A mediator-type integrated microbial electrode capable of detecting seawater biological toxicity and a preparation method and application thereof

Abstract

The application discloses a mediator type integrated microbial electrode capable of detecting seawater biological toxicity, and belongs to the field of seawater biological toxicity detection. The electrode comprises an electrode base and a microbial membrane uniformly attached to the electrode base. The microbial membrane is a thin film obtained by fixing mixed and uniform electronic mediators and microorganisms on the electrode base through a gel solution and then drying. The gel solution is formed by mixing polydiallyldimethylammonium chloride solution and chitosan solution. The electrode can be used to quickly and accurately detect seawater biological toxicity, fills the blank of the mediator type integrated microbial electrode in the field of seawater toxicity evaluation, and opens up the application of the electrochemical method in marine water quality monitoring. The application further discloses a preparation method and application of the mediator type integrated microbial electrode.

Description

Technical Field

[0001] This invention relates to the field of biotoxicity detection technology. More specifically, it relates to a mediator-type integrated microbial electrode for detecting the toxicity of marine organisms, its preparation method, and its application. Background Technology

[0002] The ocean is an important part of the Earth's surface, containing abundant mineral resources and playing a vital role in regulating climate and maintaining biodiversity, thus having significant ecological and economic importance for human survival and development. However, while utilizing marine resources, some inappropriate human activities have led to the continuous discharge of pollutants into the seawater, causing damage to the marine environment; in recent years, marine pollution incidents have also caused great harm to fisheries and marine biological resources in nearby waters.

[0003] Marine pollution is constantly driving the exploration of marine water quality monitoring technologies. Currently, most biotoxicity detection methods for marine pollution use marine animals such as fish, shrimp, and copepods as test organisms. However, these methods have problems such as high cost, long experimental cycle, and cumbersome detection process, and cannot provide timely early warning for sudden marine pollution. Therefore, there is an urgent need to develop convenient and reliable marine toxicity detection technologies to meet the needs of rapid response.

[0004] In recent years, mediator-type electrochemical microbial electrode sensors have been widely used in water toxicity research due to their advantages such as high sensitivity, ease of miniaturization, rapid response, and immunity to interference from water color and turbidity. These sensors primarily assess toxicity based on the inhibitory effect of toxins on the microbial respiratory system: electron mediators in the system (e.g., benzoquinone, potassium ferricyanide) can intervene in the microbial respiratory process, capturing and reducing electrons produced by microbial metabolism. The reduced electron mediators then undergo oxidation at the working electrode, generating a corresponding current signal. Conversely, toxins inhibit the respiration of the tested microorganisms, reducing the amount of electron mediators reduced by the microorganisms, ultimately leading to a decrease in output current. Therefore, the toxicity of the analyte can be assessed by observing changes in current. Electron mediators generally exist in two forms in microbial electrodes: dispersed systems in solution and integrated mediator systems fixed on the electrode surface. Compared to dispersed systems, integrated mediator-based systems require less electron mediator, avoiding adverse effects on organisms and secondary pollution. Furthermore, these systems attach both the test microorganism and the electron mediator to the electrode surface, eliminating the need for additional chemical reagents and offering greater flexibility in terms of personnel expertise, thus simplifying the detection process. However, research on water biotoxicity based on mediator-based electrochemical microbial sensors has primarily focused on freshwater systems. Marine systems, with their more complex composition, higher salinity, and higher electrolyte content compared to freshwater environments, present greater challenges for the construction of microbial electrochemical sensors. Additionally, current microbial assessments of marine biotoxicity rely on luminescent bacteria methods, whose results are easily affected by sample turbidity and color. The application of electrochemical methods in marine toxicity monitoring remains a gap, necessitating further research to meet the need for rapid and accurate monitoring of marine water quality. Summary of the Invention

[0005] Based on the above facts, one objective of this invention is to provide a mediator-type integrated microbial electrode for detecting marine biological toxicity, its preparation method, and its application. This electrode can be used to detect marine biological toxicity rapidly and accurately, filling the gap in the field of marine toxicity assessment based on mediator-type integrated microbial electrodes and opening up the application of electrochemical methods in marine water quality monitoring.

[0006] Traditional integrated electrochemical microbial electrodes for mediators, when applied to seawater, generally suffer from the problem that the tested microorganisms have poor salt tolerance and cannot survive in seawater. In addition, it is also necessary to overcome the impact of the high electrolyte content of seawater on the electrochemical signal intensity and detection accuracy of the detection system.

[0007] To address this problem, on the one hand, the technical solution of the present invention provides a mediator-type integrated microbial electrode that can detect the toxicity of marine organisms. The electrode includes an electrode substrate and a microbial membrane uniformly attached to the electrode substrate.

[0008] The microbial membrane is a thin film obtained by fixing a uniformly mixed electron mediator and microorganisms onto an electrode substrate through a gel solution and then drying it.

[0009] The gel solution is formed by mixing a polydiallyldimethylammonium chloride solution and a chitosan solution.

[0010] This technical solution employs a gel with a specific composition to simultaneously immobilize electron mediators and microorganisms. Poly(diallyldimethylammonium chloride) and chitosan are selected as gel components. The gel obtained by mixing these two components combines the advantages of both, exhibiting superior electrochemical performance, better stability, and higher biocompatibility compared to gels composed of a single component. Furthermore, both poly(diallyldimethylammonium chloride) and chitosan are positively charged polymers that can adsorb negatively charged electron mediators through electrostatic attraction, resulting in a stable electron-loaded membrane structure. The preparation process of the electrochemically active gel is also relatively simple. The integrated microbial electrode obtained by mixing this mixed gel with electron mediators and microorganisms can rapidly respond to seawater polluted by heavy metal ions and pesticides, and it also has good applicability in complex real-world seawater systems. It overcomes the influence of high electrolyte content in seawater on the electrochemical signal intensity and detection accuracy of the detection system, and can be applied to the acute toxicity detection of marine pollutants.

[0011] Furthermore, the solvent for the polydiallyldimethylammonium chloride solution and the chitosan solution is one or a mixture of two of water and acetic acid.

[0012] Furthermore, the electrode substrate is selected from one of glassy carbon electrode, gold electrode, platinum electrode or screen-printed electrode.

[0013] Furthermore, the electron mediator is selected from one of electron mediators such as thionine, potassium ferricyanide, benzoquinone, and menaquinone, preferably potassium ferricyanide.

[0014] Furthermore, the microorganisms are selected from salt-tolerant bacteria or marine bacteria.

[0015] Furthermore, the microorganism is selected from one or more of salt-tolerant lactic acid bacteria, Staphylococcus aureus, or Shewanella.

[0016] Furthermore, the mass ratio of polydiallyldimethylammonium chloride in the polydiallyldimethylammonium chloride solution to chitosan in the chitosan solution is 1:0.5 to 1:2.

[0017] In another aspect, the present invention provides a method for preparing the mediator-type integrated microbial electrode as described above, characterized by comprising the following steps:

[0018] The polydiallyldimethylammonium chloride solution was mixed with the chitosan solution to form a gel solution;

[0019] Add an electron mediator solution to the gel solution, mix well, and obtain an active gel solution;

[0020] The active gel solution is mixed with the microbial culture and then dropped onto the electrode surface and dried to obtain the mediator-type integrated microbial electrode.

[0021] Furthermore, the solvent for the polydiallyldimethylammonium chloride solution and the chitosan solution is one or a mixture of two of water and acetic acid.

[0022] Furthermore, the electron mediator solution is an aqueous solution of an electron mediator.

[0023] Furthermore, the polydiallyldimethylammonium chloride solution is an aqueous solution of polydiallyldimethylammonium chloride.

[0024] Furthermore, the chitosan solution is an acetic acid solution of chitosan.

[0025] Further, the concentration of the polydiallyldimethylammonium chloride solution is 0.1-0.3 g / mL, preferably 0.1-0.2 g / mL; the concentration of the chitosan solution is 1.5-3 g / L; and the volume ratio of the chitosan solution to the polydiallyldimethylammonium chloride solution is 15-25:0.24. Preferably, the concentration of the polydiallyldimethylammonium chloride solution is 0.2 g / mL; the concentration of the chitosan solution is 2.5 g / L; and the volume ratio of the chitosan solution to the polydiallyldimethylammonium chloride solution is 20:0.24. Wherein, the proportion of polydiallyldimethylammonium chloride or chitosan in the gel solution within the range of this invention can obtain a mediator-type integrated microbial electrode with better stability and conductivity.

[0026] Furthermore, the concentration of the electron mediator solution is 40-60 mmol / L; the volume ratio of the gel solution to the electron mediator solution is 3-5:1, preferably 4:1.

[0027] Furthermore, the bacterial population density of the microbial culture is 1.0 ≤ OD. 600 Bacterial solution ≤3.0. Wherein, OD 600 It is a standard indicator for characterizing the density of microorganisms in liquids, and the density of microorganisms in microbial solutions can affect the detection sensitivity.

[0028] Furthermore, when forming the gel solution, the mixing time is 1-3 hours, preferably 2 hours.

[0029] Furthermore, when forming the active gel solution, the mixing time is 10-15 hours, preferably 12 hours.

[0030] Furthermore, the volume ratio of the active gel solution to the microbial culture is 1:1. If the volume of the microbial culture is too small compared to the active gel solution, it will affect the electrode sensitivity; if the volume is too large, it will affect the fixation effect.

[0031] The microbial culture solution can be cultured as needed by those skilled in the art. For example, the microorganisms can be inoculated into a microbial liquid culture medium and then centrifuged, washed, etc.

[0032] According to a specific embodiment of the present invention, the culture conditions for the Staphylococcus aureus are as follows: cultured in a constant temperature water bath shaker at 37°C for 24 hours.

[0033] According to a specific embodiment of the present invention, the cleaning of the bacterial cells can be carried out using conventional buffer solutions or cleaning solutions to remove substances that may affect subsequent test results during bacterial culture, such as phosphate buffer, Tris-HCl buffer, HEPES buffer, borate-borax buffer, or sodium chloride solution, preferably a 0.85% (w / v) sodium chloride solution.

[0034] According to a specific embodiment of the present invention, the microbial dispersion is a phosphate buffer solution, a sodium chloride solution, or a simulated seawater solution, wherein the simulated seawater solution is prepared by dissolving sea salt in ultrapure water and has a concentration of 35 g / L.

[0035] On another front, the present invention provides the application of the mediator-type integrated microbial electrode described above in the field of marine biological toxicity detection.

[0036] Furthermore, the application includes the following steps:

[0037] Using the aforementioned integrated microbial electrode as the working electrode of a three-electrode system, the biotoxicity of the seawater sample to be tested was determined.

[0038] Furthermore, the application includes the following steps:

[0039] In an electrolytic cell, a three-electrode system consisting of a working electrode, a counter electrode, and a reference electrode is formed. A microbial respiration matrix solution is added to the electrolytic cell and a constant voltage is applied. After the current stabilizes, the sample to be tested is added. The toxicity of the sample to be tested is determined using the inhibition rate.

[0040] Furthermore, the formula for calculating the inhibition rate is as follows:

[0041] Inhibition rate (%) = (1 - i1 / i0) × 100%;

[0042] Where i0 is the steady-state current before the sample is added, and i1 is the steady-state current after the sample is added.

[0043] Furthermore, the constant voltage is 0.5V vs.

[0044] Furthermore, the preparation method of the microbial respiratory matrix solution is as follows: glucose, sodium succinate and sodium lactate are dissolved in simulated seawater solution and mixed evenly, wherein the concentrations of glucose, sodium succinate and sodium lactate are all 10 mmol / L; the simulated seawater solution is prepared by dissolving sea salt in ultrapure water and has a concentration of 35 g / L.

[0045] Those skilled in the art will understand that the mediator-type integrated microbial electrode of the present invention can also be used for biotoxicity detection in water bodies such as freshwater.

[0046] The beneficial effects of this invention are as follows:

[0047] 1. The integrated microbial electrode of the mediator type provided by the present invention can detect the toxicity of marine organisms and can respond rapidly to seawater polluted by heavy metal ions, pesticides and other pollutants. It can be widely used in the field of acute toxicity detection of marine pollutants.

[0048] 2. The water biotoxicity monitoring system prepared using the integrated microbial electrode of the mediator type that can detect the toxicity of marine organisms according to the present invention can be used to monitor changes in the toxicity of water organisms in real time and detect the magnitude of the toxicity of water organisms. It not only achieves rapid detection, but also has the characteristics of high analytical sensitivity, low cost, simple operation and easy portability.

[0049] 3. The integrated microbial electrode for detecting marine biological toxicity provided by this invention does not require the addition of additional electronic mediators, microbial solutions, or other reagents during detection, making it highly practical. It greatly reduces the impact of human operation on detection uncertainty and shows broad prospects in actual water toxicity assessment. Attached Figure Description

[0050] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0051] Figure 1 Cyclic voltammetry curves for electrodes PDDA-CHI-FC / GCE, CHI-FC / GCE, PDDA-FC / GCE, and PDDA-gelatin-FC / GCE are shown.

[0052] Figure 2 The cyclic voltammetry curves of the PDDA-CHI-FC / GCE electrode after 30 consecutive scans are shown.

[0053] Figure 3 The cyclic voltammetry curves of the electrode CHI-silane coupling agent-FC / GCE are shown after 30 consecutive scans.

[0054] Figure 4 The mediator-type integrated microbial electrode of Example 1 is shown for testing Cu-containing microorganisms. 2+Chronoamperometry curves of biotoxicity in simulated seawater solutions.

[0055] Figure 5 The mediator-type integrated microbial electrode of Example 1 is shown to be for Cu 2+ The results of the acute biotoxicity evaluation of the simulated seawater solution are shown in the figure.

[0056] Figure 6 The mediator-type integrated microbial electrode of Example 1 is shown as containing Zn 2+ The results of the acute biotoxicity evaluation of the simulated seawater solution are shown in the figure.

[0057] Figure 7 The figure shows the acute biotoxicity evaluation results of the mediator-type integrated microbial electrode of Example 1 on a simulated seawater solution containing chlorpyrifos.

[0058] Figure 8 The mediator-type integrated microbial electrode of Example 1 is shown to contain Cu 2+ Simulated seawater solution containing Cu 2+ Figure 1 shows the acute biotoxicity test results of the actual seawater solution of Qianchatong. Detailed Implementation

[0059] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0060] The simulated seawater solutions used in the following examples and experiments were prepared by dissolving sea salt in ultrapure water, with a concentration of 35 g / L.

[0061] Example 1

[0062] A method for preparing a mediator-type integrated microbial electrode for marine biotoxicity detection includes the following steps:

[0063] 1) Preparation of culture medium

[0064] Weigh 30.0g of tryptic soy peptone liquid culture medium powder and dissolve it in 1L of ultrapure water. Then dispense the solution into glass conical flasks and sterilize them in an autoclave at 120℃ for 15 minutes. After natural cooling, it is ready for use.

[0065] 2) Inoculation and culture of Staphylococcus aureus

[0066] Using a sterilized inoculation loop, pick up a small amount of Staphylococcus aureus and inoculate it into 100 mL of tryptic soy broth. Incubate in a 37°C water bath shaker for 24 h at a shaking speed of 180 rpm.

[0067] 3) Collection of Staphylococcus aureus bacterial suspension

[0068] The cultured Staphylococcus aureus bacterial suspension was centrifuged at 6000 rpm for 5 min and washed twice with 0.85% (w / v) sodium chloride solution. Subsequently, the obtained Staphylococcus aureus was dispersed in simulated seawater solution, and its absorbance value at 600 nm was adjusted using a UV spectrophotometer to achieve the desired OD value. 600 =2.5.

[0069] 4) Preparation of gel solution

[0070] Chitosan (CHI) was dissolved in acetic acid solution (0.1%, v / v) to prepare a chitosan solution with a concentration of 2.5 g / L. Then, 240 μL of PDDA (polydiallyl dimethyl ammonium chloride) (0.2 g / mL) was added dropwise to 20 mL of chitosan solution and stirred for 2 h to obtain PDDA-CHI gel solution.

[0071] 5) Preparation of active gel solution

[0072] A 50 mmol / L potassium ferricyanide (FC) aqueous solution was added dropwise to the PDDA-CHI gel solution, and the two were mixed thoroughly at a volume ratio of 1:4. The mixture was stirred for 12 h to obtain the active gel solution.

[0073] 6) Electrode pretreatment

[0074] The glassy carbon electrode (GCE) sheet was coarsely ground on 3500-grit sandpaper with 50 nm α-alumina particles, and then finely polished on 5000-grit sandpaper with the same alumina. Afterwards, it was ultrasonically cleaned for 10 min each with nitric acid solution (concentrated nitric acid: ultrapure water = 1:1), ethanol solution (ethanol: ultrapure water = 1:1), and ultrapure water.

[0075] 7) Preparation of PDDA-CHI-FC modified electrode

[0076] Take 20 μL of active gel solution and drop it onto the surface of the glassy carbon electrode. Let it air dry at room temperature to obtain the electrode modified by the active gel, denoted as PDDA-CHI-FC / GCE.

[0077] 8) Preparation of mediator-type integrated microbial electrode

[0078] Staphylococcus aureus bacterial suspension and active gel solution were mixed evenly at a volume ratio of 1:1. Then, 20 μL of the mixed solution was dropped onto the surface of the glassy carbon electrode and dried at room temperature to obtain the mediator-type integrated microbial electrode.

[0079] Example 2

[0080] A method for preparing a mediator-type integrated microbial electrode for marine biotoxicity detection includes the following steps:

[0081] 1) Preparation of culture medium

[0082] Dissolve tryptone (10 g / L), sodium chloride (10 g / L), and yeast extract (5 g / L) in an appropriate amount of ultrapure water in an Erlenmeyer flask. Adjust the pH of the solution to 7 with 0.1 M NaOH or HCl, then seal the flask and autoclave it in an autoclave (121 °C, 20 min). Cool the flask for later use.

[0083] 2) Inoculation and culture of Shewanella

[0084] Using a sterilized inoculation loop, pick up a small amount of Shewanella bacteria and inoculate it into 100 mL of culture medium. Incubate in a 30°C water bath shaker for 24 h at a shaking speed of 150 rpm.

[0085] 3) Collection of Shewanella bacterial suspension

[0086] The cultured Shewanella bacterial suspension was centrifuged at 6000 rpm for 5 min and washed twice with 0.85% (w / v) sodium chloride solution. Subsequently, the obtained Shewanella was dispersed in simulated seawater solution, and its absorbance at 600 nm was adjusted using a UV spectrophotometer to achieve the desired OD value. 600 =2.0.

[0087] 4) Preparation of gel solution

[0088] Chitosan (CHI) was weighed and dissolved in acetic acid solution (0.1%, v / v) to prepare a chitosan solution with a concentration of 3 g / L. Then, 200 μL of PDDA (0.2 g / mL) was added dropwise to 20 mL of chitosan solution and stirred for 2 h to obtain PDDA-CHI gel solution.

[0089] 5) Preparation of active gel solution

[0090] A 50 mmol / L potassium ferricyanide (FC) solution was added dropwise to the PDDA-CHI gel solution, and the two were mixed thoroughly at a volume ratio of 1:4. The mixture was stirred for 12 h to obtain the active gel solution.

[0091] 6) Electrode pretreatment

[0092] The glassy carbon electrode (GCE) sheet was coarsely ground on 3500-grit sandpaper with 50 nm α-alumina particles, and then finely polished on 5000-grit sandpaper with the same alumina. Afterwards, it was ultrasonically cleaned for 10 min each with nitric acid solution (concentrated nitric acid: ultrapure water = 1:1), ethanol solution (ethanol: ultrapure water = 1:1), and ultrapure water.

[0093] 7) Preparation of PDDA-CHI-FC modified electrode

[0094] Take 20 μL of active gel solution and drop it onto the surface of the glassy carbon electrode. Let it air dry at room temperature to obtain the electrode modified by the active gel, denoted as PDDA-CHI-FC / GCE.

[0095] 8) Preparation of mediator-type integrated microbial electrode

[0096] Shewanella bacterial suspension and active gel solution were mixed evenly at a volume ratio of 1:1. Then, 20 μL of the mixed solution was dropped onto the surface of the glassy carbon electrode and dried at room temperature to obtain the mediator-type integrated microbial electrode.

[0097] Example 3

[0098] A method for preparing a mediator-type integrated microbial electrode for marine biotoxicity detection includes the following steps:

[0099] 1) Preparation of culture medium

[0100] Add appropriate amounts of ultrapure water to dissolve peptone (10 g / L), beef extract (10 g / L), sodium acetate (5 g / L), yeast extract (5 g / L), diammonium citrate (2 g / L), dipotassium hydrogen phosphate (2 g / L), glucose (20 g / L), Tween-80 (1 mL), MgSO4·7H2O (0.5 g / L), and MnSO4·4H2O (0.25 g / L) in an Erlenmeyer flask. Adjust the solution to pH 6.2, seal it, and autoclave it in a high-pressure steam sterilizer (121 °C, 20 min). Then cool it for later use.

[0101] 2) Inoculation and culture of salt-tolerant lactic acid bacteria

[0102] Use a sterilized inoculation loop to pick up a small amount of salt-tolerant lactic acid bacteria and inoculate it into 100 mL of culture medium. Incubate in a 37°C water bath shaker for 24 h at a shaking speed of 180 rpm.

[0103] 3) Collection of salt-tolerant lactic acid bacteria culture

[0104] The cultured salt-tolerant lactic acid bacteria solution was centrifuged at 6000 rpm for 5 min and washed twice with 0.85% (w / v) sodium chloride solution. Subsequently, the obtained salt-tolerant lactic acid bacteria were dispersed in simulated seawater solution, and their absorbance at 600 nm was adjusted using a UV spectrophotometer to achieve the desired OD value. 600 =2.0.

[0105] 4) Preparation of gel solution

[0106] Chitosan (CHI) was weighed and dissolved in acetic acid solution (0.1%, v / v) to prepare a chitosan solution with a concentration of 2 g / L. Then, 270 μL of PDDA (0.15 g / mL) was added dropwise to 20 mL of chitosan solution and stirred for 2 h to obtain PDDA-CHI gel solution.

[0107] 5) Preparation of active gel solution

[0108] A 50 mmol / L potassium ferricyanide (FC) solution was added dropwise to the PDDA-CHI gel solution, and the two were mixed thoroughly at a volume ratio of 1:4. The mixture was stirred for 12 h to obtain the active gel solution.

[0109] 6) Electrode pretreatment

[0110] The glassy carbon electrode (GCE) sheet was coarsely ground on 3500-grit sandpaper with 50 nm α-alumina particles, and then finely polished on 5000-grit sandpaper with the same alumina. Afterwards, it was ultrasonically cleaned for 10 min each with nitric acid solution (concentrated nitric acid: ultrapure water = 1:1), ethanol solution (ethanol: ultrapure water = 1:1), and ultrapure water.

[0111] 7) Preparation of PDDA-CHI-FC modified electrode

[0112] Take 20 μL of active gel solution and drop it onto the surface of the glassy carbon electrode. Let it air dry at room temperature to obtain the electrode modified by the active gel, denoted as PDDA-CHI-FC / GCE.

[0113] 8) Preparation of mediator-type integrated microbial electrode

[0114] Salt-tolerant lactic acid bacteria solution and active gel solution are mixed evenly at a volume ratio of 1:1. Then, 20 μL of the mixed solution is dropped onto the surface of the glassy carbon electrode and dried at room temperature to obtain the mediator-type integrated microbial electrode.

[0115] Comparative Example 1

[0116] Same as Example 1, except that PDDA solution is not added in step 4) of preparing the gel solution. The electrode modified with the active gel is designated as CHI-FC / GCE.

[0117] Comparative Example 2

[0118] Same as Example 1, except that chitosan solution is not added in step 4) of preparing the gel solution. The electrode modified with the active gel is designated as PDDA-FC / GCE.

[0119] Comparative Example 3

[0120] Preparation of PDDA-gelatin-FC modified electrode:

[0121] Gelatin was dissolved in acetic acid solution (0.1%, v / v) and stirred at 37℃ for 30 min to ensure complete dissolution, preparing a 2.5 g / L gelatin solution. Then, 240 μL of PDDA (0.2 g / mL) was added dropwise to 20 mL of the gelatin solution, and the mixture was stirred for 2 h to obtain a PDDA-gelatin gel solution. A 50 mmol / L potassium ferricyanide (FC) solution was added dropwise to the PDDA-gelatin gel solution, and the two were mixed thoroughly at a volume ratio of 1:4. The mixture was stirred for 12 h to obtain an active gel solution. 20 μL of the active gel solution was dropped onto the surface of a cleaned and ultrasonically cleaned glassy carbon electrode and air-dried at room temperature to obtain PDDA-gelatin-FC / GCE.

[0122] Comparative Example 4

[0123] Preparation of CHI-silane coupling agent-FC modified electrode

[0124] Chitosan (CHI) was dissolved in acetic acid solution (0.1%, v / v) to prepare a 2.5 g / L chitosan solution. Then, 240 μL of 3-aminopropyltrimethoxysilane (0.2 g / mL) was added dropwise to 20 mL of gelatin solution, and the mixture was stirred at 50 °C for 6 h to obtain a CHI-silane coupling agent gel solution. A 50 mmol / L potassium ferricyanide (FC) solution was added dropwise to the CHI-silicone gel solution, and the two were mixed thoroughly at a volume ratio of 1:4. The mixture was stirred for 12 h to obtain an active gel solution. 20 μL of the active gel solution was dropped onto the surface of a cleaned and ultrasonically cleaned glassy carbon electrode and air-dried at room temperature to obtain a CHI-silane coupling agent-FC / GCE.

[0125] Experimental Example 1

[0126] Cyclic voltammetry curves of the electrodes obtained by PDDA-CHI-FC / GCE, CHI-FC / GCE (Comparative Example 1), PDDA-FC / GCE (Comparative Example 2), and PDDA-gelatin-FC / GCE (Comparative Example 3) were tested in PBS buffer solution (pH = 7.0, 0.1 mol / L) at a scan rate of 50 mV / s. The results are as follows. Figure 1 As shown.

[0127] Depend on Figure 1 It can be seen that: 1) All four electrodes exhibit a pair of redox peaks between 0.1 and 0.4 V, which are the reaction peaks of potassium ferricyanide and potassium ferrocyanide, indicating that CHI, PDDA, PDDA-CHI, and PDDA-gelatin can all fix potassium ferricyanide on the electrode surface without affecting its redox reaction. 2) The potassium ferricyanide / potassium ferrocyanide redox peaks of PDDA-FC / GCE are not as significant as those of CHI-FC / GCE, and the redox peak currents are smaller. This is because PDDA has a benzene ring in its structure, resulting in high molecular chain rigidity and poor permeability to potassium ferricyanide. 3) Compared to PDDA-FC / GCE, CHI-FC / GCE, and PDDA-gelatin / GCE, the PDDA-CHI-FC / GCE electrode has the largest peak current value. This result indicates that the active gel prepared by combining PDDA and CHI has superior electrochemical performance.

[0128] Cyclic voltammetry results of PDDA-CHI-FC / GCE in PBS buffer solution (pH = 7.0, 0.1 mol / L) for 30 consecutive scans showed that ( Figure 2 During the initial scanning process, the peak current shows a slight decrease, but gradually stabilizes in the later stages of the scan. This is mainly because some unadsorbed potassium ferricyanide exists on the electrode surface, which initially enters the solution from the electrode surface during the initial scan. However, as the unadsorbed potassium ferricyanide gradually enters the solution, the current gradually stabilizes. Therefore, potassium ferricyanide can be stably immobilized using PDDA-CHI gel through electrostatic interaction.

[0129] Figure 3 The cyclic voltammetry curves of the electrode obtained in Comparative Example 4 were obtained by continuous scanning in PBS buffer solution (pH = 7.0, 0.1 mol / L) for 30 cycles. As shown in the figure, with the increase of the number of scan cycles, the oxidation peak current and reduction peak current of potassium ferricyanide / potassium ferrocyanide decreased significantly, indicating that the CHI-silane coupling agent gel could not effectively coat the potassium ferricyanide electron mediator and could not obtain a stable electrode film structure.

[0130] Experimental Example 2

[0131] Example 1: Mediator-type integrated microbial electrode for responsive assessment of heavy metal ion biotoxicity in seawater

[0132] 1. Preparation of microbial respiration substrate solution

[0133] The microbial respiration substrate solution was prepared by dissolving glucose, sodium succinate, and sodium lactate in a simulated seawater solution, wherein the concentrations of glucose, sodium succinate, and sodium lactate were all 10 mmol / L.

[0134] 2. Preparation of heavy metal ion test solution

[0135] Preparation of Cu-containing solutions using simulated seawater solutions 2+ Seawater contaminated solution.

[0136] 3. Evaluation Methods

[0137] Acute biotoxicity testing was performed using a chronoamperometry method (0.5V vs Ag / AgCl) on an electrochemical workstation. The prepared heavy metal ion test solution and 7 mL of microbial respiration matrix solution were added to the reaction chamber, resulting in a Cu content in the chamber. 2+ A 0.1 mg / L solution was used, connected to the working electrode, counter electrode (platinum electrode), and reference electrode (Ag / AgCl electrode). The current change over time was recorded under continuous and stable stirring. After the current stabilized, a heavy metal ion test solution was added to the reaction cell, and the current change was observed.

[0138] Depend on Figure 4 It can be seen that the product contains 0.1 mg / L Cu 2+ When a seawater contaminated solution was added to the test solution (the time indicated by the arrow is the time of sample addition), the current dropped almost immediately. This phenomenon indicates that the mediator-type integrated microbial electrode is effective against Cu-containing solutions. 2+ The simulated seawater solution can respond quickly.

[0139] Furthermore, compared to the invention patent with publication number CN 104007154 A, the organic-inorganic composite gel obtained by using aminosilane coupling agent and chitosan / gelatin has a longer activation time. The current still shows a downward trend 600s after the start of the test, and it is difficult to reach a stable state. This may be due to the poor coating ability of the membrane for electron mediators or the swelling of the membrane itself in the solution.

[0140] The integrated microbial electrode of this invention exhibits a significantly shorter activation time, with the current stabilizing in approximately 300 seconds, indicating a more stable membrane structure. This stable initial current facilitates the determination of sample addition time for toxicity testing and provides more reliable quantitative results for inhibition rate calculations.

[0141] Experimental Example 3

[0142] Example 1: Mediator-type integrated microbial electrode for the treatment of heavy metal ions Cu in seawater 2+ Biotoxicity assessment

[0143] 1. Preparation of microbial respiration substrate solution

[0144] The microbial respiration substrate solution was prepared by dissolving glucose, sodium succinate, and sodium lactate in a simulated seawater solution, wherein the concentrations of glucose, sodium succinate, and sodium lactate were all 10 mmol / L.

[0145] 2. Preparation of heavy metal ion test solution

[0146] Prepared solutions containing different concentrations of Cu using simulated seawater solutions. 2+ Seawater contaminated solution.

[0147] 3. Evaluation Methods

[0148] Acute biotoxicity testing was performed using a chronoamperometry method (0.5V vs Ag / AgCl) on an electrochemical workstation. The prepared heavy metal ion test solution and 7 mL of microbial respiration matrix solution were added to the reaction chamber, resulting in a Cu content in the chamber. 2+ The concentrations were 1 mg / L, 3 mg / L, 5 mg / L, 8 mg / L, and 10 mg / L. The working electrode, counter electrode (platinum electrode), and reference electrode (Ag / AgCl electrode) were connected, and the current change over time was recorded under continuous and stable stirring. After the current stabilized, the test solution was added to the reaction cell. The formula for calculating the biotoxicity of the test sample is as follows:

[0149] Inhibition rate (%) = (1 - i1 / i0) × 100%

[0150] Where i1 is the stable current after the sample is added, and i0 is the stable current before the sample is added.

[0151] 4. Analysis and Conclusion

[0152] With Cu 2+ The data was fitted with concentration on the x-axis and inhibition rate on the y-axis (e.g., concentration on the x-axis and inhibition rate on the y-axis). Figure 5 As shown), the formula for the fitted line is y = 4.040x + 37.845, and the goodness-of-fit parameter R0 is... 2 The half-inhibition concentration (IC50) was 0.9931. 50 The value was 3.01 mg / L. This result indicates that the mediator-type integrated microbial electrode prepared in Example 1 can detect Cu. 2+ The sensitivity is high in seawater solutions, Cu 2+ The linear relationship between concentration and inhibition rate is good, and the detection results have high reliability.

[0153] Test Example 4

[0154] Example 1: Mediator-type integrated microbial electrode for the treatment of heavy metal ions Zn in seawater 2+ Biotoxicity assessment

[0155] Similar to Experiment 3, the only difference is that in step 2), the preparation of the heavy metal ion test solution involves using simulated seawater to prepare solutions containing different concentrations of Zn. 2+ Step 3) Add the prepared heavy metal ion test solution and 7 mL of microbial respiration matrix solution to the reaction tank, respectively, so that the reaction tank contains Zn. 2+ :0.1mg / L, 1mg / L, 2mg / L, 3mg / L, 5mg / L.

[0156] Analysis and Conclusion

[0157] With Zn 2+ The data was fitted with concentration on the x-axis and inhibition rate on the y-axis (e.g., concentration on the x-axis and inhibition rate on the y-axis). Figure 6 As shown in the figure, the formula for the fitted line is y = 6.385x + 41.472, and the goodness-of-fit parameter R0 is... 2 The half-inhibition concentration (IC50) was 0.9891. 50 The value was 1.34 mg / L. This result indicates that the mediator-type integrated microbial electrode prepared in Example 1 can detect Zn content. 2+ Zn has high sensitivity in seawater solutions. 2+ The linear relationship between concentration and inhibition rate is good, and the detection results have high reliability.

[0158] Experimental Example 5

[0159] Example 1: Mediator-type integrated microbial electrode for assessing the biotoxicity of pesticides in seawater.

[0160] 1. Preparation of microbial respiration substrate solution

[0161] The microbial respiration substrate solution was prepared by dissolving glucose, sodium succinate, and sodium lactate in a simulated seawater solution, wherein the concentrations of glucose, sodium succinate, and sodium lactate were all 10 mmol / L.

[0162] 2. Preparation of pesticide test solutions

[0163] The commercially available chlorpyrifos was diluted with a simulated seawater solution according to the dilution ratio required in actual use, and this concentration was recorded as 1. At the same time, to simulate the pesticide's condition in the actual environment, the pesticide concentration was also diluted by 10 times, 100 times, 500 times, and 1000 times.

[0164] 3. Evaluation Methods

[0165] Acute biotoxicity testing was performed using a chronoamperometry method (0.5V vs Ag / AgCl) on an electrochemical workstation. The prepared pesticide test solution and 7 mL of microbial respiration matrix solution were added to the reaction cell. The working electrode, counter electrode, and reference electrode were connected, and the current change over time was recorded under continuous and stable stirring. After the current stabilized, the test solution was added to the reaction cell. The formula for calculating the biotoxicity of the test sample is as follows:

[0166] Inhibition rate (%) = (1 - i1 / i0) × 100%

[0167] Where i1 is the stable current after the sample is added, and i0 is the stable current before the sample is added.

[0168] Depend on Figure 7 It is known that pesticide concentration is positively correlated with inhibition rate, and the biotoxicity of pesticides can still be detected after being diluted 1000 times, indicating that the mediator-type integrated microbial electrode of the present invention has good applicability to the detection of biotoxicity of pesticides in seawater.

[0169] Experimental Example 6

[0170] Example 1 describes a mediator-type integrated microbial electrode used for assessing the biotoxicity of actually polluted seawater.

[0171] 1. Preparation of microbial respiration substrate solution

[0172] The microbial respiration substrate solution was prepared by dissolving glucose, sodium succinate, and sodium lactate in a simulated seawater solution, wherein the concentrations of glucose, sodium succinate, and sodium lactate were all 10 mmol / L.

[0173] 2. Preparation of polluted seawater solution

[0174] Prepare the following test solutions respectively: Prepare Cu-containing solutions using simulated seawater solution. 2+ Simulated heavy metal contaminated seawater solution; preparation of pesticide-contaminated seawater solution containing chlorpyrifos using simulated seawater solution; preparation of Cu-containing seawater solution using actual seawater samples. 2+ The actual seawater solution polluted by heavy metals; the actual seawater solution containing chlorpyrifos was prepared using actual seawater samples to treat the actual seawater pollution caused by pesticides.

[0175] 3. Evaluation Methods

[0176] Acute biotoxicity testing was conducted using a chronoamperometry method (0.5V vs Ag / AgCl) on an electrochemical workstation. The prepared contaminated seawater solution and 7 mL of microbial respiration substrate solution were added to the reaction tank, respectively, so that Cu in the reaction tank... 2+For a concentration of 1 mg / L, or for a solution with a concentration of 1 mg / L, connect the working electrode, counter electrode, and reference electrode, and record the change in current over time under continuous and stable stirring. After the current stabilizes, add the test solution to the reaction cell. The formula for calculating the biotoxicity of the test sample is as follows:

[0177] Inhibition rate (%) = (1 - i1 / i0) × 100%

[0178] Where i1 is the stable current after the sample is added, and i0 is the stable current before the sample is added.

[0179] Depend on Figure 8 It can be seen that the inhibition rates of the actual seawater samples with the two types of pollution were 39.5% (heavy metals) and 45.7% (pesticides), respectively. Compared with the detection results of the samples prepared with simulated seawater solutions (heavy metals: 41.5%, pesticides: 48.1%), the relative error between the detection data is small. Therefore, the mediator-type integrated microbial electrode of the present invention has good applicability to complex actual seawater systems.

[0180] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a mediator-type integrated microbial electrode for detecting the toxicity of marine organisms, characterized in that, The electrode includes an electrode substrate and a microbial membrane uniformly attached to the electrode substrate; The microbial membrane is a thin film obtained by fixing a uniformly mixed electron mediator and microorganisms onto an electrode substrate through a gel solution and then drying it. The gel solution is formed by mixing a polydiallyldimethylammonium chloride solution and a chitosan solution; The preparation method includes the following steps: The polydiallyldimethylammonium chloride solution was mixed with the chitosan solution to form a gel solution; Add an electron mediator solution to the gel solution, mix well, and obtain an active gel solution; The active gel solution is mixed with the microbial culture and then dropped onto the electrode surface and dried to obtain the mediator-type integrated microbial electrode.

2. The preparation method according to claim 1, characterized in that, The electrode substrate is selected from one of glassy carbon electrode, gold electrode, platinum electrode or screen-printed electrode.

3. The preparation method according to claim 1, characterized in that, The electron mediator is selected from one of thionine, potassium ferricyanide, benzoquinone, and menaquinone.

4. The preparation method according to claim 1, characterized in that, The microorganisms are selected from salt-tolerant bacteria or marine bacteria.

5. The preparation method according to claim 1, characterized in that, The mass ratio of polydiallyldimethylammonium chloride in the polydiallyldimethylammonium chloride solution to chitosan in the chitosan solution is 1:0.5 to 1:

2.

6. The preparation method according to claim 1, characterized in that, The concentration of the polydiallyldimethylammonium chloride solution is 0.1-0.3 g / mL; the concentration of the chitosan solution is 1.5-3 g / L; and the volume ratio of the chitosan solution to the polydiallyldimethylammonium chloride solution is 15-25:0.

24.

7. The preparation method according to claim 1, characterized in that, The concentration of the electron mediator solution is 40-60 mmol / L; the volume ratio of the gel solution to the electron mediator solution is 3-5:

1.

8. The preparation method according to claim 1, characterized in that, The bacterial population density of the microbial culture is 1.0 ≤ OD. 600 Bacterial solution ≤ 3.

0.

9. The preparation method according to claim 1, characterized in that, The volume ratio of the active gel solution to the microbial culture is 1:

1.

10. The application of the integrated microbial electrode prepared by the preparation method according to any one of claims 1-9 in the field of marine biological toxicity detection.

11. The application according to claim 10, characterized in that, The application includes the following steps: The biotoxicity of seawater samples was detected using the aforementioned mediator-type integrated microbial electrode as the working electrode of a three-electrode system.

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