Preparation of electrochemical sensor based on poly [2-(2, 6-naphthyl)-3, 4-bis (2-ethyl-hexyloxymethoxy) thiophene] and application of electrochemical sensor in escherichia coli detection

Abstract

The invention discloses preparation of an electrochemical sensor based on poly [2-(2, 6-naphthyl)-3, 4-bis (2-ethyl-hexyloxymethoxy) thiophene] and application of the electrochemical sensor in escherichia coli detection, and relates to the technical field of electrochemical biosensors. The novel electrochemical sensor is prepared by virtue of the synergistic effect of the conductive polymer and the nano material and is used for accurately detecting the escherichia coli (such as O157: H7) in food and water sources, the technology overcomes the defects that a traditional detection method is complicated in operation, long in time consumption, easy to pollute and the like, and high-sensitivity and high-selectivity detection on the escherichia coli in the food and water sources is realized.

Description

Technical Field

[0001] This invention relates to the field of biosensing and detection technology, specifically to an electrochemical sensor based on poly[2-(2,6-naphthyl)-3,4-bis(2-ethyl-hexyloxymethoxy)thiophene] (P(Na-ProDOT)). It also relates to the preparation method of this sensor and its application in the rapid and highly sensitive detection of *Escherichia coli* (especially strain O157:H7) in food and water sources. Naphthalene possesses good electrochemical activity and a unique conjugated structure, while ProDOT exhibits good polarity and ensures polymer solubility. The conjugated structure of P(Na-ProDOT) allows for efficient electron movement within the molecule, thereby promoting electron transfer. Gold nanoparticles are modified onto the P(Na-ProDOT) layer using self-assembly technology, leveraging the large specific surface area and excellent biocompatibility of gold nanoparticles to increase the number of active electrode sites. This invention's sensor shows promising application prospects in the field of food detection. Background Technology

[0002] Food safety is a key area related to public health and social stability. Foodborne pathogens cause frequent safety problems. Among them, Escherichia coli (such as O157:H7) can produce Shiga toxin, which can cause diarrhea, hemorrhagic colitis, and in severe cases, hemolytic uremic syndrome. It poses a great threat to infants and young children and people with low immunity. It can also disrupt the balance of human intestinal flora and bring serious challenges to public health.

[0003] Currently, the main methods for detecting E. coli include traditional culture methods, polymerase chain reaction (PCR), real-time quantitative PCR, immunogold assay, and gene chip methods. However, all of these methods have significant drawbacks: traditional culture methods require complex sample pretreatment and long culture cycles, which cannot meet the needs of rapid detection; PCR methods are prone to contamination leading to false positives and require highly skilled operators; real-time quantitative PCR instruments are expensive, making it difficult to popularize in grassroots laboratories; immunogold assays have low sensitivity and insufficient quantitative accuracy; and gene chip methods are complex to operate, costly to detect, and have strict requirements for sample purity.

[0004] While organic semiconductor materials offer advantages in functionality, flexible device fabrication, transparency, and solubility, existing sensors based on them still suffer from limitations in detecting E. coli, including insufficient conductivity and signal response efficiency, weak anti-interference capabilities, and high detection limits. These limitations prevent them from efficiently meeting the practical needs of rapid screening for E. coli in food and water sources. Therefore, developing a simple, rapid, highly sensitive, selective, and cost-effective E. coli detection technology is an urgent requirement for ensuring food safety. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing E. coli detection technologies and provide an electrochemical sensor based on P(Na-ProDOT). It also provides a method for preparing this sensor and its application in E. coli detection. Modification with P(Na-ProDOT) and gold nanoparticles significantly improves the conductivity and specific surface area of ​​the electrode, accelerates the electron transfer rate, and results in a lower detection limit and higher sensitivity for E. coli. This enables rapid, highly sensitive, and highly selective detection of E. coli in food and water sources.

[0006] This invention is achieved through the following technical solution: A first aspect of the present invention provides a method for preparing P(Na-ProDOT), comprising the following steps: A certain mass of 2,6-dibromonaphthalene monomer, ProDOT, cesium carbonate (CsCO3), palladium catalyst (Pd(PPh3)4), triphenylphosphine (ligand), terpentine, and DMAC were added to a pressure-resistant flask and synthesized by heating in an oil bath. After filtration, washing, and extraction, P(Na-ProDOT) was obtained.

[0007] Preferably, the above steps yield an optimal mass of 142.98 mg 2,6-dibromonaphthalene monomer, 71.1 mg ProDOT, 203.67 mg cesium carbonate (CsCO3), 25.57 mg palladium catalyst (Pd(PPh3)4), 8.8 mg triphenylphosphine (ligand), 32.75 mg terpentine, and 5 mL DMAC.

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned P(Na-ProDOT), gold nanoparticles, and horseradish peroxidase modified electrode, comprising the following steps: The P(Na-ProDOT) solution is uniformly sprayed onto the platinum wire using a spray gun to ensure that the P(Na-ProDOT) is evenly covered on the surface of the platinum wire, thus forming a P(Na-ProDOT) electrode.

[0009] A glassy carbon electrode sprayed with P(Na-ProDOT) was immersed in a solution containing HAuCl4 at a concentration of 0.1 mmol / L. KCl was added to adjust the electrolyte environment. A voltage was applied at an electrochemical workstation to deposit gold nanoparticles onto the P(Na-ProDOT) electrode, thus fabricating an AuNPs / PTD / CE electrode.

[0010] The AuNPs / PTD / CE electrode was prepared by immersing it in a 0.1 mg / mL horseradish peroxidase (HRP) solution and incubating it at 4 °C for 2 hours.

[0011] A third aspect of the present invention provides the application of the above-described composite material in the detection of Escherichia coli. Attached Figure Description

[0012] Figure 1 Preparation steps of P(Na-ProDOT) Figure 2 Electrochemical cyclic voltammograms of HRP / AuNPs / PTD / CE electrodes Figure 3 Diagram showing the anti-interference performance of HRP / AuNPs / PTD / CE electrodes Detailed Implementation

[0013] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0014] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Unless otherwise specified, experimental conditions not detailed in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments can be obtained commercially unless otherwise specified.

[0015] Example 1 The chemical reaction process for preparing P(Na-ProDOT), gold nanoparticles, horseradish peroxidase, and modified electrodes is shown below, with specific reaction steps and conditions as follows: 142.98 mg of 2,6-dibromonaphthalene monomer, 71.1 mg of ProDOT, 203.67 mg of cesium carbonate (CsCO3), 25.57 mg of palladium catalyst (Pd(PPh3)4), 8.8 mg of triphenylphosphine (ligand), 32.75 mg of pentylamino acid, and 5 ml of DMAC were added sequentially to a dry, pressure-resistant bottle. Nitrogen gas was continuously purged into the bottle for 10 min, and then the bottle was sealed. Under anhydrous and oxygen-free conditions, the mixture was subjected to an oil bath at 120 °C for 10 h on a magnetic stirrer. The palladium catalyst catalyzed the arylation polycondensation reaction (DHAP) between 2,6-dibromonaphthalene and the ProDOT derivative, gradually forming a polymer (…). Figure 1 The synthesized product was transferred to a beaker, washed repeatedly with anhydrous ethanol, and filtered. The filter paper was wrapped with large filter paper and extracted. The extraction was completed when the liquid in the Soxhlet extractor was colorless and transparent. The product was then extracted with anhydrous ethanol, n-hexane, and chloroform, respectively. The extract was then rotary evaporated and dried in an oven for 6 hours to obtain P(Na-ProDOT).

[0016]

[0017] Figure 1Preparation steps of P(Na-ProDOT) P(Na-ProDOT) was dissolved in chloroform to prepare a solution with a concentration of 5 mg / mL. The solution was then uniformly sprayed onto a platinum wire using a spray gun, maintaining a distance of 10-15 cm between the spray gun and the wire. During the spraying process, the spray gun was moved at a constant speed to ensure that P(Na-ProDOT) was evenly covered on the surface of the platinum wire, thus forming a P(Na-ProDOT) electrode.

[0018] A glassy carbon electrode coated with P(Na-ProDOT) was immersed in a 0.1 mmol / L solution containing HAuCl4. KCl was added to adjust the electrolyte environment. An application voltage of -0.8–1.6 V was applied at room temperature, with Ag / AgCl as the reference electrode. The deposition time was 5–600 seconds. The electrode surface was then slowly rinsed with deionized water 3–5 times to remove unadsorbed gold nanoparticles. Finally, the electrode was dried with nitrogen to obtain a gold nanoparticle-modified P(Na-ProDOT)-modified carbon electrode (AuNPs / PTD / CE).

[0019] The AuNPs / PTD / CE electrode was prepared by immersing it in a 0.1 mg / mL horseradish peroxidase (HRP) solution and incubating it at 4°C for 2 hours.

[0020] Example 2 The composite material obtained in Example 1 is used as an example in the field of Escherichia coli detection.

[0021] The following examples illustrate the application process of the composite materials provided by the present invention, but the present invention is not limited to the examples given.

[0022] (1) Appearance The composite material obtained in Example 1 was uniformly sprayed onto conductive glass using a spray gun, and the shape of the gold nanoparticles was observed using a 3D stereomicroscope.

[0023] (2) Electrochemistry The composite material obtained in Example 1 was uniformly drop-coated onto a glassy carbon electrode. The glassy carbon electrode coated with the composite material was placed in a three-electrode electrolytic cell containing a PBS buffer solution with KCl solution, diluted bacterial solution, and glucose solution. The working electrode was the glassy carbon electrode coated with the composite material, the counter electrode was a platinum sheet, and the reference electrode was an Ag / AgCl electrode. Using a potentiostatic method, the voltage applied to the working electrode was adjusted by an electrochemical workstation, and the cyclic voltammetry curve was recorded to obtain the cyclic voltammetry curve of the composite material. (See figure) Figure 2 .

[0024] (3) Anti-interference performance Ten times the concentration of the interfering agent, tap water, was added to samples containing the target concentration of E. coli. The anti-interference performance of the composite material was obtained by detecting and comparing the sensor's response signals to E. coli before and after the addition of the interfering agent. (See figure). Figure 3 .

[0025] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing P(Na-ProDOT) / AuNPs composite material, characterized in that, Includes the following steps: P(Na-ProDOT) was prepared by palladium-catalyzed arylation polycondensation of 2,6-dibromonaphthalene and ProDOT. The reaction product was washed, Soxhlet extracted, and dried. Gold nanoparticles (AuNPs) were prepared by sodium citrate reduction. P(Na-ProDOT) solution was sprayed onto a platinum wire modified electrode, which was then immersed in an AuNPs solution for incubation. After rinsing and drying, P(Na-ProDOT) / AuNPs composite material was obtained. After pretreating the glassy carbon electrode, the composite material suspension was sprayed and dried to complete the modification.

2. An electrochemical sensor based on poly[2-(2,6-naphthyl)-3,4-bis(2-ethyl-hexyloxymethoxy)thiophene], characterized in that, The product is prepared by the method described in claim 1, including the synthesis of P(Na-ProDOT), the preparation of AuNPs, and the modification of a carbon electrode by both. The modification effect is verified by electrochemical characterization. It is applied in the fields of food safety monitoring and environmental water body detection to achieve the detection performance of Escherichia coli.

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