A self-powered photoelectrochemical sensor for microcystin detection and preparation method and application thereof

Abstract

Disclosed are a self-powered photoelectrochemical sensor for nodularin toxin detection and a preparation method and application thereof, wherein a TiO2-MXene suspension is added dropwise to a PWE surface, AuNPs are fixed on the TiO2-MXene / PWE surface by an electrodeposition method to obtain Au / TiO2-MXene / PWE, and a target working electrode MCH / Au / TiO2-MXene / PWE is obtained by processing with a mercapto-functionalized probe CP and MCH, so that a self-powered photoelectrochemical sensor is prepared, efficient and sensitive detection of nodularin toxin is realized, the PEC sensor prepared by the application has the remarkable characteristics of high sensitivity, low detection limit and high accuracy, has a good application prospect, and provides a favorable guarantee for food safety.

Description

Technical Field

[0001] This invention belongs to the field of photoelectrochemical detection technology, and in particular relates to a self-powered photoelectrochemical sensor for the detection of algal toxins, its preparation method, and its application. Background Technology

[0002] Currently, eutrophication leading to harmful cyanobacterial blooms is occurring frequently worldwide. The toxins produced by these harmful cyanobacteria are collectively known as cyanobacterial toxins, which have seriously endangered the safety of drinking water. Drinking water contaminated with cyanobacteria can cause acute poisoning symptoms and chronic liver disease. Nodulotoxin (NOD), one of the three most common hepatotoxins among cyanobacterial toxins, has attracted widespread attention. Nodulotoxin is a cyclic pentapeptide compound with good water solubility and thermal stability. These toxins can occur in both saline and freshwater lakes. Due to its structure similar to microcystin, NOD can also act as an effective inhibitor of protein phosphatases PP-1, PP-2A, and PP-3, further promoting the development of liver cancer. When humans ingest NOD, symptoms include depression, hemorrhagic diarrhea, and shock; in severe cases, death can occur within 24 hours.

[0003] Numerous cases of NOD poisoning in animals have been reported worldwide, posing a significant threat to human and animal safety. Currently, the US EPA has included NOD in its candidate list of drinking water contaminants. To date, various analytical techniques have been developed for NOD detection, including high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gas chromatography (GC). However, these methods require the use of hazardous reagents and expensive equipment, as well as specialized operators. Furthermore, these detection methods cannot meet the requirements for high-throughput detection. Therefore, a rapid and sensitive method for NOD detection is urgently needed.

[0004] Photoelectrochemistry, as an emerging analytical technique, possesses advantages such as simplicity, speed, and high sensitivity. In recent years, its application in monitoring water pollutants and toxins has attracted widespread research attention. While satisfactory detection results have been achieved in the detection of microcystin-LR, there are no reports on photoelectrochemical methods for detecting arthrocotoxins. Due to the significant hazard of arthrocotoxins, it is essential to develop a simple, rapid, efficient, and sensitive photoelectrochemical sensor for their detection. Summary of the Invention

[0005] To address the technical problems of high-throughput detection and high detection costs in existing technologies, this application proposes a self-powered photoelectrochemical sensor for the detection of PEC toxins, its preparation method, and its application, achieving efficient and sensitive detection of PEC toxins.

[0006] According to one aspect of the present invention, a method for preparing a self-powered photoelectrochemical sensor for detecting arthrophyllin toxins is provided, comprising the following steps:

[0007] (1) TiO2 and MXene are mixed in a solution system, wherein the amount of MXene added is 4%, to obtain a TiO2-MXene suspension;

[0008] (2) The TiO2-MXene suspension was dropped onto the surface of the paper-based working electrode PWE and dried to obtain TiO2-MXene / PWE. AuNPs were fixed on the surface of TiO2-MXene / PWE by electrodeposition to obtain Au / TiO2-MXene / PWE.

[0009] (3) The thiol-functionalized probe CP was dropped onto the surface of Au / TiO2-MXene / PWE and incubated overnight at room temperature to obtain the working electrode Au / TiO2-MXene / PWE. The working electrode was treated with MCH for 1 hour to block the unbound active sites and obtain the target working electrode MCH / Au / TiO2-MXene / PWE.

[0010] Furthermore, the synthesis method of MXene is as follows: LiF is dissolved in hydrochloric acid solution, Ti3AlC2 is added and stirred at 45°C for 24 hours, the solution is washed with ultrapure water and ethanol until the pH of the solution is >7, and the collected product is vacuum dried to obtain MXene.

[0011] Furthermore, the synthesis method of TiO2-MXene is as follows: TiO2 is fully dissolved in ultrapure water, MXene is added, ultrasonically mixed and stirred, and reacted in a polytetrafluoroethylene reactor at 110°C for 3 hours. The precipitate is washed several times by centrifugation with ultrapure water, and TiO2-MXene powder is obtained after freeze-drying.

[0012] Furthermore, the concentration of the TiO2-MXene suspension is 6 mg / mL. -1 The dosage is 10 μL.

[0013] Furthermore, the electrodeposition method is as follows: HAuCl4 solution is dropped onto the TiO2-MXene / PWE, a voltage of -0.245V is applied to the electrode and maintained for 100s, and the electrode is slowly washed several times with ultrapure water to remove the residual HAuCl4 solution on the PWE surface, and then dried to obtain the Au / TiO2-MXene / PWE.

[0014] According to a second aspect of the present invention, the application of a self-powered photoelectrochemical sensor in the detection of alginate toxins includes the following steps:

[0015] S1. Prepare AP / IS solution;

[0016] S2. NOD-R of different concentrations and the AP / IS solution were added dropwise to MCH / CP / Au / TiO2-MXene / PWE and incubated. HP1 and HP2 were added dropwise to MCH / CP / Au / TiO2-MXene / PWE and incubated for 2 hours. MB solution was added dropwise to MCH / CP / Au / TiO2-MXene / PWE and incubated. The electrode surface was rinsed with Tris-HCl buffer.

[0017] S3. Detect the PEC signal in an electrolytic cell containing PBS buffer containing ascorbic acid, using visible light for detection, and analyze the detection results.

[0018] Furthermore, the preparation method of the AP / IS solution is as follows: the aptamer chain AP and the initiator chain IS are mixed in equal amounts to obtain AP / IS, and the DNA chain is added to Tris-HCl buffer for dilution. After incubation, the solution is gradually cooled to room temperature and stored at low temperature for later use.

[0019] Furthermore, the concentrations of HP1 and HP2 are 2 μmol / L. -1 .

[0020] Furthermore, the analysis step involves recording the photocurrent intensity of NOD at different concentrations and plotting a linear curve with the logarithm of the NOD molar concentration on the x-axis and the photocurrent intensity on the y-axis.

[0021] The beneficial effects of this invention are as follows:

[0022] The self-powered photoelectrochemical sensor for detecting Arachnium toxins prepared in this invention has the following advantages compared with traditional Arachnium toxin detection methods:

[0023] (1) The combination of TiO2 and MXene effectively enhances the performance of optoelectronic materials, increases the loading capacity of the aptamer, and significantly enhances the absorption of visible light by MXene, thus expanding the application range of the material. It features simple operation, high sensitivity, good stability, and strong anti-interference ability.

[0024] (2) This invention uses the initiating chain IS and HP1 and HP2 as raw materials. The target NOD triggers the HCR reaction to initiate the amplification of double-stranded DNA. MB molecules, as signal amplifiers, can be embedded in the amplified double-stranded DNA, successfully amplifying the PEC detection signal, thereby achieving highly sensitive detection of the target NOD.

[0025] (3) HCR reaction only needs to be carried out at room temperature. The reaction time is short and the conditions are mild. It is simple to operate and does not have high requirements for the detection environment. It has good application prospects and provides favorable protection for food safety. Attached Figure Description

[0026] The accompanying drawings are included to provide a further understanding of the embodiments, and these drawings are incorporated in and constitute a part of this specification. The drawings illustrate embodiments and, together with the description, serve to explain the principles of the invention. Other embodiments and many anticipated advantages of the embodiments will be readily recognized as they become better understood through reference to the following detailed description.

[0027] Figure 1 This is a flowchart of the preparation method of the self-powered photoelectrochemical sensor of the present invention;

[0028] Figure 2 This is a flowchart illustrating the specific detection method of Arachnida toxin detection according to the present invention;

[0029] Figure 3 The image shows a SEM image of MXene prepared in Example 1 of this invention.

[0030] Figure 4 This is a SEM image of TiO2-MXene prepared in Example 1 of the present invention;

[0031] Figure 5 High-resolution XPS images of Au / TiO2-MXene and CP / Au / TiO2-MXene prepared in Example 1 of this invention;

[0032] Figure 6 These are polypropylene gel electrophoresis images of the DNAs used in the HCR reaction of Example 1 of the present invention.

[0033] Figure 7 and Figure 8 The Nyquist plot and photocurrent change diagram are respectively shown during the electrode modification process prepared in Example 1 of the present invention.

[0034] Figure 9 The photocurrent response diagram of the photoelectric chemical sensor prepared in Example 1 of this invention for detecting different concentrations of NOD;

[0035] Figure 10 The graph shows the linear relationship between the logarithm of the photocurrent and the photocurrent at different NOD concentrations.

[0036] Figure 11 The PEC response diagrams are shown for different amounts of MXene in TiO2-MXene prepared in Example 1 of this invention.

[0037] Figure 12PEC response diagrams at different HCR reaction times;

[0038] Figure 13 PEC response diagrams for different concentrations of HP1 and HP2. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] like Figure 1 As shown, a method for preparing a self-powered photoelectrochemical sensor for detecting arthrophyllin toxins is presented.

[0045] Example 1

[0046] A method for preparing a self-powered photoelectrochemical sensor for detecting arthrophyllin toxins includes the following steps:

[0047] Step 1, Synthesis of MXene: Dissolve 0.8 g of LiF in 10 mL of hydrochloric acid solution (9 mol L). -1 In the process, after stirring the solution for half an hour, 1 g of Ti3AlC2 was added and stirred at 45°C for 24 hours. The obtained product was washed several times with ultrapure water and ethanol until the pH of the solution was >7. The collected product was then vacuum dried using a freeze dryer for 12 hours to obtain MXene powder.

[0048] Figure 3 The image shows a SEM image of MXene prepared in Example 1. As can be seen from the image, MXene exhibits a well-defined layered structure.

[0049] Step 2, Synthesis of TiO2-MXene: 0.2g TiO2 was fully dissolved in 20mL of ultrapure water, a certain amount of MXene was added, ultrasonically mixed, and stirred for 1 hour; the mixture was reacted in a polytetrafluoroethylene reactor at 110℃ for 3 hours, the precipitate was washed several times by centrifugation with ultrapure water, and TiO2-MXene powder was obtained after freeze-drying.

[0050] Figure 4 The image shows a SEM image of TiO2-MXene prepared in Example 1, which shows that a large number of TiO2 particles are attached to the surface of the MXene layered structure.

[0051] Step 3, Preparation of Au / TiO2-MXene / PWE electrode: 10 μL of a 6 mg / mL solution was prepared. -1 TiO2-MXene suspension was dropped onto the PWE electrode and dried at 60°C to obtain TiO2-MXene / PWE. Au / TiO2-MXene / PWE was prepared by electrodeposition. Specifically, 15 μL of HAuCl4 solution was dropped onto TiO2-MXene / PWE to ensure that the working electrode area of ​​PWE was covered. A voltage of -0.245V was applied to the electrode and held for 100s. The electrode was then slowly washed several times with ultrapure water to remove the residual HAuCl4 solution on the surface of PWE. Finally, the electrode was dried at 60°C to obtain the Au / TiO2-MXene / PWE electrode.

[0052] Figure 5 The high-resolution XPS images of Au / TiO2-MXene and CP / Au / TiO2-MXene show that the proportion of the Au(I) peak area increased by 11.5% after CP modification, indicating that CP was successfully fixed on the surface of the sensing platform through gold thiol bonds.

[0053] Step 4, Preparation of MCH / Au / TiO2-MXene / PWE: 10 μL of 2 μmol / L... -1The thiol-functionalized probe CP was dropped onto the Au / TiO2-MXene / PWE surface and incubated overnight at room temperature. The solution was then added with 10 mmol L... -1 The prepared electrode was washed three times with Tris-HCl buffer to obtain the working electrode Au / TiO2-MXene / PWE. The working electrode was then treated with MCH for 1 hour to block unbound active sites, resulting in the target working electrode MCH / Au / TiO2-MXene / PWE.

[0054] Step 5: Prepare 100 μmol / L solution of all DNA with nuclease-free water. -1 To determine the concentration, equal volumes of aptamer strand (AP) and initiator strand (IS) were mixed to obtain AP / IS, and then diluted with the remaining DNA strands to a concentration of 10 μmol / L in Tris-HCl buffer (pH = 7.4, containing 100 mM NaCl, 10 mM MgCl2, and 10 mM KCl). -1 The prepared solutions were incubated at 95°C for 5 min and then gradually cooled to room temperature at a rate of 0.1°C / s for 60 min to prevent mismatches between DNA bases. All prepared solutions were stored at 4°C for subsequent use.

[0055] A self-powered photoelectrochemical sensor for NOD detection using Arachnium toxins:

[0056] Step 6: Mix NOD of different concentrations with AP / IS solution (0.5 μmol / L). -1 Incubate at room temperature for 30 min, then add 10 μL of each solution to MCH / CP / Au / TiO2-MXene / PWE and incubate for 1 hour; subsequently, add 10 μL of a 2 μmol / L solution. -1 HP1 and HP2 were added dropwise to the prepared working electrode and incubated for 2 hours; 10 μL of 1 mmol L... -1 MB solution was dropped onto the modified PWE and incubated for 30 minutes to embed it in the DNA double strand on the electrode surface. Then, 10 mmol / L of the solution was used. -1 The electrode surface was rinsed with Tris-HCl buffer. Using an electrochemical workstation, a solution containing 0.1 mol / L... -1 PBS buffer (containing 0.1 mol L) -1 The PEC signal was measured in an electrolytic cell containing ascorbic acid; the bias voltage was 0V, and a xenon lamp with a visible light filter was used as the light source for detection, and the photocurrent intensity of different NOD concentrations was recorded.

[0057] Figure 6 The polypropylene gel electrophoresis images of the DNAs in the HCR reaction confirm the feasibility of the HCR reaction. Figure 7 and Figure 8The Nyquist plot and photocurrent change plot are shown respectively during the electrode modification process. The results of both plots jointly confirm the feasibility of the constructed NOD aptamer sensor.

[0058] Step 7: Based on the recorded photocurrent intensities of different NOD concentrations, plot a linear curve with the logarithm of NOD molar concentration on the x-axis and photocurrent intensity on the y-axis.

[0059] Figure 9 The graph shows the photocurrent response at different NOD concentrations. It can be seen from the graph that the photocurrent increases with increasing NOD concentration. Figure 10 The graph shows the linear relationship between the logarithm of the photocurrent and the photocurrent under different NOD concentrations, exhibiting a good linear relationship.

[0060] Example 2

[0061] The preparation steps of a self-powered photoelectrochemical sensor for detecting arthrophyllin toxins are the same as in Example 1;

[0062] Step 2: To determine the optimal amount of Mxene, different amounts (mass ratios of 0%, 2%, 4%, 6%, and 8%) of Mxene were added to TiO2, and the photocurrent was measured after reacting in the solution at 110°C for 3 hours.

[0063] Figure 11 The PEC response graphs for different amounts of Mxene are shown. It can be seen from the graph that as the amount of Mxene added increases, the photoelectric signal of TiO2-MXene is enhanced, and it reaches the highest value when the amount of Mxene added reaches 4%. Therefore, 4% is selected as the optimal amount of Mxene added.

[0064] Steps 3-5 are the same as in Example 1;

[0065] Step 6: To optimize the HCR reaction, the reaction time and the concentrations of HP1 and HP2 were selected. First, the photocurrent was recorded after different reaction times (0.5, 1, 1.5, 2, 2.5, 3 hours). Under the optimal reaction time conditions, concentrations of 0.5, 1, 1.5, 2, 2.5, and 3 μmol L were used. -1 HP1 and HP2 underwent HCR reaction, and the photocurrent magnitude was recorded;

[0066] Figure 12 The graphs show the PEC response at different HCR reaction times. As can be seen from the graphs, the PEC signal gradually increases with the extension of the reaction time, and it basically stabilizes at a reaction time of 2 hours. Therefore, the optimal HCR reaction time is selected as 2 hours. Figure 13The PEC response graphs for different concentrations of HP1 and HP2 were used to determine the optimal amounts of HP1 and HP2 to achieve the best HCR reaction effect. The graphs show that the photocurrent increases with increasing HP1 and HP2 concentrations, reaching a maximum at a concentration of 2 μmol / L. -1 The concentrations then tended to stabilize; therefore, the final concentrations of HP1 and HP2 were chosen to be 2 μmol / L. -1 .

[0067] This invention proposes a self-powered photoelectrochemical sensor for the detection of phycocyanin toxins. The method generates a large amount of soluble ethers (IS) in the presence of the target NOD, which triggers HCR to form long DNA with HP1 and HP2 on a PWE. Subsequently, under visible light excitation, leuco-MB embedded in the dsDNA significantly enhances the PEC response of TiO2-MXene. The constructed biosensor exhibits a linear range of 20 fg / mL. -1 Up to 10 ng mL -1 NOD-R has a low LOD of 19.6 fg / mL. -1 The constructed PEC biosensor has the advantages of high sensitivity, good stability, and strong specificity.

[0068] Reliability assessment of the test:

[0069] The reliability and practicality of the self-powered PEC-adapted sensor for NOD detection were evaluated in real drinking water samples. Spiked samples were detected using both HPLC and the method of this invention. Furthermore, the experimental data obtained by the two methods were analyzed using SPSS software, and the results are shown in Table 1.

[0070]

[0071] * This refers to the t-test statistical analysis between the two methods. P>0.05 indicates no significant difference.

[0072] Spiked samples were analyzed using HPLC and the method of this invention. Results showed that the recovery rate of NOD in drinking water was 96.2%–103.2%, with a maximum RSD of 4.8% in repeated tests. The recovery rate of the standard HPLC method was 98.7%–102.2%, with an RSD of 4.4%–5.2%. Furthermore, the experimental data obtained by the two methods were analyzed using SPSS software, and the p-value was 0.96, indicating that there was no statistically significant difference between the self-powered PEC aptamer sensor and the HPLC method. Therefore, the aptamer sensor of this invention has high reliability for NOD detection in water samples and can be effectively applied to NOD-R detection in real-world samples.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a self-powered photoelectrochemical sensor for detecting arthrophyllin toxins, characterized in that, Includes the following steps: (1) TiO2 and MXene are mixed in a solution system, wherein the amount of MXene added is 4%, to obtain a TiO2-MXene suspension; (2) The TiO2-MXene suspension was dropped onto the surface of the paper-based working electrode PWE and dried to obtain TiO2-MXene / PWE. AuNPs were fixed on the surface of TiO2-MXene / PWE by electrodeposition to obtain Au / TiO2-MXene / PWE. (3) The thiol-functionalized probe CP was dropped onto the surface of Au / TiO2-MXene / PWE and incubated overnight at room temperature to obtain the working electrode Au / TiO2-MXene / PWE. The working electrode was treated with MCH for 1 hour to block the unbound active sites and obtain the target working electrode MCH / Au / TiO2-MXene / PWE. The synthesis method of MXene is as follows: LiF was dissolved in hydrochloric acid solution, Ti3AlC2 was added and stirred at 45°C for 24 hours. The solution was washed with ultrapure water and ethanol until the pH was >7. The collected product was dried under vacuum to obtain the MXene. The synthesis method of the TiO2-MXene is as follows: TiO2 was fully dissolved in ultrapure water, MXene was added, ultrasonically mixed and stirred, and reacted in a polytetrafluoroethylene reactor at 110°C for 3 hours. The precipitate was washed several times by centrifugation with ultrapure water and then freeze-dried to obtain TiO2-MXene powder. The electrodeposition method is as follows: HAuCl4 solution was added dropwise to the TiO2-MXene / PWE, a voltage of -0.245V was applied to the electrode and held for 100s, and the electrode was slowly washed several times with ultrapure water to remove the residual HAuCl4 solution on the surface of the PWE. The electrode was then dried to obtain the Au / TiO2-MXene / PWE.

2. The method for preparing a self-powered photoelectrochemical sensor for detecting arthrophyllin toxins according to claim 1, characterized in that, The concentration of the TiO2-MXene suspension was 6 mg / mL. -1 The dosage is 10 μL.

3. The application of the self-powered photoelectrochemical sensor prepared by the method according to any one of claims 1-2 in the detection of alginate toxins, characterized in that, The specific testing method includes the following steps: S1. Prepare AP / IS solution; S2. NOD-R of different concentrations and the AP / IS solution were added dropwise to MCH / CP / Au / TiO2-MXene / PWE and incubated. HP1 and HP2 were added dropwise to MCH / CP / Au / TiO2-MXene / PWE and incubated for 2 hours. MB solution was added dropwise to MCH / CP / Au / TiO2-MXene / PWE and incubated. The electrode surface was rinsed with Tris-HCl buffer. S3. Detect the PEC signal in an electrolytic cell containing PBS buffer containing ascorbic acid, using visible light for detection, and analyze the detection results.

4. The application of the self-powered photoelectrochemical sensor according to claim 3 in the detection of arthropoda toxins, characterized in that, The preparation method of the AP / IS solution is as follows: The aptamer strand AP and the initiator strand IS were mixed in equal amounts to obtain AP / IS. The DNA strand was added to Tris-HCl buffer to dilute the mixture. After incubation, the mixture was gradually cooled to room temperature and stored at low temperature for later use.

5. The application of the self-powered photoelectrochemical sensor according to claim 3 in the detection of arthropoda toxins, characterized in that, The concentrations of HP1 and HP2 were 2 μmol / L. -1 .

6. The application of the self-powered photoelectrochemical sensor according to claim 3 in the detection of arthropoda toxins, characterized in that, The analysis and detection results include recording the photocurrent intensity of different NOD concentrations, and plotting a linear curve with the logarithm of NOD molar concentration on the x-axis and photocurrent intensity on the y-axis.

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