A screen-printed sensitive electrode for rapid detection of food antibiotics and a preparation method thereof

Abstract

This invention relates to the field of screen-printed sensitive electrode technology, specifically to a screen-printed sensitive electrode for rapid detection of antibiotics in food and its preparation method. The sensitive electrode uses Whatman No.1 chromatographic paper as a substrate, and a sensing active layer is formed on the surface of the sensitive electrode. The sensing active layer is formed by screen printing and isothermal curing of MXene / MWCNTs-COOH nanocomposite functional ink. The beneficial effects are: by constructing a sheet-tube interwoven three-dimensional conductive network through the composite of MXene and carboxylated multi-walled carbon nanotubes in a mass ratio of 5:3, the one-dimensional carbon nanotubes interpenetrate and block the stacking of MXene sheets, and simultaneously improve the electrode conductivity and the number of electrocatalytic active sites. Combined with a special screen-printed composite ink with an addition amount of 10% and a steady-state viscosity of 8.2 Pa·s, the electrode can be mass-produced with high consistency and low cost by relying on a 120°C segmented curing process.

Description

Technical Field

[0001] This invention relates to the field of screen-printed sensitive electrode technology, specifically to a screen-printed sensitive electrode for rapid detection of antibiotics in food and its preparation method. Background Technology

[0002] The irrational use of veterinary antibiotics has led to a significant problem of excessive residues in animal-derived foods. Chloramphenicol (CAP) and metronidazole (MNZ), two prohibited / restricted veterinary antibiotics, can accumulate through the food chain, inducing hematopoietic damage, genotoxicity, and bacterial resistance in humans. my country has issued several national standards to strictly limit their food residue limits. Currently, the mainstream detection methods are divided into four categories:

[0003] Large-scale chromatography-mass spectrometry (LC-MS) technology: High performance liquid chromatography and liquid mass spectrometry have high detection accuracy, but the instruments are expensive, the pretreatment process is complicated, the detection cycle is long, and it relies on professional operators. It is only suitable for precise quantitative analysis in laboratories and cannot meet the needs of rapid screening in farmers' markets, breeding bases, and grassroots supervision sites.

[0004] Microbial detection method: low cost but the detection time is up to ten hours, the results are easily affected by matrix impurities, the detection limit is high, and it is difficult to achieve trace residue detection;

[0005] Immunochromatography: It has good specificity, but the antibody preparation cycle is long, false positives are easy to occur, it can only achieve semi-quantitative results, and it is difficult to detect multiple antibiotics simultaneously.

[0006] Traditional electrochemical sensors often employ drop-coating to modify glassy carbon electrodes, resulting in poor reproducibility and difficulty in mass production. Conventional carbon-based bare electrodes exhibit weak conductivity and a scarcity of electrocatalytic active sites. Single MXene materials suffer from layer van der Waals stacking and aggregation defects, leading to low electron transport efficiency, high detection limits for CAP and MNZ, and most systems can only detect a single component, making it impossible to achieve simultaneous quantification of two types of antibiotics.

[0007] Paper-based microfluidic analytical devices (μPADs) offer advantages such as low cost, portability, autonomous sample delivery via capillary action, and low sample consumption, making them ideal carriers for on-site detection. However, existing paper-based electrochemical electrodes have significant shortcomings: First, there is a lack of highly active conductive inks suitable for screen printing, resulting in poor consistency in drop-coating processes and hindering large-scale fabrication. Second, the use of only single carbon materials or pure MXene-modified electrodes cannot solve the stacking problem, leading to a small electrochemical active area and insufficient detection sensitivity. Third, most paper-based chips have simple structures and lack integrated pretreatment and waste liquid recovery functions, resulting in easy sample leakage and severe matrix interference. Fourth, existing detection systems struggle to effectively separate the characteristic peaks of CAP and MNZ, two nitro antibiotics, making simultaneous high-sensitivity quantification impossible.

[0008] Currently, there is no screen-printed paper-based sensitive electrode and its supporting preparation process that can simultaneously solve the four major technical challenges of MXene aggregation, batch electrode preparation, integrated microfluidic integration, and simultaneous detection of dual antibiotics at the nanomolar level. Existing technologies are unable to meet the multiple application requirements of low cost, portability, high sensitivity, and simultaneous on-site detection. Summary of the Invention

[0009] The purpose of this invention is to provide a screen-printed sensitive electrode for rapid detection of antibiotics in food and its preparation method, so as to solve the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a screen-printed sensitive electrode for rapid detection of antibiotics in food, wherein the sensitive electrode is based on Whatman No.1 chromatographic paper, and a sensing active layer is provided on the surface of the sensitive electrode. The sensing active layer is formed by screen printing and constant temperature curing of MXene / MWCNTs-COOH nanocomposite functional ink.

[0011] The MXene is A two-dimensional carbide material, wherein the MWCNTs-COOH is a carboxylated multi-walled carbon nanotube; The mass ratio of MXene to MWCNTs-COOH is 5:3. The one-dimensional tubular structure of MWCNTs-COOH is interspersed in the gaps between MXene sheets, forming a three-dimensional conductive network of sheet-tube interwoven structure, which inhibits the stacking and aggregation of MXene sheets, and improves the electrode conductivity and the number of surface electrocatalytic active sites.

[0012] The MXene / MWCNTs-COOH nanocomposite functional ink uses CI-2067 conductive carbon ink as the base, with MXene / MWCNTs-COOH composite powder added at 10% of the total ink mass. The steady-state viscosity of the ink is 8.2 Pa·s, suitable for 200-mesh screen printing. After being cured at 120℃ for 20 minutes, the composite ink forms a uniform and dense conductive film.

[0013] The sensitive electrode enables simultaneous electrochemical detection of chloramphenicol (CAP) and metronidazole (MNZ). The detection limit for chloramphenicol is 0.025 μmol / L, and the detection limit for metronidazole is 0.013 μmol / L, both reaching the nanomolar level.

[0014] Preferably, the preparation steps of the MXene / MWCNTs-COOH nanocomposite powder are as follows:

[0015] Will The powder was ultrasonically dispersed in a 1:1 volume ratio of ultrapure water and anhydrous mixed solvent; MWCNTs-COOH aqueous dispersion was added dropwise to the MXene dispersion, ultrasonicated for 30 min and then magnetically stirred for 1 h; the mixed dispersion was allowed to stand overnight to settle, the lower precipitate was taken and dried in a 60℃ oven for 12 h, and after cooling, it was ground with agate to obtain composite powder.

[0016] The composite ink showed a film peeling area of ​​≤2.8% after the cross-cut adhesion test; when printed with a 200-mesh screen, there were no defects such as ink overflow, pinholes, or screen clogging.

[0017] Preferably, the sensitive electrode is the working electrode in an integrated paper-based three-electrode system; the integrated three-electrode system is prepared by screen printing, with the reference electrode printed with CIE4250 Ag / AgCl conductive ink and the counter electrode printed with CI-2067 conductive carbon ink; the reference electrode and the counter electrode are symmetrically arranged on both sides of the sensitive electrode, with an electrode printing alignment deviation ≤0.5mm, and the three constitute a complete electrochemical detection circuit.

[0018] Preferably, the sensitive electrode is assembled inside a four-layer folded paper-based microfluidic electrochemical sensor; the four layers, from top to bottom, are a sample pretreatment layer, a fluid guiding middle layer, a three-electrode detection lower layer, and a waste liquid recovery bottom layer; the overall size of each layer after folding along the preset crease is 36mm×26mm; the pretreatment layer is composite with a 0.5μm pore size cellulose filter membrane for pre-filtration of suspended impurities in food samples; the waste liquid recovery bottom layer uses absorbent filter paper to adsorb detection waste liquid and avoid residual solution interfering with subsequent detection.

[0019] Preferably, the sensor equipped with the sensitive electrode uses 0.15 mol / L hydrochloric acid buffer as the detection medium and employs linear scanning voltammetry for detection, with the scan rate set to 50 mV / s; the characteristic peak potential difference between chloramphenicol and metronidazole reduction is 0.173 V, and the electrochemical responses of the two substances do not interfere with each other, with both having a linear detection range of 0.5–250 μmol / L.

[0020] Preferably, when amoxicillin, sulfamethoxazole, norfloxacin, ampicillin, doxycycline, and cadmium / lead / copper / zinc / calcium ion interfering substances are added at 10 times the concentration to a standard detection system containing chloramphenicol (CAP) and metronidazole (MNZ), the relative change rate of the characteristic reduction peak current of chloramphenicol (CAP) and metronidazole (MNZ) is less than 7%; it can be used for the detection of animal-derived food matrices such as milk, chicken, and fish.

[0021] Preferably, the RSD of the peak current within the same batch of 5 parallel electrodes is ≤2.1%; the RSD of the peak current between batches of 10 electrodes from different preparation batches is ≤3.5%; and after the finished electrode is stored at room temperature in a dark and sealed container for 5 weeks, the retention rate of the characteristic reduction peak current of chloramphenicol (CAP) and metronidazole (MNZ) is ≥90%.

[0022] Preferably, low, medium and high concentration spiked recovery tests were conducted on blank milk, chicken and fish samples. The recovery rates of chloramphenicol (CAP) and metronidazole (MNZ) spiked in the range of 93.8% to 104.2%, and the RSD of each group was ≤3.9%. The detection does not require large-scale chromatographic and mass spectrometric instruments and is suitable for rapid on-site screening of food.

[0023] A method for preparing a screen-printed sensitive electrode for rapid detection of antibiotics in food includes the following steps:

[0024] Step 1: Preparation of MXene / MWCNTs-COOH nanocomposite powder: Weigh out the following powders at a mass ratio of 5:3: The MWCNTs-COOH were ultrasonically mixed with solvent, stirred, allowed to settle, dried at 60°C, and ground with agate to obtain a uniform composite powder.

[0025] Step 2: Preparation of composite functional ink for screen printing: The composite powder obtained in Step 1 is mixed into CI-2067 conductive carbon ink at a ratio of 10% of the total ink mass. The mixture is stirred in an agate mortar for 30 minutes and the rheological properties are finely adjusted with anhydrous ethanol to obtain a composite ink with a viscosity of 8.2 Pa·s.

[0026] Step 3: Preparation of hydrophobic microfluidic channels by wax printing: A hydrophobic pattern was printed on Whatman No.1 chromatography paper using a Fuji Xerox 8870DN wax printer. The paper was heated to 120°C for 90 seconds, and the wax melted and penetrated the filter paper fibers to form a hydrophobic barrier. A circular hydrophilic detection area with a diameter of 4 mm was reserved. The static water contact angle of the hydrophobic area was 145°.

[0027] Step 4: Layered screen printing of three electrodes: Using a 200-mesh screen, print the Ag / AgCl reference electrode, composite ink working electrode, and conductive carbon counter electrode in the following order; cure the reference electrode at 120℃ for 15 minutes, and cure the working electrode and counter electrode at 120℃ for 20 minutes.

[0028] Step 5, Backside waterproofing: Print a wax layer on the back of the chip detection area, heat at 120℃ for 90 seconds to cure the waterproof and hydrophobic layer; cut the chip according to the design size, seal the edges with hydrophobic tape, and obtain the finished sensitive electrode.

[0029] Preferably, the width of the hydrophobic microchannel prepared in step three is 2 mm; relying on the capillary action of the filter paper, the directional transport rate of the liquid is stable at 3 μL / s, and the hydrophobic barrier can completely block the lateral leakage of the liquid without channel deformation or shrinkage.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] The present invention proposes a screen-printed sensitive electrode for rapid detection of antibiotics in food and its preparation method, using a mass ratio of 5:3. A three-dimensional conductive network of sheet-tube interwoven structure was constructed by combining MXene with carboxylated multi-walled carbon nanotubes. One-dimensional carbon nanotubes were used to interpenetrate and block the stacking of MXene sheets, simultaneously improving electrode conductivity and the number of electrocatalytic active sites. This network was then combined with a specialized screen-printing composite ink (10% addition, steady-state viscosity 8.2 Pa·s) and a 120℃ segmented curing process to achieve large-scale, high-consistency, and low-cost batch preparation of the electrodes. A novel four-layer folded integrated structure was developed, combining a 145° highly hydrophobic directional microchannel formed by wax printing with a layered screen-printed integrated three-electrode system. This structure integrates sample pre-filtration, directional flow guidance, electrochemical detection, and waste liquid recovery functions into a single micro-paper-based chip. Effective separation of chloramphenicol and metronidazole reduction peaks was achieved in the optimal detection medium of 0.15 mol / L hydrochloric acid, covering a linear range of 0.5–250 μmol / L, with detection limits as low as 0.0 μmol / L for both. The concentrations of 25 μmol / L and 0.013 μmol / L reach the nanomolar level. The peak current change rate is less than 7% under the influence of 10 times the concentration of coexisting interfering substances in the system. The relative standard deviations of intra-batch and inter-batch detection are both less than 3.5%. After 5 weeks of sealed storage at room temperature, it still retains more than 90% of the electrochemical response signal. The spiked recovery rates of three types of animal-derived foods, namely milk, chicken and fish, are stable at 93.8%~104.2%. It does not require large-scale chromatography and mass spectrometry equipment. Only a small amount of sample is needed to complete the on-site simultaneous trace screening. It comprehensively overcomes the multiple defects of existing technologies, such as easy aggregation of MXene, difficulty in mass production of electrodes, single chip function, and inability to detect two antibiotics simultaneously with high sensitivity, from four dimensions: material microstructure, electrode preparation process, microfluidic integrated architecture, and actual detection performance. It provides a low-cost, high-stability, high-selectivity, and portable complete technical solution for rapid on-site detection of food and veterinary drug residues at the grassroots level. Attached Figure Description

[0032] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the present invention clear and complete, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only some, not all, embodiments of the present invention, and are merely illustrative of the embodiments of the present invention. They are not intended to limit the embodiments of the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1: The present invention provides a technical solution: a screen-printed sensitive electrode for rapid detection of antibiotics in food and its preparation method.

[0035] 1.1 Experimental Reagents and Instruments

[0036] (1) Experimental reagents: MX powder (purity > 98%, Xinxi Technology Co., Ltd.); 8 mg / mL MWCNTs-COOH aqueous dispersion (Suzhou Carbon-Feng Technology); CI-2067 conductive carbon ink, CIE4250 silver / silver chloride ink (Yinbiao Trading Co., Ltd.); Whatman No.1 chromatography paper (Titan Corporation); anhydrous ethanol, potassium chloride, potassium ferricyanide, potassium ferrocyanide, hydrochloric acid, chloramphenicol (CAP, purity > 98%), metronidazole (MNZ, purity > 98%), and various interfering standards (amoxicillin, sulfamethoxazole, norfloxacin, ampicillin, doxycycline, cadmium / lead / copper / zinc calcium ion standard solutions) were all analytical grade, from Sinopharm Chemical Reagent / Aladdin Biochemistry; 105℃ solid wax powder (Fuji Xerox); 0.5 μm cellulose filter membrane; hydrophobic sealing tape; ultrapure water was prepared in-house.

[0037] (2) Experimental instruments: LC-FA1004 electronic balance; JP-010T ultrasonic cleaner; C-MAG HS7 magnetic stirrer; 101 / 202AS electric thermostatic drying oven; LC-LVT-1 rotational viscometer; natural agate mortar; Fuji Xerox 8870DNColor Qube wax printer; 200 mesh screen printing plate; Drop Meter A-100 optical contact angle measuring instrument; GeminiSEM560 scanning electron microscope; D-MAX 2500 X-ray diffractometer; K-Alpha+ X-ray photoelectron spectrometer; Chenhua CHI660E electrochemical workstation; SN-LSC-40S low-speed centrifuge.

[0038] 1.2 Preparation of MXene / MWCNTs-COOH nanocomposite powder:

[0039] Solvent preparation: Measure equal volumes of ultrapure water and anhydrous ethanol, and mix them to obtain a 1:1 volume ratio mixed dispersion solvent;

[0040] MXene dispersion: Weigh precisely according to the MXene:MWCNTs-COOH mass ratio of 5:3. The powder was added to a mixed solvent and ultrasonically dispersed for 20 minutes to obtain a uniform MXene dispersion.

[0041] Composite mixing: Transfer 60 mL of 8 mg / mL MWCNTs-COOH aqueous dispersion and add it dropwise at a uniform rate to the MXene dispersion system, stirring at low speed throughout to prevent local agglomeration; after the addition is completed, sonicate for 30 min, and then magnetically stir for 1 h to achieve full composite mixing;

[0042] Drying and grinding: The mixture was left to stand overnight to separate into layers. The upper clear liquid was poured off and the lower composite precipitate was collected. The mixture was placed in a constant temperature oven at 60℃ for 12 hours and then cooled to room temperature. The agate was then thoroughly ground to obtain MXene / MWCNTs-COOH nanocomposite powder with no lumps and uniform particle size.

[0043] 1.3 Preparation of MXene / MWCNTs-COOH composite functional ink:

[0044] Ingredient weighing: Using CI-2067 conductive carbon ink as the base, accurately weigh the above composite powder according to the addition ratio of 10% of the total ink mass;

[0045] Grinding and blending: Pour all the powder into the conductive carbon ink, and continuously stir and grind manually in an agate mortar for 30 minutes to ensure that the powder is not agglomerated and is evenly dispersed in the ink matrix;

[0046] Rheological control: The viscosity of the ink was finely adjusted with a small amount of anhydrous ethanol and monitored in real time with a rotational viscometer. Finally, a composite functional ink with a steady-state viscosity of 8.2 Pa·s was obtained.

[0047] Ink performance testing: The adhesion test using the cross-cut adhesion test showed that the area of ​​ink film peeling off after curing was 2.8%; the 200-mesh screen printing test showed no ink overflow, pinholes, or screen clogging defects; the ink curing process conditions were 120℃ constant temperature for 20 minutes.

[0048] 1.4 Prefabrication of paper-based hydrophobic microfluidic channels:

[0049] Pattern design: Microfluidic pattern created using Adobe Illustrator. The overall size of a single chip is 16mm × 30mm, the channel width is 2mm, the diameter of the circular injection area is 10mm, and the diameter of the circular detection area is 10mm.

[0050] Wax printing: Whatman No.1 color paper is flattened and fixed on the wax printer, the pattern is imported and wax powder is printed, and the hydrophobic area fully covers the substrate outside the preset channel;

[0051] Melting into a hydrophobic barrier: After printing, the filter paper is quickly moved into a 120℃ oven and heated at a constant temperature for 90 seconds. The wax powder melts and penetrates into the filter paper fibers to form a continuous hydrophobic network.

[0052] Hydrophobic performance verification: Five hydrophobic areas were randomly selected for testing using a contact angle meter. The average static water contact angle was 145°. When 45 μL of ultrapure water was added to the sample area, the liquid flowed into the detection area at a rate of 3 μL / s due to capillary action, with no lateral leakage or channel shrinkage or deformation.

[0053] 1.5 Integrated Three-Electrode System for Screen Printing:

[0054] Using a 200-mesh screen printing stencil, Ag / AgCl reference electrode, screen-printed sensitive electrode, and CI-2067 conductive carbon counter electrode were printed layer by layer in sequence. Each layer was cured separately after printing.

[0055] Reference electrode printing: Print CIE4250 Ag / AgCl ink, with a forming size of 2mm wide and 18mm long, and cure in an oven at 120℃ for 15min;

[0056] Working electrode printing: A 4mm diameter circular sensing active area is printed with composite ink and cured in a 120℃ oven for 20 minutes to form a uniform, dense, sheet-tube interwoven three-dimensional conductive sensing film.

[0057] Electrode printing: Print CI-2067 conductive carbon ink, with dimensions symmetrical to the reference electrode, and cure in an oven at 120℃ for 20 minutes;

[0058] Alignment control: The reference and counter electrodes are symmetrically distributed on both sides of the circular working electrode, and the alignment deviation of the electrode pattern is controlled to be ≤0.5mm;

[0059] Back-side waterproofing treatment: The chip detection area is fully covered by wax printing on the back side, and heated at 120℃ for 90 seconds to form a bottom waterproof and hydrophobic layer;

[0060] Cutting and packaging: Cut a single paper-based chip according to the 16mm×30mm specification, seal the edges with 2mm wide hydrophobic tape, remove printing dust, dry in the dark and seal for storage, and obtain the semi-finished product of the screen-printed sensitive electrode of the present invention.

[0061] 1.6 Assembly of a four-layer folded microfluidic sensor:

[0062] With the detection layer containing the sensitive electrode as the lower layer, the four layers are stacked and assembled sequentially from top to bottom:

[0063] Top layer (pretreatment layer): Whatman No.1 chromatographic substrate, integrated with a 0.5μm cellulose filter membrane, 10mm diameter circular injection area, and a hydrophobic ring printed on the edge to prevent sample diffusion; Middle layer (fluid guiding layer): Pre-fabricated 2mm wide wax-printed microchannels to receive the sample from the top layer and directionally transport it to the bottom electrode; Bottom layer (three-electrode detection layer): Printed with the screen-printed sensitive electrode of this invention and its matching reference and counter electrodes; Bottom layer (waste liquid recovery layer): Highly absorbent filter paper for adsorbing waste liquid after detection;

[0064] Folding and encapsulation: Align the four layers along the preset creases, and fix the non-functional areas with 50μm double-sided tape. After folding, the overall outer contour size is 36mm×26mm, exposing only the sample injection area and electrode lead-out end, thus obtaining a complete paper-based microfluidic electrochemical sensor.

[0065] 1.7 Pretreatment of Standard Solutions and Actual Samples:

[0066] (1) Preparation of CAP and MNZ mixed standard solution:

[0067] Single stock solution: Accurately weigh 1.620 mg of CAP and 0.860 mg of MNZ, and dilute to 10 mL with anhydrous ethanol to prepare a 0.5 mmol / L single standard stock solution. Store at 4°C protected from light.

[0068] Series of mixed working solutions: Prepare CAP / MNZ mixed standard solutions with gradients of 0.5, 1, 10, 20, 50, 100, 150, 200, and 250 μmol / L using 0.15 mol / L HCl buffer as the dilution medium. Prepare and use immediately.

[0069] (2) Pretreatment of animal-derived food samples:

[0070] Milk (liquid sample): Transfer 5 mL of pure milk, add 10 mL of acetonitrile, sonicate for 15 min, centrifuge at 4000 rpm for 30 min; take 2 mL of supernatant, dilute to 20 mL with 0.15 mol / L HCl to a final volume of 10 times for testing;

[0071] Chicken / fish (solid sample): Weigh 5g of homogenized sample, sonicate in 15mL of acetonitrile for 20min, centrifuge at 4000rpm for 30min; take 2mL of supernatant, dilute to 10mL with 0.15mol / L HCl and test 5 times.

[0072] 1.8 Electrochemical detection methods and performance testing:

[0073] Detection instrument: CHI660E electrochemical workstation; Detection medium: 0.15 mol / L hydrochloric acid buffer; Detection mode: linear sweep voltammetry (LSV); Scan range: 0 V to -0.7 V (vs Ag / AgCl); Scan rate: 50 mV / s; Single injection volume: 45 μL; After the liquid wets the electrode, let it stand for 20 s before starting the scan.

[0074] (1) Linearity and detection limit test: Gradient mixed standard solutions were tested sequentially, and a standard curve was fitted with reduction peak current as the ordinate and concentration as the abscissa:

[0075] CAP: , Detection limit ;

[0076] MNZ: , Detection limit ;

[0077] Both have a linear range of 0.5~250μmol / L, a reduction peak potential difference of 0.173V, and the signals are completely separated without cross-interference.

[0078] (2) Anti-interference performance test: The base solution is a 100μmol / L CAP+MNZ mixed solution, and 10 times the molar concentration of interfering substances are added respectively: amoxicillin, sulfamethoxazole, norfloxacin, ampicillin, doxycycline, cadmium / lead / copper / zinc ions; the relative change rate of the peak current is all <7%, which can withstand complex food matrices such as milk, chicken and fish.

[0079] (3) Reproducibility test: Within the batch: 5 electrodes from the same batch were tested in parallel, and the RSD of the CAP peak current was 2.1%, and the RSD of the MNZ peak current was 1.8%;

[0080] Inter-batch testing: One electrode was taken from each of 10 independently prepared batches for testing. The peak current RSD of CAP was 3.2%, and the peak current RSD of MNZ was 2.9%.

[0081] (4) Storage stability test: The finished electrode was stored at room temperature in a dark and sealed container, and tested every week for 5 consecutive weeks; in the 5th week, the CAP peak current retention rate was 92.8% and the MNZ peak current retention rate was 91.6%, and both retention rates were ≥90%.

[0082] (5) Spike recovery test of actual samples: Low / medium / high (10, 100, 200 μmol / L) mixed standards were added to blank milk, chicken and fish respectively, and each group was tested in parallel 3 times: CAP recovery rate 94.2%~103.5%, RSD≤3.6%; MNZ recovery rate 93.8%~104.2%, RSD≤3.9%; No large HPLC or LC-MS / MS equipment is required, only a portable electrochemical workstation can be used to complete the on-site quantitative screening.

[0083] Comparative Example 1: Pure MXene ink only, without MWCNTs-COOH composite.

[0084] The preparation process uses only the same mass of pure The powder was added to CI-2067 carbon ink at a rate of 10%, and the remaining powder, ink, printing, and chip preparation processes were completely consistent with those in Example 1.

[0085] Performance defects: MXene sheets are extensively stacked and aggregated, resulting in an electrode electrochemical active area of ​​only 0.142 cm². 2 This is significantly lower than the 0.3108 cm in Example 1. 2 ;

[0086] When tested with the same 100 μmol / L mixed standard solution, the reduction peak currents of CAP and MNZ were only 52% and 48% of those in Example 1, respectively, and the detection limits were significantly increased. The ink viscosity was 14.4 Pa·s, which exceeded the range suitable for screen printing. This resulted in screen clogging, incomplete lines, and a film adhesion peeling area of ​​7.4%, indicating poor formability.

[0087] Comparative Example 2: MWCNTs-COOH ink only, without MXene lamination:

[0088] Only an equal mass of MWCNTs-COOH powder was added to the carbon ink; all other processes were the same as in Example 1.

[0089] Performance defects: The lack of a highly conductive continuous sheet network of MXene leads to a significant increase in electrode charge transfer resistance and a low electron transport rate; the catalytic response of CAP and MNZ is weak, the baseline noise is high, the peak shape is broadened, and it is impossible to achieve simultaneous and clear quantification of two antibiotics.

[0090] Comparative Example 3: The composite powder ratio deviates from the optimal 5:3 (MXene:MWCNTs-COOH=3:3):

[0091] The powder ratio was changed to 3:3, while the other ink and chip preparation processes remained unchanged.

[0092] Performance defects: Excessive carbon nanotubes and overly segmented MXene sheets prevented the formation of continuous conductive pathways; the CAP and MNZ peak currents were only 170% of those in Example 1, resulting in a significant decrease in detection sensitivity and an ink adhesion peeling area of ​​4.9%.

[0093] Comparative Example 4: Hydrophobic microfluidic system without wax printing, direct drop sample detection:

[0094] The process of removing the hydrophobic channels of the wax imprint involves non-directional microchannels, with the sample being added directly to the filter paper detection area.

[0095] Performance defects: The liquid diffuses irregularly, the sample is not evenly dispersed, and the concentration of the analyte on the electrode surface is unstable; the inter-batch RSD is greater than 8%, which makes it impossible to achieve stable quantification and does not meet the requirements for accurate micro-volume detection on site.

[0096] In Embodiment 1 of this invention, a 5:3 ratio of MXene / MWCNTs-COOH is used to form a sheet-tube three-dimensional conductive network, which completely improves the defects of single MXene stacking, increases the electrode active area by 4 times, and significantly enhances electrocatalytic activity. The special composite ink is suitable for screen printing, enabling batch and low-cost preparation of paper-based electrodes. The adhesion and printability meet the requirements for large-scale production. The wax-printed hydrophobic channel + four-layer folded integrated structure realizes in-situ filtration, directional transport, and waste liquid recovery of samples, allowing detection of trace samples. It can simultaneously detect CAP and MNZ at the nanomolar level, with excellent anti-interference, reproducibility, and storage stability. The spiked recovery rate of complex animal food meets the national standard testing requirements, eliminating the limitations of large precision instruments and making it suitable for rapid on-site screening of food safety at the grassroots level.

[0097] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A screen-printed sensitive electrode for rapid detection of antibiotics in food, characterized in that: The sensitive electrode is based on Whatman No.1 chromatographic paper, and a sensing active layer is formed on the surface of the sensitive electrode. The sensing active layer is formed by screen printing and constant temperature curing of MXene / MWCNTs-COOH nanocomposite functional ink. The MXene is A two-dimensional carbide material, wherein the MWCNTs-COOH is a carboxylated multi-walled carbon nanotube; The mass ratio of MXene to MWCNTs-COOH is 5:

3. The one-dimensional tubular structure of MWCNTs-COOH is interspersed in the gaps between MXene sheets, forming a three-dimensional conductive network of sheet-tube interwoven structure, which inhibits the stacking and aggregation of MXene sheets, and improves the electrode conductivity and the number of surface electrocatalytic active sites. The MXene / MWCNTs-COOH nanocomposite functional ink uses CI-2067 conductive carbon ink as the base, with MXene / MWCNTs-COOH composite powder added at 10% of the total ink mass. The steady-state viscosity of the ink is 8.2 Pa·s, suitable for 200-mesh screen printing. After being cured at 120℃ for 20 minutes, the composite ink forms a uniform and dense conductive film. The sensitive electrode enables simultaneous electrochemical detection of chloramphenicol (CAP) and metronidazole (MNZ). The detection limit for chloramphenicol is 0.025 μmol / L, and the detection limit for metronidazole is 0.013 μmol / L, both reaching the nanomolar level.

2. The screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 1, characterized in that: The preparation steps of the MXene / MWCNTs-COOH nanocomposite powder are as follows: Will The powder was ultrasonically dispersed in a 1:1 volume ratio of ultrapure water and anhydrous mixed solvent; MWCNTs-COOH aqueous dispersion was added dropwise to the MXene dispersion, ultrasonicated for 30 min and then magnetically stirred for 1 h; the mixed dispersion was allowed to stand overnight to settle, the lower precipitate was taken and dried in a 60℃ oven for 12 h, and after cooling, it was ground with agate to obtain composite powder. The composite ink showed a film peeling area of ​​≤2.8% after the cross-cut adhesion test; when printed with a 200-mesh screen, there were no defects such as ink overflow, pinholes, or screen clogging.

3. The screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 2, characterized in that: The sensitive electrode is the working electrode in an integrated paper-based three-electrode system. The integrated three-electrode system is prepared by screen printing. The reference electrode is printed with CIE4250 Ag / AgCl conductive ink, and the counter electrode is printed with CI-2067 conductive carbon ink. The reference electrode and the counter electrode are symmetrically arranged on both sides of the sensitive electrode, and the electrode printing alignment deviation is ≤0.5mm. The three electrodes constitute a complete electrochemical detection circuit.

4. The screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 1, characterized in that: The sensitive electrode is assembled inside a four-layer folded paper-based microfluidic electrochemical sensor. The four layers, from top to bottom, are a sample pretreatment layer, a fluid guiding middle layer, a three-electrode detection lower layer, and a waste liquid recovery bottom layer. The overall size of each layer after folding along the preset crease is 36mm × 26mm. The pretreatment layer is composited with a 0.5μm pore size cellulose filter membrane for pre-filtration of suspended impurities in food samples. The waste liquid recovery bottom layer uses absorbent filter paper to adsorb detection waste liquid and avoid residual solution interfering with subsequent detection.

5. The screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 4, characterized in that: The sensor equipped with the aforementioned sensitive electrode uses 0.15 mol / L hydrochloric acid buffer as the detection medium and employs linear scanning voltammetry with a scan rate set to 50 mV / s. The characteristic peak potential difference between chloramphenicol and metronidazole reduction is 0.173 V, and the electrochemical responses of the two substances do not interfere with each other. The linear detection range for both is 0.5–250 μmol / L.

6. The screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 1, characterized in that: When amoxicillin, sulfamethoxazole, norfloxacin, ampicillin, doxycycline, and cadmium / lead / copper / zinc / calcium ion interfering substances are added to a standard detection system containing chloramphenicol (CAP) and metronidazole (MNZ) at 10 times the concentration, the relative change rate of the characteristic reduction peak current of chloramphenicol (CAP) and metronidazole (MNZ) is less than 7%; it can be used for the detection of animal-derived food matrices such as milk, chicken, and fish.

7. The screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 1, characterized in that: The intra-batch peak current RSD of 5 parallel electrodes in the same batch is ≤2.1%; the inter-batch peak current RSD of 10 electrodes from different preparation batches is ≤3.5%; after the finished electrodes are stored at room temperature in the dark and sealed for 5 weeks, the retention rate of characteristic reduction peak currents of chloramphenicol (CAP) and metronidazole (MNZ) is ≥90%.

8. The screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 1, characterized in that: Recovery tests were conducted on blank milk, chicken, and fish samples at low, medium, and high concentrations. The recoveries of chloramphenicol (CAP) and metronidazole (MNZ) ranged from 93.8% to 104.2%, with RSDs ≤ 3.9% for each group. The detection does not require large-scale chromatographic or mass spectrometric instruments and is suitable for rapid on-site screening of food.

9. A method for preparing a screen-printed sensitive electrode for rapid detection of antibiotics in food as described in any one of claims 1-8, characterized in that: Includes the following steps: Step 1: Preparation of MXene / MWCNTs-COOH nanocomposite powder: Weigh out the following powders at a mass ratio of 5:3: The MWCNTs-COOH were ultrasonically mixed with solvent, stirred, allowed to settle, dried at 60°C, and ground with agate to obtain a uniform composite powder. Step 2: Preparation of composite functional ink for screen printing: The composite powder obtained in Step 1 is mixed into CI-2067 conductive carbon ink at a ratio of 10% of the total ink mass. The mixture is stirred in an agate mortar for 30 minutes and the rheological properties are finely adjusted with anhydrous ethanol to obtain a composite ink with a viscosity of 8.2 Pa·s. Step 3: Preparation of hydrophobic microfluidic channels by wax printing: A hydrophobic pattern was printed on Whatman No.1 chromatography paper using a Fuji Xerox 8870DN wax printer. The paper was heated to 120°C for 90 seconds, and the wax melted and penetrated the filter paper fibers to form a hydrophobic barrier. A circular hydrophilic detection area with a diameter of 4 mm was reserved. The static water contact angle of the hydrophobic area was 145°. Step 4: Layered screen printing of three electrodes: Using a 200-mesh screen, print the Ag / AgCl reference electrode, composite ink working electrode, and conductive carbon counter electrode in the following order; cure the reference electrode at 120℃ for 15 minutes, and cure the working electrode and counter electrode at 120℃ for 20 minutes. Step 5, Backside waterproofing: Print a wax layer on the back of the chip detection area, and heat at 120℃ for 90 seconds to cure the waterproof and hydrophobic layer; The chip is cut to the designed dimensions and the edges are sealed with hydrophobic tape to obtain the finished sensitive electrode.

10. The method for preparing a screen-printed sensitive electrode for rapid detection of antibiotics in food according to claim 9, characterized in that: The hydrophobic microchannels prepared in step three have a width of 2 mm. Relying on the capillary action of the filter paper, the directional transport rate of the liquid is stable at 3 μL / s. The hydrophobic barrier can completely block the lateral leakage of the liquid, and there is no channel deformation or shrinkage.

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