Method for detecting trace elements in aquatic products

Abstract

The application discloses a kind of detection methods of trace elements of aquatic products, belongs to the technical field of trace elements detection of aquatic products;For the euryhaline fish of southeast coastal brackish water pond culture, through sample collection, pretreatment, biosensor detection and data processing steps, the detection of selenium element is realized.Pretreatment uses Tris-HCl buffer containing thiourea, Triton X-100 to treat sample, and the sample is obtained by homogenization and centrifugation;The biosensor of specific modified electrode is used in the detection process, the sensor electrode is used as the base by platinum-iridium alloy electrode, and is modified by polyethylene glycol layer, selenium cysteine lyase layer activated by glutaraldehyde, nano titanium dioxide coating and polytetrafluoroethylene microporous membrane in turn, and the electric signal is recorded by combining portable electrochemical workstation;The result is obtained by comparing with the standard curve established in advance in data processing.The application can complete detection in about 30 minutes, and meet the needs of on-site detection in coastal aquaculture area.

Description

Technical Field

[0001] This invention belongs to the field of trace element detection technology for aquatic products, specifically a method for detecting trace elements in aquatic products. Background Technology

[0002] Euryhaline fish species, such as tilapia, sea bass, and mullet, raised in brackish water ponds along my country's southeastern coast, are affected by environmental factors including the periodic exchange of fresh and salt water, frequent red tides, significant tidal action, and high humidity. Consequently, selenium in their muscle tissue often forms complex complexes with marine-derived humus and algal toxins. Selenium, an essential antioxidant trace element for the human body, is a crucial indicator for evaluating the nutritional value of aquatic products. The selenium content in these euryhaline fish directly impacts food safety supervision, aquaculture environment assessment, and product market competitiveness. However, in brackish water environments along the coast, sulfides released from pond sediments readily react with selenium in redox reactions, severely interfering with detection results. Current technology for selenium detection uses the 2,3-diaminonaphthalene fluorescence method, which requires a complex digestion process: fish muscle tissue is first digested at high temperatures with a nitric acid-perchloric acid mixture to release selenium from the complex, followed by reduction treatment with hydrochloric acid before fluorescence measurement. This digestion process is not only cumbersome, requiring laboratory conditions and taking over 3 hours, making it unsuitable for on-site testing in coastal aquaculture areas lacking large-scale equipment, but also exposes the selenium to the reaction of sulfides in the brackish water, coupled with the high humidity causing deliquescence of the fluorescent reagent, increasing detection errors and severely impacting accuracy. The aforementioned problems mean that existing methods cannot achieve rapid detection, nor can they guarantee the accuracy of the detection results, making it difficult to meet the need for rapid and accurate detection of selenium in euryhaline fish farmed in brackish water ponds along the southeast coast. Summary of the Invention

[0003] The purpose of this invention is to provide a method for detecting trace elements in aquatic products, in order to solve the problems of existing detection methods, such as complex digestion, large interference from specific coastal environments, long detection cycles, and low detection accuracy, which cannot meet the needs of rapid and accurate on-site detection.

[0004] The objective of this invention is achieved through the following technical solution: A method for detecting trace elements in aquatic products, comprising the following steps: S1. Sample collection: Fresh euryhaline fish from brackish water ponds in the southeast coastal area were selected. The scales, internal organs and bones were removed and the muscle tissue from the back, abdomen and lateral line were collected and mixed as test samples. S2. Sample pretreatment: Cut the tissue sample into small pieces, put them into a tissue homogenizer, add Tris-HCl buffer containing thiourea and Triton X-100 with a pH of 7.0-7.5, mix and centrifuge, and take the supernatant as the test solution; S3. Detection process: The test solution is dropped onto the biosensor to ensure full contact between the test solution and the surface of the sensor electrode; at the same time, a portable electrochemical workstation is connected to record the changes in the electrical signal generated by the biosensor. The steps for fabricating the sensor electrodes are as follows: First, a layer of polyethylene glycol was modified on the surface of the platinum-iridium alloy electrode. Then, the polyethylene glycol layer was preactivated with glutaraldehyde. Next, selenocysteine ​​lyase was immobilized. Then, a nano-titanium dioxide coating was coated on the enzyme layer. Finally, a polytetrafluoroethylene microporous membrane was coated on the coating surface. S4. Data Processing: Using a portable data processing terminal, the electrical signal generated by the sensor is compared with the pre-established standard curve of selenium element in euryhaline fish in the southeastern coastal area to calculate the selenium content in the test solution.

[0005] The present invention discloses a method for detecting trace elements in aquatic products, which is specifically designed for euryhaline fish raised in brackish water ponds along the southeast coast. Through specific sample collection, pretreatment, biosensor detection, and data processing steps, the method enables the detection of selenium.

[0006] Samples were collected from a mixture of dorsal, ventral, and lateral line muscle tissues of fresh euryhaline fish. Pretreatment involved treating the samples with a Tris-HCl buffer solution containing thiourea and Triton X-100, followed by homogenization and centrifugation to obtain the test solution. The detection process employed a biosensor with a specific structure. Its electrode consisted of a platinum-iridium alloy electrode sequentially modified with a polyethylene glycol layer, a selenocysteine ​​lyase layer activated and immobilized with glutaraldehyde, a nano-titanium dioxide coating, and a polytetrafluoroethylene microporous membrane. Electrical signals were recorded using a portable electrochemical workstation. Data processing involved comparing the results with a pre-established standard curve.

[0007] The specific testing principle is as follows: Organically bound selenium in the test solution is specifically recognized and catalyzed by selenocysteine ​​lyase after passing through a polytetrafluoroethylene microporous membrane and a nano-titanium dioxide coating, generating an electroactive selenium-containing small molecule. This substance undergoes a reduction reaction on the surface of a platinum-iridium alloy inert electrode, generating a current signal positively correlated with the selenium concentration. Free inorganic selenium in the sample can directly generate a response on the electrode surface. The above electrical signal is positively correlated with the total selenium concentration and can quantitatively reflect the selenium content in the sample.

[0008] Furthermore, the functions of each component in the buffer solution are as follows: Thiourea: It can form a weak adsorption layer on the electrode surface, inhibiting the oxidation reaction of sulfur ions on the electrode surface, eliminating the interference current generated by sulfides, and simultaneously complexing Cu. 2+ Pb 2+ It contains heavy metal ions, which significantly improves the accuracy of selenium detection.

[0009] Triton X-100: As a nonionic surfactant, it can disrupt the colloidal structure of humic substances, causing humic substances to separate from selenium, releasing free selenium, and ensuring that the detected value reflects the true content. Tris-HCl buffer: Maintains the test solution at a suitable pH, ensures the activity of selenocysteine ​​lyase, adapts to possible pH fluctuations in coastal environments, and provides a stable chemical environment for detection. The functions of each component of the electrode are as follows: Platinum-iridium alloy electrodes: They exhibit excellent corrosion resistance and conductivity in coastal high-salt-spray environments, providing a stable carrier for electrical signal transmission and preventing electrode corrosion from affecting detection. Polyethylene glycol: forms steric hindrance on the electrode surface, reduces the adsorption of algal toxins and proteins on the electrode surface, and reduces background interference.

[0010] Immobilized selenocysteine ​​lyase after activation with glutaraldehyde: The surface activity of polyethylene glycol layer is low, and the binding is not strong when the enzyme is directly immobilized, and the enzyme is easy to fall off, which affects the detection sensitivity and stability. Immobilized selenocysteine ​​lyase after activation with glutaraldehyde: Glutaraldehyde, as a bifunctional cross-linking agent, activates the terminal hydroxyl groups of polyethylene glycol, and then forms a stable covalent bond with the amino group of the enzyme molecule, which enhances the immobilization strength and stability of the enzyme in the high temperature environment of the coastal area.

[0011] Nano-titanium dioxide coating: Located on the inner side of the polytetrafluoroethylene microporous membrane, it provides a smooth channel for the transfer of selenium through the porous nanostructure (not gradient filtration), while stabilizing the enzyme layer, enhancing interfacial conductivity, supporting the outer membrane structure, preventing membrane collapse, and improving the stability and service life of the electrode in the high humidity and eutrophic environment of the coast.

[0012] Polytetrafluoroethylene microporous membrane: As the outermost breathable and hydrophobic membrane, it prevents water vapor from condensing inside the electrode in high humidity environments, ensuring the stability of the detection system; its microporous structure allows selenium to pass through efficiently, while intercepting large molecular impurities such as proteins, lipids, and colloids on the outermost side of the electrode, avoiding internal pore blockage and enzyme layer contamination.

[0013] The electrode components of this invention are designed for actual coastal environments such as high salt spray, high humidity, eutrophication, and frequent red tides. Through the synergistic effects of corrosion resistance, conductivity, impurity blocking, enzyme stabilization for selenium identification, and hydrophobicity and breathability, they form a rapid detection system suitable for coastal sites, ensuring accurate and stable selenium detection even in complex environments.

[0014] As one possible implementation method of this application, before modifying polyethylene glycol, the surface of the platinum-iridium alloy electrode is first roughened by sandblasting, and then the roughened electrode surface is activated by silane coupling agent.

[0015] The adhesion between the platinum-iridium alloy electrode and the polyethylene glycol (PEG) layer is relatively weak. During frequent sample addition and rinsing, the PEG layer is prone to detachment, leading to weakened steric hindrance and an increased probability of interfering substances such as algal toxins binding to the enzyme, thus increasing detection errors. Therefore, before modifying the PEG, this invention first roughens the surface of the platinum-iridium alloy electrode by sandblasting to increase its roughness. Then, a silane coupling agent is used to activate the roughened electrode surface, allowing the PEG to bind to the electrode surface through covalent bonds. This significantly improves the adhesion of the PEG layer and reduces detachment.

[0016] As some possible embodiments of this application, the nano-titanium dioxide coating has a thickness of 50–200 nm and a pore size of 150–300 nm.

[0017] As one possible implementation of this application, nano-silicon particles are incorporated during the preparation of the titanium dioxide coating.

[0018] Simple nano-titanium dioxide coatings are prone to cracking and peeling during long-term use. Adding nano-silicon particles can enhance the toughness and adhesion of the coating, making it more resistant to wear from frequent inspections.

[0019] As one possible implementation of this application, the surface of the polytetrafluoroethylene microporous membrane is provided with hydrophilic groups.

[0020] Untreated polytetrafluoroethylene microporous membranes have a hydrophobic surface, which easily adsorbs macromolecules in the test solution, clogging the micropores and causing a decrease in the selenium element throughput, thus affecting the detection signal. Introducing hydrophilic groups can effectively solve this problem.

[0021] As some possible embodiments of this application, the pore size of the polytetrafluoroethylene microporous membrane is 80–150 nm.

[0022] As some possible implementations of this application, in step S3, the change in electrical signal generated by the biosensor is recorded using cyclic voltammetry, with the peak current as the characteristic signal value; the magnitude of the peak current is positively correlated with the selenium content; the scanning speed is 60-80mV / s, and the detection potential range is -0.5~1.1V.

[0023] As some possible implementation methods of this application, in step S4, when establishing the standard curve, samples of euryhaline fish cultured in brackish water ponds with different salinity and red tide cycles in the southeast coastal area are selected. At least 30 samples are collected under each condition, and 3 parallel samples are set for each sample. The selenium content is detected by the aforementioned method for detecting trace elements in aquatic products and by inductively coupled plasma mass spectrometry. After removing outliers using the Grubbs test, the detection results of inductively coupled plasma mass spectrometry are used as the abscissa, and the electrical signal value in step S3 is used as the ordinate. A univariate linear regression method is used to plot the standard curve and determine the curve equation.

[0024] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention does not require a complicated digestion process and can complete the detection in about 30 minutes (about 10 minutes for homogenization + about 15 minutes for centrifugation + about 5 minutes for detection), which is suitable for the on-site detection needs of coastal aquaculture areas and solves the problems of existing technologies that rely on laboratory digestion and have long detection cycles. 2. The detection method of this invention, through the synergistic effect of multiple components in the buffer solution and electrode, effectively resists interference from sulfides, algal toxins, high humidity, and high salt spray in coastal waters containing sulfides, thereby improving detection accuracy and stability (after 10 consecutive tests, the RSD of the detection value fluctuation is ≤3.5%), overcoming the shortcomings of large detection errors in existing technologies. 3. The detection equipment of this invention is portable and easy to operate, which reduces the detection threshold and cost, and is conducive to its promotion in aquaculture production areas. It solves the problems of existing technologies that rely on large equipment and are complicated to operate. Detailed Implementation

[0025] Example 1 S1. Sample Collection: Fresh tilapia cultured in a brackish water pond in Shanwei, Guangdong Province, were selected. The pond had a salinity of 5 wt‰, a sulfide content of 0.03 mg / L, a humic substance content of 1.0 mg / L, and had not experienced red tides in the past 20 days. The relative humidity was 78%, and the temperature was 30℃. Scales, viscera, and bones were removed, and 5g each of muscle tissue from the back, abdomen, and lateral line were collected and mixed thoroughly as test samples.

[0026] S2. Sample Pretreatment: Cut the test sample into 1cm square pieces and place them in a tissue homogenizer. Add 20 mL of 50 mM Tris-HCl buffer (pH=7.2) containing 0.2wt% thiourea and 0.03wt% Triton X-100. The ratio of test sample mass (g): buffer volume (mL) is 1:4. Homogenize for 10 min to obtain a homogenate. Pour the homogenate into a centrifuge tube and centrifuge at 8000 rpm for 15 min. Use the supernatant as the test solution. S3. Detection process: Add the test droplet to the above biosensor, connect it to a portable electrochemical workstation, and use cyclic voltammetry (CV) for detection. Set the scan rate to 70mV / s and the detection potential range to -0.5V~1.1V. Record the peak current of the reduction peak as the characteristic signal. The method for fabricating the sensor electrodes is as follows: S31. Take a platinum-iridium alloy electrode (5 mm in diameter), ultrasonically clean it with anhydrous ethanol for 5 min, and then air dry it. S32. Take a 5 wt% polyethylene glycol solution (molecular weight 2000), add 10 μL to the electrode surface using a micropipette, and let it stand for 30 min at 25±2℃ and normal pressure to form a polyethylene glycol modified layer. S33. First, immerse the electrode in 0.1wt% glutaraldehyde aqueous solution for 15 min to activate it. After removing it, rinse it three times with deionized water to remove unreacted glutaraldehyde. Then, add 8 μL of selenocysteine ​​lyase solution (1 mg / mL, wherein the selenocysteine ​​lyase is a recombinant expression of E. coli, commercially available, with an enzyme activity ≥50 U / mg), and incubate at 37℃ for 2 h to form an enzyme layer. Afterward, wash the electrode three times with PBS buffer for 1 min each time to remove unfixed enzymes and residual reagents. S34. Tetrabutyl titanate, ethanol, and water were mixed in a volume ratio of 3:10:1, and the pH was adjusted to 2.0. The mixture was then magnetically stirred at 500 rpm for 30 min to obtain a sol. 5 μL of the sol was then dropped onto the surface of the enzyme layer, air-dried at room temperature, and cured in a 45°C oven for 30 min to form a coating with a thickness of 200 nm and a pore size of 150–180 nm. S35. Cut a polytetrafluoroethylene microporous membrane with a pore size of 100 nm, wet it with deionized water, cover it on the surface of the titanium dioxide coating, and seal the edges with epoxy glue to fix it, thus obtaining the sensor electrode.

[0027] S4. Data processing: The electrical signal generated by the sensor is compared with the standard curve established according to the method of this invention using a portable data processing terminal; the linear range is 0.05–1.0 mg / kg, and the detection limit is 0.02 mg / kg; the user can establish the standard curve by following the steps in the instruction manual.

[0028] The method for establishing the standard curve is as follows: We selected brackish water ponds in different areas of the southeast coast (Guangdong and Zhejiang), covering salinity gradients of 4wt‰, 8wt‰, 12wt‰, 16wt‰, and 20wt‰, as well as three cycles: before, during, and after red tide. Under each condition, we collected 30 euryhaline fish (10 tilapia, 10 sea bass, and 10 barracuda). Each fish was treated according to steps S1-S2 of Example 1 to obtain the test solution, and the selenium content was detected by the detection method of the present invention (step S3) and inductively coupled plasma mass spectrometry (ICP-MS). The x-axis represents the ICP-MS detection values ​​( x (Unit: mg / kg). The peak value of the electrical signal in the method of this invention is used as the ordinate (y, unit: μA). Linear regression is performed to obtain the standard curve equation: y = 12.5 x + 0.32 (R²=0.985), this curve covers the detection range of 0.05-1.0 mg / kg.

[0029] Note: This standard curve was established for the muscle tissue of euryhaline fish (tilapia, sea bass, and mullet) cultured in brackish water ponds in the southeastern coastal region. When applied to other fish species or non-muscle tissues, a new standard curve needs to be established. The linear range of the method of this invention is 0.05-1.0 mg / kg. Samples below the detection limit need to be concentrated or treated with other pretreatment methods.

[0030] Example 2 S1. Sample Collection: Freshwater perch cultured in a brackish water pond in Wenzhou, Zhejiang Province, were selected. The pond had a salinity of 15 wt‰, a sulfide content of 0.05 mg / L, and a humic substance content of 1.3 mg / L. The samples were collected on the 5th day after the red tide occurred, with a relative humidity of 80% and an air temperature of 32℃. Scales, viscera, and bones were removed, and 5g each of muscle tissue from the back, abdomen, and lateral line were collected and mixed thoroughly as test samples. S2. Sample Pretreatment: Cut the test sample into 1cm square pieces and place them in a tissue homogenizer. Add 20 mL of 50 mM Tris-HCl buffer (pH=7.2) containing 0.2wt% thiourea and 0.03wt% Triton X-100. The ratio of test sample mass (g): buffer volume (mL) is 1:4. Homogenize for 10 min to obtain a homogenate. Pour the homogenate into a centrifuge tube and centrifuge at 8000 rpm for 15 min. Use the supernatant as the test solution. S3. Detection process: Add the test droplet to the above biosensor, connect it to a portable electrochemical workstation, and use cyclic voltammetry (CV) for detection. Set the scan rate to 65mV / s and the detection potential range to -0.5V to 1.1V. Record the peak current of the reduction peak as the characteristic signal.

[0031] The method for fabricating the sensor electrodes is as follows: S31. Take a platinum-iridium alloy electrode (5 mm in diameter), ultrasonically clean it with anhydrous ethanol for 5 min, and then air dry it. S32. Take a 6 wt% polyethylene glycol solution (molecular weight 2000), add 12 μL to the electrode surface using a micropipette, and let it stand for 30 min at 25±2℃ and normal pressure to form a polyethylene glycol modified layer; S33. First, immerse the electrode in 0.1wt% glutaraldehyde aqueous solution for 15 min to activate it. After removing it, rinse it three times with deionized water to remove unreacted glutaraldehyde. Then, add 8 μL of selenocysteine ​​lyase solution (1 mg / mL, wherein the selenocysteine ​​lyase is a recombinant expression of E. coli, commercially available, with an enzyme activity ≥50 U / mg). Incubate at 37℃ for 2 h to form an enzyme layer. Then, wash the electrode three times with PBS buffer for 1 min each time to remove unfixed enzymes and residual reagents. S34. Tetrabutyl titanate, ethanol, and water were mixed in a volume ratio of 3:12:1, and the pH was adjusted to 2.1. The mixture was then magnetically stirred at 500 rpm for 30 min to obtain a sol. 6 μL of the sol was then dropped onto the surface of the enzyme layer, air-dried at room temperature, and cured in a 45°C oven for 30 min to form a coating with a thickness of 200 nm and a pore size of 150–180 nm. S35. Cut a polytetrafluoroethylene microporous membrane with a pore size of 100 nm, wet it with deionized water, cover it on the surface of the titanium dioxide coating, and seal the edges with epoxy glue to fix it, thus obtaining the sensor electrode. S4. Data Processing: Using a portable data processing terminal, the electrical signal was compared with the standard curve established in Example 1, revealing that the selenium content in the fish muscle tissue was 0.31 mg / kg. ICP-MS detected the selenium content in the example to be 0.32 mg / kg.

[0032] Example 3 S1. Sample collection: Same as in Example 1.

[0033] S2. Sample pretreatment: Same as in Example 1. S3. Detection process: Same as in Example 1.

[0034] The method for fabricating the sensor electrodes is as follows: S31. Take a platinum-iridium alloy electrode (5 mm in diameter), ultrasonically clean it with anhydrous ethanol for 5 min and then air dry it; then sandblast it with 100 mesh alumina sand at 0.3 MPa pressure for 30 seconds, rinse it with deionized water, and then immerse it in 5 wt% 3-aminopropyltriethoxysilane ethanol solution for 40 min to activate it, rinse it with distilled water and then air dry it. S32-S35: Same as Example 1.

[0035] S4. Data processing: The electrical signal was compared with the standard curve established in Example 1 using a portable data processing terminal, and the selenium content was found to be 0.28 mg / kg.

[0036] Example 4 S1. Sample collection: Same as in Example 3.

[0037] S2. Sample pretreatment: Same as in Example 3. S3. Detection process: Same as in Example 3. The method for fabricating the sensor electrodes is as follows: S31-S33: Same as Example 3. S34. Tetrabutyl titanate, ethanol, and water were mixed in a volume ratio of 3:10:1. Nano-silicon particles (50 nm in diameter) accounting for 5% of the mass of tetrabutyl titanate were added. The pH was adjusted to 2.0, and the mixture was stirred for 30 min to obtain a sol. Then, 5 μL of the sol was dropped onto the surface of the enzyme layer, air-dried at room temperature, and cured in an oven at 45 °C for 30 min to form a nano-titanium dioxide-silicon composite coating with a thickness of 200 nm and a pore size of 150–180 nm. S35: Same as Example 3. S4. Data processing: The electrical signal was compared with the standard curve established in Example 1 using a portable data processing terminal, and the selenium content was found to be 0.29 mg / kg. Example 5 S1. Sample collection: Same as in Example 4.

[0038] S2. Sample pretreatment: Same as in Example 3. S3. Detection process: Same as in Example 4. The method for fabricating the sensor electrodes is as follows: S31~S34: Same as Example 4; S35. Cut a polytetrafluoroethylene microporous membrane with a pore size of 100 nm, treat it with plasma (argon:oxygen = 9:1, power 80W, time 60 seconds) to introduce hydrophilic groups on the surface of the polytetrafluoroethylene microporous membrane and reduce macromolecular adsorption. Then, wet it with deionized water and cover it with a titanium dioxide coating. Seal the edges with epoxy glue to fix it and obtain the sensor electrode. S4. Data processing: The electrical signal was compared with the standard curve established in Example 1 using a portable data processing terminal, and the selenium content was found to be 0.29 mg / kg.

[0039] Comparative Example 1 The 2,3-diaminonaphthalene fluorescence method mentioned in the background section was used for detection.

[0040] Comparative Example 2 Compared to Example 1, no titanium dioxide layer is added to the electrode, while the other parameters and steps are the same as in Example 1.

[0041] Comparative Example 3 Compared to Example 1, the electrode does not contain a polytetrafluoroethylene microporous membrane, while the other parameters and steps are the same as in Example 1.

[0042] Experimental Example I. Electrode effect verification (single test comparison), the results are shown in Table 1.

[0043] The selenium content detected in Examples 1, 3, 4, 5 and Comparative Examples 1-3 was recorded. The deviation (%) of this content from the ICP-MS value was also tested. Furthermore, the resistance to algal toxin interference and high humidity stability of the electrodes in Examples 1, 3, 4, 5 and Comparative Examples 1-3 were assessed. Specific results are shown in Table 1.

[0044] Table 1: Note: The resistance to algal toxin interference in Table 1 is calculated based on the signal of the blank sample without algal toxins as 100%, and the relative signal value is calculated.

[0045] As shown in Table 1, the detection methods of the various embodiments of the present invention exhibit excellent performance in terms of accuracy, resistance to algal toxin interference, and stability against high humidity, significantly outperforming Comparative Example 1 (2,3-diaminonaphthalene fluorescence method) and Comparative Examples 2-3. Example 1, as a basic scheme, effectively addresses the interference from complex coastal environments. Further optimization of electrode surface treatment, coating composition, enzyme immobilization method, and membrane structure (i.e., Examples 3-5) further enhances performance, with progressively stronger anti-interference capabilities and stability, fully demonstrating the advantages of the detection methods of the present invention in adapting to the detection needs of brackish water environments in coastal areas.

[0046] II. Stability Verification.

[0047] Electrodes from Examples 1, 3, 4, and 5, and Comparative Examples 2-3 were selected. The tilapia test solution (selenium content 0.28 mg / kg) from Example 1 was used as the test object. Ten consecutive tests were conducted in a brackish water pond environment in Shanwei, Guangdong (salinity 5 wt‰, humidity 78%, temperature 30℃). [After each test, the electrodes were rinsed with Tris-HCl buffer (pH 7.0-7.5).] Each test was conducted at 5-minute intervals. The relative standard deviation (RSD) of the ten tests was calculated, and changes in the appearance of the electrode surfaces (such as the polyethylene glycol layer, enzyme layer, titanium dioxide coating, and polytetrafluoroethylene film) were observed (e.g., peeling, cracking, deposition, microbial growth, etc.). The results are shown in Table 2.

[0048] Table 2: Table 2 shows that: After 10 consecutive tests, the detection values ​​of the electrodes of this invention (Examples 1, 3, 4, and 5) showed minimal fluctuations (RSD ≤ 3.5%). Comparative Example 2, lacking titanium dioxide, was susceptible to microbial contamination, resulting in a significant decrease in the detection value on the 10th test. Comparative Example 3, lacking a polytetrafluoroethylene membrane, experienced significant decreases in detection values ​​due to moisture interference. This demonstrates that the multilayer structure of the electrodes of this invention is crucial for stability, and that high stability and repeatability are maintained even in continuous testing in a sulfide-containing coastal environment.

[0049] It is worth noting that in actual testing, operations such as sample replacement and equipment calibration will result in longer intervals (usually ≥30 min), allowing the electrode sufficient time to recover its activity (such as enzyme conformation reset and natural membrane permeability). However, this experiment uses only a short interval of 5 min to test the electrode's fatigue resistance under extreme conditions of high frequency and no recovery time, which better highlights the stability of the structural design of this invention.

Claims

1. A method for detecting trace elements in aquatic products, characterized in that, Includes the following steps: S1. Sample collection: Fresh euryhaline fish from brackish water ponds in the southeast coastal area were selected. The scales, internal organs and bones were removed and the muscle tissue from the back, abdomen and lateral line were collected and mixed as test samples. S2. Sample pretreatment: Cut the tissue sample into small pieces, put them into a tissue homogenizer, add Tris-HCl buffer containing thiourea and Triton X-100 with a pH of 7.0-7.5, mix and centrifuge, and take the supernatant as the test solution; S3. Detection process: The test solution is dropped onto the biosensor to ensure full contact between the test solution and the surface of the sensor electrode; at the same time, a portable electrochemical workstation is connected to record the changes in the electrical signal generated by the biosensor. The fabrication steps for the sensor electrodes are as follows: First, a layer of polyethylene glycol was modified on the surface of the platinum-iridium alloy electrode. Then, the polyethylene glycol layer was preactivated with glutaraldehyde. Next, selenocysteine ​​lyase was immobilized. Then, a nano-titanium dioxide coating was coated on the enzyme layer. Finally, a polytetrafluoroethylene microporous membrane was coated on the coating surface. S4. Data Processing: Using a portable data processing terminal, the electrical signal generated by the sensor is compared with the pre-established standard curve of selenium element in euryhaline fish in the southeastern coastal area to calculate the selenium content in the test solution.

2. The method for detecting trace elements in aquatic products according to claim 1, characterized in that, Before modifying with polyethylene glycol, the surface of the platinum-iridium alloy electrode was roughened by sandblasting, and then the roughened electrode surface was activated by silane coupling agent.

3. The method for detecting trace elements in aquatic products according to claim 1, characterized in that, The nano-titanium dioxide coating has a thickness of 50–200 nm and a pore size of 150–300 nm.

4. The method for detecting trace elements in aquatic products according to claim 3, characterized in that, Nano-silicon particles are incorporated during the preparation of the titanium dioxide coating.

5. The method for detecting trace elements in aquatic products according to claim 1, characterized in that, The surface of the polytetrafluoroethylene microporous membrane is incorporating hydrophilic groups.

6. The method for detecting trace elements in aquatic products according to claim 1, characterized in that, The pore size of the polytetrafluoroethylene microporous membrane is 80–150 nm.

7. The method for detecting trace elements in aquatic products according to claim 1, characterized in that, In step S3, the changes in electrical signals generated by the biosensor are recorded using cyclic voltammetry, with the peak current used as the characteristic signal value. The peak current is positively linearly correlated with the selenium content; the scanning speed is 60-80 mV / s, and the detection potential range is -0.5~1.1V.

8. The method for detecting trace elements in aquatic products according to claim 1, characterized in that, In step S4, when establishing the standard curve, samples of euryhaline fish cultured in brackish water ponds with different salinity and red tide cycles along the southeast coast were selected. At least 30 samples were collected under each condition, and 3 parallel samples were set up for each sample. The selenium content was detected by the aforementioned method for detecting trace elements in aquatic products and by inductively coupled plasma mass spectrometry. After removing outliers using the Grubbs test, the detection results of inductively coupled plasma mass spectrometry were used as the abscissa, and the electrical signal value from step S3 was used as the ordinate. A univariate linear regression method was used to plot the standard curve and determine the curve equation.

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