CN117147640B - Method, system and use for producing microbial electrochemical sensors - Google Patents
Method, system and use for producing microbial electrochemical sensors Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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Abstract
The invention discloses a method, a system and application for preparing a microbial electrochemical sensor, wherein the method comprises the following steps: (1) Culturing a bioanode comprising a biofilm carrier to which a target strain is attached to obtain an initial sensor; (2) And injecting a target toxic substance into the initial sensor, and carrying out toxic stimulation on the initial sensor so as to obtain the microbial electrochemical sensor. According to the invention, after conventional domestication is finished and before on-machine test, the toxicity stimulation treatment of the sensor is added, so that the sensitivity of the sensor to toxic substances is improved, and the toxicity response time of the sensor during on-machine test is shortened, so that the sensitivity of the sensor to toxic substances is higher and the response speed is higher, and the problems of low sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow response speed (the toxicity response signal starts to appear after a test is performed for hours or even days) of the current sensor are solved.
Description
Technical Field
The invention belongs to the technical field of biological sensors, and particularly relates to a method, a system and application for preparing a microbial electrochemical sensor.
Background
In recent years, with the development of heavy industry, heavy metal pollution has become one of the most harmful water pollution problems. Heavy metal pollution monitoring is an important means for knowing the current situation of water pollution, and has very important significance for guaranteeing the safety of human life and property and carrying out subsequent water treatment work in a targeted manner.
The traditional physicochemical analysis method, such as ultraviolet spectrophotometry, atomic absorption method, atomic fluorescence method, inductively coupled plasma method, X fluorescence spectrum, inductively coupled plasma mass spectrometry, anodic stripping voltammetry and the like, has the test result that the biotoxicity of heavy metal in water cannot be directly reflected, and in practical application, large-scale and expensive instruments and equipment are often needed, a large amount of chemical reagents and heavy metal standard solutions are needed to be prepared, the equipment maintenance amount is large, the test time is long, the cost is high, a large amount of toxic substances exist in the test waste liquid, and secondary pollution is caused to the environment, so the traditional physicochemical analysis method cannot meet the biological safety early warning requirement of the heavy metal in water.
The biological analysis method adopts organisms, biological tissues and the like as biological sensors, when the biological sensors are impacted by toxic substances, the biological metabolism activities of the organisms, the biological tissues and the like are influenced, for example, the luminous intensity is reduced when luminous bacteria are impacted by toxic substances, and the electricity generation intensity is reduced when electricity generating bacteria are impacted by toxic substances, so that the biological toxicity of the water body can be judged by monitoring the corresponding physiological and biochemical index changes before and after the biological sensors are impacted by toxic substances. The microbial electrochemical sensor (Microbial Electrochemical Sensor, MES) takes electrochemical active microorganisms as cores, has the advantages of low cost, no secondary pollution and the like, and plays an increasingly important role in water environment safety monitoring. However, the existing MES has the problems of low heavy metal test sensitivity (the response concentration of most sensors is several to tens of mg/L), low response speed (the toxicity response signal starts to appear after several hours or even days of test) and the like, and cannot realize the high-sensitivity, rapid and low-cost monitoring of low-concentration heavy metals in water.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, the object of the present invention is to propose a method, a system and a use for manufacturing a microbial electrochemical sensor. According to the invention, after conventional domestication is finished and before on-machine test, the toxicity stimulation treatment of the sensor is added, so that the sensitivity of the sensor to toxic substances is improved, and the toxicity response time of the sensor during on-machine test is shortened, so that the sensitivity of the sensor to toxic substances is higher and the response speed is higher, and the problems of low sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow response speed (the toxicity response signal starts to appear after a test is performed for hours or even days) of the current sensor are solved.
In one aspect of the invention, a method of making a microbial electrochemical sensor is provided. According to an embodiment of the invention, the method comprises:
(1) Culturing a bioanode comprising a biofilm carrier to which a target strain is attached to obtain an initial sensor;
(2) And injecting a target toxic substance into the initial sensor, and carrying out toxic stimulation on the initial sensor so as to obtain the microbial electrochemical sensor.
According to the method for preparing the microbial electrochemical sensor, disclosed by the embodiment of the invention, the toxicity stimulation treatment of the sensor is added after the conventional domestication is finished and before the on-line test, so that the sensitivity of the sensor to toxic substances is improved, the toxicity response time of the sensor during the on-line test is shortened, the sensitivity of the sensor to toxic substances is higher, the response speed is faster, and the problems that the current sensor is low in heavy metal test sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow in response speed (the toxicity response signal starts to appear after a test is performed for hours or even days) are solved. Therefore, high-sensitivity, rapid and low-cost monitoring of low-concentration heavy metals in the water body can be realized.
In addition, the method according to the above embodiment of the present invention may further have the following additional technical features:
in some embodiments of the invention, in step (2), it is determined whether the toxic stimulus is completed by monitoring the current change of the initial sensor during the toxic stimulus.
In some embodiments of the invention, step (2) comprises:
(2-1) injecting a second fresh culture solution of carbon source into the initial sensor to replace the culture solution of carbon source in the initial sensor, and recording the output current I of the sensor 1 Wherein the carbon source concentration of the second carbon source fresh culture solution is less than the carbon source concentration of the carbon source culture solution in the initial sensor;
(2-2) injecting a carbon source culture solution containing a target toxic substance into the sensor to replace the second carbon source fresh culture solution in the sensor, performing toxic stimulation on the sensor for a preset time, and recording the output current I of the sensor after the preset time 2 Wherein the carbon source concentration of the carbon source culture solution containing the target toxic substance is less than the carbon source concentration of the carbon source culture solution in the initial sensor;
(2-3) calculating the toxic stimulus current suppression rate r= (I) 1 -I 2 ）/I 1 ；
Repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, and the R value deviation is not more than +/-10% for three times, and judging that the toxicity stimulation is completed.
In some embodiments of the present invention, before step (2-1), further comprising:
(2-0) injecting a first carbon source fresh culture solution into the initial sensor to replace the carbon source culture solution in the initial sensor, and then the second carbon source fresh culture solution injected into the sensor in the step (2-1) is used for replacing the first carbon source fresh culture solution in the sensor, wherein the carbon source concentration of the second carbon source fresh culture solution is smaller than that of the first carbon source fresh culture solution.
In some embodiments of the present invention, in step (2-1), after the replacement of the first carbon source fresh culture solution in the sensor is completed, the sensor is left for a preset time, and then the output current I of the sensor is recorded 1 The method comprises the steps of carrying out a first treatment on the surface of the In the step (2-2), after the fresh culture solution of the second carbon source in the sensor is replaced, the sensor is left for a preset time to perform toxic stimulation on the sensor, and then the output current I of the sensor after the preset time is recorded 2 ；
Or in the step (2-1), after the first carbon source fresh culture solution in the sensor is replaced, continuously introducing the second carbon source fresh culture solution into the sensor for a preset time, and then recording the output current I of the sensor 1 The method comprises the steps of carrying out a first treatment on the surface of the In the step (2-2), after the fresh culture solution of the second carbon source in the sensor is replaced, continuously introducing the culture solution of the carbon source containing the target toxic substance into the sensor for a preset time to perform toxic stimulation on the sensor, and then recording the output current I of the sensor after the preset time 2 。
In some embodiments of the present invention, the concentration of the target toxic substance in the carbon source culture solution containing the target toxic substance is 0.05mg/L to 1.0mg/L; and/or in the step (2-2), the time of single toxicity stimulation is 15-60 min; and/or in the step (2-1), after the first carbon source fresh culture solution in the sensor is replaced, standing for 20-40 min, or continuously introducing the second carbon source fresh culture solution into the sensor for 20-40 min; and/or in the step (2-0), before the step (2-1), injecting the first carbon source fresh culture solution into the initial sensor 10-14 h in advance.
In some embodiments of the invention, the first carbon source fresh culture broth is the same as the fresh culture broth that incubated the initial sensor; and/or the carbon source concentration of the second carbon source fresh culture solution is 5-50% of that of the first carbon source fresh culture solution, and other components and conditions of the second carbon source fresh culture solution are the same as those of the first carbon source fresh culture solution; and/or the other components and conditions of the carbon source culture solution containing the target toxic substance except the target toxic substance are the same as those of the second carbon source fresh culture solution; and/or the target toxic substance comprises Zn 2+ 、Cu 2+ 、Pb 2+ 、Ni 2+ 、Tl 2+ 、Hg 2+ And Cd 2+ At least one of them.
In some embodiments of the invention, a carbon source culture solution containing specific single heavy metal ions is adopted to carry out toxicity stimulation on the sensor, the steps (2-1) - (2-3) are repeated until R is more than or equal to 15% and less than or equal to 75%, and the deviation of the R value is not more than +/-10% three times continuously, so that the toxicity stimulation is judged to be completed;
or, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific multi-element composite heavy metal ions, repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, and judging that the toxicity stimulation is finished if the R value deviation is not more than +/-10% three times continuously;
Or, firstly, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific single heavy metal ions, and repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, wherein the R value deviation is not more than +/-10% for three times continuously; and then, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific multi-element composite heavy metal ions, repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, and judging that the toxicity stimulation is finished if the R value deviation is not more than +/-10% three times continuously.
In a second aspect of the invention, the invention provides a system for performing the toxic stimulation in the method described in the examples above. According to an embodiment of the invention, the system comprises:
a sensor;
the first liquid storage device is used for storing the second carbon source fresh culture solution;
at least one second liquid storage device for storing the carbon source culture solution containing the target toxic substance;
the liquid inlet of the first multi-way valve flow dividing element is respectively connected with the liquid outlet of the first liquid storage device and the liquid outlet of the second liquid storage device, and the first liquid outlet of the first multi-way valve flow dividing element is communicated with the liquid inlet of the sensor;
The first liquid outlet of the second multi-way valve flow dividing element is respectively connected with the liquid inlet of the first liquid storage device and the liquid inlet of the second liquid storage device, and the liquid inlet of the second multi-way valve flow dividing element is connected with the liquid outlet of the sensor;
the first waste liquid collecting device is connected with the second liquid outlet of the first multi-way valve flow dividing element;
the second waste liquid collecting device is connected with a second liquid outlet of the second multi-way valve flow dividing element;
a pumping device disposed on a line between the first multi-way valve split element and the sensor;
the data acquisition and signal control unit is connected with the sensor, the first multi-way valve shunt element, the second multi-way valve shunt element and the pumping device through electric signals respectively.
Therefore, the system can realize the process of carrying out toxic stimulation on the sensor, thereby improving the sensitivity of the sensor to toxic substances, shortening the toxic response time when the sensor is subjected to on-machine test, leading the sensor to have higher sensitivity and faster response speed to the toxic substances, and solving the problems of low heavy metal test sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow response speed (the toxic response signal starts to appear after testing for hours or even days) of the current sensor. In addition, in the on-line test process of the response performance of the sensor to heavy metal, the test environment is in a flowing water state, and in the process of realizing the toxic stimulation to the sensor, the sensor is in a continuous flowing state by the system, so that the sensor with the toxic stimulation is more suitable for the scene of the on-line test, and the sensitivity of the sensor to toxic substances can be further improved.
In a third aspect of the invention, the invention provides the use of the method of the above embodiments in biotoxicity detection. Therefore, the method for carrying out toxicity stimulation on the sensor which is mature in incubation is used for biological toxicity detection, so that the sensitivity of the sensor to toxic substances is improved, the toxicity response time of the sensor during on-machine test is shortened, the sensitivity of the sensor to toxic substances is higher, the response speed is higher, and the problems that the current sensor is low in heavy metal test sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow in response speed (the toxicity response signal starts to appear after a test is carried out for hours or even days) are solved. Therefore, high-sensitivity, rapid and low-cost monitoring of low-concentration heavy metals in the water body can be realized.
Additional aspects and 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 the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of a system for performing a toxic stimulation process in accordance with the present invention;
FIG. 2 is a graph showing the trend of the single heavy metal on-line test inhibition rate change of the sensor prepared in comparative example 1 of the present invention;
FIG. 3 is a graph showing the trend of the inhibition rate of the on-machine test of the composite heavy metal of the sensor prepared in comparative example 1;
FIG. 4 is a graph showing the trend of the single heavy metal on-line test inhibition rate of the sensor prepared in example 2 of the present invention;
FIG. 5 is a graph showing the trend of the inhibition rate of the on-machine test of the composite heavy metal of the sensor prepared in the embodiment 2 of the invention;
FIG. 6 is a graph showing the trend of the single heavy metal on-line test inhibition rate of the sensor prepared in example 3 of the present invention;
FIG. 7 is a graph showing the trend of the inhibition rate of the on-machine test of the composite heavy metal of the sensor prepared in the embodiment 3 of the invention;
FIG. 8 is a graph showing the trend of the single heavy metal on-line test inhibition rate of the sensor prepared in example 4 of the present invention;
FIG. 9 is a graph showing the trend of the inhibition rate of the on-load test of the composite heavy metal of the sensor prepared in example 4 of the present invention.
The device comprises a 1-sensor, a 2-second multi-way valve flow dividing element, a 3-second waste liquid collecting device, a 4-first liquid storage device, a 5-second liquid storage device, a 6-first waste liquid collecting device, a 7-first multi-way valve flow dividing element, an 8-pumping device and a 9-data acquisition and signal control unit.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
In one aspect of the invention, a method of making a microbial electrochemical sensor is provided. According to an embodiment of the invention, a method comprises: (1) Culturing a bioanode comprising a biofilm carrier to which a target strain is attached to obtain an initial sensor; (2) And injecting a target toxic substance into the initial sensor, and carrying out toxic stimulation on the initial sensor so as to obtain the microbial electrochemical sensor. Therefore, the invention increases the toxicity stimulation treatment of the sensor after the conventional domestication is finished and before the on-machine test, namely, the sensor is subjected to toxicity stimulation after the incubation is mature, so that the sensitivity of the sensor to toxic substances is improved, the toxicity response time of the sensor during the on-machine test is shortened, the sensor has higher sensitivity to toxic substances and higher response speed, and the problems of low sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow response speed (the toxicity response signal starts to appear after a test is hours or even days) of the current sensor are solved. Therefore, high-sensitivity, rapid and low-cost monitoring of low-concentration heavy metals in the water body can be realized.
The method for preparing the microbial electrochemical sensor according to the present invention is described in detail as follows:
the method for preparing the microbial electrochemical sensor comprises the following steps:
s100: culturing a bioanode comprising a biofilm carrier with target bacteria attached thereto to obtain an initial sensor.
In this step, the specific method of preparing the above-mentioned initial sensor is not particularly limited as long as the mature sensor can be incubated. As some specific examples, step S100 includes:
s110: assembling an electrochemical active microbial membrane incubation sensor;
in the embodiment of the present invention, the specific structure of the sensor is not particularly limited, and for example, a single-chamber sensor, a dual-chamber sensor, a three-chamber sensor or a combination sensor (i.e., multiple sets of bioelectrodes are assembled in the same chamber) may be used. As a specific example, each sensor comprises an electrolytic cell on which 1 bioanode WE, 1 reference electrode RE and 1 counter electrode CE are provided. The electrolytic cell is also provided with a liquid inlet and a liquid outlet for installing a liquid inlet pipe and a liquid outlet pipe; and the liquid inlet pipe and the liquid outlet pipe are provided with control valves capable of controlling the on/off of the flow paths. The components of the sensor are assembled.
S120: preparing fresh culture solution;
in this step, the culture medium comprises salts, trace elements, vitamins, carbon sources, etc. The mother liquor of each part can be prepared independently, and then the mother liquor of each part is taken according to the set proportion, mixed and diluted by deionized water. Then placing the mixture in a constant temperature incubator, and introducing nitrogen gas while aerating nitrogen gas.
The specific kinds of the above salts, trace elements, vitamins and carbon sources are not particularly limited, for example, phosphate buffer substances for maintaining pH stability of the culture solution, other buffer substances such as HEPES buffer substances, naHCO buffer substances, etc. may be selected 3 /H 2 CO 3 Etc. For example, sodium acetate which provides a carbon source may be sodium lactate, glucose, or the like. For example, the ammonium chloride that provides a nitrogen source may be an inorganic ammonium salt such as ammonium sulfate or ammonium nitrate, or may be a substance that can provide organic nitrogen such as yeast powder or peptone. The specific concentrations of the above salts, trace elements, vitamins and carbon sources are not particularly limited.
S130: inoculating a target strain;
in the step, the fresh culture solution prepared in the step S120 and the seed source water are mixed according to a preset proportion to obtain an inoculation bacterial suspension, and the inoculation bacterial suspension is filled into a liquid storage bottle.
S140: a connecting pipeline;
In the step, a liquid taking pipe is inserted into the liquid storage bottle cap, the other end of the liquid taking pipe is connected with a peristaltic pump, and an outlet of the peristaltic pump is connected with a water inlet of the sensor; the sensor water outlet pipe is prolonged and inserted into the liquid storage bottle. The flow path controllers on the water inlet pipe and the water outlet pipe of the sensor are connected, and the peristaltic pump is started to rotate, so that the culture solution circularly flows in the sensor-liquid storage bottle.
S150: connecting an electrochemical workstation, and starting culture;
in this step, to increase the cultivation efficiency, a multi-channel electrochemical workstation may be used, in which a plurality of sensors are connected to each channel of the electrochemical workstation, one channel being connected to each sensor. Starting a potentiostatic electrochemical control mode to start culture, and gradually enriching electrochemically active microorganisms in the inoculating bacterial suspension onto the biological anode; meanwhile, new high-activity electrochemical active microorganisms are continuously generated in a nutrient-rich system, the current of each channel sensor can be observed to be gradually increased, and the current of each channel sensor is recorded in real time.
S160: changing liquid;
in step S150, as the cultivation time is prolonged, nutrient substances in the cultivation system are consumed, and the sensor current rises to reach a first peak value (I 1 ) And then rapidly decreases. When all channel currents are reduced to 10 -3 When the mA level is achieved, the culture solution in the liquid storage bottle is directly replaced by the fresh culture solution prepared in the step S120, after the replacement of the culture solution, the current of each channel can be rapidly increased, and the first peak value (I) is reached in a period of time 1 ) Thereafter, the culture was carried out in the second stage, and the current was at the first peak (I 1 ) Gradually and slowly rising on the basis of the above. When the sensor current rises to a second peak value (I 2 ) Then the nutrient consumption is rapidly reduced again, and when the current of all channels is reduced to 10 -3 When the mA level is reached, the culture solution in the liquid storage bottle is straightened againThe fresh culture solution prepared in the step S120 is replaced; after the liquid change, the current of each channel can rise sharply again, and reach a second peak value (I 2 ) Thereafter, the culture proceeds to the third stage. And repeating the operation, and judging that the sensor is mature in incubation when the peak current value is different by not more than +/-10% after the last 3 continuous liquid changes.
S200: and injecting a target toxic substance into the initial sensor, and carrying out toxic stimulation on the initial sensor so as to obtain the microbial electrochemical sensor.
In the step, a target toxic substance is injected into the initial sensor, the initial sensor is subjected to toxic stimulation, whether the toxic stimulation is finished or not is judged by monitoring the current change of the initial sensor in the toxic stimulation process, and the microbial electrochemical sensor is obtained after the toxic stimulation is finished.
In the embodiment of the present invention, the manner of stimulation of the toxic substance during the toxic stimulation is not particularly limited, and first, the kind of the toxic substance: the method can be carried out by adding the toxic substances in multiple times, only adding one toxic substance at a time, or mixing multiple toxic substances and adding the mixed substances at one time. Second, toxic stimulation time: the stimulation may be performed only once or a small number of times to extend the time of single toxic stimulation, or may be performed in a plurality of time periods to shorten the time period of each stimulation.
In the embodiment of the invention, the injection mode of toxic substances is not particularly limited in the process of toxic stimulation, (1) a high-concentration toxic substance mother solution is directly and rapidly injected into a sensor containing a culture solution at one time by adopting a syringe manually (without any solution overflow), and then the sensor is evenly shaken manually; (2) Or mixing the mother solution of toxic substances with the culture solution, disassembling the sensor, pouring out the original culture solution in the sensor, and finally directly and rapidly injecting the culture solution containing toxic substances into the sensor by using tools such as a syringe; (3) Alternatively, the mother solution of toxic substance is mixed with the culture solution, and then the connecting pipeline adopts an automatic pumping device to slowly inject the toxic substance.
According to some embodiments of the invention, step S200 further comprises the steps of:
s201: injecting a first carbon source fresh culture solution into the initial sensor to replace the carbon source culture solution in the initial sensor. It should be noted that, the sensors cultured in batch in step S100 do not need to develop toxic stimulation immediately, i.e., the sensors can be stored for a period of time (not more than two weeks) in a suitable environment (0 ℃ to 30 ℃) after finishing culturing according to a conventional scheme, and then develop toxic stimulation as required. If sensors produced in different batches are adopted, the microbial film states in the sensors may be different, so that the subsequent toxic stimulation process needs to increase the stimulation times to achieve a consistent effect. It is therefore necessary to inject a first fresh culture broth of carbon source into the initial sensor before performing the toxic stimulus to replace the carbon source culture broth in the initial sensor, so that each set of sensors is in the same culture broth environment, thereby enabling to improve the consistency between each set of sensors. As some specific embodiments, before step S210 is performed, the first carbon source fresh culture solution is injected into the initial sensor in advance by 10 h-14 h.
In the embodiment of the invention, the components and the contents of the first carbon source fresh culture solution and the fresh culture solution for incubating the initial sensor may be the same or different, and the carbon source concentration of the first carbon source fresh culture solution may float 30% up and down on the basis of the carbon source concentration of the fresh culture solution of the initial sensor. Preferably, the first carbon source fresh culture solution is the same as the fresh culture solution for incubating the initial sensor, that is, the first carbon source fresh culture solution and the fresh culture solution for incubating the initial sensor contain the same components including salts, trace elements, vitamins, carbon sources and the like, and the contents of the components are the same, so that the initial sensor placed for a period of time can be restored to the state just after incubation and maturation as much as possible.
As some specific examples, the carbon source concentration of the first carbon source fresh culture solution may be 1-10 mmol/L.
S210: injecting a fresh culture solution of a second carbon source into the sensor to replace the first carbon source in the sensorFresh culture solution of carbon source, record output current I of sensor 1 The concentration of the carbon source of the second carbon source fresh culture solution is smaller than that of the first carbon source fresh culture solution, the culture solution in the sensor is replaced by the high-concentration first carbon source fresh culture solution to the low-concentration second carbon source fresh culture solution, so that the sensor is more controllable, and the consistency among the groups of sensors is further improved.
As some specific examples, the carbon source concentration of the second carbon source fresh culture solution is 5% -50% of the carbon source concentration of the first carbon source fresh culture solution, and other components and conditions of the second carbon source fresh culture solution are the same as those of the first carbon source fresh culture solution, namely, the second carbon source fresh culture solution and the first carbon source fresh culture solution comprise the same kinds of components, including salts, trace elements, vitamins, carbon sources and the like, and the concentrations of other components are the same except for the carbon source concentration.
Step S210 includes the following two embodiments: the first way is: after the first carbon source fresh culture solution in the sensor is replaced, standing for a preset time, and then recording the output current I of the sensor 1 The method comprises the steps of carrying out a first treatment on the surface of the As some specific examples, the time of standing after the replacement of the fresh culture solution of the first carbon source in the sensor is 20min to 40min. The second way is: after the first carbon source fresh culture solution in the sensor is replaced, continuously introducing the second carbon source fresh culture solution into the sensor for a preset time, and then recording the output current I of the sensor 1 . As some specific examples, after the first carbon source fresh culture solution in the sensor is replaced, the second carbon source fresh culture solution is continuously introduced into the sensor for 20-40 min. Either of the above two embodiments may be selected, and the second embodiment is preferable because: in the on-machine test process of the response performance of the sensor to heavy metals, the test environment is in a flowing water state, and the sensor is in a continuous flowing state in the process of toxic stimulation in advance, so that the sensor with the completed toxic stimulation is more suitable for the scene of the on-machine test and can enter The sensitivity of the sensor to toxic substances is improved in one step.
S220: injecting a carbon source culture solution containing a target toxic substance into the sensor to replace the second carbon source fresh culture solution in the sensor, carrying out toxic stimulation on the sensor for a preset time, and recording the output current I of the sensor after the preset time 2 Wherein the carbon source concentration of the carbon source culture solution containing the target toxic substance is smaller than the carbon source concentration of the carbon source culture solution in the initial sensor.
Step S220 includes the following two embodiments: the first way is: after the fresh culture solution of the second carbon source in the sensor is replaced, standing for a preset time to carry out toxic stimulation on the sensor, and recording the output current I of the sensor after the preset time 2 The method comprises the steps of carrying out a first treatment on the surface of the As some specific examples, the time of standing (i.e., the time of a single toxic stimulus) is 15min to 60min. The second way is: after the second carbon source fresh culture solution in the sensor is replaced, continuously introducing the carbon source culture solution containing the target toxic substance into the sensor for a preset time to perform toxic stimulation on the sensor, and then recording the output current I of the sensor after the preset time 2 . As some specific examples, the time of continuously introducing the carbon source culture solution containing the target toxic substance into the sensor (i.e., the time of single toxic stimulation) is 15 min-60 min. Either of the above two embodiments may be selected, and the second embodiment is preferable because: in the on-machine test process of the response performance of the sensor to heavy metals, the test environment is in a flowing water state, and the sensor is in a continuous flowing state in the toxic stimulation process in advance, so that the sensor with the completed toxic stimulation is more suitable for the scene of the on-machine test, and the sensitivity of the sensor to toxic substances can be further improved.
As some specific examples, the other components and conditions of the carbon source culture solution containing the target toxic substance other than the target toxic substance are the same as those of the second carbon source fresh culture solution, that is, the carbon source culture solution containing the target toxic substance other than the target toxic substance contains the same components and contents as those of the second carbon source fresh culture solution, thereby avoiding the carbon source concentration change during the replacement of the second carbon source fresh culture solution in the sensor by the carbon source culture solution containing the target toxic substance and further improving the controllability of the toxicity stimulating process. That is, the carbon source concentration of the carbon source culture solution containing the target toxic substance is 5% -50% of the carbon source concentration of the first carbon source fresh culture solution, and the sensor has obvious toxicity stimulation effect in the carbon source concentration range.
In the embodiment of the present invention, the specific type of the target toxic substance is not particularly limited, and a person skilled in the art can select a corresponding toxic substance according to the test requirements, for example, to acclimate heavy metal Cu 2+ The microorganism electrochemical sensor with high sensitivity and high response speed is selected to contain Cu 2+ Stimulating toxic substances; to be acclimatized to heavy metal Pb 2+ The microorganism electrochemical sensor with high sensitivity and high response speed selects Pb-containing material 2+ The toxic substances are stimulated. As some specific examples, the target toxic substances include, but are not limited to, zn 2+ 、Cu 2+ 、Pb 2+ 、Ni 2+ 、Tl 2+ 、Hg 2+ And Cd 2+ At least one of them.
It should be noted that there are many kinds of heavy metal soluble salt components, and it is not necessary to use a specific kind of heavy metal soluble salt, as long as it can provide the corresponding target ion. For example, heavy metal zinc ions (Zn 2+ ) Zinc chloride (ZnCl) can be used 2 ) Zinc nitrate (Zn (NO 3 ) 2 ) Or zinc sulfate (ZnSO) 4 ) Etc., so long as it can provide Zn 2+ And (3) obtaining the product.
According to some embodiments of the invention, the concentration of the target toxic substance in the carbon source culture solution containing the target toxic substance may be 0.05mg/L to 1.0mg/L.
According to still other embodiments of the present invention, in step S220, the time of the single toxic stimulus may be 15min to 60min.
S230: calculating the toxicity stimulation current inhibition rate R= (I) of the sensor 1 -I 2 ）/I 1 The method comprises the steps of carrying out a first treatment on the surface of the And (3) repeating the steps S210-S230 until R is more than or equal to 15% and less than or equal to 75%, and the R value deviation is not more than +/-10% three times continuously, so as to judge that the toxic stimulation is completed. R is too small and is ineffective at < 15%; too large, greater than 75% may be too heavy to handle the stimulation process, potentially resulting in too deep a majority of the cell death biofilm being difficult to repair. Preferably, R is 30% or more and 60% or less.
According to further embodiments of the present invention, the concentration of the target toxic substance in the carbon source culture solution containing the target toxic substance is 0.05mg/L to 1.0mg/L, preferably 0.1mg/L to 0.5mg/L.
According to still other embodiments of the present invention, in step S220, the time for the single toxic stimulus is 15min to 60min, preferably 20min to 45min.
In the embodiment of the invention, when the microbial electrochemical sensor with high sensitivity and high response speed to specific single heavy metal ions is to be domesticated, a carbon source culture solution containing the specific single heavy metal ions is adopted to carry out toxicity stimulation on the sensor, and the steps S210-S230 are repeated until R is more than or equal to 15% and less than or equal to 75%, and the R value deviation is not more than +/-10% three times continuously, so that the microbial electrochemical sensor with high sensitivity to the specific single heavy metal ions and high response speed is obtained.
When the microbial electrochemical sensor with high sensitivity and high response speed to the specific multi-element composite heavy metal ions needs to be domesticated, carbon source culture solution containing the specific multi-element composite heavy metal ions can be used for carrying out toxicity stimulation on the sensor, the steps S210-S230 are repeated until R is more than or equal to 15% and less than or equal to 75%, and the deviation of the R value is not more than +/-10% for three times continuously, so that the completion of the toxicity stimulation is judged. Or, firstly, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific single heavy metal ions, and repeating the steps S210-S230 until R is more than or equal to 15% and less than or equal to 75%, wherein the R value deviation is not more than +/-10% for three times continuously; and then, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific multi-element composite heavy metal ions, repeating the steps S210-S230 until R is more than or equal to 15% and less than or equal to 75%, and judging that the toxicity stimulation is finished if the R value deviation is not more than +/-10 three times continuously. Thus, the microbial electrochemical sensor with high sensitivity and high response speed to specific multielement composite heavy metal ions is obtained.
The method for preparing the microbial electrochemical sensor according to the embodiment of the invention has at least the following advantages:
(1) The microorganism electrochemical sensor prepared by the method has higher speed for detecting heavy metals. The sensor after toxicity stimulation can send out toxicity alarm when in the 1 st toxicity impact in the on-machine monitoring process, and the time from the installation of the sensor to the successful alarm can be controlled within 1 h;
(2) The microbial electrochemical sensor prepared by the method has lower detection limit and can detect heavy metal pollution with lower concentration in water. In the on-machine monitoring process of the sensor after toxicity stimulation, the heavy metal with the concentration of 0.24mg/L can be detected by first toxicity detection, and different heavy metals have toxicity superposition, namely the total concentration of the several heavy metals reaches 0.24mg/L, and the sensor can also be effectively monitored.
In a word, the invention provides a preparation method of the water quality biotoxicity on-line monitoring sensor with high sensitivity and low cost.
In a second aspect of the invention, the invention provides a system for performing the toxic stimulation in the method described in the examples above. According to an embodiment of the invention, referring to fig. 1, the system comprises: a sensor 1; a first liquid storage device 4, wherein the first liquid storage device 4 is used for storing the second carbon source fresh culture solution; at least one second liquid storage device 5, wherein the second liquid storage device 5 is used for storing the carbon source culture solution containing the target toxic substances; the liquid inlet of the first multi-way valve diversion element 7 is respectively connected with the liquid outlet of the first liquid storage device 4 and the liquid outlet of the second liquid storage device 5, and the first liquid outlet of the first multi-way valve diversion element 7 is communicated with the liquid inlet of the sensor 1; the first liquid outlet of the second multi-way valve flow dividing element 2 is respectively connected with the liquid inlet of the first liquid storage device 4 and the liquid inlet of the second liquid storage device 5, and the liquid inlet of the second multi-way valve flow dividing element 2 is connected with the liquid outlet of the sensor 1; a first waste liquid collection device 6, wherein the first waste liquid collection device 6 is connected with a second liquid outlet of the first multi-way valve diversion element 7; a second waste liquid collection device 3, wherein the second waste liquid collection device 3 is connected with a second liquid outlet of the second multi-way valve diversion element 2; pumping means 8, said pumping means 8 being arranged on the line between said first multi-way valve split element 7 and said sensor 1; the data acquisition and signal control unit 9 is connected with the sensor 1, the first multi-way valve shunt element 7, the second multi-way valve shunt element 2 and the pumping device 8 through electric signals respectively.
According to the system for toxicity stimulation according to the embodiment of the invention, the sensor 1 is connected with the first liquid storage device 4 storing the second carbon source fresh culture solution, the second liquid storage device 5 storing the carbon source culture solution containing target toxic substances, the first waste liquid collecting device 6 and the second waste liquid collecting device 3 by adopting the pumping device 8, the first multi-way valve flow dividing element 7 and the second multi-way valve flow dividing element 2, the pumping device 8 pumps the second carbon source fresh culture solution or the carbon source culture solution containing target toxic substances into the sensor 1 at a stable flow rate, and the first multi-way valve flow dividing element 7 and the second multi-way valve flow dividing element 2 can control whether the solution pumped into the sensor 1 is sourced from the first liquid storage device 4 or the second liquid storage device 5, and can also selectively control whether the effluent of the sensor 1 flows back into the first liquid storage device 4, the second liquid storage device 5 or is directly discharged into the waste liquid collecting device.
Therefore, the system can realize the process of carrying out toxic stimulation on the sensor 1, thereby improving the sensitivity of the sensor to toxic substances, shortening the toxic response time when the sensor is tested on machine, leading the sensor to have higher sensitivity to toxic substances and faster response speed, and solving the problems of low heavy metal test sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow response speed (the toxic response signal starts to appear after testing for hours or even days) existing in the current sensor. In addition, in the on-line test process of the response performance of the sensor to heavy metal, the test environment is in a flowing water state, and in the process of realizing the toxic stimulation to the sensor, the sensor is in a continuous flowing state by the system, so that the sensor with the toxic stimulation is more suitable for the scene of the on-line test, and the sensitivity of the sensor to toxic substances can be further improved.
Specifically, the first liquid storage device 4 is used for storing a second carbon source fresh culture solution, the second liquid storage device 5 is used for storing a carbon source culture solution containing a target toxic substance, and when only the microbial electrochemical sensor 1 with high sensitivity and high response speed to a specific single heavy metal ion is required to be domesticated, one second liquid storage device 5 is arranged, and the second liquid storage device 5 is filled with the carbon source culture solution containing the specific single heavy metal ion. When the microbial electrochemical sensor 1 with high sensitivity and high response speed to specific single heavy metal ions is required to be domesticated, and meanwhile, when the microbial electrochemical sensor 1 with high sensitivity and high response speed to specific multi-element composite heavy metal ions is required to be domesticated, two second liquid storage devices 5 are arranged, wherein one second liquid storage device 5 is filled with a carbon source culture solution containing the specific single heavy metal ions, and the other second liquid storage device 5 is filled with a carbon source culture solution containing the specific multi-element composite heavy metal ions.
Referring to fig. 1, the toxic stimulation of the sensor 1 using the system described above proceeds as follows:
1) The first liquid storage device 4 is filled with a second carbon source fresh culture solution, and the second liquid storage device 5 is filled with a carbon source culture solution containing target toxic substances.
2) The sensor 1 is rinsed for the first time. The first multi-way valve diversion element 7 is communicated with the first liquid storage device 4, the second multi-way valve diversion element 2 is communicated with the second waste liquid collection device 3, the pumping device 8 is started to pump the second carbon source fresh culture liquid into the sensor 1 at a set flow rate, and the duration is 3-10min.
3) The sensor 1 signal is restored. The first multi-way valve diversion element 7 and the second multi-way valve diversion element 2 are both communicated with the first liquid storage device 4, and the pumping device 8 is started to set the flow velocity to be transmittedPumping a second carbon source fresh culture solution into the sensor 1, and immediately recording the output current I of the sensor 1 after the duration time is 20-40 min 1 。
4) Stopping pumping and standing for 20-40 min.
5) The sensor 1 is rinsed a second time. The first multi-way valve diversion element 7 is communicated with the second liquid storage device 5, the second multi-way valve diversion element 2 is communicated with the second waste liquid collection device 3, the pumping device 8 is started to pump the carbon source culture solution containing the target toxic substances into the sensor 1 at a set flow rate, and the duration is 3-10min.
6) Injecting a toxic substance culture solution. The first multi-way valve flow dividing element 7 and the second multi-way valve flow dividing element 2 are both connected with the second liquid storage device 5, the pumping device 8 is started to pump the carbon source culture solution containing the target toxic substances into the sensor 1 at a set flow rate, and the output current I of the sensor 1 is recorded immediately after the duration time is 20-40 min 2 。
7) The sensor 1 is rinsed, as in step 2).
8) Stopping pumping and standing for 20-40 min, and calculating the inhibition rate R= (I) of toxic stimulation current 1 -I 2 ）/ I 1 A value that is positive, a greater value indicating a greater degree of toxic stimulation of the sensor 1.
9) Repeating the steps 2) -8) until the R value reaches 15% -75%, and the R value deviation is not more than +/-10% three times continuously, and judging that the toxic stimulation is completed. R is too small and is ineffective at < 15%; too large, > 75% may be too heavy to handle the stimulation process, possibly resulting in too deep a majority of the cell death biofilm in sensor 1 being difficult to repair. After the stimulation is finished, the R value is qualified and can be used for on-machine test.
In an embodiment of the invention, the first waste liquid collection device 6 may be used to collect biofilm debris in the sensor 1, and the sensor 1 may enter solution from the bottom and exit solution from the top for a long period of time, which may cause that some biofilm settled on the bottom of the sensor 1 cannot be discharged. After precipitation appears in the sensor 1, the first multi-way valve diverting element 7 is connected with the first waste liquid collecting device 6, and the pumping device 8 is reversed, so that the precipitation at the bottom of the sensor 1 is discharged into the first waste liquid collecting device 6, and the signal of the sensor 1 can be more stable by timely discharging the precipitation.
In the embodiment of the invention, the data acquisition and signal control unit 9 comprises a data acquisition unit and a control unit, wherein the data acquisition unit is used for acquiring the current of the microbial electrochemical sensor in the operation process; and the control unit is used for controlling the on/off of each channel of the multi-way valve, the starting/stopping, the forward/reverse rotation time, the speed, the stopping time, the toxicity stimulation times and the like of the pumping device.
The experimental operator can manually and independently control the pumping device, the multi-way valve channel switch and the like through the control unit to gradually finish the sensor stimulation process, one or more toxic stimulation processes can be preset through the control unit, only a required preset scheme is selected, clicking is started, and the control unit automatically performs toxic stimulation according to the preset scheme.
In a third aspect of the invention, the invention provides the use of the method of the above embodiments in biotoxicity detection. Therefore, the method for carrying out toxicity stimulation on the sensor which is mature in incubation is used for biological toxicity detection, so that the sensitivity of the sensor to toxic substances is improved, the toxicity response time of the sensor during on-machine test is shortened, the sensitivity of the sensor to toxic substances is higher, the response speed is higher, and the problems that the current sensor is low in heavy metal test sensitivity (the response concentration of most sensors is several to tens of mg/L) and slow in response speed (the toxicity response signal starts to appear after a test is carried out for hours or even days) are solved. Therefore, high-sensitivity, rapid and low-cost monitoring of low-concentration heavy metals in the water body can be realized.
The following detailed description of embodiments of the invention is provided for the purpose of illustration only and is not to be construed as limiting the invention. In addition, all reagents employed in the examples below are commercially available or may be synthesized according to methods herein or known, and are readily available to those skilled in the art for reaction conditions not listed, if not explicitly stated.
Comparative example 1
In the comparative example, the mature biological film is incubated according to the conventional electrochemical active microbial film domestication method, and the signals are stable after the biological film is domesticated and mature, and the biological film is directly assembled into the microbial electrochemical sensor for on-machine test. Comprises 6 steps, the details are as follows:
step 1: and assembling the electrochemical active microbial membrane hatching sensor. Each sensor comprises an electrolytic cell, and 1 bioanode WE, 1 reference electrode RE and 1 counter electrode CE are arranged on the electrolytic cell. The electrolytic cell is also provided with a liquid inlet and a liquid outlet for installing a liquid inlet pipe and a liquid outlet pipe; and the liquid inlet pipe and the liquid outlet pipe are provided with control valves capable of controlling the on/off of the flow paths. The components of the sensor are assembled.
Step 2: fresh culture solution is prepared. The culture solution comprises four parts of salts, trace elements, vitamins and carbon sources. The four parts of mother liquor are prepared separately, 200mL of salt mother liquor, 2mL of trace element mother liquor, 0.2mL of vitamin mother liquor and 5mL of carbon source mother liquor (component: sodium acetate) are respectively taken, and the mother liquor is diluted to 3.2L by deionized water, so that the carbon source concentration of the fresh culture solution is 5mmol/L. Placing the mixture in a constant temperature incubator, and introducing nitrogen with the purity of more than 99.5% at the rate of 500mL/min, and keeping the temperature while nitrogen is being introduced.
Step 3: inoculating target strain. Mixing the fresh culture solution obtained in the step 2 with seed source effluent at a ratio of 1:1 to obtain inoculation bacterial suspension, and filling the inoculation bacterial suspension into a liquid storage bottle.
Step 4: and a connecting pipeline. A liquid taking pipe is inserted into the liquid storage bottle cap, the other end of the liquid taking pipe is connected with a peristaltic pump, and an outlet of the peristaltic pump is connected with a water inlet of the sensor; the sensor water outlet pipe is prolonged and inserted into the liquid storage bottle. And (3) connecting a flow path controller on the water inlet pipe and the water outlet pipe of the sensor, starting a peristaltic pump to rotate, and circularly flowing the culture solution in the sensor-liquid storage bottle.
Step 5: and connecting an electrochemical workstation, and starting culture. To increase the efficiency of the culture, a multi-channel electrochemical workstation is used, wherein a plurality of sensors are respectively connected to each channel of the electrochemical workstation, and one channel is connected with one sensor. Starting a potentiostatic electrochemical control mode to start culture, and gradually enriching electrochemically active microorganisms in the inoculating bacterial suspension onto the biological anode; meanwhile, new high-activity electrochemical active microorganisms are continuously generated in a nutrient-rich system, the current of each channel sensor can be observed to be gradually increased, and the current of each channel sensor is recorded in real time.
Step 6: and (5) liquid exchange. In step 5, as the culture time is prolonged, nutrients in the culture system are consumed, and the sensor current rises to reach a first peak value (I 1 ) And then rapidly decreases. When all channel currents are reduced to 10 -3 When the mA level is achieved, the culture solution in the liquid storage bottle is directly replaced by the fresh culture solution in the step 2, after the replacement of the culture solution, the current of each channel can be rapidly increased, and the first peak value (I) is reached within 10min 1 ) Thereafter, the culture was carried out in the second stage, and the current was at the first peak (I 1 ) Gradually and slowly rising on the basis of the above. When the sensor current rises to a second peak value (I 2 ) Then the nutrient consumption is rapidly reduced again, and when the current of all channels is reduced to 10 -3 When the mA level is achieved, the culture solution in the liquid storage bottle is directly replaced by the fresh culture solution in the step 2; after the liquid change, the current of each channel can rise sharply again, and reach a second peak value (I in 10min 2 ) Thereafter, the culture proceeds to the third stage. And repeating the operation, and judging that the sensor is mature in incubation when the peak current value is different by not more than +/-10% after the last 3 continuous liquid changes.
And (3) performing on-machine test: 3 out of the incubated mature sensors are randomly selected for on-machine testing, and the on-machine testing is used for evaluating the response performance of the sensors to heavy metals. The water quality toxicity online analyzer is set as blank calibration, zero point check and toxic substance standard liquid check, and the test is automatically started at fixed points per hour, and the test is continuously performed for 10 days, 6 times per day of zero point check and 6 times of toxic substance standard liquid check. On days 1-7, single heavy metal toxicity test is carried out, and the standard solution of toxic substances is 0.24mg/L Cu 2+ A standard substance solution; on day 8-10, compound multiple heavy metal test is carried out, and the total concentration of toxic substance standard solution is 0.24mg/L heavy metal (0.08 mg/L Cu) 2+ + 0.08mg/L Pb 2+ + 0.08mg/L Cd 2+ ) Standard substance solution. Only daily maintenance such as adding empty water, replacing reagent, replacing toxic substance standard solution and the like is carried out during the test. TestingThe data are shown in figures 2 and 3.
Example 1 (toxicity stimulation procedure toxic substance contact time test)
The method comprises the following steps of:
step 1: mature sensors were incubated in steps 1-6 of comparative example 1.
Step 2: toxicity stimulation pretreatment. Step 1 of incubating the sensors of comparative example 1, in which a sufficient amount (generally 3 times the volume of the sensor lumen, relative to the volume of the sensor lumen) of fresh culture broth of the sensors was injected into each sensor by a syringe 12h in advance, to ensure that the sensors were filled with fresh culture broth, which was a high carbon source culture broth (C 0 ) (i.e., the first carbon source fresh culture broth) having a carbon source concentration of 5mmol/L.
Step 3: toxicity stimulates process solution preparation. Preparing fresh culture solution (C) with low carbon source 1 ) (i.e., a second carbon source fresh culture solution), wherein the carbon source is a high carbon source culture solution (C 0 ) 25% of the total, other ingredients and conditions are the same; preparing a low-carbon source fresh culture solution (C) 2 ) In a fresh medium (C) 1 ) Adding standard solution mother liquor of toxic substance, preparing Cu in this example 2+ Fresh culture broth (C) of low carbon source containing toxic substance at final concentration of 0.3mg/L 2 ）。
Step 4: a toxic stimulation process. (1) A syringe was used to fill each sensor with 3 volumes of fresh medium (C) 1 ) The injection process is not more than 3min, after the injection is finished, the water inlet and the water outlet of the sensor are closed, the sensor is kept stand for a period of time T, and the output current I of the sensor after the standing is recorded 1 (2) injecting 3 times of fresh culture solution (C) containing toxic substances into each sensor by using a syringe 2 ) The injection process is not more than 3min, after the injection is completed, the water inlet and the water outlet of the sensor are closed, and the sensor is kept stand for a period of time T 0 (i.e. single stimulus time), the sensor output current I after rest is recorded 2 (3) calculating the inhibition ratio R= (I) of toxic stimulation current 1 -I 2 ）/ I 1 The value of the sum of the values,the value is positive, and the greater the value is, the greater the toxicity stimulation degree of the sensor is; (4) Repeating the steps (1) - (3) until the R value reaches 15% -75%, and the R value deviation is not more than +/-10% three times continuously, and judging that the toxic stimulation is completed.
The present example compares the effect of stimulation in different time periods in a single stimulus, the toxic substance contact time T in the above procedure 0 The test data are shown in Table 1, and the test data are set to 15min, 20min, 30min, 45min and 60min, respectively.
TABLE 1
As can be seen from Table 1, the too long and too short toxic stimulation times are detrimental to achieving the stimulation effect rapidly, and according to the above data, the stimulation effect can be achieved in a single toxic stimulation time of 20min-45min, wherein 30min is relatively optimal. The detailed data were analyzed as follows:
1) The R values of the two sensors show gradually slowly rising trend after single stimulation for 15min, and the R values do not reach a steady state until the 10 th time; it may be that the single toxic stimulus time is too short, requiring more cyclic combinations to reach stimulus homeostasis.
2) The stimulation effect can be achieved by single stimulation for 20min, 30min and 45 min. Wherein, the R value of two sensors exceeds 15% for the first time after the single stimulation for 20min and after the stimulation for 5-6 times, the steady state is reached at the 7 th-8 th time; after the single stimulation is performed for 30min, the R values of the two sensors exceed 15% after the first stimulation, the three times reach a steady state, and the R values of the two sensors are close to each other and are stable and good all the time after reaching the steady state; the R values of the two sensors exceed 15% after the first stimulus for 45min after the single stimulus, and the steady state is reached from 6 th to 7 th.
3) The single stimulation is carried out for 60min, the R values of the two sensors reach the required range (15% -75%) when the first stimulation is carried out, but the R1 results have larger difference; and the R values of the two sensors are unstable in the subsequent stimulation process; it may be that the single toxicity stimulus is too long and the sensor is too deep to recover.
Example 2 (toxicity stimulation procedure toxicity substance concentration test)
The method comprises the following steps of:
step 1: mature sensors were incubated in steps 1-6 of comparative example 1.
Step 2: as in example 1.
Step 3: the only difference from example 1 is that: gradient concentration Cu was formulated in this example 2+ The final concentration is 0.05mg/L, 0.1mg/L, 0.3mg/L, 0.5mg/L and 1.0mg/L of low-carbon source fresh culture solution containing toxic substances respectively.
Step 4: the procedure of the toxic stimulation was the same as in example 1, with a single toxic stimulation time fixed at 30min. The test data are shown in Table 2.
TABLE 2 toxicity concentration test data sheet
As can be seen from table 2, the toxicity stimulation process test results: relatively optimal toxic substances (Cu) 2+ ) The concentration was 0.3mg/L and the detailed data were analyzed as follows:
1) The concentration of toxic substances is too low (0.05 mg/L) to show obvious stimulation effect, and too high concentration (1.0 mg/L) can easily lead to too deep poisoning of the sensor to be recovered;
2) The intermediate concentration is 0.1mg/L to 0.5mg/L, and the toxicity stimulation effect can be achieved, wherein the R value is the most stable in the continuous toxicity stimulation process of 0.3 mg/L.
The sensor after manual stimulation of 0.3mg/L single heavy metal in this example was selected for the on-machine test, and the method of the on-machine test and the data analysis method were the same as those of comparative example 1. The test data are shown in figures 4 and 5.
Example 3 (toxicity stimulation protocol test-Manual injection)
The method comprises the following steps of:
step 1: mature sensors were incubated in steps 1-6 of comparative example 1.
Step 2: as in example 1.
Step 3: as in example 1. The implementation isThe prepared low-carbon source fresh culture solutions containing toxic substances respectively contain 0.3mg/L Cu 2+ Fresh low-carbon source culture solution containing 0.1mg/L Cu 2+ + 0.1mg/L Pb 2+ + 0.1mg/L Cd 2+ Low carbon source fresh culture broth of (c).
Step 4: a toxic stimulation process. The procedure is as in example 1, with a single toxicity stimulation time fixed at 30min. The method comprises two stages, wherein the first stage adopts single toxic substance for stimulation, and the first stage adopts Cu solution containing 0.3mg/L 2+ And (3) performing toxicity stimulation on the low-carbon source fresh culture solution until the R value reaches 0.15-0.75, and determining that the first-stage toxicity stimulation is finished after the R value is not more than +/-10% in three continuous times. If R is more than 0.75 continuously in the stimulation process or R value is still less than 0.15 after 5 groups of toxic stimulation, judging that the sensor is invalid, and selecting a proper sensor to re-develop the toxic stimulation. The second stage adopts compound heavy metal toxic substances for stimulation, and adopts Cu containing 0.1mg/L 2+ + 0.1mg/L Pb 2+ + 0.1mg/L Cd 2+ And (3) carrying out toxicity stimulation on the low-carbon source fresh culture solution until the R value reaches 15% -75% again, and judging that the second-stage toxicity stimulation is finished when the R value is not more than +/-10% after three continuous times. If R is more than 75% in the stimulation process or R value is still less than 15% after 5 groups of toxic stimulation, judging that the sensor is invalid, and re-selecting a proper sensor and re-developing the toxic stimulation from the first-stage toxic stimulation.
The sensor after the toxicity stimulus of this example was subjected to the on-machine test, and the method of the on-machine test and the data analysis method were the same as those of comparative example 1. The test data are shown in figures 6 and 7.
Example 4 (toxicity stimulation mode test-cycle injection)
The method comprises the following steps of:
step 1: mature sensors were incubated in steps 1-6 of comparative example 1.
Step 2: as in example 1.
Step 3: same as in example 3.
Step 4: the toxic stimulation process is the same as that of example 3, and the method for judging the stimulation effect is the same as that of example 3. Unlike example 3, this example uses an automatic cyclic toxicity stimulation scheme, using a system as shown in fig. 1, comprising: a sensor 1; a first liquid storage device 4, wherein the first liquid storage device 4 is used for storing the second carbon source fresh culture solution; at least one second liquid storage device 5, wherein the second liquid storage device 5 is used for storing the carbon source culture solution containing the target toxic substances; the liquid inlet of the first multi-way valve diversion element 7 is respectively connected with the liquid outlet of the first liquid storage device 4 and the liquid outlet of the second liquid storage device 5, and the first liquid outlet of the first multi-way valve diversion element 7 is communicated with the liquid inlet of the sensor 1; the first liquid outlet of the second multi-way valve flow dividing element 2 is respectively connected with the liquid inlet of the first liquid storage device 4 and the liquid inlet of the second liquid storage device 5, and the liquid inlet of the second multi-way valve flow dividing element 2 is connected with the liquid outlet of the sensor 1; a first waste liquid collection device 6, wherein the first waste liquid collection device 6 is connected with a second liquid outlet of the first multi-way valve diversion element 7; a second waste liquid collection device 3, wherein the second waste liquid collection device 3 is connected with a second liquid outlet of the second multi-way valve diversion element 2; pumping means 8, said pumping means 8 being arranged on the line between said first multi-way valve split element 7 and said sensor 1; the data acquisition and signal control unit 9 is connected with the sensor 1, the first multi-way valve shunt element 7, the second multi-way valve shunt element 2 and the pumping device 8 through electric signals respectively.
The specific operation steps are as follows:
1) The first stock solution bottle is filled with fresh culture solution (C) 1 ) The second liquid storage bottle is filled with Cu 2+ Fresh culture broth (C) of low carbon source containing toxic substance at final concentration of 0.3mg/L 2 ) The third liquid storage bottle is filled with Cu with the final concentration of 0.1mg/L 2+ + 0.1mg/L Pb 2+ + 0.1mg/L Cd 2+ ) Is a low-carbon source fresh culture solution (C) 3 ）。
2) The sensor 1 is rinsed for the first time.The first multi-way valve flow dividing element 7 is connected with the first liquid storage bottle, the second multi-way valve flow dividing element 2 is connected with the second waste liquid collecting device 3, and the pumping device 8 is started to pump the culture solution C into the sensor 1 at the flow rate of 10mL/min 1 The duration was 6min.
3) The sensor 1 signal is restored. The first multi-way valve flow dividing element 7 and the second multi-way valve flow dividing element 2 are both connected with the first liquid storage bottle, and the pumping device 8 is started to pump the culture solution C into the sensor 1 at the flow rate of 10mL/min 1 Immediately after a duration of 30min the output current I of the sensor 1 was recorded 1 。
4) Stopping pumping and standing for 30min.
5) The sensor 1 is rinsed a second time. The first multi-way valve diversion element 7 is communicated with the second liquid storage bottle, the second multi-way valve diversion element 2 is communicated with the second waste liquid collection device 3, and the pumping device 8 is started to pump Cu-containing liquid into the sensor 1 at a flow rate of 10mL/min 2+ Low carbon source culture solution C 2 The duration was 6min.
6) Injecting a toxic substance culture solution. The first multi-way valve flow dividing element 7 and the second multi-way valve flow dividing element 2 are both connected with the second liquid storage bottle, and the pumping device 8 is started to pump Cu-containing liquid into the sensor 1 at the flow rate of 10mL/min 2+ Low carbon source culture solution C 2 Immediately after a duration of 30min the output current I of the sensor 1 was recorded 2 。
7) The sensor 1 is rinsed, as in step 2).
8) Stopping pumping and standing for 30min, and calculating the inhibition rate R= (I) of toxic stimulation current 1 -I 2 ）/ I 1 Values.
9) Repeating the steps 2) -8) until the R value reaches 15% -75%, and the R value deviation is not more than +/-10% three times continuously, and judging that the toxic stimulation is completed. After the stimulation is finished, the R value is qualified and can be used for on-machine test.
The method of the on-machine test and the data analysis method are the same as comparative example 1. The test data are shown in fig. 8 and 9. In the on-press tests of comparative example 1 and examples 2 to 4, the single heavy metal test was conducted for 7 days using 0.24mg/L Cu 2+ A single heavy metal standard solution; the composite heavy metal test is continued after the single heavy metal test is finishedFor 3 days, adopting mixed toxic substance standard solution containing three heavy metals Cu 2+ 、 Pb 2+ 、 Cd 2+ Each concentration was 0.08mg/L, totaling 0.24mg/L.
In summary, the operation process of comparative example 1 includes: domesticating the mature biological membrane; and (5) testing on the machine. The operation process of embodiment 1 includes: domesticating the mature biological membrane; stimulation time was explored. The operation process of embodiment 2 includes: domesticating the mature biological membrane; stimulus concentration exploration; and (5) testing on the machine. The operation process of embodiment 3 includes: domesticating the mature biological membrane; after the single toxic substance is injected manually for stabilization, the compound toxic substance is injected manually for stimulation; and (5) testing on the machine. The operation process of embodiment 4 includes: domesticating the mature biological membrane; after the automatic circulation injection of the single toxic substance is stable, the automatic circulation injection of the composite toxic substance is performed for stimulation; and (5) testing on the machine.
Remarks: in FIGS. 2-9, dz1, dz2, dz3, sc1, sc2, sc3 are synonymous, and each combination was tested in parallel in 3 groups; dz1, dz2 and dz3 respectively represent zero verification inhibition rates of the sensors 1, 2 and 3; sc1, sc2, sc3 represent the test inhibition rates of toxic substances by the sensors 1, 2, 3, respectively.
As can be seen from fig. 2, in the single heavy metal test of the sensor prepared in comparative example 1, the fluctuation range of the zero-point check inhibition rate of the first and the 3 channels is relatively large, and the fluctuation ranges are between-10% and 15%; secondly, the toxicity response speed is low, wherein the toxicity alarm limit value is reached after the 15 th and 18 th heavy metals are respectively tested by the two channels; one of the channels can reach the toxicity alarm limit value after the first test, but the toxicity alarm cannot be identified after the subsequent tests, and finally the toxicity alarm cannot be identified hundred percent until the 26 th test; thirdly, the inhibition rate of the standard solution checking test of the toxic standard substance solutions of the 3 channels is gradually increased, the whole is smaller, the fluctuation of unstable data is larger, and the range of 0-45%.
As can be seen from fig. 3, in the composite heavy metal test of the sensor prepared in comparative example 1, the fluctuation ranges of the zero point check inhibition rates of the first and the third channels are relatively large and fluctuate between-10% to 15%; secondly, the toxicity response speed is low, and the toxicity alarm limit value is reached after the 3 channels respectively test the 10 th, 12 th and 14 th heavy metals; thirdly, after all toxic substances are identified and alarming is started, the standard solution checking and testing inhibition rate of the 3-channel toxic standard substance solution gradually increases and is smaller as a whole, and the fluctuation of unstable data is larger between 0 and 36 percent although the alarm limit can be reached.
As can be seen from fig. 4, in the single heavy metal test of the sensor prepared in example 2, the first and third channel zero point check tests are more stable than the sensor without toxicity stimulation, and the inhibition rate fluctuates between 10% and 15%; secondly, the toxicity response speed is high, the 1 st heavy metal test reaches the toxicity alarm limit value, and all toxicity tests in 7 days can identify toxic substances and alarm; thirdly, the inhibition rate of the standard solution of the toxicity standard substance is gradually increased, the whole inhibition rate is larger than that of comparative example 1 and is between 20 and 65 percent, but the test is not stable and the fluctuation range is larger.
As can be seen from fig. 5, in the composite heavy metal test of the sensor prepared in example 2, the first and third channel zero point check tests are more stable than the sensor without toxicity stimulation, and the inhibition rate fluctuates between 10% and 15%; secondly, the toxicity response speed is low, and the toxicity alarm limit value is reached after the 3 channels are respectively tested for the 4 th, 5 th and 7 th heavy metals; thirdly, the inhibition rate of the standard solution of the toxicity standard substance is gradually increased, the whole inhibition rate is larger than that of the comparative example, the unstable fluctuation range of the test is larger between 0 and 55 percent.
As can be seen from fig. 6, in the single heavy metal test of the sensor prepared in example 3, the first, 3 channel zero-point check test is more stable than the sensor without toxicity stimulation, and the inhibition rate fluctuates between 10% and 15%; secondly, the toxicity response speed is high, the 1 st heavy metal test reaches the toxicity alarm limit value, and all toxicity tests in 7 days can identify toxic substances and alarm; thirdly, the inhibition rate of the standard solution examination of the toxicity is gradually increased, the whole inhibition rate is larger than that of the example 2 and is between 20 and 70 percent, but the test is not stable.
As can be seen from fig. 7, in the composite heavy metal test of the sensor prepared in example 3, the first and third channel zero point check tests are more stable than the sensor without toxicity stimulation, and the inhibition rate fluctuates between 10% and 15%; secondly, the toxicity response speed is high, the 1 st heavy metal test reaches the toxicity alarm limit value, and all toxicity tests in 3 days can identify toxic substances and alarm; thirdly, the inhibition rate of the standard solution verification of the toxicity is stable in a certain range in the early stage and in the later stage with a gradual increasing trend, the whole is larger than that of the embodiment 2, and some fluctuation still exists between 40% and 70%.
As can be seen from fig. 8, in the single heavy metal test of the sensor prepared in example 4, the first and third channel zero point check tests are all stable and the inhibition rate is within ±5% compared with the sensor without toxic stimulation and the sensor with manual injection stimulation; secondly, the toxicity response speed is high, the 1 st heavy metal test reaches the toxicity alarm limit value, and all toxicity tests in 7 days can identify toxic substances and alarm; thirdly, the standard solution of the toxicity is stable in checking and testing, and the data set is between 45% and 55%.
As can be seen from fig. 9, in the composite heavy metal test of the sensor prepared in example 4, the first and third channel zero point check tests are all stable and the inhibition rate is within ±5% compared with the sensor without toxic stimulation and the sensor with manual injection stimulation; secondly, the toxicity response speed is high, the 1 st heavy metal test reaches the toxicity alarm limit value, and all toxicity tests in 3 days can identify toxic substances and alarm; thirdly, the standard solution of the toxicity is stable in checking and testing, and the data set is between 45% and 55%.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (8)
1. A method of making a microbial electrochemical sensor comprising:
(1) Culturing a bioanode comprising a biofilm carrier to which a target strain is attached to obtain an initial sensor;
(2) Before the initial sensor on-board test, performing a sensor toxicity stimulation process comprising:
(2-1) injecting a second fresh culture solution of carbon source into the initial sensor to replace the culture solution of carbon source in the initial sensor, and recording the output current I of the sensor 1 Wherein the carbon source concentration of the second carbon source fresh culture solution is less than the carbon source concentration of the carbon source culture solution in the initial sensor;
(2-2) injecting a carbon source culture solution containing a target toxic substance into the sensor to replace the second carbon source fresh culture solution in the sensor, performing toxic stimulation on the sensor for a preset time, and recording the output current I of the sensor after the preset time 2 Wherein the carbon source concentration of the carbon source culture solution containing the target toxic substance is less than the carbon source concentration of the carbon source culture solution in the initial sensor;
(2-3) calculating the toxic stimulus current suppression rate r= (I) 1 -I 2 ）/I 1 ；
Repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, and the R value deviation is not more than +/-10% for three times, and judging that the toxic stimulation is completed;
the concentration of the target toxic substance in the carbon source culture solution containing the target toxic substance is 0.05 mg/L-1.0 mg/L;
in the step (2-2), the time of single toxic stimulation is 15-60 min.
2. The method of claim 1, further comprising, prior to step (2-1):
(2-0) injecting a first carbon source fresh culture solution into the initial sensor to replace the carbon source culture solution in the initial sensor, and then the second carbon source fresh culture solution injected into the sensor in the step (2-1) is used for replacing the first carbon source fresh culture solution in the sensor, wherein the carbon source concentration of the second carbon source fresh culture solution is smaller than that of the first carbon source fresh culture solution.
3. The method according to claim 2, wherein in step (2-1), after the fresh culture medium of the first carbon source in the sensor is replaced, the culture medium is left for a predetermined period of time, and then the output current I of the sensor is recorded 1 The method comprises the steps of carrying out a first treatment on the surface of the In the step (2-2), after the fresh culture solution of the second carbon source in the sensor is replaced, the sensor is left for a preset time to perform toxic stimulation on the sensor, and then the output current I of the sensor after the preset time is recorded 2 ；
Or in the step (2-1), after the first carbon source fresh culture solution in the sensor is replaced, continuously introducing the second carbon source fresh culture solution into the sensor for a preset time, and then recording the output current I of the sensor 1 The method comprises the steps of carrying out a first treatment on the surface of the In the step (2-2), after the fresh culture solution of the second carbon source in the sensor is replaced, continuously introducing the culture solution of the carbon source containing the target toxic substance into the sensor for a preset time to perform toxic stimulation on the sensor, and then recording the output current I of the sensor after the preset time 2 。
4. The method according to claim 3, wherein in the step (2-1), after the replacement of the first carbon source fresh culture solution in the sensor is completed, the time for standing is 20min to 40min, or the time for continuously introducing the second carbon source fresh culture solution into the sensor is 20min to 40min;
and/or in the step (2-0), before the step (2-1), injecting the first carbon source fresh culture solution into the initial sensor 10-14 h in advance.
5. The method of claim 2, wherein the first carbon source fresh culture broth is the same as the fresh culture broth that incubated the initial sensor;
and/or the carbon source concentration of the second carbon source fresh culture solution is 5-50% of that of the first carbon source fresh culture solution, and other components and conditions of the second carbon source fresh culture solution are the same as those of the first carbon source fresh culture solution;
and/or the other components and conditions of the carbon source culture solution containing the target toxic substance except the target toxic substance are the same as those of the second carbon source fresh culture solution;
and/or the target toxic substance comprises Zn 2+ 、Cu 2+ 、Pb 2+ 、Ni 2+ 、Tl 2+ 、Hg 2+ And Cd 2+ At least one of them.
6. The method according to claim 2, wherein the sensor is subjected to toxicity stimulation by using a carbon source culture solution containing specific single heavy metal ions, and the steps (2-1) - (2-3) are repeated until R is 15% -75%, and the deviation of the R value is not more than +/-10% three times continuously, so that the toxicity stimulation is judged to be completed;
or, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific multi-element composite heavy metal ions, repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, and judging that the toxicity stimulation is finished if the R value deviation is not more than +/-10% three times continuously;
Or, firstly, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific single heavy metal ions, and repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, wherein the R value deviation is not more than +/-10% for three times continuously; and then, carrying out toxicity stimulation on the sensor by adopting a carbon source culture solution containing specific multi-element composite heavy metal ions, repeating the steps (2-1) - (2-3) until R is more than or equal to 15% and less than or equal to 75%, and judging that the toxicity stimulation is finished if the R value deviation is not more than +/-10% three times continuously.
7. A system for performing the toxic stimulation of any one of claims 1-6, comprising:
a sensor;
the first liquid storage device is used for storing a second carbon source fresh culture solution;
at least one second liquid storage device for storing a carbon source culture solution containing a target toxic substance;
the liquid inlet of the first multi-way valve flow dividing element is respectively connected with the liquid outlet of the first liquid storage device and the liquid outlet of the second liquid storage device, and the first liquid outlet of the first multi-way valve flow dividing element is communicated with the liquid inlet of the sensor;
The first liquid outlet of the second multi-way valve flow dividing element is respectively connected with the liquid inlet of the first liquid storage device and the liquid inlet of the second liquid storage device, and the liquid inlet of the second multi-way valve flow dividing element is connected with the liquid outlet of the sensor;
the first waste liquid collecting device is connected with the second liquid outlet of the first multi-way valve flow dividing element;
the second waste liquid collecting device is connected with a second liquid outlet of the second multi-way valve flow dividing element;
a pumping device disposed on a line between the first multi-way valve split element and the sensor;
the data acquisition and signal control unit is connected with the sensor, the first multi-way valve shunt element, the second multi-way valve shunt element and the pumping device through electric signals respectively.
8. Use of the method of any one of claims 1-6 in a biotoxicity assay.
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| CN114199966A (en) * | 2021-12-27 | 2022-03-18 | 南开大学 | Preparation of novel biological cathode water source water quality toxicity sensing system |
| CN116042472A (en) * | 2022-12-30 | 2023-05-02 | 中国科学院成都生物研究所 | Pseudomonas and application thereof in water toxicity monitoring |
| CN116555085A (en) * | 2023-04-12 | 2023-08-08 | 中国科学院成都生物研究所 | Biological film sensor and method for detecting biological toxicity by using same |
| CN116429858A (en) * | 2023-06-15 | 2023-07-14 | 广东盈峰科技有限公司 | Method and use for acclimatizing electrochemically active biofilms |
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