PP2A / ZnO-PDA / GCE electrochemical biosensor and preparation method and application thereof

Abstract

The invention provides a PP2A / PDA-ZnO / GCE electrochemical biosensor as well as a preparation method and application of the PP2A / PDA-ZnO / GCE electrochemical biosensor. The sensor comprises a glassy carbon electrode, an immobilized enzyme layer is arranged on the surface of the glassy carbon electrode, and the immobilized enzyme layer comprises protein phosphatase 2A and nano-zinc oxide coated with polydopamine; the mass ratio of the protein phosphatase 2A in the immobilized enzyme layer to the polydopamine-coated nano-zinc oxide is 5: 8-5: 2, and the molar ratio of dopamine hydrochloride used as raw materials of the polydopamine-coated nano-zinc oxide to the nano-zinc oxide is 1: 3-1: 2. The invention also provides a preparation method of the electrochemical biosensor. The electrochemical biosensor can be used for detecting diarrhetic shellfish poison in seawater and marine organisms, especially okadaic acid and dinosporin; the polydopamine-coated nano zinc oxide and the protein phosphatase 2A have a synergistic effect, so that the binding force is strong, the enzyme activity is good, the performance is stable, the reutilization is realized, and the cost is low.

Description

Technical Field

[0001] This invention relates to the technical field of electrochemical biosensors, and in particular to a PP2A / ZnO-PDA / GCE electrochemical biosensor, its preparation method, and its application. Background Technology

[0002] Red tides, also known as harmful algae blooms, are a harmful ecological phenomenon caused by the explosive proliferation or high aggregation of certain phytoplankton, protozoa, or bacteria in seawater under specific physicochemical and nutritional conditions, resulting in water discoloration and damage to the structure and function of the ecosystem. Red tides have become a serious ecological disaster globally. The increasing number of red tide events not only causes enormous economic losses but also triggers serious marine ecological and food safety problems. Common red tide toxins include paralytic shellfish poisoning (DSP), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (DSP), amnesic shellfish poisoning (ASPP), and sicca toxins. Among these, DSP, represented by okadaic acid (OA) and its natural derivative dinophysistoxins (DTXs), is one of five known algal toxins that can cause symptoms such as diarrhea, vomiting, and abdominal pain. These toxins can be transmitted through the food chain and can cause varying degrees of toxicity to ecosystems, marine fisheries, the marine environment, and human health.

[0003] Currently, domestic and international researchers utilize various methods for detecting DSP toxin, including the Mouse Bioassay (MBA), chemical analysis, immunoassay, cellular analysis, and biosensor methods. While the MBA method is simple to operate and can directly determine sample usability, it suffers from long detection cycles, low sensitivity, significant individual variability, and has consistently faced protests from animal rights activists. Among chemical analysis methods, high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) has garnered significant attention due to its high-performance qualitative and quantitative analysis; however, this method requires expensive reference materials, incurs high instrument maintenance costs, and demands stringent operator skills. Immunoassays based on antigen-antibody reactions offer high specificity and sensitivity, but specific monoclonal antibodies are expensive and difficult to obtain. Cellular analysis, while offering advantages such as high throughput, low cost, and good reproducibility, remains complex for cell detection.

[0004] Biosensor methods have attracted much attention in recent years. These methods utilize immobilized enzyme membranes as biorecognition elements. These biosensors capture measurable signals generated by the reaction between the analyte and the biorecognition element through various physical and chemical signal transducers. These signals are proportional to the concentration of the analyte, thus enabling quantitative determination of the analyte. For example, Chinese patent CN104977332A, published on October 14, 2015, discloses a biosensor and its preparation method for detecting daphniacin. This biosensor includes an insulating substrate and an electrode system formed on the substrate, comprising a working electrode, a reference electrode, and a counter electrode. A bioenzyme layer, including protein phosphatase 2A and an electron transporter (sodium p-nitrophenyl phosphate), is immobilized on the working electrode using photocrosslinking or agarose embedding. This biosensor provides intuitive detection results, high specificity and accuracy, and a short detection cycle, meeting the needs for immediate and rapid detection. However, the active ingredient in the enzyme layer of this biosensor is protein phosphatase 2A, which has low enzyme activity and poor stability, and can only be stored for a short period of time. Moreover, protein phosphatase 2A is fixed on the working electrode by photocrosslinking or agarose embedding, which is cumbersome to operate, has weak binding force, and cannot be reused. As a result, the detection cost of this biosensor is high and its performance is limited. Summary of the Invention

[0005] The purpose of this invention is to provide a PP2A / ZnO-PDA / GCE electrochemical biosensor, its preparation method, and its application, aiming to solve the problem that existing biosensors suffer from poor stability and high detection costs due to low activity and weak binding force of the bioenzyme layer, thus limiting their application scope.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is implemented as follows.

[0007] In one aspect, an electrochemical biosensor of the present invention, comprising a working electrode, wherein the working electrode is a glassy carbon electrode; an immobilized enzyme layer is disposed on the surface of the working electrode, the immobilized enzyme layer comprising protein phosphatase 2A and polydopamine-coated nano-zinc oxide; the mass ratio of protein phosphatase 2A to polydopamine-coated nano-zinc oxide in the immobilized enzyme layer is 5:8-5:2, and the molar ratio of dopamine hydrochloride used as a raw material for polydopamine-coated nano-zinc oxide to nano-zinc oxide is 1:3-1:2.

[0008] The PP2A / ZnO-PDA / GCE electrochemical biosensor of this invention uses a glassy carbon electrode as the working electrode, and an immobilized enzyme layer is fixed on the surface of the working electrode. This immobilized enzyme layer contains protein phosphatase 2A and polydopamine-coated nano-zinc oxide. The polydopamine-coated nano-zinc oxide and protein phosphatase 2A work synergistically. The catechol functional groups in polydopamine can firmly bind protein phosphatase 2A without the need for additional cross-linking agents, thus avoiding toxicity issues. Due to the large specific surface area of ​​the nano-zinc oxide, the polydopamine-coated nano-zinc oxide can bind more... The addition of protein phosphatase 2A increases the concentration of protein phosphatase 2A per unit area, thereby improving the sensitivity of the electrochemical biosensor. Simultaneously, the excellent biomimetic adhesion of polydopamine and the superior antibacterial properties of nano-zinc oxide create a milder microenvironment for protein phosphatase 2A, which helps maintain its enzyme activity. This enhances the biocompatibility and environmental stability of the electrochemical biosensor, making it more suitable for use in seawater environments. Furthermore, it exhibits stable performance, is reusable, and has low detection costs, making it suitable for detecting various red tide toxins in seawater.

[0009] This invention is a PP2A / ZnO-PDA / GCE electrochemical biosensor suitable for complex marine matrices. The enzyme activity of this immobilized enzyme layer is stable and the binding force is strong. The resulting electrochemical biosensor has stable performance, high reusability, and a wide detection range. It is particularly suitable for the rapid and efficient detection of oota-sponge acid and fin algae toxins in red tide toxins in seawater.

[0010] In another aspect, a method for preparing a PP2A / ZnO-PDA / GCE electrochemical biosensor according to the present invention includes the following steps: S1 Preparation of immobilized enzyme solution 1) Take nano-zinc oxide and dopamine hydrochloride, the molar ratio of dopamine hydrochloride and nano-zinc oxide is 1:3-1:2, add to a buffer solution with a molar concentration of 8-12 mmol / L and a pH of 7-8, the molar ratio of nano-zinc oxide to buffer is 0.006:1-0.01:1, stir, react at room temperature for 100-150 min, centrifuge, wash, and obtain polydopamine-coated nano-zinc oxide biomimetic material; 2) Take the polydopamine-coated nano-zinc oxide biomimetic material obtained in step 1), add to a buffer solution with a molar concentration of 8-12 mmol / L and a pH of 7-8, to obtain a biomimetic material solution; take the biomimetic material solution, add enzyme with an activity of 8-12 Protein phosphatase 2A (U / mL) was added, with a mass ratio of protein phosphatase 2A to polydopamine-coated zinc oxide nanoparticles of 5:8-5:2. The mixture was shaken and fixed at 10-40 °C for 8-16 h. After centrifugation, washing, and adding buffer solution to achieve a polydopamine-coated zinc oxide nanoparticle concentration of 2-4 mg / mL, an immobilized enzyme solution was obtained. Preparation of the S2 electrochemical biosensor: 3) A glassy carbon electrode was taken, polished, washed, and dried to obtain the working electrode. 4) The immobilized enzyme solution obtained in step 2) was dropped onto the surface of the working electrode and placed at 3-5 °C for 3-5 h. After washing with buffer solution, the PP2A / ZnO-PDA / GCE electrochemical biosensor was obtained.

[0011] The preparation method of the PP2A / ZnO-PDA / GCE electrochemical biosensor of the present invention involves firstly, coating dopamine hydrochloride onto nano-zinc oxide via in-situ polymerization. The nano-zinc oxide (ZnO) and polydopamine (PDA) are strongly bonded together. The nano-zinc oxide possesses a large specific surface area and excellent bactericidal properties, while the polydopamine exhibits good biomimetic adhesion, increasing the polydopamine content per unit area. Next, protein phosphatase 2A (PP2A) is immobilized on the polydopamine-coated nano-zinc oxide. The catechol functional groups in the polydopamine are firmly bound to protein phosphatase 2A, eliminating the need for additional cross-linking agents and avoiding toxicity issues. Finally, the polydopamine-coated nano-zinc oxide containing protein phosphatase 2A is immobilized on a glassy carbon electrode, thereby obtaining the PP2A / ZnO-PDA / GCE electrochemical biosensor. This electrochemical biosensor exhibits good biocompatibility, superior corrosion resistance, and excellent durability. It demonstrates high enzyme activity of protein phosphatase 2A per unit area, stable performance, reusability, and low detection cost, making it suitable for detecting various red tide toxins in seawater. Furthermore, the biological preparation method for this electrochemical biosensor is simple, with a short process flow, mild conditions, and is easily industrialized.

[0012] In this invention, protein phosphatase 2A is covalently immobilized on polydopamine-coated zinc oxide nanoparticles. Both the immobilization temperature and time significantly influence the reactivity of the biosensor during detection. As the immobilization temperature increases, the reactivity of the biosensor initially increases and then decreases during detection; this temperature setting achieves both high reactivity and low energy consumption. Similarly, as the immobilization time increases, the reactivity of the biosensor initially increases and then decreases during detection; this duration also achieves high reactivity and high energy efficiency.

[0013] In a preferred embodiment, step 2) involves washing with ultrapure water, sodium chloride solution, and a buffer solution sequentially, 3-5 times. The ultrapure water washing removes impurities such as free enzymes and byproducts generated during the reaction; the sodium chloride solution washing eliminates physical adsorption via ionic bonds; and the buffer solution washing provides an optimal stable environment for the immobilized enzyme and removes salt residue from the sodium chloride solution washing. Through multiple washings, impurities, free enzymes, and byproducts generated during the reaction are thoroughly removed from the immobilized enzyme solution, thereby improving the purity of the immobilized enzyme solution.

[0014] In a preferred embodiment, in step 1), the buffer solution is a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution or a phosphate buffer solution. The buffer solution in this invention primarily provides a suitable pH environment and disperses the nano-zinc oxide and dopamine hydrochloride, increasing the contact area between the nano-zinc oxide and dopamine hydrochloride, and enhancing the interaction between them. A tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution is preferred.

[0015] The nano zinc oxide of the present invention can be commercially available nano zinc oxide or nano zinc oxide synthesized on-site. The synthesis method of nano zinc oxide is the commonly used hydrothermal synthesis method, which is simple, easy to operate, and easy to industrialize.

[0016] In a preferred embodiment, in step 1), the washing is performed using ultrapure water, and the washing is repeated 3-5 times. This invention uses ultrapure water to wash the nano-zinc oxide product coated with dopamine hydrochloride via in-situ polymerization, primarily to remove the buffer solution during the reaction process. The buffer solution mainly provides the reaction environment. After polymerization is complete, the buffer solution is removed, and the polydopamine-coated nano-zinc oxide is purified.

[0017] In a preferred embodiment, step 3) involves grinding and polishing using alumina powders with particle sizes of 1.0, 0.3, and 0.05 μm, respectively. In this invention, the glassy carbon electrode typically has a diameter of 4 mm. Grinding with alumina powders of different particle sizes removes surface impurities, polishes the surface to a mirror finish, improves the electrode's flatness and smoothness, and also removes potential contaminants and oxide layers, preventing signal attenuation and potential shift, thus improving result reproducibility.

[0018] In a preferred embodiment, step 3) involves washing with ultrapure water and anhydrous ethanol sequentially, using ultrasonic washing for 8-12 minutes. In this invention, the glassy carbon electrode, after grinding and polishing, is ultrasonically washed with ultrapure water and anhydrous ethanol to remove the polishing powder—alumina powder—residual on the surface of the glassy carbon electrode during the grinding and polishing process. Ethanol, under ultrasonic cavitation, can better wash away the polishing powder from the crevices, and can also effectively dissolve organic and inorganic matter. It is also easily volatile, resulting in rapid drying of the glassy carbon electrode after washing, thus quickly removing residual alumina powder and other impurities from the surface of the glassy carbon electrode, leaving it bright.

[0019] In a preferred embodiment, in step 3), the drying is performed by natural drying at room temperature. After polishing and washing, the glassy carbon electrode of the present invention is typically dried naturally at room temperature; this drying method is convenient, easy to operate, and does not generate secondary organisms during the drying process, thus fully ensuring the cleanliness of the glassy carbon electrode surface.

[0020] In another aspect, the present invention relates to the application of a PP2A / ZnO-PDA / GCE electrochemical biosensor in detecting diarrhetic shellfish toxins in seawater and marine organisms.

[0021] This electrochemical biosensor exhibits good biocompatibility, superior corrosion resistance, and excellent durability. It boasts high enzyme activity of protein phosphatase 2A per unit area, making it particularly suitable for applications in marine environments. It can detect various red tide toxins in seawater, especially diarrhetic shellfish poisoning in seawater and marine organisms. During the detection of diarrhetic shellfish poisoning in seawater or marine organisms, this electrochemical biosensor can be reused eight times, maintaining over 85% of the enzyme activity of the immobilized enzyme layer. Furthermore, it can be quickly reused through washing, simplifying the process and reducing operating costs.

[0022] As a preferred embodiment, the PP2A / ZnO-PDA / GCE electrochemical biosensor is used in the detection of ootan echinoclamtic acid and algal toxins. The PP2A / ZnO-PDA / GCE electrochemical biosensor of this invention can detect not only ootan echinoclamtic acid in seawater or marine organisms but also algal toxins in seawater or marine organisms. It has a wide detection range and strong versatility, greatly reducing the time required for laboratory sample analysis and testing, improving detection speed, and achieving rapid and efficient detection.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The PP2A / ZnO-PDA / GCE electrochemical biosensor of the present invention uses a glassy carbon electrode as the working electrode, and an immobilized enzyme layer is fixed on the surface of the working electrode. This immobilized enzyme layer contains protein phosphatase 2A and polydopamine-coated nano-zinc oxide. The polydopamine-coated nano-zinc oxide and protein phosphatase 2A work synergistically, and the catechol functional groups in polydopamine can firmly bind protein phosphatase 2A. Due to the large specific surface area of ​​the nano-zinc oxide, the polydopamine-coated nano-zinc oxide can bind more protein phosphatase 2A, increasing the concentration of protein phosphatase 2A per unit area, thereby improving the electrochemical biosensor's efficiency. The sensor's sensitivity is enhanced; simultaneously, due to the excellent biomimetic adhesion of polydopamine and the superior antibacterial properties of nano-zinc oxide, protein phosphatase 2A enjoys a milder microenvironment, which helps maintain its enzyme activity, thus improving the biocompatibility and environmental stability of the electrochemical biosensor. This makes it more suitable for use in seawater environments, and it also offers low detection costs. It is suitable for detecting various red tide toxins in seawater, particularly diarrhetic shellfish poisoning in seawater and marine organisms. This electrochemical biosensor can be reused eight times in the detection of diarrhetic shellfish poisoning in seawater or marine organisms, and the enzyme activity of the immobilized enzyme layer can still be maintained at over 85%, significantly reducing detection costs. The preparation method of this electrochemical biosensor is simple, with a short process flow, mild conditions, and easy industrialization. Attached Figure Description

[0024] Figure 1 A graph showing the trend of stability test results for different electrochemical biosensors;

[0025] Figure 2 This is a graph showing the trend of repeatability test results for different electrochemical biosensors.

[0026] Figure 3 This is a graph showing the activity of OA-inhibiting immobilized enzymes.

[0027] Figure 4 The curves show the activity of DXT-inhibited immobilized enzymes. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The present invention discloses an electrochemical biosensor for PP2A / ZnO-PDA / GCE, comprising a working electrode, wherein the working electrode is a glassy carbon electrode; an immobilized enzyme layer is disposed on the surface of the working electrode, the immobilized enzyme layer comprising protein phosphatase 2A and polydopamine-coated nano-zinc oxide; the mass ratio of protein phosphatase 2A to polydopamine-coated nano-zinc oxide in the immobilized enzyme layer is 5:8-5:2, and the mass ratio of dopamine hydrochloride used as a raw material for polydopamine-coated nano-zinc oxide to nano-zinc oxide is 1:3-1:2.

[0030] The present invention discloses a method for preparing a PP2A / ZnO-PDA / GCE electrochemical biosensor, comprising the following steps:

[0031] Preparation of S1 immobilized enzyme solution

[0032] 1) Take nano zinc oxide and dopamine hydrochloride, with a molar ratio of 1:3-1:2, add them to a buffer solution with a molar concentration of 8-12 mmol / L and a pH of 7-8, and a molar ratio of nano zinc oxide to buffer of 0.006:1-0.01:1. Stir and react at room temperature for 100-150 min. Centrifuge and wash to obtain polydopamine-coated nano zinc oxide biomimetic material.

[0033] 2) Take the polydopamine-coated zinc oxide nano-bionic material obtained in step 1) and add it to a buffer solution with a molar concentration of 8-12 mmol / L and a pH of 7-8 to obtain a biomimetic material solution; take the biomimetic material solution and add protein phosphatase 2A with an enzyme activity of 8-12 U / mL. The mass ratio of protein phosphatase 2A to polydopamine-coated zinc oxide nano-bionic material is 5:8-5:2. Shake and fix at a temperature of 10-40 ℃ for 8-16 h. Centrifuge, wash, and add buffer solution to make the concentration of polydopamine-coated zinc oxide nano-bionic material 2-4 mg / mL to obtain an immobilized enzyme solution.

[0034] Fabrication of S2 electrochemical biosensor

[0035] 3) Take the glassy carbon electrode, grind and polish it, wash it, and dry it to obtain the working electrode;

[0036] 4) The immobilized enzyme solution obtained in step 2) is dropped onto the surface of the working electrode, placed at 3-5 ℃ for 3-5 h, and washed with buffer solution to obtain the PP2A / ZnO-PDA / GCE electrochemical biosensor.

[0037] Preferably, in step 2), the washing is performed by sequentially washing with ultrapure water, sodium chloride solution and buffer solution, and the number of washing cycles is 3-5.

[0038] Preferably, in step 1), the buffer solution is a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution or a phosphate buffer solution.

[0039] Preferably, in step 1), the washing is performed using ultrapure water, and the number of washing cycles is 3-5.

[0040] Preferably, in step 3), the grinding and polishing are performed sequentially using alumina powder with particle sizes of 1.0, 0.3 and 0.05 μm.

[0041] Preferably, in step 3), the washing process involves sequentially washing with ultrapure water and anhydrous ethanol, using ultrasonic washing, and the washing time is 8-12 minutes.

[0042] Preferably, in step 3), the drying is performed by natural drying at room temperature.

[0043] The present invention relates to the application of a PP2A / ZnO-PDA / GCE electrochemical biosensor, specifically its application in detecting diarrhetic shellfish poisoning in seawater and marine organisms.

[0044] Preferably, the PP2A / ZnO-PDA / GCE electrochemical biosensor is used in the detection of daphniacin and fin algae toxins.

[0045] Example

[0046] The present invention discloses a method for preparing a PP2A / ZnO-PDA / GCE electrochemical biosensor, comprising the following steps:

[0047] Preparation of S1 immobilized enzyme solution

[0048] 1) Take 2.5 g of zinc acetate dihydrate, 15 mL of PEG 400, 65 mL of anhydrous ethanol and 6 g of sodium hydroxide, mix them, and sonicate them at room temperature for 40 min to obtain a mixture; then, transfer it to a 100 mL stainless steel autoclave with a polytetrafluoroethylene liner and keep it at 120 ℃ for 12 h; cool down, take it out, wash the product three times each with ultrapure water and anhydrous ethanol alternately, and dry it in a vacuum oven at 60 ℃ to obtain nano zinc oxide;

[0049] 2) Take 20 mg of nano zinc oxide and 20 mg of dopamine hydrochloride obtained in step 1) and add them to 10 mL of Tris-HCl buffer solution with a molar concentration of 10 mmol / L and a pH of 7.5. Stir and react at room temperature for 120 min. Centrifuge and wash to obtain polydopamine-coated nano zinc oxide biomimetic material.

[0050] 3) Take 1 mg of the polydopamine-coated zinc oxide nano-bionic material obtained in step 1) and add it to a Tris-HCl buffer solution with a molar concentration of 10 mmol / L and a pH of 7.5 to obtain a biomimetic material solution; take 40 μL of the biomimetic material solution, add 50 μg of protein phosphatase 2A with an enzyme activity of 10 U / mL, and fix it by shaking at 30 ℃ for 12 h. Centrifuge at 6000 r / min, wash several times with ultrapure water, NaCl solution and Tris-HCl buffer, and add a Tris-HCl buffer solution with a molar concentration of 10 mmol / L and a pH of 7.6 to make the concentration of polydopamine-coated zinc oxide nano-bionic material 3 mg / mL to obtain an immobilized enzyme solution;

[0051] Fabrication of S2 electrochemical biosensor

[0052] 4) Take a glassy carbon electrode (GCE) with a diameter of 4 mm, polish it with alumina powder of 1.0, 0.3 and 0.05 μm respectively, and ultrasonically wash it in ultrapure water and anhydrous ethanol for 10 min respectively. Then, let it dry naturally at room temperature to obtain the working electrode.

[0053] 5) Using a pipette, 10 μL of the immobilized enzyme solution obtained in step 2) was dropped onto the surface of the working electrode and placed at 4°C for 4 h. The electrode was then washed with a 0.1 M phosphate buffer solution with a pH of 7.0 to obtain the PP2A / ZnO-PDA / GCE electrochemical biosensor.

[0054] Comparative Example

[0055] A method for preparing a PP2A / PDA / GCE electrochemical biosensor includes the following steps:

[0056] Preparation of S1 immobilized enzyme solution

[0057] 1) Take 20 mg of dopamine hydrochloride, add it to 10 mL of Tris-HCl buffer solution with a molar concentration of 10 mmol / L and a pH of 7.5, stir, and react at room temperature for 120 min. Centrifuge, wash, and obtain polydopamine.

[0058] 2) Take 1 mg of polydopamine obtained in step 1) and add it to a Tris-HCl buffer solution with a molar concentration of 10 mmol / L and a pH of 7.5 to obtain a polydopamine solution; take 40 μL of the polydopamine solution and add 50 μg of protein phosphatase 2A with an enzyme activity of 10 U / mL, fix it by shaking at 30 ℃ for 12 h, centrifuge at 6000 r / min, wash several times with ultrapure water, NaCl solution and Tris-HCl buffer, and add a Tris-HCl buffer solution with a molar concentration of 10 mmol / L and a pH of 7.6 to make the concentration of polydopamine 3 mg / mL to obtain an immobilized enzyme solution;

[0059] Fabrication of S2 electrochemical biosensor

[0060] 3) Take a glassy carbon electrode (GCE) with a diameter of 4 mm, polish it with alumina powder of 1.0, 0.3 and 0.05 μm respectively, and ultrasonically wash it in ultrapure water and anhydrous ethanol for 10 min respectively. Then, let it dry naturally at room temperature to obtain the working electrode.

[0061] 4) Using a pipette, 10 μL of the immobilized enzyme solution obtained in step 2) was dropped onto the surface of the working electrode and placed at 4°C for 4 h. The electrode was then washed with a 0.1 M phosphate buffer solution with a pH of 7.0 to obtain the PP2A / PDA / GCE electrochemical biosensor, which is the control sample.

[0062] Performance Test 1

[0063] Stability tests were conducted on the PP2A / ZnO-PDA / GCE electrochemical biosensor obtained in the embodiments of the present invention and the control sample obtained in the comparative example. Eight samples each of the electrochemical biosensor obtained in the embodiments and the control sample obtained in the comparative example were placed in a refrigerator at 0-4 ℃ for 0, 1, 2, 5, 8, 12, 16, and 20 days, respectively, and then removed to measure enzyme activity. The enzyme activity was measured by placing the electrochemical biosensor in 20 µL of a 1 mmol / L p-nitrophenyl phosphate (PNPP) solution and reacting at room temperature for 20 min, then measuring the content of the product p-nitrophenol (PNP). The enzyme activity retention rate, representing relative stability, was obtained by dividing the PNP content generated on day 0 by the PNP content generated on day 0 at other storage times, with the PNP content generated on day 0 as 100%.

[0064] Depend on Figure 1It can be seen that the PP2A / ZnO-PDA / GCE electrochemical biosensor obtained in this invention maintains a relative stability of over 80% during 8 days of storage, and still maintains a relative stability of over 50% after 16 days of storage. However, the control sample's relative stability begins to fall below 80% after 5 days of storage, and is less than 40% after 16 days. Therefore, during storage, the PP2A / ZnO-PDA / GCE electrochemical biosensor of this invention exhibits high enzyme activity retention, slow decay, high relative stability, and a longer service life.

[0065] Performance Test 2

[0066] The PP2A / ZnO-PDA / GCE electrochemical biosensor obtained in the embodiments of the present invention and the control sample obtained in the comparative example were subjected to repeatability tests. The PP2A / ZnO-PDA / GCE electrochemical biosensor obtained in the embodiments of the present invention and the control sample obtained in the comparative example were placed in 20 µL of a 1 mmol / L p-nitrophenyl phosphate (PNPP) solution and reacted at room temperature for 20 min. The product was then removed, and the p-nitrophenol (PNP) content was measured. The electrochemical biosensor was washed with ultrapure water and Tris-HCl buffer solution. This operation was repeated 9 times. The PNP content generated in the first test was taken as 100%. The PNP content generated in other different test runs was divided by the PNP content generated in the first test to obtain the enzyme activity retention rate, which was used to represent relative reusability.

[0067] Depend on Figure 2 It can be seen that the PP2A / ZnO-PDA / GCE electrochemical biosensor obtained in this invention maintains a relative reusability of over 85% after 8 reuses; however, the control sample shows a relative reusability close to 85% after 5 reuses, but begins to fall below 85% after 6 reuses, and the relative reusability decreases faster with each reuse, reaching only 70% after 8 reuses. Therefore, during reuse, the PP2A / ZnO-PDA / GCE electrochemical biosensor of this invention exhibits high relative reusability, high enzyme activity retention, slow decay, and can be reused more times.

[0068] Performance Test 3

[0069] Take eight 2 mL aliquots of 50 mmol / L Tris-HCl buffer solution, and add 4 µL of 1 mol / L MnCl2 aqueous solution to each aliquot; one aliquot is the blank solution, and the other seven are the experimental solutions. Add 20 μL of methanol to the blank solution, and add 5 × 10⁻⁶ MnCl2 aqueous solution to each of the seven experimental solutions. -1 mg / mL, 5×10-2 mg / mL, 5×10 -3 mg / mL, 5×10 -4 mg / mL, 5×10 -5 mg / mL, 5×10 -6 mg / mL and 5×10 -7 20 µL of OA standard solution at a concentration of mg / mL was added. Eight PP2A / ZnO-PDA / GCE electrochemical biosensors obtained in the embodiments of the present invention were placed in blank solution and experimental solution, respectively; the reaction was carried out at room temperature for 10 min; then, 20 µL of PNPP solution at a concentration of 1 mmol / L was added, and the reaction was continued for another 20 min; based on the change in electrochemical signal, the IC50 value of OA inhibition of immobilized enzyme PP2A / ZnO-PDA was obtained. Each concentration experiment was repeated 3 times, and the average value was taken.

[0070] From the appendix Figure 3 It can be seen that the immobilized enzyme (PP2A / ZnO-PDA enzyme) can react with OA, significantly reducing the amount of substrate PNPP converted to PNP. OA is a positive inhibitor of the immobilized enzyme, with an IC50 of 16.22 ng / L. This is because PP2A can catalyze the conversion of the substrate—p-nitrophenyl phosphate (PNPP)—to the reaction product p-nitrophenol (PNP). Therefore, the presence of OA inhibits the activity of the immobilized enzyme, leading to a reduction in the amount of PNP produced.

[0071] Performance Test 4

[0072] Take eight 2 mL aliquots of 50 mmol / L Tris-HCl buffer solution, and add 4 µL of 1 mol / L MnCl2 aqueous solution to each aliquot; one aliquot is a blank solution, and the other seven are experimental solutions. Add 20 μL of methanol to the blank solution, and add methanol at concentrations of 1 mg / mL, 0.1 mg / mL, and 1 × 10⁻⁶ mg / mL to the seven experimental solutions, respectively. -2 mg / mL, 1×10 -3 mg / mL, 1×10 -4 mg / mL, 1×10 -5 mg / mL and 1×10 -620 µL of DTX1 standard solution at a concentration of mg / mL was added. Eight PP2A / ZnO-PDA / GCE electrochemical biosensors obtained in the embodiments of the present invention were placed in blank solution and experimental solution, respectively; the reaction was carried out at room temperature for 10 min; then, 20 µL of PNPP solution at a concentration of 1 mmol / L was added, and the reaction was continued for another 20 min; the IC50 value of DTX1 inhibition of immobilized enzyme was obtained according to the change of electrochemical signal. Each concentration experiment was repeated 3 times, and the average value was taken.

[0073] From the appendix Figure 4 It can be seen that the immobilized enzyme (PP2A / ZnO-PDA enzyme) can react with DTX1, reducing the amount of substrate PNPP converted to PNP. DTX1 is a positive inhibitor of the immobilized enzyme, with an IC50 of 154.88 ng / L. This is because PP2A can catalyze the conversion of the substrate p-nitrophenyl phosphate (PNPP) to the reaction product p-nitrophenol (PNP); therefore, the presence of DTX1 inhibits the activity of the immobilized enzyme, leading to a reduction in the amount of PNP produced.

[0074] From the appendix Figure 3 and attached Figure 4 It can be seen that OA has a stronger inhibitory effect on immobilized enzymes, about 9 times that of DTX1, meaning that immobilized enzymes are more sensitive to the concentration of OA.

[0075] Performance Test 5

[0076] Practical applications of electrochemical biosensors in seawater

[0077] (1) Seawater pretreatment

[0078] Three seawater samples of 2000 mL each were collected from Shilaoren Beach in Laoshan District, Qingdao City, Shandong Province, and filtered using a 0.45 µm glass fiber membrane.

[0079] Activation of the HLB solid-phase extraction was performed using 5 mL of methanol and 5 mL of ultrapure water, followed by enrichment of seawater at a rate of 1 mL / min. After enrichment, the extraction column was rinsed with 5 mL of 15% methanol aqueous solution, and air was evacuated for 5 min. The solid-phase extraction column was then eluted with 15 mL of methanol solution containing 1% ammonia to obtain the extract. Subsequently, the extract was evaporated to dryness using a rotary evaporator at 40 °C, yielding three portions of dried seawater for later use.

[0080] (2) Testing for DSP toxins

[0081] Add 9 mL of a Tris-HCl buffer solution containing MnCl2 (wherein the concentration of MnCl2 is 2 mmol / L and the concentration of Tris-HCl is 50 mmol / L; the preparation method is the same as performance test 3 and performance test 4) to the dried seawater obtained in step (1), and make up to 10 mL. Soluble the solution to obtain 3 seawater samples. Take another 10 mL of Tris-HCl buffer solution with a molar concentration of 10 mmol / L as a blank control sample. Take 4 PP2A / ZnO-PDA / GCE electrochemical biosensors obtained in the embodiments of the present invention, and put them into the seawater sample and blank control sample respectively, and react for 10 min. Then, add 20 µL of 1 mmol / L PNPP solution to each sample and continue to react for 20 min to determine the inhibitory effect of DSP toxin in seawater. The experimental results are listed in Table 1.

[0082] Table 1. Experimental results of seawater application on different samples

[0083]

[0084] As shown in Table 1, with the relative enzyme activity of the blank control solution as 100%, the PP2A / ZnO-PDA / GCE electrochemical biosensor obtained in this invention, after reacting with seawater samples, showed that the immobilized enzyme bound to the DSP toxin in the seawater samples, resulting in a decrease in the content of PNP generated from the substrate PNPP catalyzed by the immobilized enzyme. In other words, the seawater samples exhibited a strong inhibitory effect on the immobilized enzyme, with an inhibition rate of 55.21%.

[0085] Performance Test 6

[0086] Practical applications of electrochemical biosensors in marine organisms

[0087] (1) Pretreatment of marine organisms

[0088] Purchase commercially available mussels, and accurately weigh out 3 portions of 2.0 g mussel meat. Place the mussel meat in 20 mL of 90% methanol aqueous solution for homogenization. Then, centrifuge the crude extract (6000 rpm, 15 min), collect the supernatant, and wash with 5 mL of n-hexane. After centrifugation, filter the clear lower layer through a 0.45 µm glass fiber membrane and dry it under nitrogen to obtain dried mussel meat for later use.

[0089] (2) Testing for DSP toxins

[0090] The dried mussel species obtained in step (1) were added to 9 mL of a Tris-HCl buffer solution containing MnCl2 (wherein the concentration of MnCl2 was 2 mmol / L and the concentration of Tris-HCl was 50 mmol / L; the preparation method was the same as performance test 3 and performance test 4), and the volume was adjusted to 10 mL. The mixture was then reconstituted by sonication to obtain 3 mussel samples. Another 10 mL of a Tris-HCl buffer solution with a molar concentration of 10 mmol / L was used as a blank control sample. Four PP2A / ZnO-PDA / GCE electrochemical biosensors obtained in this invention were placed into the mussel samples and blank control samples, respectively, and reacted for 10 min. Then, 20 µL of 1 mmol / L substrate PNPP toxin was added to each sample, and the reaction was continued for 20 min to determine the inhibitory effect of DSP toxin in the mussels. The experimental results are listed in Table 2.

[0091] Table 2 Results of seawater application experiments on different samples

[0092]

[0093] As shown in Table 2, with the relative enzyme activity of the blank control solution as 100%, the PP2A / ZnO-PDA / GCE electrochemical biosensor obtained in this invention, after reacting with mussel samples, immobilized enzymes bound to DSP toxins in the mussel samples, resulting in a decrease in the content of PNP generated from the substrate PNPP catalyzed by the immobilized enzymes. That is, the mussel samples have a certain inhibitory effect on the immobilized enzymes, and the inhibition rate of the immobilized enzymes is 37.23%.

[0094] In summary, compared with the prior art, the beneficial effects of the present invention are as follows: The PP2A / ZnO-PDA / GCE electrochemical biosensor of the present invention uses a glassy carbon electrode as the working electrode, and an immobilized enzyme layer is fixed on the surface of the working electrode. This immobilized enzyme layer contains protein phosphatase 2A and polydopamine-coated nano-zinc oxide. The polydopamine-coated nano-zinc oxide and protein phosphatase 2A work synergistically, and the catechol functional groups in polydopamine can firmly bind protein phosphatase 2A. Due to the large specific surface area of ​​the nano-zinc oxide, the polydopamine-coated nano-zinc oxide can bind more protein phosphatase 2A, increasing the concentration of protein phosphatase 2A per unit area, thereby improving the electrochemical biosensor. The sensor's sensitivity is enhanced; simultaneously, due to the excellent biomimetic adhesion of polydopamine and the superior antibacterial properties of nano-zinc oxide, protein phosphatase 2A enjoys a milder microenvironment, which helps maintain its enzyme activity, enhancing the biocompatibility and environmental stability of the electrochemical biosensor. This makes it more suitable for use in seawater environments, and its low detection cost has led to its widespread application. It is suitable for detecting various red tide toxins in seawater, particularly diarrhetic shellfish poisoning in seawater and marine organisms. In the detection of diarrhetic shellfish poisoning in seawater or marine organisms, this electrochemical biosensor can be reused eight times, maintaining over 85% of the enzyme activity of the immobilized enzyme layer, significantly reducing detection costs. The preparation method of this electrochemical biosensor is simple, with a short process flow, mild conditions, and easy industrialization.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A PP2A / ZnO-PDA / GCE electrochemical biosensor, characterized in that: Includes a working electrode, wherein the working electrode is a glassy carbon electrode; An immobilized enzyme layer is provided on the surface of the working electrode, the immobilized enzyme layer including protein phosphatase 2A and polydopamine-coated nano zinc oxide. The mass ratio of protein phosphatase 2A to polydopamine-coated nano-zinc oxide in the immobilized enzyme layer is 5:8-5:2, and the molar ratio of dopamine hydrochloride to nano-zinc oxide used in the polydopamine-coated nano-zinc oxide is 1:3-1:

2.

2. The method for preparing the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 1, characterized in that, Includes the following steps: Preparation of S1 immobilized enzyme solution 1) Take nano zinc oxide and dopamine hydrochloride, with a molar ratio of dopamine hydrochloride to nano zinc oxide of 1:3-1:2, add them to a buffer solution with a molar concentration of 8-12 mmol / L and a pH of 7-8, and a molar ratio of nano zinc oxide to buffer of 0.006:1-0.01:1, stir, react at room temperature for 100-150 min, centrifuge, wash, and obtain polydopamine-coated nano zinc oxide biomimetic material; 2) Take the polydopamine-coated zinc oxide nano-bionic material obtained in step 1) and add it to a buffer solution with a molar concentration of 30-60 mmol / L and a pH of 7-8 to obtain a biomimetic material solution; take the biomimetic material solution and add protein phosphatase 2A with an enzyme activity of 8-12 U / mL. The mass ratio of protein phosphatase 2A to polydopamine-coated zinc oxide nano-bionic material is 5:8-5:

2. Shake and fix at a temperature of 10-40 ℃ for 8-16 h. Centrifuge, wash, and add buffer solution to make the concentration of polydopamine-coated zinc oxide nano-bionic material 2-4 mg / mL to obtain an immobilized enzyme solution. Fabrication of S2 electrochemical biosensor 3) Take the glassy carbon electrode, grind and polish it, wash it, and dry it to obtain the working electrode; 4) The immobilized enzyme solution obtained in step 2) is dropped onto the surface of the working electrode, placed at 3-5 ℃ for 3-5 h, and washed with buffer solution to obtain the PP2A / ZnO-PDA / GCE electrochemical biosensor.

3. The method for preparing the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 2, characterized in that: In step 2), the washing process involves sequentially washing with ultrapure water, sodium chloride solution, and buffer solution, with the washing cycle repeated 3-5 times.

4. The method for preparing the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 2, characterized in that: In step 1), the buffer solution is tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution or phosphate buffer solution.

5. The method for preparing the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 2, characterized in that: In step 1), the washing is done with ultrapure water, and the washing is performed 3-5 times.

6. The method for preparing the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 2, characterized in that: In step 3), the grinding and polishing are carried out by sequentially grinding and polishing with alumina powder with particle sizes of 1.0, 0.3 and 0.05 μm respectively.

7. The method for preparing the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 2, characterized in that: In step 3), the washing process involves sequentially washing with ultrapure water and anhydrous ethanol, using ultrasonic washing, and the washing time is 8-12 minutes.

8. The method for preparing the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 2, characterized in that: In step 3), drying is done naturally at room temperature.

9. The application of the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 1, characterized in that: Application of the PP2A / ZnO-PDA / GCE electrochemical biosensor in the detection of diarrhetic shellfish toxins in seawater and marine organisms.

10. The application of the PP2A / ZnO-PDA / GCE electrochemical biosensor according to claim 9, characterized in that: Application of the PP2A / ZnO-PDA / GCE electrochemical biosensor in the detection of datian sclerotium acid and fin algae toxins.

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