation;

[0023] S13 monitors the current of the microbial electrolytic cell type sensor reactor, and when the current is lower than 0.1 mA, replaces the culture solution of the microbial electrolytic cell type sensor reactor;

[0024] S14 operates the sensor reactor in a batch mode for 1 to 2 weeks, and when the current is stabilized at more than 20 mA for more than two cycles, a first intermediate working electrode with a stable electroactive biofilm is obtained.

[0025] In the method for preparing the working electrode of the heavy metal ion toxicity sensor, the concentration of the dopamine hydrochloride solution is 1-10 g / L.

[0026] In the above-mentioned method for preparing the working electrode of the heavy metal ion toxicity sensor, the concentration of the heavy metal ion solution is 0-100 ppm; the heavy metal ions in the heavy metal ion solution are copper ions, silver ions or iron ions.

[0027] The method for preparing the working electrode of the heavy metal ion toxicity sensor mentioned above further includes between step S3 and step S4: placing the third intermediate working electrode in a sensor reactor and operating it in a batch mode until the current is stabilized at more than 20 mA for more than two cycles.

[0028] In another aspect, the present invention provides a heavy metal ion toxicity sensor, comprising:

[0029] The reactor body is a container with one end open;

[0030] A cover body, used for covering the opening of the reactor body;

[0031] A working electrode is disposed inside the reactor body; wherein the working electrode is the working electrode described above;

[0032] A counter electrode is disposed inside the reactor body;

[0033] A plurality of holes are provided on the cover and the reactor body.

[0034] The above-mentioned heavy metal ion toxicity sensor further includes: a baffle plate for fixing the counter electrode.

[0035] In the above-mentioned heavy metal ion toxicity sensor, the counter electrode is a stainless steel mesh.

[0036] In the above-mentioned heavy metal ion toxicity sensor, the material of the reactor body is resin.

[0037] In the above-mentioned heavy metal ion toxicity sensor, the reactor body is made by 3D printing.

[0038] The heavy metal ion toxicity sensor mentioned above has three holes, namely, a water replacement and poisoning hole, a working electrode connection opening, and a counter electrode connection opening.

[0039] Compared with the existing heavy metal ion toxicity sensor, the present invention has the following beneficial effects:

[0040] (1) The present invention protects the electroactive biofilm of the anode by using a polydopamine wrapping method, which not only realizes real-time online monitoring with simple operation, stable results and fast feedback, but also effectively increases the number of continuous detections;

[0041] (2) The heavy metal ion toxicity sensor of the present invention avoids the problem of uneven electric field strength in traditional microbial electrolytic cell toxicity sensors, and can also achieve multiple detections of small amounts of toxic substances;

[0042] (3) The heavy metal ion toxicity sensor designed in the present invention can significantly improve the stability, detection range and reusability of the microbial electrolytic cell type heavy metal ion toxicity sensor, and can realize real-time online monitoring and continuous multiple detection of heavy metal ion toxicity with simple operation, stable results and fast feedback. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0044] Figure 1 It is a schematic diagram of the structure of a microbial electrolytic cell type toxicity sensor reactor;

[0045] Figure 2 The present invention and the conventional microbial electrolytic cell type toxicity sensor are used for Cu 2+ Comparison of standard curves for toxicity detection, where the solid line is the experimental result of the present invention and the dotted line is the experimental result of the traditional microbial electrolytic cell type toxicity sensor;

[0046] Figure 3 The present invention and the conventional microbial electrolytic cell type toxicity sensor are used for Ag + Comparison of standard curves for toxicity detection, where the solid line is the experimental result of the present invention and the dotted line is the experimental result of the traditional microbial electrolytic cell type toxicity sensor;

[0047] Figure 4 The present invention and the conventional microbial electrolytic cell type toxicity sensor are used for Fe 3+ Comparison of standard curves for toxicity detection, where the solid line is the experimental result of the present invention and the dotted line is the experimental result of the traditional microbial electrolytic cell type toxicity sensor;

[0048] Figure 5 The present invention is used for Ag + Result graph of continuous toxicity testing;

[0049] Fig. 6Ais a scanning electron microscope image of the electroactive biofilm at the anode of the microbial electrolytic cell type toxicity sensor of the present invention;

[0050] Figure 6B This is a scanning electron microscope image of the electroactive biofilm at the anode of a conventional microbial electrolytic cell-based toxicity sensor. DETAILED DESCRIPTION

[0051] In order to fully understand the purpose, features and effects of the present invention, the present invention is described in detail through the following specific embodiments. Except for the following contents, the process method of the present invention adopts conventional methods or devices in the art. Unless otherwise specified, the following terms have the meanings commonly understood by those skilled in the art.

[0052] The terms "working electrode" and "anode" used in this article have the same meaning, "counter electrode" and "cathode" have the same meaning, "microbial electrolytic cell type heavy metal ion toxicity sensor", "heavy metal ion toxicity sensor", "toxicity sensor", "sensor" and "microbial electrolytic cell" have the same meaning. Terms with the same meaning are used interchangeably in this article. The terms "first", "second", etc. do not indicate any order or importance, but are used to distinguish one element from another. When a numerical range is disclosed herein, the above range is deemed to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values.

[0053] Specifically, the working electrode of the heavy metal ion toxicity sensor provided by the present invention comprises:

[0054] Base material;

[0055] a biofilm attached to the surface of the substrate;

[0056] A protective layer is attached to the surface of the electroactive biofilm.

[0057] The present invention can effectively protect the activity of microorganisms on the biofilm by wrapping a protective layer on the surface of the biofilm, thereby improving the working stability and detection times of the working electrode.

[0058] In some preferred embodiments, the substrate used in the present invention is a carbon brush.

[0059] In some preferred embodiments, the electroactive microbial species on the biofilm are one or more of Shewanella, Geobacter sulfurreducens, Rhodopseudomonas palustris, and Geobacter anodereducens.

[0060] Further preferably, the electroactive microbial species are a mixture of Shewanella and Geobacter sulfurreducens.

[0061] In some preferred embodiments, the protective layer is polydopamine.

[0062] The polydopamine used in the present invention is a polymer with good biocompatibility. It can be self-polymerized and adhered to the surface of the electroactive biofilm under mild conditions. When heavy metal ion poisons are added, the polydopamine protective layer will play a certain protective role on the internal electroactive biofilm, thereby preventing the electroactive biofilm from being inactivated or even killed due to the impact of the poison.

[0063] In another aspect, the present invention provides a method for preparing a working electrode of a heavy metal ion toxicity sensor, comprising:

[0064] 1) Inoculation of electroactive biofilm

[0065] The first intermediate working electrode is obtained by inoculating an electroactive biofilm on a substrate, which can be prepared by existing methods or obtained by commercial purchase.

[0066] In some preferred embodiments, the first intermediate working electrode can be obtained by immersing the substrate in phosphate buffer, inoculating electroactive microbial strains, and operating in a batch mode for 1 to 2 weeks to ensure the formation of a stable electroactive biofilm to obtain the first intermediate working electrode.

[0067] The batch mode is run for 1 to 2 weeks and specifically includes: operating the microbial electrolysis cell in the form of an applied DC voltage, connecting the positive electrode to the working electrode, and monitoring the current in real time. The current will show a trend of first increasing, then stabilizing, and finally decreasing. After the current drops below the threshold, the sodium acetate culture medium is replaced, and the current is continuously monitored in real time. This process is repeated 1 to 2 times.

[0068] In some preferred embodiments, the batch mode is run for 1 to 2 weeks and specifically includes: operating the microbial electrolysis cell in the form of an applied 0.7V DC voltage, connecting the positive electrode to the working electrode, and monitoring the current in real time. The current will show a trend of first increasing, then stabilizing, and finally decreasing. After the current drops below 0.1mA, the culture medium with a sodium acetate concentration of 1g / L is replaced, and the current is continued to be monitored in real time. This process is repeated 1 to 2 times.

[0069] Preferably, the substrate is immersed in the phosphate buffer for 12-36 hours, more preferably 24 hours; the concentration of the phosphate buffer is 10-100 mmol / L, more preferably 50 mmol / L.

[0070] Wherein, the phosphate buffer comprises: 4.576 g / L Na 2 HPO 4 , 2.132 g / L NaH 2 PO 4 、0.31g / LNH4 Cl, 0.13 g / L KCl.

[0071] In some preferred embodiments, the preparation of the first intermediate working electrode further comprises the following steps:

[0072] S11 uses the substrate soaked in phosphate buffer as the anode of the heavy metal ion toxicity sensor, puts the culture solution in the main container, and uses nitrogen and carbon dioxide mixed gas aeration for deoxygenation;

[0073] S12 inoculates the culture solution with electroactive microbial strains and continues aeration and deoxygenation;

[0074] S13 monitors the current of the microbial electrolytic cell type sensor reactor, and when the current is lower than 0.1 mA, replaces the culture solution of the microbial electrolytic cell type sensor reactor;

[0075] S14 operates the sensor reactor in a batch mode for 1 to 2 weeks, and when the current is stabilized at more than 20 mA for more than two cycles, a first intermediate working electrode with a stable electroactive biofilm is obtained.

[0076] The culture solution includes: 50mmol phosphate buffer, 12.5mL / L trace mineral element solution, 5mL / L vitamin solution and 1g / L sodium acetate.

[0077] The inoculation source of the electroactive microbial strains is a mixed bacteria of Shewanella and Geobacter sulfurreducens in a dry weight ratio of 1:1 to 1:5 combined with 10% (V / V) marine sediment extract.

[0078] Before inoculating the electroactive microbial strains, nitrogen and carbon dioxide mixed gas was used for aeration and deoxygenation for 30 minutes; after inoculation, aeration was continued for 10 minutes.

[0079] 2) In situ modification of polydopamine protective layer

[0080] When the sensor reactor current is stable at more than 20 mA for more than two cycles, the electroactive biofilm surface of the first intermediate working electrode is wrapped with a dopamine hydrochloride solution to obtain a second intermediate working electrode. A high-concentration heavy metal ion solution is then used for extreme impact to obtain a third intermediate working electrode. Finally, the sensor reactor is operated in a batch mode until the current is stable at more than 20 mA for more than two cycles, and then wrapped with a dopamine hydrochloride solution again to obtain a working electrode.

[0081] In some preferred embodiments, the concentration of the dopamine hydrochloride solution is 1-10 g / L, more preferably 2 g / L.

[0082] In some preferred embodiments, a Tris-HCl buffer system is added to the dopamine hydrochloride solution to make its pH weakly alkaline (preferably, pH=8.5).

[0083] In some preferred embodiments, the concentration of the heavy metal ion solution is 0-100 ppm (more preferably 100 ppm); the heavy metal ions in the heavy metal ion solution are copper ions, silver ions or iron ions. When copper ions are used, the upper limit of the concentration can be increased to 120 ppm.

[0084] The purpose of the present invention to use heavy metal ion solution to perform extreme toxic shock on the second intermediate working electrode is to select the electroactive biofilm community, thereby optimizing the community composition of the electroactive biofilm and making it more suitable for the requirements of being a toxic sensor.

[0085] Among them, the step of "running in a batch mode until the current of the sensor reactor is stable at more than 20mA for more than two cycles" specifically includes: running the microbial electrolysis cell in the form of an applied DC voltage, connecting the positive electrode to the working electrode, and monitoring the current in real time. The current will show a trend of first increasing, then stabilizing, and finally decreasing. When the current drops below the threshold, the sodium acetate culture medium is replaced and the current is continued to be monitored in real time. During this batch cycle, the current in the stable period will increase. It is observed that the stable period current of two cycles exceeds 20mA, and the subsequent steps are carried out.

[0086] In some preferred embodiments, the step of "running in a batch mode until the current of the sensor reactor is stable at more than 20 mA for more than two cycles" specifically includes: running the microbial electrolysis cell in the form of an applied 0.7V DC voltage, connecting the positive electrode to the working electrode, and monitoring the current in real time. The current will show a trend of first increasing, then stabilizing, and finally decreasing. When the current drops to below 0.1mA, replace the culture medium with a sodium acetate concentration of 1g / L, and continue to monitor the current in real time. During this batch cycle, the current in the stable period will increase, and it is observed that the stable period current of two cycles exceeds 20mA, and then proceed to the subsequent steps.

[0087] Among them, the present invention uses dopamine hydrochloride solution for secondary wrapping again, thereby ensuring that the carbon brush anode electroactive biofilm modified with polydopamine in situ has a strong toxicity tolerance upper limit.

[0088] On the other hand, Figure 1 As shown, the present invention also provides a heavy metal ion toxicity sensor, comprising:

[0089] The reactor body 1 is a container with one end open;

[0090] A cover 2, used to cover the opening of the reactor body 1;

[0091] A working electrode 3 is arranged inside the reactor body 1;

[0092] A counter electrode 4 is arranged inside the reactor body 1;

[0093] A plurality of holes 5 are provided on the cover 2 and the reactor body 1 .

[0094] In some preferred embodiments, the heavy metal ion toxicity sensor further includes: a baffle 6 for fixing the counter electrode 4 .

[0095] In some preferred embodiments, the number of the holes 5 is three, namely a water changing and poisoning hole 51 , a working electrode connection opening 52 , and a counter electrode connection opening 53 .

[0096] In some preferred embodiments, the counter electrode 4 is a stainless steel mesh, which is curled into a barrel shape and placed between the baffle 6 and the inner wall of the reactor body 1, and the working electrode 3 is a carbon brush, which is fixed to the working electrode connection opening 52 through the handle of the carbon brush. Thereby, when a DC power supply is passed, the reactor obtains a cylindrical uniform electric field, which further makes the formed electroactive biofilm more stable.

[0097] Optionally, the counter electrode 4 is connected to an external power source via a titanium wire.

[0098] Optionally, the material of the reactor body 1 is resin, and the reactor body 1 is manufactured by 3D printing.

[0099] Example

[0100] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions. The raw materials used in the following examples are all conventionally purchased.

[0101] The main body of the sensor reactor is cylindrical and is divided into two parts: the bottle body and the bottle cap, which are connected by threads. The inner diameter of the reactor is 5cm, the effective depth is 8cm, and the effective volume is 120mL. The inside of the bottle body is designed with a fence-like baffle with a diameter of 4cm to fix the counter electrode, and a hole with a diameter of 0.8cm is opened on the side of the bottle body for connecting the counter electrode to the external power supply. A 0.8cm hole is opened in the center of the top of the bottle cap for connecting the working electrode carbon brush to the external power supply, and a 0.8cm hole is opened on the side of the top of the bottle cap for updating the culture medium and adding poisons. The working electrode is a carbon brush with a diameter of 4cm and an effective length of 7cm. The working electrode is connected to the external power supply through the carbon brush handle. The counter electrode is a stainless steel mesh with a length of 15.7cm and a width of 7.5cm, curled into a barrel shape and placed between the baffle and the inner wall. The counter electrode is connected to the external power supply through a titanium wire, and the external power supply is a 0.7V DC power supply.

[0102] Use 50mmol / L phosphate buffer (4.576g / L Na 2 HPO 4 , 2.132 g / L NaH 2 PO 4 、0.31g / LNH 4 Cl, 0.13g / L KCl) soak the sensor reactor for 24 hours. The sensor reactor uses a mixed bacteria with a dry weight ratio of Shewanella and Geobacter sulfurreducens of 1:1 to 1:5 and 10% (V / V) marine sediment leaching solution as the inoculation source, 1.2g / L sodium acetate as the carbon source, and strictly controls the anaerobic environment. A nitrogen and carbon dioxide mixed gas is used for aeration and deoxygenation for 30 minutes before inoculation, and the sensor reactor is aerated for 10 minutes after inoculation. Monitor the current of the microbial electrolytic cell sensor reactor. When the current is lower than 0.1mA, replace the culture medium of the microbial electrolytic cell sensor reactor. The culture medium uses 50mmol phosphate buffer, 12.5mL / L trace mineral element solution, 5mL / L vitamin solution and 1g / L sodium acetate, and runs for 1 to 2 weeks to ensure the formation of a stable electroactive biofilm on the working electrode carbon brush.

[0103] When the current of the sensor reactor is stable at more than 20 mA for more than two cycles, the surface of the electroactive biofilm of the carbon brush anode is wrapped with a 2 g / L dopamine hydrochloride solution in a Tris-HCl buffer system (pH = 8.5), and the wrapped carbon brush anode is put back into the sensor reactor. A high concentration of 100 ppm heavy metal ion solution is used for extreme toxic shock to select the electroactive biofilm community of the carbon brush anode. The sensor reactor is then operated in a batch mode until the current of the sensor reactor is stable at more than 20 mA for more than two cycles, and then the dopamine hydrochloride solution is used for secondary wrapping to obtain the working electrode of the polydopamine-protected microbial electrolytic cell type heavy metal ion toxicity sensor.

[0104] Another set of clean reactor bodies, covers, counter electrodes and baffles are taken, and the working electrode prepared by the present invention is placed in the sensor reactor for assembly to obtain the improved heavy metal ion toxicity sensor of the present invention.

[0105] Test 1

[0106] The polydopamine-protected microbial electrolytic cell type heavy metal ion toxicity sensor of the present invention is used for heavy metal ion Cu 2+ The toxicity test was performed and compared with the test results of the traditional microbial electrolytic cell type toxicity sensor. The reactor configuration and operation mode of the traditional microbial electrolytic cell type toxicity sensor are consistent with those of the present invention.

[0107] When the sensor reactor current is stable, different concentrations of CuSO 4 The solution was added to the sensor reactor, the current decay during the change process was recorded, and △I was calculated according to formula 1 to obtain the relationship between △I and Cu 2+ The concentration correlation curve, such as Figure 2 As shown, the ΔI obtained using the toxicity sensor of the present invention and its corresponding Cu 2+ The concentration has a strong linear relationship in the range of 0 to 120 ppm, while the △I obtained using the traditional toxicity sensor and its corresponding Cu 2+ There is a certain exponential relationship in the concentration range of 0-100 ppm. The theoretical detection upper limit (concentration corresponding to △I=100%) deduced from the standard curve is 236 ppm (the present invention) and 123 ppm (the traditional sensor).

[0108]

[0109] Where:

[0110] △I——current change percentage, used to indicate the change of current;

[0111] I ave ——The average current value in the current stabilization stage before adding heavy metal poisons;

[0112] I min ——The minimum current value within five minutes after adding heavy metal poison.

[0113] Test 2

[0114] The polydopamine-protected microbial electrolytic cell type heavy metal ion toxicity sensor of the present invention is used for heavy metal ion Ag + The toxicity test was performed and compared with the test results of the traditional microbial electrolytic cell type toxicity sensor. The reactor configuration and operation mode of the traditional microbial electrolytic cell type toxicity sensor are consistent with those of the present invention.

[0115] When the current of the sensor reactor is stable, different concentrations of AgNO 3 The solution was added to the sensor reactor, the current decay during the change process was recorded, and △I was calculated according to formula 1 to obtain the relationship between △I and Ag. + The concentration correlation curve, such as Figure 3 As shown, the ΔI obtained using the toxicity sensor of the present invention and its corresponding Ag + The concentration has a strong linear relationship in the range of 0 to 100 ppm, while the △I obtained using traditional toxicity sensors and its corresponding Ag +There is a certain linear relationship in the concentration range of 0-100 ppm. The theoretical upper limit of detection (the corresponding concentration when △I=100%) deduced from the standard curve is 317 ppm (the present invention) and 147 ppm (the traditional sensor).

[0116] Test 3

[0117] In this embodiment, a polydopamine-protected microbial electrolytic cell-type heavy metal ion toxicity sensor of the present invention is used for heavy metal ion Fe 3+ The toxicity test was performed and compared with the test results of the traditional microbial electrolytic cell type toxicity sensor. The reactor configuration and operation mode of the traditional microbial electrolytic cell type toxicity sensor are consistent with those of the present invention.

[0118] When the sensor reactor current is stable, different concentrations of FeCl 3 The solution was added to the sensor reactor, the current decay during the change process was recorded, and △I was calculated according to formula 1 to obtain the relationship between △I and Fe 3+ The concentration correlation curve, such as Figure 4 As shown, the ΔI obtained using the toxicity sensor of the present invention and its corresponding Fe 3+ The concentration has a strong linear relationship in the range of 0 to 100 ppm, while the △I obtained using the traditional toxicity sensor and its corresponding Fe 3+ There are different linear relationships in the concentration range of 0-50ppm and 70-100ppm. The theoretical upper detection limit (concentration corresponding to △I=100%) deduced from the standard curve is 337ppm (the present invention) and 246ppm (the traditional sensor).

[0119] Test 4

[0120] In this embodiment, a polydopamine-protected microbial electrolytic cell-type heavy metal ion toxicity sensor of the present invention is used for heavy metal ion Ag + The continuous toxicity test uses 30ppm of AgNO every 10 minutes when the current of the sensor reactor is stable. 3 The solution is added to the sensor reactor, the current decay during the change process is recorded, and △I is calculated according to formula 1 to obtain the △I change curve over time, such as Figure 5 As shown, the present invention can realize 10 consecutive detections of heavy metal ion toxicity within 100 minutes without renewing the culture medium.

[0121] Test 5

[0122] In this embodiment, scanning electron microscope was used to take images of the anode electroactive biofilm of a polydopamine protected microbial electrolytic cell type heavy metal ion toxicity sensor of the present invention and a conventional microbial electrolytic cell type toxicity sensor. Fig. 6A and Figure 6B As shown, compared with the anode electroactive biofilm of the traditional sensor in which a large number of rod-shaped microorganisms are exposed on the surface, it can be clearly seen that the anode electroactive biofilm of the present invention is wrapped by polydopamine.

[0123] The present invention has been disclosed above with preferred embodiments, but those skilled in the art should understand that these embodiments are only used to describe the present invention and should not be understood as limiting the scope of the present invention. It should be noted that all changes and substitutions equivalent to these embodiments should be deemed to be included in the scope of the claims of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined in the claims.

Claims

1. A working electrode for a heavy metal ion toxicity sensor, characterized in that: include: Base material; a biofilm attached to the surface of the substrate; A protective layer is attached to the surface of the electroactive biofilm.

2. The working electrode according to claim 1, characterized in that The substrate is a carbon brush.

3. The working electrode according to claim 1, characterized in that The electroactive microbial species on the biofilm are one or more of Shewanella, Geobacter sulfurreducens, Rhodopseudomonas palustris, and Geobacter anodereducens.

4. The working electrode according to claim 1, characterized in that The protective layer is polydopamine.

5. The method for preparing a working electrode according to any one of claims 1 to 4, characterized in that: include: S1: immersing the substrate in a phosphate buffer and inoculating electroactive microbial strains to obtain a first intermediate working electrode; S2: immersing the first intermediate working electrode in a dopamine hydrochloride solution, so that dopamine generates polydopamine on the surface of the first intermediate working electrode, thereby obtaining a second intermediate working electrode; S3: placing the second intermediate working electrode into a heavy metal ion toxicity sensor, and subjecting the second intermediate working electrode to an extreme impact using a heavy metal ion solution to obtain a third intermediate working electrode; S4: immersing the third intermediate working electrode in a dopamine hydrochloride solution, so that dopamine regenerates polydopamine on the surface of the third intermediate working electrode to obtain a working electrode.

6. The preparation method according to claim 5, characterized in that: In step S1, the substrate is immersed in the phosphate buffer for 12-36 hours; the concentration of the phosphate buffer is 10-100 mmol / L.

7. The preparation method according to claim 5, characterized in that: The preparation of the first intermediate working electrode in step S1 further includes the following steps: S11 uses the substrate as the anode of the heavy metal ion toxicity sensor, puts the culture solution in the main container, and uses nitrogen and carbon dioxide mixed gas aeration to deoxygenate; S12 inoculates the culture solution with electroactive microbial strains and continues aeration and deoxygenation; S13 monitors the current of the microbial electrolytic cell type sensor reactor, and when the current is lower than 0.1 mA, replaces the culture solution of the microbial electrolytic cell type sensor reactor; S14 operates the sensor reactor in a batch mode for 1 to 2 weeks, and when the current is stabilized at more than 20 mA for more than two cycles, a first intermediate working electrode with a stable electroactive biofilm is obtained.

8. The preparation method according to claim 5, characterized in that: The concentration of the dopamine hydrochloride solution is 1-10 g / L.

9. The preparation method according to claim 5, characterized in that: The concentration of the heavy metal ion solution is 0-100 ppm; the heavy metal ions in the heavy metal ion solution are copper ions, silver ions or iron ions.

10. The preparation method according to claim 5, characterized in that: The method between step S3 and step S4 also includes: placing the third intermediate working electrode in a sensor reactor and operating in a batch mode until the current is stabilized at more than 20 mA for more than two cycles.

11. A heavy metal ion toxicity sensor, characterized in that: include: The reactor body is a container with one end open; A cover body, used for covering the opening of the reactor body; A working electrode is disposed inside the reactor body; wherein the working electrode is the working electrode according to any one of claims 1 to 4; A counter electrode is disposed inside the reactor body; A plurality of holes are provided on the cover and the reactor body.

12. The heavy metal ion toxicity sensor according to claim 11, characterized in that: Also includes: A baffle is used to fix the pair of electrodes.

13. The heavy metal ion toxicity sensor according to claim 11, characterized in that: The counter electrode is a stainless steel mesh.

14. The heavy metal ion toxicity sensor according to claim 11, characterized in that: The material of the reactor body is resin.

15. The heavy metal ion toxicity sensor according to claim 14, characterized in that: The reactor body is manufactured by 3D printing.

16. The heavy metal ion toxicity sensor according to claim 11, characterized in that: The number of the holes is 3, which are a water changing and poisoning hole, a working electrode connection opening, and a counter electrode connection opening.

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