Biotoxicity sensor of calcium-magnesium synergetic cross-linked sodium alginate and preparation method of biotoxicity sensor

Abstract

The invention discloses a biotoxicity sensor of calcium-magnesium synergetic cross-linked sodium alginate and a preparation method, and belongs to the field of biosensing, the sensor comprises a reactor, a working electrode fixed with electroactive microorganisms, a counter electrode, a reference electrode, a sample introduction system and a constant potential system. The core of the working electrode is that calcium and magnesium ions are synergistically cross-linked with sodium alginate to fix electroactive microorganisms, and a porous structure is formed in sodium alginate hydrogel through the unsteady cross-linking effect of the magnesium ions and the sodium alginate, so that the mass transfer of toxic pollutants and electron transfer mediators is improved, and the sensitivity of the sensor is improved. The problem that a traditional sodium alginate immobilized microbial sensor is insufficient in sensitivity is effectively solved, complex microbial modification is not needed, and a new way is provided for rapid, sensitive and stable monitoring of water body biotoxicity.

Description

Technical Field

[0001] The present invention relates to a biotoxicity detection sensor and a preparation method thereof, and in particular to a calcium-magnesium synergistically cross-linked sodium alginate biotoxicity sensor and a preparation method thereof. Background Art

[0002] Toxic pollutants in the water environment (such as heavy metals, organic poisons and emerging pollutants) pose a serious threat to ecosystems and human health. Although traditional physical and chemical detection methods (such as chromatography-mass spectrometry) have high accuracy, they have limitations such as expensive equipment, complex pre-treatment, and inability to reflect comprehensive biological toxic effects in real time. Microbial sensors based on bioelectrochemical principles convert the toxic effects of pollutants into quantifiable electrical signals through the metabolic activity and electron transfer characteristics of electroactive microorganisms. They have the advantages of online monitoring, rapid response and controllable costs, and have become a research hotspot in the field of sudden water pollution warning. Electrochemical sensors with electroactive microorganisms (such as Shewanella and Geobacter) as sensing elements can achieve label-free detection of a wide spectrum of toxic pollutants by detecting changes in current signals generated by toxic pollutants interfering with the microbial respiratory chain, but their practical application still faces many challenges.

[0003] Traditional microbial electrochemical sensors are mainly based on suspension culture or electrode surface biofilm construction. Suspension culture is prone to cause the loss of electroactive microorganisms due to the weak retention capacity of microorganisms at the solid-liquid interface. Although biofilms can be fixed in situ through microbial colonization, they require a directional acclimation cycle of 7-21 days to form functional biofilms with stable electrochemical responses, which makes it difficult to meet the rapid monitoring needs of sudden water pollution incidents. To this end, researchers introduced sodium alginate ion cross-linking immobilization technology, using calcium ions to complex with the carboxyl groups of sodium alginate to form a three-dimensional hydrogel network to immobilize electroactive microorganisms. Although this method shortens the sensor startup time to 1-48 hours, the gel network structure is dense (pore size is about 10-50nm), which leads to the obstruction of the diffusion of toxic pollutant molecules and the reduction of microorganism-toxin contact efficiency, thereby limiting the sensitivity of the sensor. At the same time, the dense gel hinders the direct electron transfer between microorganisms and electrodes, and relies on mediator molecules to indirectly transfer electrons, resulting in a decrease in signal stability. In order to break through the sensitivity bottleneck, some research has turned to genetically engineering microorganisms (such as overexpressing cytochrome proteins or toxicant-responsive promoters). Although this can improve the detection limit, there are problems such as complex genetic manipulation, high risk of environmental release of engineered bacteria, and limited broad-spectrum detection capabilities, which seriously restrict its practical application.

[0004] Therefore, there is an urgent need to develop a new method that can effectively improve the detection sensitivity of biotoxicity sensors based on immobilized electroactive microorganisms by optimizing the physicochemical properties of the immobilized matrix without relying on complex microbial modifications, so as to meet the demand for fast, sensitive, stable and cost-effective biotoxicity detection technology in practical applications. Summary of the Invention

[0005] The purpose of the present invention is to provide a biological toxicity sensor of calcium-magnesium synergistically cross-linked sodium alginate and a preparation method thereof, so as to solve the problem of insufficient sensitivity of traditional sodium alginate immobilized microorganism sensors.

[0006] To achieve the above objectives, one aspect of the present invention provides a calcium-magnesium synergistically cross-linked sodium alginate biotoxicity sensor, comprising:

[0007] The reactor is composed of a working electrode, a counter electrode, a reference electrode, a water inlet and a water outlet; the water inlet and the water outlet are placed on the surface of the reactor, and the working electrode, the counter electrode and the reference electrode are placed inside the reactor;

[0008] The sampling system consists of a nutrient solution sampling device and a water sample sampling device to be tested; the nutrient solution sampling device includes a nutrient solution air inlet, a nutrient solution storage device and a nutrient solution water inlet pump, one end of the nutrient solution water inlet pump is connected to the nutrient solution storage device, and the other end is connected to the water inlet; the water sample sampling device to be tested includes a water sample storage device to be tested, a water sample air inlet and a water sample water inlet pump to be tested, one end of the water sample water inlet pump to be tested is connected to the water sample storage device to be tested, and the other end is connected to the water inlet; the nutrient solution air inlet and the water sample air inlet to be tested are respectively connected to the nutrient solution storage device and the water sample storage device to be tested;

[0009] Constant potential system, the constant potential system includes an electrochemical workstation and a computer.

[0010] Furthermore, the working electrode is composed of a sodium alginate hydrogel that is synergistically cross-linked by calcium and magnesium and immobilizes electroactive microorganisms.

[0011] Furthermore, the conductive substrate material of the working electrode is a titanium mesh with a mesh size range of 20 to 200 meshes; the nutrient solution is an optimized LB liquid culture medium, which consists of: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 5.0 g / L NaCl.

[0012] Furthermore, the working electrode and the counter electrode are placed in parallel; the reference electrode is a saturated calomel electrode or an Ag / AgCl electrode, and the counter electrode is a platinum electrode, a carbon electrode or a titanium electrode.

[0013] Furthermore, the nutrient solution inlet pump and the water sample inlet pump pump the nutrient solution and the water sample to be tested into the reactor at a constant flow rate through the water inlet, and the flow rate ratio of the nutrient solution and the water sample to be tested is 1:4; the nutrient solution air inlet and the water sample air inlet both pump nitrogen into the nutrient solution and the water sample to be tested at the same flow rate.

[0014] Another aspect of the present invention provides a method for preparing a biological toxicity sensor of calcium-magnesium synergistically cross-linked sodium alginate, comprising the steps of:

[0015] S1: Construct sensor A, which includes reactor A, a sampling system, and a constant potential system; wherein reactor A includes a counter electrode, a reference electrode, a water inlet, and a water outlet;

[0016] S2: Activate, purify, and expand electroactive microorganisms. The electroactive bacteria Shewanella oneiden sisMR-1 strain stored at -80°C is revived in a 30°C water bath. The revived bacteria are then inoculated into sterilized LB liquid medium, and the LB liquid medium is cultured in a constant temperature shaker until the logarithmic growth phase. After reaching the logarithmic growth phase for the first time, single colonies with good morphology and vigorous growth are selected and inoculated into freshly sterilized LB liquid medium, which is then expanded and cultured in a constant temperature shaker. After the bacterial liquid reaches the logarithmic growth phase again, the LB liquid medium is stored at 4°C for short-term use.

[0017] S3: Preparation and standardization of bacterial suspension: The Shewanella oneidensis MR-1 bacterial suspension amplified in S2 was centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded to obtain a bacterial pellet; the bacterial pellet was then washed with 0.85% (w / v, mass / volume percentage) sterile saline, and this step was repeated three times to remove residual culture medium components; the washed bacteria were then resuspended with 0.85% sterile saline, and the optical density (OD) of the bacterial suspension was adjusted. 600 ) to 0.08 to normalize the concentration of electroactive microorganisms;

[0018] S4: Preparation of sodium alginate-bacteria suspension mixture, OD 600 The electroactive bacterial suspension with a concentration of 0.08 was uniformly mixed with a 1.5% (w / v) sodium alginate aqueous solution at a volume ratio of 1:1, and the mixture was then ultrasonicated and allowed to stand at 4°C for at least 4 hours to remove bubbles;

[0019] S5: Coating and cross-linking of the working electrode: 100 μL of the sodium alginate-bacteria suspension mixture is accurately pipetted and evenly coated on the surface of the pretreated conductive substrate. Subsequently, the conductive substrate coated with the mixture is immersed in a mixed salt solution of MgCl2 for calcium-magnesium synergistic cross-linking to form a hydrogel layer immobilized with electroactive microorganisms, thereby completing the preparation of the working electrode.

[0020] S6: Assemble the prepared working electrode into the reactor A provided by S1, and then connect the working electrode and the counter electrode to the working electrode clamp and the counter electrode clamp respectively in the reactor A; then continuously pump the nutrient solution into the reactor at a constant flow rate through the injection system, and under the control of the electrochemical workstation, apply a preset constant potential to the working electrode, and continuously monitor the current signal in the reactor circuit until the current value increases and reaches a relatively stable baseline level. At this time, it can be considered that the biotoxicity sensor has been successfully started and is in a stable working state, thereby completing the preparation of the biotoxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate.

[0021] Furthermore, in step S5, the concentration of CaCl2 is set to 1.5%; the concentration of MgCl2 is set to 0.15% to 2%; and the cross-linking time of the calcium-magnesium synergistic cross-linked sodium alginate is in the range of 5 to 300 seconds.

[0022] Furthermore, in step S5, the conductive substrate material of the working electrode is a titanium mesh that is stable at a high electrode potential, with a mesh size range of 20 to 200 meshes. The pretreatment step includes sequentially ultrasonicating with 1 M HCl, 1 M NaOH, anhydrous ethanol and ultrapure water at 60°C for 30 minutes, rinsing with ultrapure water three times in the middle, and finally drying at 30°C.

[0023] Furthermore, the composition of the LB liquid culture medium includes: tryptone: 10.0 g / L; yeast extract: 5.0 g / L; NaCl: 1.0 g / L.

[0024] In summary, the present invention has the following beneficial effects compared to the prior art:

[0025] (1) Effectively improve sensor sensitivity: The present invention proposes a method based on calcium and magnesium ions (Ca 2+ / Mg 2+ ) synergistically cross-linked sodium alginate to regulate the structure of electroactive microbial hydrogels. The core of this method is to use Mg 2+ Non-steady-state cross-linking characteristics with sodium alginate: 2+ In the process of synergistic cross-linking of sodium alginate, Mg 2+ The non-steady-state cross-linking facilitates the formation of channels within the hydrogel that facilitate the diffusion of toxic pollutants and electron transfer mediators. This structure not only enables the efficient immobilization of electroactive microorganisms and enhances the mass transfer efficiency of toxic pollutants, but also helps improve the efficiency of mediated electron transfer and the stability of signal response, thereby significantly improving the detection sensitivity of the biotoxicity sensor.

[0026] (2) Simplified preparation process, avoiding complex microbial modification: The present invention improves the performance of biotoxicity sensors by regulating the physicochemical properties of the immobilized matrix, eliminating the need for complex genetic engineering of electroactive microorganisms (such as overexpressing cytochrome proteins or constructing toxicant-responsive promoters). This not only simplifies the sensor preparation process, lowers technical barriers and costs, but also avoids the environmental release risks and limited broad-spectrum detection capabilities that may be associated with genetically engineered bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 Schematic diagram of the biotoxicity sensor of calcium-magnesium synergistically cross-linked sodium alginate;

[0029] Figure 2 The circuit current of the biotoxicity sensor of calcium-magnesium synergistic cross-linking and single calcium cross-linking sodium alginate at the start-up stage;

[0030] Figure 3 The loop current of the biotoxicity sensor of calcium-magnesium synergistic cross-linking and single calcium cross-linking sodium alginate when detecting Zn(II) during the operation stage;

[0031] Figure 4 The loop current of the biotoxicity sensor of calcium-magnesium synergistic cross-linking and single calcium cross-linking sodium alginate when detecting formaldehyde during the operation stage;

[0032] Figure 5 Scanning electron micrographs of the anode electrodes of biotoxicity sensors with calcium-magnesium synergistic cross-linking and single calcium-cross-linked sodium alginate.

[0033] The above drawings include the following reference numerals:

[0034] 1. Counter electrode; 2. Working electrode; 3. Water inlet; 4. Nutrient solution inlet pump; 5. Nutrient solution storage device; 6. Nutrient solution air inlet; 7. Test water sample storage device; 8. Test water sample air inlet; 9. Test water sample inlet pump; 10. Reactor; 11. Reference electrode; 12. Working electrode holder; 13. Counter electrode holder; 14. Electrochemical workstation; 15. Computer; 16. Water outlet. DETAILED DESCRIPTION

[0035] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form can also include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0037] Unless otherwise specifically stated, the relative arrangement of the parts and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention. At the same time, it should be understood that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to actual proportional relationships. The techniques, methods and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the techniques, methods and equipment should be considered as part of the authorized specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as being merely exemplary and not as limitations. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following figures, and therefore, once an item is defined in one figure, it does not need to be further discussed in subsequent figures.

[0038] See also Figure 1 As shown, the present invention provides a calcium-magnesium synergistically cross-linked sodium alginate biotoxicity sensor, comprising:

[0039] Reactor 10, which consists of a working electrode 2 (for fixing electroactive microorganisms), a counter electrode 1, a reference electrode 11, a water inlet 3, and a water outlet 16. The water inlet 3 and the water outlet 16 are placed on the surface of the reactor 10, while the working electrode 2, the counter electrode 1, and the reference electrode 11 are placed inside the reactor 10.

[0040] The sampling system consists of a nutrient solution sampling device and a water sample sampling device. The nutrient solution sampling device includes a nutrient solution air inlet 6, a nutrient solution storage device 5, and a nutrient solution water inlet pump 4. The nutrient solution water inlet pump 4 is connected to the nutrient solution storage device 5 at one end and to the water inlet 3 at the other end, thereby pumping the nutrient solution in the nutrient solution storage device 5 into the reactor 10.

[0041] The test water sample inlet device includes a test water sample storage device 7, a test water sample air inlet 8, and a test water sample inlet pump 9. Similarly, the test water sample inlet pump 9 is connected to the test water sample storage device 7 at one end and to the water inlet 3 at the other end, thereby achieving the purpose of pumping the test water sample in the test water sample storage device 7 into the interior of the reactor 10.

[0042] The nutrient solution air inlet 6 and the test water sample air inlet 8 are connected to the nutrient solution storage device 5 and the test water sample storage device 7 respectively, thereby providing nitrogen to the nutrient solution storage device 5 and the test water sample storage device 7 to create an anaerobic environment for the electroactive bacteria.

[0043] The constant potential system includes an electrochemical workstation 14 and a computer 15.

[0044] One end of the electrochemical workstation 14 is connected to the working electrode clamp 12, the counter electrode clamp 13 and the reference electrode 11 through a copper wire, which is used to maintain a constant potential of the working electrode 2 and monitor the current of the biotoxicity sensor loop. The other end of the electrochemical workstation 14 is connected to a computer 15, which is used to record and store data on the current of the biotoxicity sensor loop. The working electrode 2 is composed of a sodium alginate hydrogel with calcium and magnesium synergistic cross-linking to fix electroactive microorganisms. Compared with the single calcium cross-linked working electrode 2, the calcium and magnesium ion synergistic cross-linking method utilizes Mg 2+ The non-stable cross-linking properties of sodium alginate make it 2+ During the synergistic cross-linking of sodium alginate, channels that are conducive to the diffusion of toxic pollutants and electron transfer mediators can be formed inside the hydrogel, significantly improving the mass transfer efficiency of toxic pollutants and electron mediators, thereby improving the detection sensitivity of the biological toxicity sensor.

[0045] As a preference, the flow rate ratio of the nutrient solution to the water sample to be tested in the sampling system is 1:4, and the nutrient solution is an optimized LB liquid culture medium, which consists of: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 5.0 g / L NaCl.

[0046] Preferably, the working electrode 2 is placed parallel to the counter electrode 1; a reference electrode 11 is disposed near the working electrode 2 and serves as a reference for maintaining the potential of the working electrode 2 in the electrochemical workstation 14. The reference electrode 11 is a saturated calomel electrode or an Ag / AgCl electrode, and the counter electrode 1 is a platinum electrode, a carbon electrode, or a titanium electrode.

[0047] As a preference, the water inlet 3 is arranged at the bottom position of the side of the reactor 10 , and the water outlet 16 is arranged at the top position of the side of the reactor 10 .

[0048] Preferably, the nutrient solution inlet pump 4 and the test water sample inlet pump 9 pump the nutrient solution and the test water sample into the reactor 10 at a constant flow rate through the water inlet 3. The nutrient solution air inlet 6 and the test water sample air inlet 8 both pump nitrogen into the nutrient solution and the test water sample at the same flow rate.

[0049] Another aspect of the present invention provides a method for preparing a biological toxicity sensor of calcium-magnesium synergistically cross-linked sodium alginate, comprising the steps of:

[0050] S1: Construct biotoxicity sensor A. Biotoxicity sensor A includes reactor A, a sample injection system, and a constant potential system (applying a constant working potential and collecting loop current). Reactor A includes a counter electrode 1, a reference electrode 11, a water inlet 3, and a water outlet 16.

[0051] S2: Activate, purify, and expand electroactive microorganisms. Resuscitate the electroactive bacteria Shewanella oneiden sis MR-1 strain stored at -80°C in a 30°C water bath. Then, inoculate the revived bacteria into sterilized LB liquid medium, and culture the LB liquid medium in a constant temperature shaker until the logarithmic growth phase. After reaching the logarithmic growth phase for the first time, select single colonies with good morphology and vigorous growth, inoculate them into fresh sterilized LB liquid medium, and expand them in a constant temperature shaker. After the bacterial liquid reaches the logarithmic growth phase again, store the LB liquid medium at 4°C for short-term use.

[0052] Sterilize the sterilized LB liquid medium and LB solid medium by autoclaving at 121°C for 30 minutes. The LB liquid medium consists of: 10.0 g / L tryptone; 5.0 g / L yeast extract; and 1.0 g / L NaCl. The LB solid medium also consists of: 10.0 g / L tryptone; 5.0 g / L yeast extract; and 1.0 g / L NaCl, and 15.0 g / L agar.

[0053] The temperature range of the constant temperature shaker was set to 25-35°C and the speed was set to 150-300 rpm.

[0054] S3: Preparation and standardization of bacterial suspension: The Shewanella oneidensis MR-1 bacterial suspension amplified in S2 was centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded to obtain a bacterial pellet; the bacterial pellet was then washed with 0.85% (w / v) sterile saline, and this step was repeated three times to remove residual culture medium components. Subsequently, the washed bacterial cells were resuspended with 0.85% sterile saline, and the optical density (OD) of the bacterial suspension was adjusted. 600 ) to 0.08 to normalize the electroactive microbial concentration.

[0055] S4: Preparation of sodium alginate-bacteria suspension mixture, OD 600 The electroactive bacterial suspension of 0.08% was uniformly mixed with a 1.5% (w / v) sodium alginate aqueous solution at a volume ratio of 1:1, and the mixture was then ultrasonically treated and allowed to stand at 4°C for at least 4 hours to remove bubbles.

[0056] S5: Coating and cross-linking of working electrode 2: Accurately pipette 100 μL of the above sodium alginate-bacteria suspension mixture and evenly coat it on the surface of the pretreated conductive substrate. Then, immerse the conductive substrate coated with the mixture in a solution containing a specific concentration of Ca 2+ and Mg 2+ The mixed salt solution is cross-linked for 5 to 300 seconds to form a hydrogel layer with electroactive microorganisms fixed thereon, which is the working electrode 2.

[0057] As a preferred embodiment, in S5, Ca 2+ and Mg 2+ Ca in mixed salt solution 2+ and Mg 2+ The calcium and magnesium synergistic cross-linking of sodium alginate is carried out for a time range of 5 to 300 seconds.

[0058] As a preferred embodiment, the conductive base material of the working electrode 2 is a titanium mesh with a mesh size range of 20 to 200 meshes. The pretreatment step includes ultrasonication with 1M HCl, 1M NaOH, anhydrous ethanol and ultrapure water at 60°C for 30 minutes respectively, rinsing with ultrapure water three times in the middle, and finally drying at 30°C.

[0059] S6: Assemble the prepared working electrode 2 into the reactor A provided by S1, and then connect the working electrode 2 and the counter electrode 1 to the working electrode electrode clamp 12 and the counter electrode electrode clamp 13 in the reactor A, thereby obtaining the reactor 10. The nutrient solution is then continuously pumped into the reactor 10 at a constant flow rate through the injection system. Under the control of the electrochemical workstation 14, a preset constant potential is applied to the working electrode 2. The current signal in the reactor 10 circuit is continuously monitored until the current value increases and reaches a relatively stable baseline level. At this time, it can be considered that the biotoxicity sensor has been successfully started and is in a stable working state, thereby completing the preparation of the biotoxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate.

[0060] As a preference, the working electrode clamp 12 and the counter electrode clamp 13 are platinum electrode clamps or carbon electrode clamps.

[0061] The steps in actual use are:

[0062] (1) Start the biotoxicity sensor. The water sample to be tested in the sampling system is set to deionized water, and the nutrient solution is set to the optimized LB liquid culture medium. The nutrient solution air inlet 6 and the water sample air inlet 8 are both set to introduce nitrogen at a flow rate of 10 to 200 mL / min. The flow rates of the nutrient solution inlet pump 4 and the water sample inlet pump 9 are set to 0.1 to 10 mL / min and 0.4 to 40 mL / min respectively. The constant potential of the working electrode 2 is set to -0.1 to 0.2 V (relative to a saturated calomel electrode). The above-mentioned biotoxicity sensor is placed at room temperature of 24°C to 28°C and started to operate until the loop current value increases and reaches a relatively stable baseline level.

[0063] The optimized LB liquid culture medium composition was as follows: tryptone: 10.0 g / L; yeast extract: 5.0 g / L; NaCl: 5.0 g / L.

[0064] (2) To detect the biological toxicity of water samples, the deionized water of the water sample to be tested is replaced with a water sample containing toxic pollutants, and then the sensor is continuously operated for 10 to 600 minutes. The current suppression rate is calculated based on the change in the loop current before and after the biotoxicity sensor is poisoned.

[0065] After the sensor stabilizes, the sample is continuously pumped into reactor 10 at a constant flow rate using the sample injection system. After the sample is exposed for a specified period of time, the changes in the current in reactor 10's circuit before and after the introduction of the sample are recorded and compared. The current inhibition rate is used to quantitatively characterize the biotoxicity level of the sample.

[0066] The calculation formula of current suppression rate is as follows:

[0067]

[0068] Where, I nor I is the average value of the loop current during a period of stable operation of the biotoxicity sensor before the addition of toxic pollutants; tox After the toxic pollutant is added for a specified time, the average loop current of the biotoxicity sensor during the period before the addition of poisoning is completed.

[0069] Example 1:

[0070] Step 1: Construct a biotoxicity sensor, such as Figure 1 As shown, the reactor 10 is a single-chamber, three-electrode sealed system consisting of a borosilicate glass cylinder (φ4.7 cm) and a polytetrafluoroethylene lid. It has a capacity of 50 mL and a working volume of 35 mL. The water inlet 3 is located 1.0 cm from the bottom of the cylinder, and the water outlet 16 is located 2.5 cm from the top of the cylinder. Both the water inlet 3 and the water outlet 16 are barbed borosilicate pipe fittings with an inner diameter of 4.0 mm.

[0071] Step 2: Activation, purification and expansion of electroactive microorganisms. Activation of strains: The electroactive bacteria Shewanella oneidensis MR-1 strain stored in a -80°C glycerol tube was quickly revived in a 30°C water bath. The revived strain was then inoculated into 20 mL of sterilized LB liquid culture medium and cultured in a constant temperature shaker at 30°C and 200 rpm until the logarithmic growth phase. Strain purification and expansion: To ensure the purity of the strain, the activated bacterial suspension was separated and purified on a sterilized LB solid culture medium using the partition plate streak method. Single colonies with good morphology and vigorous growth were selected, inoculated into 20 mL of freshly sterilized LB liquid culture medium, and placed in a constant temperature shaker for expansion and culture. After the bacterial solution reaches the logarithmic growth phase again, it is stored at 4°C for short-term use.

[0072] LB liquid medium composition: Tryptone: 10.0 g / L; Yeast extract: 5.0 g / L; NaCl: 1.0 g / L. LB solid medium composition: Tryptone: 10.0 g / L; Yeast extract: 5.0 g / L; NaCl: 1.0 g / L; Agar: 15.0 g / L. LB liquid and solid medium were sterilized by autoclaving at 121°C for 30 minutes.

[0073] Step 3: Prepare a working electrode 2 for calcium-magnesium synergistic cross-linked sodium alginate to immobilize electroactive microorganisms. Preparation and standardization of bacterial suspension: The Shewanella oneidensis MR-1 bacterial solution amplified and cultured in S2 was centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded. The bacterial precipitate was washed with 0.85% (w / v) sterile saline, and this step was repeated three times to remove residual culture medium components. Subsequently, the washed bacteria were resuspended with 0.85% sterile saline, and the optical density value (OD 600 ) to 0.08 to standardize the concentration of electroactive microorganisms. Preparation of sodium alginate-bacteria suspension mixture: OD 600 The electroactive bacterial suspension of 0.08% was evenly mixed with 1.5% (w / v) sodium alginate aqueous solution in a volume ratio of 1:1. The mixture was then ultrasonicated and allowed to stand at 4°C for at least 4 hours to remove bubbles. Coating and cross-linking of working electrode 2: 100 μL of the above sodium alginate-bacteria suspension mixture was accurately pipetted and evenly coated on the surface of a pretreated titanium mesh (2.0 cm × 2.0 cm, 60 mesh). Subsequently, the titanium mesh coated with the mixture was immersed in a solution containing Ca 2+ and Mg 2+ The mixed salt solution (CaCl2 concentration: 1.5% (w / v), MgCl2 concentration: 0.75% (w / v)) was cross-linked for 19 seconds to form a hydrogel layer with fixed electroactive microorganisms, which was the working electrode 2.

[0074] The pretreatment steps of the titanium mesh are as follows: the titanium mesh is ultrasonically treated with 1M HCl, 1M NaOH, anhydrous ethanol and ultrapure water at 60°C for 30 minutes respectively, rinsed with ultrapure water three times in the middle, and finally placed in a forced air drying oven at 30°C for drying.

[0075] Step 4: Set up the electrodes. Connect the prepared working electrode 2 and counter electrode 1 (platinum sheet: 1.0 cm × 1.0 cm) to the working electrode holder 12 and counter electrode holder 13, respectively. Both the working electrode holder 12 and counter electrode holder 13 are platinum electrode holders (with built-in platinum sheet: 9.0 mm × 12.0 mm × 0.1 mm, and welded platinum wire: 0.7 mm × 10.0 mm). Then, place the working electrode 2 and counter electrode 1 parallel to each other in the reactor 10, with a spacing of 2.0 cm. Place the reference electrode 11 (saturated calomel electrode) next to the working electrode 2, with a spacing of 1.0 cm.

[0076] Step 5: Set up the constant potential system. Connect one end of an electrochemical workstation 14 (VSP, BioLogic, France) to the working electrode holder 12, the counter electrode holder 13, and the reference electrode 11 via copper wires. This is used to maintain a constant potential at the working electrode 2 and monitor the biotoxicity sensor loop current. The other end of the electrochemical workstation 14 is connected to a computer 15 to record and store data on the biotoxicity sensor loop current.

[0077] Step 6: Set up the sample inlet system. Both the nutrient solution inlet pump 4 and the test water sample inlet pump 9 are peristaltic pumps (Huiyu Weiye Fluid Equipment Co., Ltd., BT100F-1A). The flow rates of the nutrient solution inlet pump 4 and the test water sample inlet pump 9 are set to 0.4 mL / min and 1.6 mL / min, respectively. Nitrogen is pumped into the nutrient solution 5 and test water sample 7 at a flow rate of 20 mL / min through the nutrient solution air inlet 6 and the test water sample air inlet 8.

[0078] Step 7: Start the biotoxicity sensor. Set the nutrient solution 5 to optimized LB medium, whose composition is: tryptone: 10.0 g / L; yeast extract: 5.0 g / L; NaCl: 5.0 g / L. Set the water sample 7 to deionized water. Set the constant potential of the working electrode 2 to 0.1 V (relative to a saturated calomel electrode). Place the biotoxicity sensor at room temperature (24°C to 28°C) and start operating until the loop current increases and reaches a relatively stable baseline level.

[0079] Step 8: Sensor Detection of Zn(II) Toxicity. Replace the deionized water in test sample 7 with a solution containing 125.0 mg / L Zn(II). (Since the flow rate ratio of the nutrient solution to the test sample is 1:4, the actual detected Zn(II) concentration is 100.0 mg / L.) The sensor is run continuously for 53 minutes (calculated as three times the residence time based on the working volume and the sample flow rate). The current suppression rate is then calculated based on the stored loop current data.

[0080] Step 9: Calculate the current inhibition rate. The current inhibition rate is used to reflect the biological toxicity of toxic pollutants. The calculation formula is as follows:

[0081]

[0082] Where, I nor I is the average loop current of the biotoxicity sensor during 10 minutes of stable operation before the addition of toxic pollutants; tox The average loop current of the biotoxicity sensor within 10 minutes after the toxic pollutant is added for 53 minutes before the poisoning ends.

[0083] Example 2:

[0084] This example describes a biotoxicity sensor for formaldehyde detection based on calcium-magnesium synergistically cross-linked sodium alginate. The operating conditions remained the same as in Example 1, except that the Zn(II) solution in step eight of Example 1 was replaced with a 0.0156% formaldehyde solution (due to a 1:4 flow rate ratio between the nutrient solution and the test water sample, the actual measured formaldehyde concentration was 0.0125%).

[0085] Comparative Example 1:

[0086] This comparative example is a biotoxicity sensor based on single calcium ion cross-linked sodium alginate to detect Zn(II). 2+ and Mg 2+ The mixed salt solution (CaCl2 concentration: 1.5% (w / v), MgCl2 concentration: 0.75% (w / v)) was replaced with Ca 2+ The saline solution (CaCl2 concentration: 1.5% (w / v)) and other operating conditions were consistent with those in Example 1.

[0087] Comparative Example 2:

[0088] This comparative example is a biotoxicity sensor for detecting formaldehyde based on a single calcium ion cross-linked sodium alginate. 2+ and Mg 2+ The mixed salt solution (CaCl2 concentration: 1.5% (w / v), MgCl2 concentration: 0.75% (w / v)) was replaced with Ca 2+Salt solution (CaCl2 concentration: 1.5% (w / v)), and the Zn(II) solution in step eight of Example 1 was replaced with 0.0156% formaldehyde solution (because the flow rate ratio of the nutrient solution to the water sample to be tested is 1:4, the actual detected formaldehyde concentration is 0.0125%), and the other operating conditions are consistent with Example 2.

[0089] Results: During the startup phase, the loop current of the calcium-magnesium synergistic cross-linked biotoxicity sensor and the single calcium cross-linked biotoxicity sensor changed with time. Figure 2 After approximately 16 hours of operation, the loop current of the calcium-magnesium synergistic cross-linked biotoxicity sensor gradually increased and stabilized at 0.898±0.002μA, significantly higher than the stable current of the single calcium cross-linked biotoxicity sensor of 0.728±0.002μA, indicating that the calcium-magnesium synergistic cross-linking strategy helps improve the power generation performance of the sodium alginate-based biotoxicity sensor. This may be attributed to the calcium-magnesium synergistic cross-linking forming channels in the sodium alginate hydrogel matrix that are conducive to the efficient mass transfer of nutrients and electron transfer mediators, thereby enhancing the sensor's loop current.

[0090] During the operation phase, the changes in the loop current when the calcium-magnesium synergistic cross-linked biotoxicity sensor and the single calcium cross-linked biotoxicity sensor detect Zn(II) and formaldehyde are as follows: Figure 3 and Figure 4 When detecting 100.0 mg / L Zn(II), the loop current of the calcium-magnesium synergistic cross-linked biotoxicity sensor decreased significantly from the initial 0.896±0.002μA to 0.499±0.005μA. The corresponding current suppression rate was calculated to be 44.3±1.8%, which was much higher than the current suppression rate of the single calcium cross-linked biotoxicity sensor of 22.3±1.6% (the current decreased from 0.728±0.002μA to 0.566±0.004μA) ( Figure 3 Similarly, when detecting 0.0125% formaldehyde solution, the loop current of the calcium-magnesium synergistic cross-linked biotoxicity sensor decreased from 0.914±0.003μA to 0.595±0.006μA, and the corresponding current suppression rate was calculated to be 34.9±2.1%, which was also significantly higher than the current suppression rate of the single calcium cross-linked biotoxicity sensor of 26.4±1.8% (the current decreased from 0.690±0.002μA to 0.508±0.005μA) ( Figure 4 The above results show that the calcium-magnesium synergistic cross-linked biotoxicity sensor has higher sensitivity than the single calcium cross-linked biotoxicity sensor.

[0091] To further explore the intrinsic mechanism of calcium-magnesium synergistic cross-linking of sodium alginate to improve sensor performance, scanning electron microscopy was performed on the sodium alginate hydrogels with calcium-magnesium synergistic cross-linking and single calcium cross-linking. Figure 5Scanning electron microscopy images show that compared to alginate hydrogels cross-linked with calcium alone, alginate hydrogels cross-linked with magnesium and calcium exhibit a more pronounced porous structure, likely due to the non-steady-state cross-linking properties of magnesium ions with sodium alginate. It is speculated that these porous structures not only facilitate the diffusion and mass transfer of toxic pollutants but also improve the mass transfer efficiency of nutrients and electron transfer mediators within the hydrogel matrix, thereby contributing to the enhanced sensitivity of the biotoxicity sensor.

[0092] In summary, the above results show that compared with the single calcium-crosslinked sodium alginate biotoxicity sensor, the biotoxicity sensor prepared by the calcium-magnesium synergistic crosslinking strategy has higher sensitivity.

[0093] For ease of description, spatially relative terms such as "above", "above", "on the upper surface of", "above", etc. may be used herein to describe the spatial positional relationship of a device or feature to other devices or features as shown in the figures. It should be understood that spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation described in the figures. For example, if the device in the drawings is inverted, the device described as "above other devices or structures" or "above other devices or structures" will be positioned as "below other devices or structures" or "below other devices or structures". Thus, the exemplary term "above" can include both "above" and "below". The device can also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatially relative descriptions used here are interpreted accordingly.

[0094] In addition, it should be noted that the use of terms such as "first" and "second" to limit components is only for the convenience of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore cannot be understood as limiting the scope of protection of the present invention.

[0095] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A biotoxicity sensor of calcium-magnesium synergistically cross-linked sodium alginate, characterized in that: include: A reactor (10), the reactor (10) comprising a working electrode (2), a counter electrode (1), a reference electrode (11), a water inlet (3) and a water outlet (16); the water inlet (3) and the water outlet (16) are disposed on the surface of the reactor (10), and the working electrode (2), the counter electrode (1) and the reference electrode (11) are disposed inside the reactor (10); The sampling system is composed of a nutrient solution sampling device and a water sample sampling device to be tested; the nutrient solution sampling device comprises a nutrient solution air inlet (6), a nutrient solution storage device (5) and a nutrient solution water inlet pump (4); one end of the nutrient solution water inlet pump (4) is connected to the nutrient solution storage device (5), and the other end is connected to the water inlet (3); the water sample sampling device to be tested comprises a water sample storage device to be tested (7), a water sample air inlet (8) and a water sample water inlet pump (9); one end of the water sample water inlet pump (9) to be tested is connected to the water sample storage device to be tested (7), and the other end is connected to the water inlet (3); the nutrient solution air inlet (6) and the water sample air inlet (8) to be tested are connected to the nutrient solution storage device (5) and the water sample storage device to be tested (7), respectively; A constant potential system comprises an electrochemical workstation (14) and a computer (15).

2. The biotoxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate according to claim 1, characterized in that: The working electrode (2) is composed of a sodium alginate hydrogel that is synergistically cross-linked with calcium and magnesium and immobilizes electroactive microorganisms.

3. The biotoxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate according to claim 1, characterized in that: The conductive base material of the working electrode (2) is a titanium mesh with a mesh size range of 20 to 200 meshes; the nutrient solution is an optimized LB liquid culture medium, which comprises: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 5.0 g / L NaCl.

4. The biotoxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate according to claim 1, characterized in that: The working electrode (2) is placed in parallel with the counter electrode (1); the reference electrode (11) is a saturated calomel electrode or an Ag / AgCl electrode, and the counter electrode (1) is a platinum electrode, a carbon electrode or a titanium electrode.

5. The biotoxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate according to claim 1, characterized in that: The nutrient solution inlet pump (4) and the water sample inlet pump (9) pump the nutrient solution and the water sample to be tested into the reactor (10) at a constant flow rate through the water inlet (3), and the flow rate ratio of the nutrient solution to the water sample to be tested is 1:4; the nutrient solution air inlet (6) and the water sample air inlet (8) both pump nitrogen into the nutrient solution and the water sample to be tested at the same flow rate.

6. A method for preparing a biotoxicity sensor of calcium-magnesium synergistically cross-linked sodium alginate, which is used to prepare the biotoxicity sensor of calcium-magnesium synergistically cross-linked sodium alginate according to any one of claims 1 to 5, comprising the steps of: S1: Constructing a sensor A, wherein the sensor A comprises a reactor A, the injection system and the constant potential system; wherein, The reactor A comprises the counter electrode (1), the reference electrode (11), the water inlet (3) and the water outlet (16); S2: Activate, purify, and expand electroactive microorganisms. The electroactive bacteria Shewanella oneidensis MR-1 strain stored at -80°C was revived in a 30°C water bath. The revived bacteria were then inoculated into sterilized LB liquid medium, and the LB liquid medium was cultured in a constant temperature shaker until the logarithmic growth phase. After reaching the logarithmic growth phase for the first time, single colonies with good morphology and vigorous growth were selected and inoculated into freshly sterilized LB liquid medium, which was then expanded and cultured in a constant temperature shaker. After the bacterial liquid reached the logarithmic growth phase again, the LB liquid medium was stored at 4°C for short-term use. S3: Preparation and standardization of bacterial suspension: The Shewanella oneidensis MR-1 bacterial suspension amplified in S2 was centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded to obtain a bacterial pellet; the bacterial pellet was then washed with 0.85% sterile saline, and this step was repeated three times to remove residual culture medium components; the washed bacteria were then resuspended with 0.85% sterile saline, and the optical density (OD) of the bacterial suspension was adjusted. 600 to 0.08 to normalize the concentration of electroactive microorganisms; S4: Preparation of sodium alginate-bacteria suspension mixture, OD 600 The electroactive bacterial suspension with a concentration of 0.08 was uniformly mixed with a 1.5% sodium alginate aqueous solution at a volume ratio of 1:1, and then the mixture was ultrasonically treated and allowed to stand at 4°C for 4 hours to remove bubbles; S5: coating and cross-linking of the working electrode (2): accurately pipetting 100 μL of the sodium alginate-bacteria suspension mixture and uniformly coating it on the surface of the pretreated conductive substrate material; then, immersing the conductive substrate material coated with the mixture in a mixed salt solution MgCl2 for calcium-magnesium synergistic cross-linking to form a hydrogel layer immobilized with electroactive microorganisms; S6: Assemble the prepared working electrode (2) into the reactor A provided by S1, and then connect the working electrode (2) and the counter electrode (1) to the working electrode electrode clamp (12) and the counter electrode electrode clamp (13) in the reactor A, respectively, to form a reactor (10); then continuously pump the nutrient solution into the reactor (10) at a constant flow rate through the injection system, and under the control of the electrochemical workstation (14), apply a preset constant potential to the working electrode (2), and continuously monitor the current signal in the reactor (10) circuit until the current value increases and reaches a stable baseline level. At this time, the biotoxicity sensor has been successfully started and is in a stable working state, thereby completing the preparation of a high-sensitivity biotoxicity sensor based on calcium-magnesium synergistic cross-linked sodium alginate.

7. The method for preparing the biological toxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate according to claim 6, characterized in that: In step S5, the CaCl2 concentration is set to 1.5%; the MgCl2 concentration is set to 0.15% to 2%; and the cross-linking time range of the calcium-magnesium synergistic cross-linking sodium alginate is 5 to 300 seconds.

8. The method for preparing the biological toxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate according to claim 6, characterized in that: In step S5, the conductive base material of the working electrode (2) is a titanium mesh with a mesh size range of 20 to 200 meshes. The pretreatment step includes sequentially ultrasonicating with 1M HCl, 1M NaOH, anhydrous ethanol and ultrapure water at 60°C for 30 minutes, rinsing with ultrapure water three times in the middle, and finally drying at 30°C.

9. The method for preparing the biological toxicity sensor of calcium-magnesium synergistic cross-linked sodium alginate according to claim 6, characterized in that: The LB liquid culture medium includes: tryptone: 10.0 g / L; yeast extract: 5.0 g / L; NaCl: 1.0 g / L.

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