A method for detecting polychlorinated biphenyls using an electrochemical aptamer sensor

Abstract

The application discloses a method for detecting polychlorinated biphenyl by using an electrochemical aptamer sensor. x The sensor comprises a glassy carbon electrode, a surface of a detection end of the glassy carbon electrode is decorated with an electroactive nanomaterial, the electroactive nanomaterial comprises Ti3C2T 0000001 Nanosheet, a conductive polymer material is embedded in an interlayer structure of the nanosheet and forms a composite nanomaterial, and gold nanoparticles are deposited on a surface of the composite nanomaterial; a capture chain is self-assembled on the electroactive nanomaterial, a specific aptamer probe is combined on the capture chain, a DNA double-stranded structure is formed, and an electroactive label is introduced. In the application, the sensor has the advantages of wide detection range, low detection limit, strong anti-interference ability, good stability and the like, can realize specific detection of polychlorinated biphenyl, has the advantages of low cost, short time consumption, high accuracy, good adaptability and the like, and has important significance for rapid detection of polychlorinated biphenyl.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensing technology and relates to a method for detecting polychlorinated biphenyls using an electrochemical aptamer sensor. Background Technology

[0002] Methods for detecting polychlorinated biphenyls (PCBs) mainly include chemiluminescence immunoassay, high-performance liquid chromatography (HPLC), colorimetry, and chromatography coupled with other techniques. While each method has its advantages, they also have limitations, such as complex sample preparation, long processing times, expensive instruments, and insufficient detection limits. Therefore, developing a simple, highly sensitive, and selective method for the quantitative detection of PCBs in the environment is essential.

[0003] In recent years, electrochemical aptamer sensors based on nucleic acid aptamers have found wide application in detecting polychlorinated biphenyls (PCBs) in the environment due to the high affinity and selectivity of nucleic acid aptamers for PCBs. In electrochemical aptamer sensors, nanomaterials are typically deposited on the surface of the electrode detection end. These nanomaterials can serve not only as carriers for immobilizing biorecognition molecules but also as labeling matrices for biomolecules, significantly increasing signal strength and improving the ability to recognize analytes, enabling them to interact with the analytes. However, existing electrochemical aptamer sensors primarily utilize nanomaterials including carbon nanomaterials, noble metal nanomaterials, metal oxide nanomaterials, and composite materials derived from them. These materials still suffer from the following drawbacks: (a) high cost. For example, graphene, as a nanomaterial, suffers from high cost and difficult preparation. Similarly, the preparation cost of noble metal nanoparticles is relatively high, especially when using composite materials derived from multiple nanomaterials; (b) few active sites. Noble metal nanoparticles tend to aggregate and have poor dispersibility, resulting in a small number of active sites and ultimately hindering the improvement of detection sensitivity. A common improvement measure is to disperse noble metal nanoparticles on a carrier material. However, existing conventional carrier materials still suffer from few active sites and poor conductivity, making it difficult to improve the dispersibility of noble metal nanoparticles and enhance detection performance. Therefore, obtaining a nanomaterial with excellent conductivity, good dispersibility, and high stability is crucial for promoting the widespread application of electrochemical aptamer sensors in detecting polychlorinated biphenyls (PCBs) in the environment.

[0004] MXenes are a class of two-dimensional transition metal carbides / nitrides with a layered structure, and their general formula is M. n+1 X n T x Where M represents an early transition metal element (such as Ti, V, Cr, Zr, Nb, Mo), X represents carbon or nitrogen, and T represents... xRepresents surface functional groups (such as -O, -OH, -F), where n is 1, 2, 3, or 4. Ti3C2T x As an emerging type of MXene material, Ti3C2T possesses numerous excellent properties such as large specific surface area, high conductivity, and good hydrophilicity, making it highly suitable as an electrode modification material for biosensors and attracting widespread attention in the field of electrochemistry. However, Ti3C2T... x The thin film is prone to stacking during drying and film formation, and Ti3C2T x The edges and surfaces of Ti3C2T are prone to electrochemical oxidation, transforming into insulating oxides such as TiO2, leading to decreased conductivity and signal attenuation. These factors all contribute to a decrease in electrochemical signal, affecting the stability of electrochemical sensing detection; simultaneously, Ti3C2T x The strong hydrophilicity of the surface can also lead to non-specific adsorption of some biomolecules, interfering with detection specificity and limiting its practical application. These drawbacks make it difficult for existing Mxene-based electrochemical aptamer sensors to quickly and accurately detect polychlorinated biphenyls (PCBs) in the environment.

[0005] For the reasons stated above, this invention is proposed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for detecting polychlorinated biphenyls using an electrochemical aptamer sensor that is low in cost, short in time, highly accurate and adaptable.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for detecting polychlorinated biphenyls (PCBs) using an electrochemical aptamer sensor is disclosed. The method utilizes an electrochemical aptamer sensor to detect PCBs in water. The electrochemical aptamer sensor includes a glassy carbon electrode used as the working electrode in a three-electrode system. The surface of the detection end of the glassy carbon electrode is modified with an electroactive nanomaterial. A trapping strand is self-assembled on the electroactive nanomaterial, and a specific aptamer probe for specific recognition of PCBs is bound to the trapping strand. The trapping strand and the specific aptamer probe strand are complementary to form a DNA double-stranded structure. An electroactive tag is introduced into the DNA double-stranded structure. The electroactive nanomaterial includes Ti3C2T. x Nanosheets, the Ti3C2T x Conductive polymer materials are embedded in the interlayer structure of nanosheets to form composite nanomaterials, and gold nanoparticles are deposited on the surface of the composite nanomaterials; the conductive polymer material is a polymer composed of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate.

[0008] A further improvement to the above method is the preparation method of the working electrode, which includes the following steps: S1. Add HCl solution to the reaction vessel, add LiF in portions while stirring, add Ti3AlC2 to react, centrifuge, wash, and obtain multilayer Ti3C2T. x ; S2, the multilayer Ti3C2T prepared in step S1 x Add to deionized water, sonicate to disperse, centrifuge, collect the supernatant, freeze-dry to obtain Ti3C2T x Nanosheets; S3, the Ti3C2T prepared in step S2 x Nanosheets were added to deionized water and ultrasonically dispersed. Then, conductive polymer solution and dimethyl sulfoxide solution were added separately and stirred to obtain a composite nanomaterial dispersion. S4. The composite nanomaterial dispersion prepared in step S3 is drop-coated onto the detection end surface of the glassy carbon electrode and dried to obtain a glassy carbon electrode modified with composite nanomaterial. S5. The glassy carbon electrode modified with composite nanomaterials prepared in step S4 is immersed in HAuCl4 solution for electrodeposition to obtain a glassy carbon electrode modified with electroactive nanomaterials.

[0009] In a further improvement to the above method, in step S1, the reaction is carried out at a temperature of 35°C; the reaction time is 12h to 36h; and the washing is performed by washing the centrifuged solid product with deionized water until the pH of the washing solution is 6 to 7.

[0010] In a further improvement to the above method, in step S2, the ultrasonic dispersion time is 0.5 h to 1 h; and the freeze-drying time is 24 h to 36 h.

[0011] The above method is further improved in step S3, where the Ti3C2T x The ratio of nanosheets to deionized water is 0.5–3 mg:1 mL; the ultrasonic dispersion time is 5–20 min; the amount of conductive polymer solution added is 5%–15% of the total volume of the composite nanomaterial dispersion; the conductive polymer solution is a PEDOT:PSS solution; the amount of dimethyl sulfoxide added is 5%–15% of the total volume of the composite nanomaterial dispersion; the stirring time is 5 h.

[0012] In a further improvement to the above method, in step S4, the concentration of the composite nanomaterial dispersion is 0.5 mg / mL; the drying time is 1 h; and the drying temperature is 40 °C.

[0013] In a further improvement to the above method, in step S5, the concentration of the HAuCl4 solution is 1% W / V; the electrodeposition time is 20 s to 100 s; and the electrodeposition potential is -0.2 V.

[0014] A further improvement to the above method is that the preparation method of the working electrode further includes the following steps: S6. The trapping chain solution is dropped onto the detection end surface of the glassy carbon electrode modified with electroactive nanomaterials obtained in step S5 and incubated to allow the trapping chain to be modified on the detection end surface of the glassy carbon electrode, thus obtaining a glassy carbon electrode modified with trapping chains. S7. Add the sealing agent solution dropwise to the detection end surface of the glassy carbon electrode modified with the trapping chain obtained in step S6 and incubate it to seal the excess active sites and clean the electrode surface. S8. The specific aptamer probe solution is dropped onto the detection end surface of the glassy carbon electrode obtained in step S7 after incubation with the blocking agent solution, and incubated to modify the specific aptamer probe on the detection end surface of the glassy carbon electrode, thereby obtaining a glassy carbon electrode modified with a trapping chain and a specific aptamer probe. S9. Immerse the glassy carbon electrode modified with the trapping chain and specific aptamer probe obtained in step S8 in an electroactive tag solution, and introduce an electroactive tag on the surface of the detection end of the glassy carbon electrode to obtain the working electrode.

[0015] In a further improvement to the above method, in step S6, the concentration of the chain-capturing solution is 0.5 μM to 3 μM; the incubation time is 10 h to 14 h; and the incubation temperature is 0 to 5 °C.

[0016] In a further improvement to the above method, in step S7, the concentration of the blocking agent solution is 0.1 mM; the blocking agent in the blocking agent solution is MCH; and the incubation time is 1 h to 3 h.

[0017] In a further improvement to the above method, in step S8, the concentration of the specific aptamer probe solution is 0.5 μM to 3 μM; the incubation time is 1 h to 3 h; and the incubation temperature is 0 to 5 °C.

[0018] In a further improvement to the above method, in step S9, the electroactive tag solution is a phosphate buffer containing an electroactive tag; the concentration of the electroactive tag in the electroactive tag solution is 0.1 mM; the electroactive tag is methylene blue; the phosphate buffer is Tris-HCl buffer; the soaking time is 5 min to 25 min; after the soaking is completed, the following step is also included: immersing the glassy carbon electrode in Tris-HCl buffer to remove unbound electroactive tag molecules.

[0019] A further improvement to the above method, utilizing an electrochemical aptamer sensor to detect polychlorinated biphenyls (PCBs) in water, includes the following steps: (1) The detection end of the working electrode is immersed in the test solution to react, and the polychlorinated biphenyls in the test solution are specifically identified and captured to obtain a working electrode that captures polychlorinated biphenyls; (2) The working electrode, counter electrode and reference electrode that capture polychlorinated biphenyls are placed in a buffer solution to establish a three-electrode system. The electrical signal of the working electrode is collected by differential pulse voltammetry to obtain the current value of the solution to be tested. (3) Input the current value of the test solution into the detection linear regression equation constructed by the relationship between different concentrations and current changes, and calculate the concentration of polychlorinated biphenyls in the test solution.

[0020] In a further improvement to the above method, in step (3), when the polychlorinated biphenyls in the test solution are PCB77, the detection linear regression equation constructed based on the relationship between different concentrations and current changes is as follows: I(μA) = 1.8556 lgC(ng·L) -1 )+3.7433(1); In equation (1), I represents the difference in peak current change, in μA; C is the concentration of PCB77 in the test solution, in ng / L; and the correlation coefficient R0 is... 2 =0.9939, the detection linear range is 0.05 ng / L~10 4 The detection limit is 0.012 ng / L.

[0021] In a further improvement to the above method, in step (1), when the polychlorinated biphenyl in the test solution is PCB77, the capture chain in the working electrode has a nucleotide sequence as shown in SEQ ID NO.1, and the specific aptamer probe has a nucleotide sequence as shown in SEQ ID NO.2.

[0022] In a further improvement to the above method, in step (1), the test solution is lake or river water containing polychlorinated biphenyls (PCBs); the concentration of PCBs in the test solution is 0.05 ng / L to 10 ng / L. 4 ng / L; the pH value of the test solution is 6.0 to 8.5.

[0023] In a further improvement to the above method, in step (1), the reaction time is 10 min to 50 min.

[0024] Compared with the prior art, the advantages of the present invention are as follows: (1) In view of the shortcomings of existing electrochemical aptamer sensors, such as high cost, few active sites, easy aggregation, and poor stability of electroactive nanomaterials, and the resulting defects such as difficulty in reducing detection costs, poor detection effect and poor reusability, this invention creatively proposes a method for detecting polychlorinated biphenyls using an electrochemical aptamer sensor. The electrochemical aptamer sensor includes a glassy carbon electrode used as the working electrode in a three-electrode system. The surface of the detection end of the glassy carbon electrode is modified with electroactive nanomaterials, including Ti3C2T x Nanosheets in Ti3C2T x Conductive polymer materials are embedded in the interlayer structure of nanosheets to form composite nanomaterials. Gold nanoparticles are deposited on the surface of the composite nanomaterials. The conductive polymer material is a polymer composed of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate. Compared with conventional conductive polymers (such as polypyrrole), the present invention uses a polymer (PP) composed of poly(3,4-ethylenedioxythiophene) (PEDOT) and polystyrene sulfonate (PSS) as the conductive polymer material, which has the following advantages: (a) PEDOT:PSS has intrinsically high conductivity, excellent aqueous solution processability and film-forming flexibility, and can be used with Ti3C2T x (a) More uniform composite formation, forming a more stable and continuous conductive network; (b) The contained PSS anions can more effectively suppress Ti3C2T through intercalation and steric hindrance. x (c) The recombination of nanosheets maintains the active specific surface area to a greater extent; the tight composite interface of PEDOT and PSS can better block water and oxygen, significantly enhancing the oxidation resistance and long-term cycling stability of the material. Sensors prepared using this composite material can obtain better stability, and therefore it is used as a conductive polymer spacer inserted into Ti3C2T. x The nanosheets form a stable network structure, which not only inhibits layer stacking, maintains a high specific surface area, and enhances the mechanical toughness and ion transport efficiency of the film, but also allows for partial encapsulation of Ti3C2T due to their excellent conductivity and film-forming properties. x At the edges of the lamellar plates, a physical and electrochemical barrier is formed, delaying the degradation of Ti3C2T. x The electrochemical oxidation process maintains the long-term conductivity stability and signal reproducibility of the electrode. Simultaneously, PP can modulate the electrochemical oxidation process of Ti3C2T. x The surface interfacial microenvironment, with its uniform coverage, can reduce Ti3C2T xThe direct exposure of strongly hydrophilic functional groups on the surface reduces non-specific bioadsorption, providing a more compatible and ordered immobilization interface for gold nanoparticles (AuNPs) and biorecognition elements, thus enhancing sensor stability. In particular, it significantly increases the loading capacity of gold nanoparticles. On the one hand, gold nanoparticles possess excellent biocompatibility, which is beneficial for aptamer immobilization; on the other hand, it also provides more active sites for aptamer immobilization. Compared with conventional Ti3C2T… x Compared to composite materials, the electroactive nanomaterials used in this invention exhibit better conductivity, mechanical stability, and a greater number of active sites. Based on this, a glassy carbon electrode modified with electroactive nanomaterials is used as a substrate to co-assemble a trapping chain and a polychlorinated biphenyl (PCB) aptamer (specific aptamer probe). The resulting working electrode possesses advantages such as high conductivity, high specificity, strong binding ability, and high stability, making it a highly interference-resistant recognition element. Specifically, it not only significantly improves electrode stability but also reduces non-specific adsorption, thereby enhancing the detection repeatability and lifespan of the electrochemical aptamer sensor. Furthermore, the specific binding between the PCB aptamer and PCBs ensures that the sensor can accurately identify and respond to target molecules in complex biological samples, achieving high-sensitivity detection of PCBs. In addition, the hybridization of the trapping chain and the aptamer chain, along with the introduction of the electroactive tag, further enhances the intensity of the detection signal, enabling the sensor to accurately detect the presence of PCBs at low concentrations. Taking PCB77 as an example, the detection range of the electrochemical aptamer sensor used in this invention is 0.05 ng / L to 10 ng / L. 4 With a detection limit of 0.012 ng / L, it covers a wide range from low to high concentrations. It effectively overcomes the drawbacks of existing detection technologies, such as expensive equipment, complex processes, long processing times, and difficulty in field application. It boasts advantages such as good stability, long service life, strong anti-interference ability, wide detection range, and low detection limit. As a high-performance new sensor, it can be widely used to detect polychlorinated biphenyls (PCBs) in water. The entire detection process is short, making it suitable for rapid diagnosis and high-throughput screening. It offers advantages such as low cost, short processing time, high accuracy, and good adaptability, making it significant for the rapid detection of PCBs.

[0025] (2) The method of the present invention uses a working electrode preparation method that is relatively simple, does not require complex equipment and operation, has low preparation cost and high preparation efficiency, and is conducive to large-scale production and application. Attached Figure Description

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0027] Figure 1 The Ti3C2T prepared in Example 1 of this invention x / GCE、PP / Ti3C2T x / GCE、Au NPs / PP / Ti3C2T x Comparison of CV currents for / GCE.

[0028] Figure 2 The working electrode (Au NPs / PP / Ti3C2T) prepared in Example 1 of this invention x / GCE、cDNA / Au NPs / PP / Ti3C2T x / GCE、MCH / cDNA / Au NPs / PP / Ti3C2T x / GCE、Apt / MCH / cDNA / Au NPs / PP / Ti3C2T x A comparison chart of impedance test results for the bare GCE electrode and the blank electrode ( / GCE).

[0029] Figure 3 This is a current response diagram of the electrochemical aptamer sensor in Example 1 of the present invention when detecting standard solutions of different concentrations.

[0030] Figure 4 This is a graph of the detection linear regression equation constructed based on the relationship between different concentrations and current changes in the electrochemical aptamer sensor in Example 1 of the present invention.

[0031] Figure 5 This is a graph showing the effect of the electrochemical aptamer sensor on the peak current under different pH conditions in Example 1 of the present invention.

[0032] Figure 6 This is a graph showing the effect of different concentrations of specific aptamer probes on the peak current of the electrochemical aptamer sensor in Example 1 of the present invention.

[0033] Figure 7 This is a graph showing the effect of the electrochemical aptamer sensor on the current difference under different reaction time conditions in Example 1 of the present invention.

[0034] Figure 8 This is the current response diagram of the electrochemical aptamer sensor in Embodiment 2 of the present invention under the condition of the presence of interfering substances.

[0035] Figure 9 This is a stability comparison chart of the electrochemical aptamer sensor in Example 3 of the present invention. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0037] In the following embodiments of the present invention, unless otherwise specified, the materials and instruments used are commercially available, the equipment used is conventional equipment, and the data obtained are the average values ​​of more than three repeated experiments.

[0038] Example 1 A method for detecting polychlorinated biphenyls (PCBs) using an electrochemical aptamer sensor, specifically, using an electrochemical aptamer sensor to detect PCB77 in water, includes the following steps: (1) Immerse the detection end of the working electrode into the test solution (the solution contains PCB77 and has a pH of 7.4) for 30 min to specifically identify and capture PCB77 in the test solution to obtain a working electrode with captured PCB77.

[0039] In this step, the test solution consisted of Taozi Lake water and tap water containing PCB77. Before testing, the pH was adjusted to 7.4 using phosphate buffer solution. The concentration of the target substance (PCB77) in the sample is shown in Table 1.

[0040] (2) The working electrode, counter electrode (platinum electrode) and reference electrode (Ag / AgCl electrode) containing PCB77 were placed in a buffer solution to establish a three-electrode system and complete the preparation of the electrochemical aptamer sensor. The electrical signal of the working electrode was collected by differential pulse voltammetry to obtain the current value of the solution to be tested. (3) Input the current value of the test solution into the detection linear regression equation constructed by the relationship between different concentrations and current changes, and calculate the concentration of PCB77 in the test solution.

[0041] In this embodiment, a glassy carbon electrode is used as the working electrode. Electroactive nanomaterials are modified on the surface of the detection end of the glassy carbon electrode. A capture chain for recognizing and capturing PCB77 is self-assembled on the electroactive nanomaterials. A specific aptamer probe for specifically recognizing PCB77 is bound to the capture chain. The capture chain and the specific aptamer probe chain are complementary to form a DNA double-stranded structure. An electroactive tag is introduced into the DNA double-stranded structure. The electrochemical signal is measured by differential pulse voltammetry to realize the detection of polychlorinated biphenyls (such as PCB77).

[0042] In this embodiment, the electroactive nanomaterials used include Ti3C2T x Nanosheets in Ti3C2T xConductive polymer materials are embedded in the interlayer structure of nanosheets to form composite nanomaterials. Gold nanoparticles are deposited on the surface of the composite nanomaterials. The conductive polymer material is a polymer (PP) composed of poly(3,4-ethylenedioxythiophene) (PEDOT) and polystyrene sulfonate (PSS), which is a commercially available raw material.

[0043] In this embodiment, Ti3C2T is used. x Nanosheets were prepared by acid etching method, PP / Ti3C2T x The nanocomposite material Au NPs / PP / Ti3C2T was prepared by self-assembly. x Through PP / Ti3C2T x Preparation of surface electrodeposition Au NPs In this embodiment, the capture chain used has a nucleotide sequence as shown in SEQ ID NO.1, specifically: 5´-SH-CACGGGCCATCGGAA-3´.

[0044] In this embodiment, the specific aptamer probe used for specific recognition of PCB77 has a nucleotide sequence as shown in SEQ ID NO.2, specifically: 5´-GGCGGGGCTACGAAGTAGTGATTTTTTCCGATGGCCCGTG-3´.

[0045] In this embodiment, the electroactive tag used is methylene blue (MB).

[0046] In this embodiment, the method for preparing the working electrode includes the following steps: S1. Add HCl solution to the reaction vessel, add LiF in portions, stir well, add Ti3AlC2, stir well, react at 35℃ for 18 h, centrifuge the obtained product, wash, discard the supernatant, until the pH of the washing liquid is 6-7, to obtain multilayer Ti3C2T x .

[0047] S2, the multilayer Ti3C2T prepared in step S1 x Add to deionized water, sonicate for 1 hour, centrifuge, collect the supernatant, freeze-dry for 24 hours to obtain Ti3C2T x Nanosheets.

[0048] S3, Add 10 mg of Ti3C2T prepared in step S2. xNanosheets were added to 10 mL of deionized water and ultrasonically dispersed for 10 min. Then, 1 mL of conductive polymer solution (PEDOT:PSS solution) and 1 mL of dimethyl sulfoxide (DMSO) were added. The mixture was stirred at room temperature for 5 h to allow the conductive polymer material to be embedded into Ti3C2T. x Composite nanomaterials (PP / Ti3C2T) are formed in the interlayer structure of nanosheets. x ), to obtain a dispersion of composite nanomaterials.

[0049] S4. Drop-coat the 0.5 mg / mL composite nanomaterial dispersion prepared in step S3 onto the detection end surface of the glassy carbon electrode (GCE), and dry it at 40°C for 1 h to allow the PP / Ti3C2T to mature. x Composite nanomaterials were modified onto the detection end surface of a glassy carbon electrode (GCE) to obtain a glassy carbon electrode modified with composite nanomaterials, denoted as PP / Ti3C2T. x / GCE.

[0050] S5. Immerse the glassy carbon electrode modified with composite nanomaterials prepared in step S4 into a 1% W / V HAuCl4 solution, and perform electrodeposition for 30 s at a potential of -0.2 V. Gold nanoparticles are deposited in situ on the composite nanomaterials on the surface of the glassy carbon electrode to form electroactive nanomaterials (Au NPs / PP / Ti3C2T). x A glassy carbon electrode modified with electroactive nanomaterials was obtained, denoted as Au NPs / PP / Ti3C2T. x / GCE.

[0051] S6. Add 5 μL of a 2 μM trapping chain solution to the detection end surface of the glassy carbon electrode modified with electroactive nanomaterials obtained in step S5. Incubate at 4°C for 12 h to allow the trapping chains to be fixed to the detection end surface of the glassy carbon electrode via gold-thiol bonds. Wash the electrode surface with buffer to remove excess trapping chains, obtaining the glassy carbon electrode modified with trapping chains, denoted as cDNA / Au NPs / PP / Ti3C2T. x / GCE.

[0052] S7. Add 5 μL of 0.1 mM blocking agent (MCH) solution to the detection end surface of the glassy carbon electrode modified with the capture strand obtained in step S6. Incubate at room temperature for 1 h to block excess active sites. Wash the electrode surface with buffer to remove excess blocking agent. The resulting glassy carbon electrode is labeled MCH / cDNA / Au NPs / PP / Ti3C2T. x / GCE.

[0053] S8. Add 5 μL of a 2 μM specific aptamer probe solution to the detection end surface of the glassy carbon electrode obtained in step S7 after incubation with the blocking agent solution. Incubate at 37°C for 1 h to allow the specific aptamer probe to form a DNA double-stranded structure through complementarity with the capture strand. Wash the electrode surface with buffer to remove excess specific aptamer, obtaining a glassy carbon electrode modified with the capture strand and specific aptamer probe, denoted as Apt / MCH / cDNA / Au NPs / PP / Ti3C2T. x / GCE.

[0054] S9. Immerse the glassy carbon electrode modified with the capture strand and specific aptamer probe obtained in step S8 in 10 mL of electroactive tag solution (the electroactive tag solution is a phosphate buffer containing an electroactive tag, the concentration of the electroactive tag in the electroactive tag solution is 0.1 mM, wherein the electroactive tag is methylene blue, the phosphate buffer is Tris-HCl buffer, the concentration of KCl in the Tris-HCl buffer is 1.0 M, pH=7.4) for 15 min, so that the electroactive tag is introduced on the detection end surface of the glassy carbon electrode by binding with the DNA double strand. Then, immerse it in Tris-HCl buffer (the concentration of KCl in the Tris-HCl buffer is 1.0 M, pH=7.4) for 10 min to remove unbound MB molecules, and obtain the working electrode.

[0055] Control group: In step S3, no conductive polymer solution was added, and the corresponding glassy carbon electrode was denoted as Ti3C2T. x / GCE.

[0056] The Au NPs / PP / Ti3C2T prepared in Example 1 of this invention x Transmission electron microscopy, elemental mapping image analysis, and X-ray photoelectron spectroscopy were performed on the composite material. The results showed that PP can be used without damaging Ti3C2T. x A layered structure was successfully introduced, and gold nanoparticles could be well deposited on PP / Ti3C2T. x The surface of the composite nanomaterials indicates that Au NPs / PP / Ti3C2T x Successful preparation.

[0057] Figure 1 The Ti3C2T prepared in Example 1 of this invention x / GCE、PP / Ti3C2T x / GCE、Au NPs / PP / Ti3C2T x Comparison of CV currents for / GCE. When PP is introduced, Ti3C2T... xThe current was significantly increased, which can be attributed to the good solution processability and film forming ability of PP, enabling it to be used in Ti3C2T. x Continuous charge transport channels are formed between the layers, improving interfacial contact and reducing interfacial contact resistance. Further modification with gold nanoparticles, due to their excellent metallic conductivity and surface plasmon resonance, can further promote rapid collection and transport of interfacial charges, enhancing the electron migration efficiency of the material. This also explains the superior performance of the Au NPs / PP / Ti3C2T material of this invention. x Composite materials (electroactive nanomaterials) have a superior conductive network structure.

[0058] The working electrode (Au NPs / PP / Ti3C2T) prepared in Example 1 of this invention x / GCE、cDNA / Au NPs / PP / Ti3C2T x / GCE、MCH / cDNA / Au NPs / PP / Ti3C2T x / GCE、Apt / MCH / cDNA / Au NPs / PP / Ti3C2T x / GCE) and a blank electrode bare GCE in a 5.0 mM ferricyanide solution containing 0.1 mM KCl ([Fe(CN)6] ) 3- / 4- Impedance testing was performed in ), and the results are as follows: Figure 2 As shown.

[0059] Figure 2 The working electrode (Au NPs / PP / Ti3C2T) prepared in Example 1 of this invention x / GCE、cDNA / Au NPs / PP / Ti3C2T x / GCE、MCH / cDNA / Au NPs / PP / Ti3C2T x / GCE、Apt / MCH / cDNA / Au NPs / PP / Ti3C2T x A comparison chart of impedance test results for the bare GCE electrode and the blank electrode ( / GCE).

[0060] Depend on Figure 2 It can be seen that when Au NPs / PP / Ti3C2T x When modified on the electrode surface, the resistance is relatively low. When biological materials such as cDNA, MCH, MCH, and Apt are progressively modified onto Au NPs / PP / Ti3C2T, the resistance is significantly reduced. x After being applied to the / GCE surface, its resistance gradually increases again. This experimental phenomenon indicates that the aptamer molecule and its complementary chain have been successfully modified onto the electrode.

[0061] In this embodiment, the method for constructing the detection linear regression equation based on the relationship between different concentrations and current changes includes the following steps: (3.1) Immerse the detection end of the working electrode in different concentrations (0.05 ng / L to 10 ng / L). 4 The reaction was carried out in 20 ng / L standard solutions (these standard solutions were PCB77 solutions, all with a pH of 7.4) for 30 min to specifically identify and capture PCB77 in standard solutions of different concentrations, thus obtaining working electrodes with different concentrations of captured PCB77.

[0062] (3.2) The working electrode, counter electrode and reference electrode containing different concentrations of PCB77 were placed in a buffer solution to establish a three-electrode system. The electrical signal of the working electrode was collected by differential pulse voltammetry to obtain the current value of the standard solution with different concentrations.

[0063] (3.3) Using standard solutions with different logarithmic concentrations as the abscissa and the corresponding electrical signal difference (compared with the blank control) as the ordinate, a standard curve was plotted to obtain the detection linear regression equation constructed by the relationship between different concentrations and current changes.

[0064] Figure 3 This is a current response graph showing the detection of standard solutions of different concentrations by the electrochemical aptamer sensor in Example 1 of the present invention. Figure 3 It can be seen that, within a certain concentration range, the difference in current change increases with the increase of PCB77 concentration.

[0065] Figure 4 This is a graph of the detection linear regression equation constructed based on the relationship between different concentrations and current changes in the electrochemical aptamer sensor in Example 1 of the present invention.

[0066] Depend on Figure 4 It can be determined that the polychlorinated biphenyl in the test solution is PCB77. The corresponding linear regression equation for the detection relationship between different concentrations and current changes is as follows: I(μA) = 1.8556 lgC(ng·L) -1 )+3.7433(1); In equation (1), I represents the difference in peak current change, in μA; C is the concentration of PCB77 in the test solution, in ng / L; and the correlation coefficient R0 is... 2 =0.9939, the detection linear range is 0.05 ng / L~10 4 The detection limit is 0.012 ng / L.

[0067] Therefore, it can be seen that in this invention, Au NPs / PP / Ti3C2T xModified glassy carbon electrode (Au NPs / PP / Ti3C2T) x Electrochemical aptamer sensor (Apt / MCH / cDNA / Au NPs / PP / Ti3C2T) prepared by / GCE x The GCE (Gen PCB 77) can be used to detect PCB77 and can calculate the concentration of PCB77 based on a linear regression equation. It has the advantages of wide detection range and low detection limit.

[0068] The test results of PCB77 in Taozi Lake water and tap water are shown in Table 1.

[0069] Table 1. Validation results of the recovery rate of the test solution

[0070] As can be seen from Table 1, the electrochemical aptamer sensor (Apt / MCH / cDNA / Au NPs / PP / Ti3C2T) of the present invention... x Within the measurable concentration range ( / GCE), the recovery rate is generally between 96.02% and 101%, indicating accurate measurement results. Therefore, compared to traditional detection techniques, the detection method based on an electrochemical aptamer sensor in this invention is simple and rapid to operate.

[0071] As shown in Table 1, Au NPs / PP / Ti3C2T x Electrochemical aptamer sensor (Apt / MCH / cDNA / Au NPs / PP / Ti3C2T) prepared by GCE x The / GCE can be used to detect PCB77 in water, and can achieve good detection accuracy.

[0072] In this embodiment, the effect of the electrochemical aptamer sensor on the peak current under different pH values ​​(6.0, 6.5, 7.0, 7.4, 8.0, 8.5) was also investigated. Specifically, the electrochemical aptamer sensor from Example 1 was used to detect a PCB77 solution with a concentration of 5 ng / L. The results are as follows: Figure 5 As shown.

[0073] Figure 5 This is a graph showing the effect of the electrochemical aptamer sensor in Example 1 of the present invention on the peak current under different pH conditions. Figure 5 It is known that the current response signal value of the prepared electrochemical aptamer sensor continuously increases with increasing pH, reaching a peak at pH 7.4, after which its current response signal value begins to decrease sharply. Therefore, in this invention, the optimal pH value is selected as 7.4.

[0074] In this embodiment, the effect of different concentrations of specific aptamer probes on the peak current of the electrochemical aptamer sensor was also investigated. Specifically, the preparation method in Example 1 was used to prepare electrochemical aptamer sensors using specific aptamer probe solutions of different concentrations (0.5 μM to 2.0 μM). These electrochemical aptamer sensors were then used to detect a PCB77 solution with a concentration of 5 ng / L. The results are as follows. Figure 6 As shown.

[0075] Figure 6 This is a graph showing the effect of different concentrations of specific aptamer probes on the peak current of the electrochemical aptamer sensor in Example 1 of the present invention. Figure 6 It can be seen that the DPV signal detected by the electrochemical aptamer sensor increases significantly with the concentration of the specific aptamer probe in the range of 0.5 μM to 2.0 μM, and then the current response reaches its maximum value at 2.0 μM, indicating that the reaction has approached saturation at this point. Therefore, the optimal concentration of the specific aptamer probe solution is 2.0 μM.

[0076] In this embodiment, the effect of the electrochemical aptamer sensor on the current difference under different reaction time conditions was also investigated. Specifically, in step (1), the reaction time was 0 min to 30 min, that is, the current response of the electrochemical aptamer sensor to PCB77 solution with a concentration of 5.0 ng / L was investigated under different reaction times.

[0077] Figure 7 This is a graph showing the effect of the electrochemical aptamer sensor in Example 1 of the present invention on the current difference under different reaction time conditions. Figure 7 It can be seen that as the reaction time increases from 0 to 30 min, the current response signal value of the electrochemical aptamer sensor continuously increases, and then remains essentially constant after 30 min. This experimental phenomenon indicates that the optimal incubation and binding reaction time between the prepared electrochemical aptamer sensor and PCB77 is 30 min. Therefore, in this invention, all electrochemical detections are initiated after 30 minutes of electrode incubation.

[0078] Example 2 The electrochemical aptamer sensor (Apt / MCH / cDNA / Au NPs / PP / Ti3C2T) of Example 1 was examined. x The anti-interference capability of / GCE is specifically demonstrated by using the electrochemical aptamer sensor in Example 1 to detect PCB77 at a concentration of 10 ng / L and PCB77 at a concentration of 10 ng / L, respectively. 3 Interfering substances at concentrations of ng / L include PCB118, PCB138, 2,4,6-trichlorophenol (2,4,6-TCP), benzidine (BZD), and Pb. 2+ Hg 2+Nitrobenzene (NB) was tested (the determination method is as described in Example 1), and the test results are as follows: Figure 8 As shown.

[0079] Figure 8 This is the current response diagram of the electrochemical aptamer sensor in Embodiment 2 of the present invention under the condition of the presence of interfering substances. Figure 8 It can be seen that the electrochemical aptamer sensor (Apt / MCH / cDNA / Au NPs / PP / Ti3C2T) prepared in Example 1 of this invention... x The / GCE sensor exhibits good current response to PCB77 and virtually no current response to other interfering components, indicating that the electrochemical aptamer sensor of this invention has good anti-interference capability.

[0080] Example 3 The electrochemical aptamer sensor (Apt / MCH / cDNA / Au NPs / PP / Ti3C2T) of Example 1 was examined. x The stability of / GCE was specifically assessed by testing PCB77 (10 ng / L) with the electrochemical aptamer sensor from Example 1 under the same conditions. The prepared electrochemical aptamer sensor was placed at 4°C, and the current response was measured on days 4, 7, 12, and 15. During this period, the current signal remained almost constant. The test results are as follows: Figure 9 As shown.

[0081] Figure 9 This is a stability comparison chart of the electrochemical aptamer sensor in Example 3 of the present invention. Figure 9 It can be seen that the response current did not change significantly during this period, indicating that the electrochemical aptamer sensor of the present invention has good stability.

[0082] The above test results indicate that in this invention, Au NPs / PP / Ti3C2T x The prepared electrochemical aptamer sensor has advantages such as wide detection range, low detection limit, strong anti-interference ability, and good stability. It is a high-performance electrochemical sensor that can achieve specific detection of polychlorinated biphenyls and has great application value and prospects.

[0083] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting polychlorinated biphenyls using an electrochemical aptamer sensor, characterized in that, The method utilizes an electrochemical aptamer sensor to detect polychlorinated biphenyls (PCBs) in water. The electrochemical aptamer sensor includes a glassy carbon electrode used as the working electrode in a three-electrode system. The detection end surface of the glassy carbon electrode is modified with electroactive nanomaterials. A trapping strand is self-assembled on the electroactive nanomaterials, and a specific aptamer probe for specific recognition of PCBs is bound to the trapping strand. The trapping strand and the specific aptamer probe strand are complementary, forming a DNA double-stranded structure. An electroactive tag is introduced into the DNA double-stranded structure. The electroactive nanomaterials include Ti3C2T. x Nanosheets, the Ti3C2T x Conductive polymer materials are embedded in the interlayer structure of nanosheets to form composite nanomaterials, and gold nanoparticles are deposited on the surface of the composite nanomaterials; the conductive polymer material is a polymer composed of poly(3,4-ethylenedioxythiophene) and polystyrene sulfonate.

2. The method according to claim 1, characterized in that, The method for preparing the working electrode includes the following steps: S1. Add HCl solution to the reaction vessel, add LiF in portions while stirring, add Ti3AlC2 to react, centrifuge, wash, and obtain multilayer Ti3C2T. x ; S2, the multilayer Ti3C2T prepared in step S1 x Add to deionized water, sonicate to disperse, centrifuge, collect the supernatant, freeze-dry to obtain Ti3C2T x Nanosheets; S3, the Ti3C2T prepared in step S2 x Nanosheets were added to deionized water and ultrasonically dispersed. Then, conductive polymer solution and dimethyl sulfoxide solution were added separately and stirred to obtain a composite nanomaterial dispersion. S4. The composite nanomaterial dispersion prepared in step S3 is drop-coated onto the detection end surface of the glassy carbon electrode and dried to obtain a glassy carbon electrode modified with composite nanomaterial. S5. The glassy carbon electrode modified with composite nanomaterials prepared in step S4 is immersed in HAuCl4 solution for electrodeposition to obtain a glassy carbon electrode modified with electroactive nanomaterials.

3. The method according to claim 2, characterized in that, In step S1, the reaction is carried out at a temperature of 35°C; the reaction time is 12h to 36h; the washing is performed by washing the centrifuged solid product with deionized water until the pH of the washing solution is 6 to 7. In step S2, the ultrasonic dispersion time is 0.5 h to 1 h; the freeze-drying time is 24 h to 36 h. In step S3, the Ti3C2T x The ratio of nanosheets to deionized water is 0.5–3 mg:1 mL; the ultrasonic dispersion time is 5–20 min; the amount of conductive polymer solution added is 5%–15% of the total volume of the composite nanomaterial dispersion; the conductive polymer solution is a PEDOT:PSS solution; the amount of dimethyl sulfoxide added is 5%–15% of the total volume of the composite nanomaterial dispersion; the stirring time is 5 h. In step S4, the concentration of the composite nanomaterial dispersion is 0.5 mg / mL; the drying time is 1 h; and the drying temperature is 40 °C. In step S5, the concentration of the HAuCl4 solution is 1% W / V; the electrodeposition time is 20 s to 100 s; and the electrodeposition potential is -0.2 V.

4. The method according to claim 3, characterized in that, The method for preparing the working electrode further includes the following steps: S6. The trapping chain solution is dropped onto the detection end surface of the glassy carbon electrode modified with electroactive nanomaterials obtained in step S5 and incubated to allow the trapping chain to be modified on the detection end surface of the glassy carbon electrode, thus obtaining a glassy carbon electrode modified with trapping chains. S7. Add the sealing agent solution dropwise to the detection end surface of the glassy carbon electrode modified with the trapping chain obtained in step S6 and incubate it to seal the excess active sites and clean the electrode surface. S8. The specific aptamer probe solution is dropped onto the detection end surface of the glassy carbon electrode obtained in step S7 after incubation with the blocking agent solution, and incubated to modify the specific aptamer probe on the detection end surface of the glassy carbon electrode, thereby obtaining a glassy carbon electrode modified with a trapping chain and a specific aptamer probe. S9. Immerse the glassy carbon electrode modified with the trapping chain and specific aptamer probe obtained in step S8 in an electroactive tag solution, and introduce an electroactive tag on the surface of the detection end of the glassy carbon electrode to obtain the working electrode.

5. The method according to claim 4, characterized in that, In step S6, the concentration of the chain capture solution is 0.5 μM to 3 μM; the incubation time is 10 h to 14 h; and the incubation temperature is 0 to 5 °C. In step S7, the concentration of the blocking agent solution is 0.1 mM; the blocking agent in the blocking agent solution is MCH; and the incubation time is 1 h to 3 h. In step S8, the concentration of the specific aptamer probe solution is 0.5 μM to 3 μM; the incubation time is 1 h to 3 h; and the incubation temperature is 0 to 5 °C. In step S9, the electroactive tag solution is a phosphate buffer containing an electroactive tag; the concentration of the electroactive tag in the electroactive tag solution is 0.1 mM; the electroactive tag is methylene blue; the phosphate buffer is Tris-HCl buffer; the soaking time is 5 min to 25 min; after the soaking is completed, the following step is also included: immersing the glassy carbon electrode in Tris-HCl buffer to remove unbound electroactive tag molecules.

6. The method according to any one of claims 1 to 5, characterized in that, The detection of polychlorinated biphenyls (PCBs) in water using an electrochemical aptamer sensor includes the following steps: (1) The detection end of the working electrode is immersed in the test solution to react, and the polychlorinated biphenyls in the test solution are specifically identified and captured to obtain a working electrode that captures polychlorinated biphenyls; (2) The working electrode, counter electrode and reference electrode that capture polychlorinated biphenyls are placed in a buffer solution to establish a three-electrode system. The electrical signal of the working electrode is collected by differential pulse voltammetry to obtain the current value of the solution to be tested. (3) Input the current value of the test solution into the detection linear regression equation constructed by the relationship between different concentrations and current changes, and calculate the concentration of polychlorinated biphenyls in the test solution.

7. The method according to claim 6, characterized in that, In step (3), when the polychlorinated biphenyls in the test solution are PCB77, the detection linear regression equation constructed based on the relationship between different concentrations and current changes is as follows: I (μA) = 1.8556 lgC (ng L) -1 )+3.7433(1); In equation (1), I represents the difference in peak current change, in μA; C is the concentration of PCB77 in the test solution, in ng / L; and the correlation coefficient R0 is... 2 =0.9939, the detection linear range is 0.05 ng / L~10 4 The detection limit is 0.012 ng / L.

8. The method according to claim 7, characterized in that, In step (1), when the polychlorinated biphenyl in the test solution is PCB77, the capture chain in the working electrode has a nucleotide sequence as shown in SEQ ID NO.1; the specific aptamer probe has a nucleotide sequence as shown in SEQ ID NO.

2.

9. The method according to claim 6, characterized in that, In step (1), the test solution is lake or river water containing polychlorinated biphenyls (PCBs); the concentration of PCBs in the test solution is 0.05 ng / L to 10 ng / L. 4 ng / L; the pH value of the test solution is 6.0 to 8.

5.

10. The method according to claim 9, characterized in that, In step (1), the reaction time is 10 min to 50 min.

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