A portable electrochemical test strip and its preparation method and application

Abstract

The application provides a portable electrochemical test strip and a preparation method and application thereof, prepares Au / RGO / SPCE electrodes and TiO2 QDs@MXene / SPCE electrodes, dissolves molecularly imprinted polymer MIP powder prepared, and drops and coats the two electrodes to obtain the portable electrochemical test strip. Compared with the prior art, the application uses a nanocomposite modified screen-printed electrode test strip (SPCE), combines a molecular imprinting technology, and constructs a screen-printed electrode electrochemical test strip based on a molecular imprinting type. The application first constructs a screen-printed electrode electrochemical test strip based on a molecular imprinting type, realizes high selectivity, portable and sensitive detection of sylazine, and the portable test strip shows a wide linear range, a low detection limit, and good selectivity and stability for detection of sylazine. The application has low cost, is maintenance-free, easy to operate and carry, and is suitable for on-site detection of non-professionals.

Description

Technical Field

[0001] This invention relates to the field of food testing, and in particular to a portable electrochemical test strip, its preparation method, and its application. Background Technology

[0002] China's dairy product imports have also shown a rapid growth trend in recent years. Milk powder imports surged from 110,000 tons in 2005 to 850,000 tons in 2013, a cumulative increase of 107% over four years. The rapid growth of liquid milk imports is equally noteworthy, with imports doubling from 3,781 tons in 2005 to 180,000 tons in 2013. According to data from the General Administration of Customs, in 2020, China imported a total of 3.4783 million tons of various dairy products, amounting to US$12.599 billion, representing year-on-year increases of 11.07% and 6.22%, respectively. Therefore, strengthening the testing and supervision of dairy product quality and safety is of great significance for improving the quality and safety of dairy products and promoting the healthy and rapid development of my country's dairy industry.

[0003] In the production process of milk and dairy products, whether it's the preparation of raw materials (milk), such as the administration of medication to lactating dairy cows and the addition of antibiotics to feed to enhance their immunity, or the production process of dairy products, such as the addition of antibiotics to prevent milk spoilage, some antibiotics will remain in milk and dairy products. This can pose significant health risks, such as allergic reactions, increased drug resistance, and imbalances in the human gut microbiota. In 2019, a total of 1945 batches were sampled, of which 18 batches were found to be substandard. Therefore, how to quickly and accurately detect antibiotic residues in milk and dairy products is a pressing issue that the dairy testing industry needs to address.

[0004] Xylazine (XYL, C) 12 H 16 Xylazine (N2S) is a thiazide derivative, a potent α2-adrenergic agonist used for sedation, analgesia, muscle relaxation, and resuscitation. Xylazine is registered as a veterinary drug for use in farm animals such as cattle and horses, as well as in other animals such as sheep, goats, llamas, and deer. Xylazine can be administered via various routes (e.g., intravenous, intramuscular, subcutaneous, and oral), is absorbed, metabolized, and broken down in the body, and up to 8% of the dose is not metabolized in its original form, resulting in xylazine residues in dairy products. Although xylazine is rapidly metabolized in all animals, overdose by some illegal farmers can lead to remineralization of xylazine in the body. There are currently reported cases of xylazine poisoning. Individuals who have consumed food containing xylazine residues for more than one year may experience vomiting, nausea, weakness, and numbness in the extremities; overdose can also cause coma and bradycardia. Drug control is currently the most effective method for managing poisoning cases.

[0005] To protect consumer health, my country stipulates that xylazine residues must not be detected in milk. Currently, there are various analytical methods for detecting xylazine, including gas chromatography (GC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). These methods are fast, sensitive, provide stable and accurate results, but they involve complex sample pretreatment, expensive instruments, and require specialized operators, resulting in high analysis costs and long processing times, making them unsuitable for real-time on-site monitoring in dairy enterprises. With increasing demand for high-quality dairy products, dairy safety testing is characterized by large sample volumes and high time sensitivity. Traditional methods can no longer meet the requirements of rapid, accurate, portable, sensitive, and low-cost food safety testing. Rapid detection technologies, with their advantages of fast detection speed, portable devices, and ease of online detection, play a crucial role in food safety. Therefore, developing efficient, rapid, and accurate rapid detection technologies for application in dairy production is of great significance for achieving efficient management of the dairy production process and improving dairy product quality. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology, such as high analysis cost, long time consumption, and unsuitability for on-site testing, by providing a portable electrochemical test strip, its preparation method, and its application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] One aspect of the present invention provides a method for preparing a portable electrochemical test strip, comprising the following steps:

[0009] S1: Xylazine, methacrylic acid and chloroform are mixed and then subjected to ultrasonic treatment to prepolymerize, resulting in a prepolymerized solution;

[0010] S2: Add the crosslinking agent and initiator to the prepolymerization solution described in step S1; blow the bottle with a cold nitrogen stream, and then immediately seal the water bath; grind and sieve the obtained bulk product to obtain molecularly imprinted polymer (MIP) powder;

[0011] S3: Disperse the above MIP powder in a mixture of ethylene glycol and Nafion@117, drop the mixture containing the dispersed MIP onto the electrode, and dry it to obtain the modified electrode;

[0012] S4: Use a methanol-acetic acid mixed solution to elute the xylazine in the modified electrode described in step S3, and the resulting xylazine-free molecularly imprinted polymer electrode is the portable electrochemical test strip.

[0013] Further, the ratio of xylazine, methacrylic acid and chloroform used in step S1 is 1 mmol: 4 mmol: 70 mL.

[0014] Further, the crosslinking agent in step S2 is ethylene glycol dimethacrylate, and the initiator is 2,2'-azobisisobutyronitrile.

[0015] Furthermore, the ratio of the crosslinking agent to the initiator is 5 mmol: 4 mmol.

[0016] Furthermore, the ultrasonication time in step S1 is 5-15 min; the prepolymerization temperature is 1-8℃.

[0017] Furthermore, the blowing time in step S2 is 4-6 minutes; the water bath temperature is 55-65℃, and the water bath time is 20-30 hours.

[0018] Further, in step S3, the ratio of MIP, ethylene glycol, and Nafion@117 is 10 mg: 1 mL: 10 μL.

[0019] Furthermore, the electrode preparation method described in step S3 is as follows:

[0020] GO suspension was dropped onto a screen-printed carbon electrode SPCE, and then RGO / SPCE was obtained by electrochemical reduction using cyclic voltammetry. Subsequently, HAuCl4 was dropped onto RGO / SPCE, and gold nanoparticles were electrodeposited using potentiostatic technology to finally obtain the Au / RGO / SPCE electrode.

[0021] or,

[0022] Pour HCl and LiF into a polytetrafluoroethylene beaker and stir well. Then add Ti3AlC2 and stir magnetically in a water bath. After the reaction is complete, wash twice with HCl, then wash with deionized water by centrifugation until the pH of the supernatant is ≤6. Take the lower black clay-like precipitate, add an appropriate amount of deionized water, shake mechanically, and sonicate in an ultrasonic machine. Centrifuge the mixture to obtain the upper layer solution, which is the few-layer or monolayer Ti3C2T. x The MXene dispersion was prepared by filling the solution with argon gas and storing it in a light-proof, sealed container. MXene prepared above was then added to CTAB and stirred under argon protection. TiCl3 was added while stirring, and the mixture was transferred to a reaction vessel for reaction. The product was washed three times each with ethanol and water, and finally freeze-dried to obtain TiO2QDs@MXene powder. The above TiO2 QDs@MXene powder was dissolved in the dispersion and ultrasonically dispersed to obtain a uniform dispersion. The dispersion was then drop-coated onto the surface of an SPCE electrode and vacuum-dried to obtain TiO2QDs@MXene / SPCE.

[0023] Furthermore, the initial voltage of the cyclic voltammetry electrochemical reduction (CV) technique is -1.4V, the termination voltage is 0.6V, and the scan rate is 50mV / s; the electrodeposition voltage is -0.2V.

[0024] Furthermore, the ratio of HCl, LiF and Ti3AlC2 is 0.35-0.37 mol: 3-3.4 g: 1-3 g; the ratio of MXene, CTAB and TiCl3 is 40-60 mg: 1.2-1.6 g: 15-25 μL.

[0025] Furthermore, when the electrode is an Au / RGO / SPCE electrode, the modified electrode is a MIP / Au / RGO / SPCE electrode, and the molecularly imprinted polymer electrode mentioned in step S4 is a rMIP / Au / RGO / SPCE electrode.

[0026] Furthermore, when the electrode is a TiO2 QDs@MXene / SPCE electrode, the modified electrode is MIP / TiO2 QDs@MXene / SPCE, and the molecularly imprinted polymer electrode in step S4 is rMIP / TiO2QDs@MXene SPCE.

[0027] The second aspect of this invention provides a portable electrochemical test strip prepared by the above-described method.

[0028] The third aspect of this invention provides the application of the aforementioned portable electrochemical test strip in detecting xylazine concentration in dairy products.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) A screen-printed electrode test strip (SPCE) modified with nanocomposite materials was constructed using molecular imprinting technology to create a molecular imprinted electrochemical test strip. The excellent conductivity and superior electrochemical performance of the nanocomposite materials significantly improved the current signal.

[0031] (2) The differential pulse voltammetry (DPV) method is used to quantitatively detect trace substances on the electrode surface. Compared with other electrochemical techniques, it reduces the background current and exhibits higher detection sensitivity.

[0032] (3) This invention is the first to construct a molecularly imprinted screen-printed electrode electrochemical test strip, achieving highly selective, portable, and sensitive detection of xylazine. The test strip exhibits a wide linear range, a low detection limit, and good selectivity and stability for the detection of xylazine using a portable test strip.

[0033] (4) This invention is low in cost, maintenance-free, disposable, plug-and-play, and has no complicated pretreatment process; the electrode batch difference is small, and the detection results are more reliable; only one drop of sample is needed to complete the detection; independent detection, no electrolyte contamination; high sensitivity, simple operation, low cost, easy miniaturization; easy to operate and carry, suitable for on-site detection by non-professionals. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the portable electrochemical test strip used for the rapid detection of xylazine in raw milk in Example 1 of the present invention;

[0035] Figure 2 This is a TEM image of Au / RGO prepared in Example 1 of the present invention;

[0036] Figure 3 The images show the XRD patterns of RGO and Au / RGO prepared in Example 1 of this invention.

[0037] Figure 4 The rMIP / Au / RGO / SPCE test strip prepared in Example 1 of this invention contains 0.1-10 5 DPV curve in ug / L xylazine solution;

[0038] Figure 5 The rMIP / Au / RGO / SPCE test strip prepared in Example 1 of this invention contains 0.1-10 5 Linear calibration curve in ug / L xylazine solution;

[0039] Figure 6 This is a SEM image of TiO2 QDs@MXene prepared in Example 2 of this invention;

[0040] Figure 7 The image shows the XRD pattern of TiO2 QDs@MXene prepared in Example 2 of this invention;

[0041] Figure 8 The rMIP / TiO2 QDs@MXene test strips prepared in Example 2 of this invention contain 0.1-10 5 DPV curve in ug / L xylazine solution;

[0042] Figure 9 The rMIP / TiO2 QDs@MXene test strips prepared in Example 2 of this invention contain 0.1-10 5 Linear calibration curve in ug / L xylazine solution. Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0044] Example 1

[0045] 100 μL of a 1 mg / mL GO suspension was dropped onto the SPCE, followed by electrochemical reduction using cyclic voltammetry to obtain the RGO / SPCE. The initial voltage for CV was -1.4 V, the termination voltage was 0.6 V, and the scan rate was 50 mV / s. Subsequently, 100 μL of 1 mmol / L HAuCl4 was dropped onto the RGO / SPCE, and gold nanoparticles were electrodeposited using potentiostatic deposition at a voltage of -0.2 V, ultimately yielding the Au / RGO / SPCE electrode.

[0046] 0.1 mmol of xylazine template molecule and 0.4 mmol of functional monomer methacrylic acid were added to a glass bottle containing 7 mL of chloroform. The mixture was sonicated for 10 min and prepolymerized overnight at 4 °C. 0.3 mmol of ethylene glycol dimethacrylate crosslinking agent and 0.24 mmol of 2,2'-azobisisobutyronitrile initiator were added. After blowing the bottle with a stream of cold nitrogen for 5 min to remove air, it was immediately sealed and shaken in a 60 °C water bath for 24 h. The mixture was ground and sieved to obtain molecularly imprinted polymer (MIP) powder. 10 mg of MIP was dispersed in a mixture of 1 mL of ethylene glycol and 10 μL of Nafion@117. 100 μL of the mixture was drop-coated onto an Au / RGO / SPCE electrode and dried to obtain the modified electrode MIP / Au / RGO / SPCE. The template molecules were eluted with a methanol / acetic acid (9:1, v / v) mixture to obtain a molecularly imprinted polymer electrode (rMIP / Au / RGO / SPCE) with the template molecules removed, which is the portable electrochemical test strip. As a control, a non-molecularly imprinted polymer-modified electrode (NIP / Au / RGO / SPCE) was simultaneously synthesized without the addition of template molecules.

[0047] Figure 2 The image shows a TEM image of Au / RGO. It reveals that the reduced graphene oxide modified on the electrode surface exhibits a wrinkled, sheet-like two-dimensional morphology, and a large number of gold nanoparticles are present on the surface of the reduced graphene oxide substrate. Therefore, the Au / RGO composite material was successfully synthesized. Loading gold nanoparticles can accelerate electron transfer, thereby improving the intensity of the response current signal.

[0048] The crystal structure of the material was investigated using X-ray diffraction (XRD). The broad diffraction peaks of 2θ in the range of 20–30° correspond to the (002) crystal plane of the stacked RGO. Figure 3In the Au / RGO nanocomposite, apart from the characteristic diffraction peak of the (002) crystal plane of RGO, the other diffraction peaks correspond to the (111), (200), (220), and (311) crystal planes of Au (face-centered cubic, JCPDS 04-0784), indicating that the electrochemical method successfully reduced HAuCl4 to Au nanoparticles. Compared with pure RGO, the RGO diffraction peak intensity is reduced in the Au / RGO nanocomposite, which is because the Au nanoparticles prevent the RGO sheets from reforming.

[0049] Differential pulse voltammetry (DPV) can be used to determine the reversibility of electrode processes and study adsorption behavior on electrode surfaces. Compared to other electrochemical techniques, DPV has lower background current and higher detection sensitivity, and is therefore often used for the quantitative detection of trace substances. This work uses DPV for the quantitative detection of xylazine, recording the peak DPV current of the sensor in solutions containing xylazine gradient concentrations of 0.1, 1, 10, 100, 1000, 10000, and 100000 μg / L. Figure 4 The current decreases with increasing xylazine concentration, and its linear fitting equation is I = 250.2929 - 21.6397 × lgC(R). 2 =0.9931)( Figure 5 )

[0050] The testing process is as follows Figure 1 As shown, a detection system consisting of a computer, an electrochemical workstation, and a modified screen-printed carbon electrode is constructed. Utilizing the specific recognition property of template molecules by molecular imprinting technology, a portable screen-printed electrochemical test strip with specific detection capability for xylazine is produced. When xylazine in dairy products such as milk is recognized by rMIP / Au / RGO / SPCE, the xylazine binds to the cavity in the rMIP, creating steric hindrance and resulting in a decrease in peak current. The measured current corresponds to the concentration of xylazine, thus achieving rapid and sensitive detection of xylazine.

[0051] Example 2

[0052] Measure 40 mL of 9M HCl solution and pour it into a polytetrafluoroethylene beaker. Weigh 3.2 g of LiF and slowly and evenly pour it into the beaker, stirring until homogeneous. Then weigh 2 g of Ti3AlC2 and slowly and evenly pour it into the beaker. Stir magnetically in a 40℃ water bath for 36 h. After the reaction has finished for 36 h, wash twice with 1M HCl, then wash with deionized water by centrifugation until the pH of the supernatant is ≤6. Take the lower layer of black clay-like precipitate, add an appropriate amount of deionized water, and mechanically shake well. Place it in an ultrasonic machine (240W) and sonicate for 1 h. Centrifuge the mixture at 3500 rpm for 1 h to obtain the upper layer solution, which is the few-layer or monolayer Ti3C2T. xThe dispersion was prepared by purging the solution with argon gas and storing it in a light-protected, sealed container. 50 mL of the prepared MXene (1 mg / mL) was added, along with 1.4 g of CTAB. The mixture was stirred for 30 min under argon protection. Then, 20 μL of TiCl3 (15% wt) was added while stirring. The mixture was transferred to a reaction vessel and reacted at 200 °C for 6 h. The product was washed three times each with ethanol and water, and finally freeze-dried to obtain TiO2 QDs@MXene. 10 mg of the above TiO2 QDs@MXene powder was weighed, dissolved in 1 mL of ethylene glycol, and 10 μL of Nafion@117 solution was added. The mixture was ultrasonically dispersed to ensure uniform dispersion. 10 μL of the dispersion was drop-coated onto the surface of an SPCE electrode and dried under vacuum at 40 °C to obtain TiO2 QDs@MXene / SPCE.

[0053] 0.1 mmol xylazine template molecule and 0.4 mmol functional monomer methacrylic acid were added to a glass bottle containing 7 mL chloroform. The mixture was sonicated for 10 min and prepolymerized overnight at 4 °C. 0.3 mmol ethylene glycol dimethacrylate crosslinking agent and 0.24 mmol 2,2'-azobisisobutyronitrile initiator were added. After blowing the bottle with a cold nitrogen stream for 5 min to remove air, it was immediately sealed and shaken in a 60 °C water bath for 24 h. The mixture was ground and sieved to obtain molecularly imprinted polymer (MIP) powder. 10 mg of MIP was dispersed in a mixture of 1 mL ethylene glycol and 10 μL Nafion@117. 100 μL of the mixture was drop-coated onto a TiO2 QDs@MXene / SPCE electrode and dried to obtain the modified electrode MIP / TiO2 QDs@MXene / SPCE. Template molecules were eluted with a methanol / acetic acid (9:1, v / v) mixture to obtain a molecularly imprinted polymer electrode (rMIP / TiO2QDs@MXene / SPCE) with template molecules removed. As a control, a non-molecularly imprinted polymer-modified electrode (NIP / TiO2 QDs@MXene / SPCE) was simultaneously synthesized without template molecules.

[0054] Figure 6 The image shows a SEM image of TiO2 QDs@MXene. As can be seen from the image, the composite material retains the two-dimensional morphology of MXene. The small particles on the MXene surface are TiO2 QDs grown in situ on the MXene surface. Figure 7 The XRD patterns of TiO2, MXene, and TiO2QDs@MXene composite materials are shown. The diffraction pattern of TiO2 corresponds to the standard card of anatase. MXene shows a diffraction peak at 2θ = 5°, which corresponds to the (002) crystal plane of MXene, indicating that a single layer of MXene material was successfully prepared by chemical etching. The diffraction peaks of TiO2 and MXene appear in the composite material without shift, indicating the successful preparation of the composite material.

[0055] The DPV method was used for quantitative detection of xylazine. The peak DPV current of the sensor was recorded in solutions containing xylazine concentration gradients of 0.1, 1, 10, 100, 1000, 10000, and 100000 μg / L. Figure 8 The current decreased with increasing xylazine concentration, and its linear fitting equation was I = 0.2375 + 0.00796lgC(R). 2 =0.9925)( Figure 9 ).

[0056] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method of making a portable electrochemical test strip, characterized by, Includes the following steps: S1: Xylazine, methacrylic acid and chloroform are mixed and then subjected to ultrasonic treatment to prepolymerize, resulting in a prepolymerized solution; S2: Add the crosslinking agent and initiator to the prepolymerization solution described in step S1; blow the bottle with a cold nitrogen stream, and then immediately seal the water bath; grind and sieve the obtained bulk product to obtain molecularly imprinted polymer (MIP) powder; S3: Disperse the above MIP powder in a dispersion, drop the mixture containing the dispersed MIP onto the electrode, and dry to obtain the modified electrode; S4: Use a methanol-acetic acid mixed solution to elute the xylazine in the modified electrode described in step S3, and the resulting xylazine-free molecularly imprinted polymer electrode is the portable electrochemical test strip. The electrode preparation method described in step S3 is as follows: GO suspension was dropped onto a screen-printed carbon electrode SPCE, and then rGO / SPCE was obtained by electrochemical reduction using cyclic voltammetry. Subsequently, HAuCl4 was dropped onto rGO / SPCE, and gold nanoparticles were electrodeposited using potentiostatic technology to finally obtain an Au / rGO / SPCE electrode. or, HCl, LiF, and Ti3AlC2 were magnetically stirred in a water bath. After the reaction was complete, the mixture was washed twice with HCl, then centrifuged with deionized water until the pH of the supernatant was ≤6. The lower black clay-like precipitate was collected, and an appropriate amount of deionized water was added and mechanically shaken. The mixture was then sonicated. The mixture was centrifuged to obtain the upper layer solution, which is the few-layer or monolayer Ti3C2T. x The MXene dispersion was prepared by filling the solution with argon gas and storing it in a light-proof, sealed container. MXene prepared above was then added to CTAB and stirred under argon protection. TiCl3 was added under stirring, and the mixture was transferred to a reaction vessel for reaction. The product was washed three times each with ethanol and water, and finally freeze-dried to obtain TiO2 QDs@MXene powder. The dispersion of TiO2 QDs@MXene powder was drop-coated onto the surface of an SPCE electrode and vacuum-dried to obtain TiO2QDs@MXene / SPCE.

2. The method of claim 1, wherein the method further comprises, The initial voltage of the cyclic voltammetry electrochemical reduction (CV) technique is -1.4 V, the termination voltage is 0.6 V, and the scan rate is 50 mV / s; the electrodeposition voltage is -0.2 V; the ratio of HCl, LiF, and Ti3AlC2 is 0.35-0.37 mol: 3-3.4 g: 1-3 g; and the ratio of MXene, CTAB, and TiCl3 is 40-60 mg: 1.2-1.6 g: 15-25 μL.

3. The method of claim 1, wherein the method further comprises, The crosslinking agent in step S2 is ethylene glycol dimethacrylate, and the initiator is 2,2'-azobisisobutyronitrile.

4. The method of claim 1, wherein the method further comprises, The ratio of xylazine, methacrylic acid and chloroform used in step S1 is 1 mmol: 4 mmol: 70 mL; the ratio of crosslinking agent and initiator used in step S2 is 5 mmol: 4 mmol.

5. The method of claim 1, wherein the method further comprises, The ultrasonication time in step S1 is 5-15 min; the prepolymerization temperature is 1-8 ℃; the blow molding time in step S2 is 4-6 min; the water bath temperature is 55-65 ℃, and the water bath time is 20-30 h; the dispersion in step S3 is a mixture of ethylene glycol and Nafion 117, wherein the ratio of MIP, ethylene glycol and Nafion 117 is 10 mg: 1 mL: 10 μL.

6. The method of claim 1, wherein the method further comprises, When the electrode is an Au / rGO / SPCE electrode, the modified electrode is a MIP / Au / rGO / SPCE electrode, and the molecularly imprinted polymer electrode in step S4 is a rMIP / Au / rGO / SPCE electrode.

7. The method of claim 1, wherein the method further comprises, When the electrode is a TiO2 QDs@MXene / SPCE electrode, the modified electrode is MIP / TiO2 QDs@MXene / SPCE, and the molecularly imprinted polymer electrode in step S4 is rMIP / TiO2 QDs@MXene / SPCE.

8. A portable electrochemical test strip prepared according to the method of any one of claims 1 to 7.

9. Use of a portable electrochemical test strip according to claim 8 for detecting the concentration of xipirron in a dairy product.

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