Aminoglycoside antibiotic dual-mode visual detection card as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemical sensing, in particular to an aminoglycoside antibiotic dual-mode visual detection card and a preparation method and application thereof, and the detection card is composed of two parts: an aminoglycoside antibiotic dual-mode labeling reagent and qualitative filter paper. The dual-mode labeling reagent is formed by self-assembling ninhydrin and lanthanide metal ions, and is used for providing a color development / ratio fluorescence dual-mode detection signal; the qualitative filter paper is used for efficient adsorption and enrichment of aminoglycoside antibiotics, and can significantly enhance color development and fluorescence signals. The detection card provided by the invention can realize double-mode (color development and fluorescence) visual detection and identification of aminoglycoside antibiotics only by using one labeling reagent, and is good in response selectivity, high in sensitivity, strong in anti-interference capability, mild in condition and easy to popularize. The detection accuracy and reliability can be remarkably improved through cross validation of double-signal-channel visual detection results.

Description

Technical Field

[0001] This invention relates to the field of biochemical sensing technology, specifically to a dual-mode visual detection card for aminoglycoside antibiotics, its preparation method, and its application. Background Technology

[0002] Antimicrobial agonists (AGs), as potent and broad-spectrum antibiotics, are commonly used to treat bacterial infections and are widely applied in clinical practice, animal husbandry, and aquaculture. However, studies have reported a close association between AGs and ototoxicity and nephrotoxicity. Misuse and abuse of AGs may increase human health risks through the food chain and prolonged environmental exposure. AG residues in environmental water bodies can also accelerate bacterial resistance and lead to the emergence of superbugs. Therefore, the detection of AG residues in food and environmental samples is of great significance.

[0003] Because agglutinosa (AGs) lack chromophores and have poor light absorption, traditional UV-Vis spectroscopy is difficult to use directly for AG detection. High-performance liquid chromatography (HPLC), mass spectrometry (MS), chromatographic-mass spectrometry tandem (LC-MS / GC-MS), electrochemical detection, microbiological techniques, and immunoassays remain the primary methods for AG detection. While these methods can yield satisfactory results, they are highly dependent on bulky instruments and skilled technicians, have high technical barriers, poor portability, cumbersome pretreatment processes, and are time-consuming, making them unsuitable for rapid on-site detection.

[0004] A visualization-based detection strategy using colorimetric analysis is one feasible approach to address the aforementioned problems. Existing technologies disclose a CH3COOAg material with laccase-like activity for AG detection, using 2,4-dichlorophenol as a substrate and 4-aminoantipyrine as a chromogenic agent. Five AGs exhibit varying degrees of inhibition of the laccase-like activity of CH3COOAg in different buffer systems. Based on this, a four-channel colorimetric sensor array was designed, employing four solutions as sensing channels for efficient identification and detection of AGs. Existing technologies also disclose a compartmentalized dual nanoenzyme cascade complex (AmPC) for AG colorimetric detection. AmPC possesses activities similar to both glucose oxidase and peroxidase. AGs with sugar structures serve as the initial substrate for the reaction, while the generated H2O2 acts as the second substrate for colorimetric detection. This cascade reaction can effectively detect AGs. Ninhydrin (Nin) has good colorimetric properties for amine compounds, especially primary amines. Due to its simple operation, it is also widely used for visual colorimetric determination of AGs. For example, the detection of tobramycin in eye drops was achieved by combining ninhydrin colorimetry and thin-layer chromatography; amikacin was determined by combining ninhydrin colorimetry with spectrophotometry.

[0005] However, the aforementioned visualization detection methods all suffer from the problem of signal monotony. This type of colorimetric analysis based on a single color is easily affected by environmental factors. In addition, the ninhydrin colorimetric reaction also suffers from problems such as unstable color development, high reaction temperature (often above 90 °C), long reaction time (usually more than 30 minutes), poor selectivity (it can develop color for various amine compounds), and susceptibility to interference from impurities (such as amino acids). As a result, the reliability of colorimetric analysis results based on this reaction is generally poor, and it is also unable to distinguish between different types of AGs.

[0006] Compared to single-color-based colorimetric methods, multi-signal / mode colorimetry is attracting increasing attention. This strategy not only improves detection throughput but also enhances the accuracy and reliability of colorimetric analysis through cross-validation between different detection signals / modes. For example, a nano-gold (AuNPs) colorimetric sensor array is used for the simultaneous detection of multiple agglutinants (AGs). AuNPs of different particle sizes are used as different channels in the array sensor, resulting in different colors appearing in different AGs, thus achieving multi-color visualization detection. However, sensor arrays based on AuNPs are not only costly to use, but the tendency of AuNPs to aggregate can also lead to false positives. A comparative study was conducted on 10 commercially available colloidal gold rapid detection products for malachite green (MG) in terms of false positive / negative rates, detection rates, and stability. The results showed that only 3 products could accurately detect MG in 84 fish paste samples, while the remaining products showed false positives or false negatives, with significant differences between different batches. A comprehensive evaluation was conducted on 5 commercially available colloidal gold test strips for fungal toxins in corn, wheat, and brown rice matrices in terms of accuracy, precision, quantitative range, detection time, and cost. The results showed that the recovery rates of these products for the detection of several common fungal toxins in corn, wheat, and brown rice matrices ranged from 61% to 178%, with relative standard deviations ranging from 4% to 38%, and significant differences between different brands.

[0007] It is evident that multi-signal / mode colorimetric strategies possess many unique advantages in the visual detection of agglutinated AGs. However, to achieve diverse colorimetric signals, it is often necessary to prepare multiple colorimetric channels or arrays composed of different colorimetric schemes / signal modes. This not only complicates the technical approach but also increases the cost. Developing a rapid visual detection and identification method for multi-signal / mode agglutinated AGs based on a single labeled reagent could overcome these technical bottlenecks, but no relevant reports have been found to date.

[0008] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0009] The purpose of this invention is to solve the problems that AGs colorimetric analysis based on single-color display is easily affected by environmental factors, and that existing multi-signal visualization detection systems are highly dependent on the construction of multiple colorimetric channels or arrays, which leads to complicated technical routes and high usage costs. This invention provides a dual-mode visualization detection card for aminoglycoside antibiotics, its preparation method and its application.

[0010] To achieve the above objectives, this invention discloses a method for preparing a dual-mode visual detection card for aminoglycoside antibiotics, comprising the following steps:

[0011] S1, ninhydrin and Ln 3+ Dissolved in an aqueous ethanol solution containing acetic acid and sodium acetate, a dual-mode labeling reagent for aminoglycoside antibiotics is obtained;

[0012] S2, prepare standard samples of aminoglycoside antibiotics with concentration gradients, adsorb them with qualitative filter paper, and after adsorption, use the aminoglycoside antibiotic dual-mode labeling reagent obtained in step S1 to label and develop color. Arrange the colored filter paper according to the concentration gradient of aminoglycoside antibiotics to obtain a visual colorimetric card.

[0013] S3. Place the visual colorimetric card obtained in step S2 into an excitation light source to excite fluorescence, take a picture to record the result, and arrange the filter paper according to the concentration gradient of aminoglycoside antibiotics to obtain the fluorescence colorimetric card.

[0014] In step S1, Ln 3+ For La 3+ Eu 3+ Gd 3+ 、Tb 3+ Any one of the lanthanide metal ions.

[0015] In step S1, the aminoglycoside antibiotic dual-mode labeling reagent contains: ninhydrin 10 mg / mL, acetic acid 0.5 M, sodium acetate 0.25 M, and... 10 mg / mL or 2.5 mg / mL.

[0016] In step S2, the adsorption of the filter paper is either physical adsorption or chemical adsorption.

[0017] In step S2, the visual colorimetric chart includes 10 color blocks for each antibiotic, and each color block corresponds to the concentration of the corresponding aminoglycoside antibiotic.

[0018] In step S2, the concentration gradient of the aminoglycoside antibiotic standard samples is 0, 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, and 10 μg / mL.

[0019] In step S3, the contrast color of each antibiotic in the fluorescent colorimetric card includes 10 color blocks, and each color block corresponds to the concentration of the corresponding aminoglycoside antibiotic.

[0020] The present invention also discloses a dual-mode visualization detection card for aminoglycoside antibiotics prepared by the above preparation method.

[0021] This invention also discloses the application of the aforementioned dual-mode visual detection card for aminoglycoside antibiotics in the quantitative and semi-quantitative visual detection and identification of aminoglycoside antibiotic content in food, environmental, or clinical samples. During detection, the color / fluorescence of the test strip after processing the actual sample is compared with the colorimetric card to semi-quantitatively determine the antibiotic concentration; or the ratio value is calculated by measuring the fluorescence spectrum and substituted into a standard curve for quantitative analysis.

[0022] In this invention, the filter paper not only extracts and pre-concentrates alginates (AGs) to enhance colorimetric and fluorescence signals, but also transfers AGs from the solution onto the paper, thus avoiding the influence of the aqueous substrate on the fluorescence of the composite labeled reagent. This involves solid-phase microextraction, colorimetric development, and Ln... 3+ Based on multiple technical fields such as fluorescence emission due to antenna effect; Ln in this invention 3+ It self-assembles with ninhydrin to form a composite labeling reagent (abbreviated as Nin@Ln). 3+ It provides dual labeling signals of color and fluorescence, but the two signals are interrelated. Specifically, the deeper the color, the weaker the fluorescence, and vice versa. The principle of generating this dual signal is based on the color reaction between ninhydrin and primary amino groups and the fluorescence quenching phenomenon based on the internal filtration effect.

[0023] Compared with existing technologies, the beneficial effects of this invention are as follows: Compared with traditional ninhydrin colorimetric methods, under the same conditions, this detection card significantly improves the colorimetric effect and increases the detection sensitivity. The detection limit for AGs is as low as 0.08 μg / mL, far below the maximum limits in relevant national standards such as 'GB8979-1996 Integrated Wastewater Discharge Standard' and 'GB31650-2019 National Food Safety Standard Maximum Residue Limits for Veterinary Drugs in Food'. This invention's detection card also integrates both colorimetric and ratiometric fluorescence signal modes. This dual-mode colorimetric detection effectively improves the reliability of visualized detection results. Furthermore, this invention's detection card greatly reduces the temperature required for the ninhydrin colorimetric reaction (from over 90℃ to 20-40℃), significantly shortens the colorimetric time (from over 30 minutes to 3-5 minutes), and improves anti-interference performance (substances such as amino acids that are colorimetric in traditional ninhydrin colorimetric reactions are not colorimetric under the conditions of this invention), making the detection conditions milder while significantly improving colorimetric performance, greatly enhancing practicality. Attached Figure Description

[0024] Figure 1This is a schematic diagram illustrating the principle of the colorimetric card preparation route of the present invention;

[0025] Figure 2 In the image, (A) is a scanning electron microscope image of bare filter paper; (B) is the UV-Vis spectrum of Nin solution in the presence of different AGs; (C) is an image of a mixed solution of Nin and different AGs, and filter paper with or without AGs extracted after Nin color development. The combination order of Nin and AGs 1-7 in (C) is consistent with the top-to-bottom arrangement of the legend in (B).

[0026] Figure 3 In the table, (A) represents the solution levels Nin and Nin@Eu. 3+ and Nin@Tb 3+ Fluorescence spectra before and after incubation with NEO, (B) showing Nin and Nin@Eu on filter paper. 3+ and Nin@Tb 3+ Fluorescence spectra before and after incubation with NEO; (C) is Nin@Eu 3+ / Nin@Tb 3+ The photostability of the droplet on filter paper after 60 minutes of continuous irradiation; (A) Inset showing Nin, Nin@Eu 3+ and Nin@Tb 3+ The fluorescence digital photographs of the solution (excitation wavelength 365 nm) are arranged from left to right in the same order as the top-to-bottom order in Figure (A); Figure (B) shows the appearance (top) and fluorescence (bottom, 365 nm excitation) photographs of different filter papers, and their layout matches the top-to-bottom order in Figure (B).

[0027] Figure 4 In the image, (A) shows the color development effect of ninhydrin solution prepared with different solvents, and (B) shows the color development effect of ninhydrin solution prepared with different ethanol concentrations. In (A), the solvents from left to right are ethanol, isobutanol, methanol, DMSO, acetonitrile, and DMF.

[0028] Figure 5 In the diagram, (A) shows the effect of Nin dosage on the color development effect of filter paper before NEO adsorption; (B) shows the effect of Nin dosage on the color development effect of filter paper after NEO adsorption; (C) shows the effect of heating temperature on the color development effect; (D) shows the effect of heating time on the color development effect; and (E) shows the effect of NEO dosage on the color development effect of ninhydrin. The paper used in the left columns of (A) and (C) is bare filter paper, while the paper used in the right columns of (B, D, E) and (C) is filter paper that has adsorbed NEO.

[0029] Figure 6 In the figure, (A) represents Nin@Tb under different pH conditions. 3+ Fluorescence spectra of labeled bare filter paper; (B) Nin@Tb under different pH conditions. 3+Fluorescence spectra of labeled NEO extraction filter paper; (C) Nin@Tb under different pH conditions 3+ The curves showing the FL strength of labeled bare filter paper and NEO-extracted filter paper as a function of pH, where the filter paper used is bare filter paper (left) and NEO-extracted filter paper (right), respectively; (D) shows Nin@Tb under different pH conditions. 3+ The marked filter paper shows the FL strength ratio before and after NEO bonding; (E) represents the bare filter paper in Nin@Tb. 3+ Digital photographs of the appearance (left) and fluorescence (right) after staining, showing the changes at pH values ​​from 2.5 to 12.5; (F) shows the NEO extraction filter paper in Nin@Tb 3+ Digital photographs of the appearance (left) and fluorescence (right) after staining show the changes in pH range 2.5 to 12.5; (E, F) Each group was repeated 5 times, with NEO concentration set at 1.5 μg / mL, NEO extraction time at 30 minutes, and NEO concentration at 1.5 μg / mL.

[0030] Figure 7 In the image, (A) represents bare filter paper in Nin@Tb 3+ Fluorescence spectrum after 30 minutes of incubation; (B) NEO anchoring filter paper in Nin@Tb 3+ Fluorescence spectrum after 30 minutes of incubation; (C) shows the fluorescence intensity as a function of Tb. 3+ The functional relationship of concentration change; (D) is the bare filter paper in Nin@Eu 3+ Fluorescence spectrum after 30 minutes of incubation; (E) shows the NEO-anchored filter paper in Nin@Eu 3+ Fluorescence spectrum after 30 minutes of incubation; (F) shows the fluorescence intensity as a function of Eu. 3+ Functional relationship of concentration change;

[0031] Figure 8 In the image, (A) represents bare filter paper in Nin@Tb 3+ via Nin@Tb 3+ (Left) and Nin@Eu 3+ (Right) Digital photograph of the fluorescent color after marking; (B) Filter paper with NEO binding after Nin@Tb 3+ (Left) and Nin@Eu 3+ (Right) Digital photograph of the fluorescent color after marking; (C) Bare filter paper in Nin@Tb 3+ via Nin@Tb 3+ (Left) and Nin@Eu 3+ (Right) Digital photograph of apparent color after marking; (D) Filter paper with NEO bonded to Nin@Tb 3+ (Left) and Nin@Eu3+ (Right) A digital photograph of the apparent color after marking;

[0032] Figure 9 In the middle, (A) represents paper with different antibiotic loadings after Nin@Eu 3+ (B) Fluorescence spectra after labeling; Nin@Eu preloaded with different antibiotics 3+ The fluorescence intensity ratio of the labeled paper; (C) represents the fluorescence intensity ratio of paper loaded with different antibiotics after Nin@Tb treatment. 3+ (D) Fluorescence spectra after labeling; Nin@Tb preloaded with different antibiotics. 3+ The fluorescence intensity ratio of the marked paper; (Nin@Eu) 3+ I marking the paper 618 / I 696 Nin@Tb 3+ I marking the paper 546 / I 621 );

[0033] Figure 10 In the middle, (A) is related to Nin@Eu 3+ Fluorescence images (top three) and appearance (bottom three) of paper loaded with different aminoglycoside antibiotics after incubation; (B) is for Nin@Tb 3+ Fluorescence images (top three) and appearance (bottom three) of papers loaded with different aminoglycoside antibiotics after incubation; among them, the antibiotics used in (1~18) are... Figure 9 The x-coordinates in (B) and (D) correspond in order;

[0034] Figure 11 In the middle, (A) represents the effect of commonly used food additives on Nin@Eu 3+ The effect of labeling on the fluorescence intensity of filter paper; (B) the effect of common food additives on Nin@Eu 3+ The effect of the fluorescence ratio of labeled filter paper; (C) is the effect of common food additives on Nin@Tb 3+ The effect of labeling on the fluorescence intensity of filter paper; (D) is the effect of common food additives on Nin@Tb 3+ The effect of the fluorescence ratio of the labeled filter paper; the papers used in (A~D) (1~13) were NEO anchoring paper, phosphate buffer incubation paper (control), and paper loaded with DL-malic acid, citric acid, high fructose syrup, acesulfame potassium, potassium sorbate, sodium benzoate, stevia hydrate, sucralose, sucrose, Glc and food flavoring, respectively.

[0035] Figure 12 In the middle, (A) represents the effect of commonly used food additives on Nin@Eu 3+ The effect of labeling on the fluorescence intensity of filter paper; (B) the effect of common food additives on Nin@Eu 3+The effect of the fluorescence ratio of labeled filter paper; (C) is the effect of common food additives on Nin@Tb 3+ The effect of labeling on the fluorescence intensity of filter paper; (D) is the effect of common food additives on Nin@Tb 3+ The effect of the fluorescence ratio of the labeled filter paper; the papers used in (A~D) (1~13) were NEO anchoring paper, phosphate buffer incubation paper (control), and paper loaded with DL-malic acid, citric acid, high fructose syrup, acesulfame potassium, potassium sorbate, sodium benzoate, stevia hydrate, sucralose, sucrose, Glc and food flavoring, respectively.

[0036] Figure 13 In the diagram, (A) represents the potential environmental disturbance to Nin@Eu. 3+ The effect of labeled filter paper fluorescence intensity; (B) the effect of potential environmental interfering substances on Nin@Eu 3+ The effect of the fluorescence ratio of the labeled filter paper; (C) represents the effect of potential environmental interfering substances on Nin@Tb. 3+ The effect of labeled filter paper fluorescence intensity; (D) on the effect of potential environmental interfering substances on Nin@Tb 3+ The effect of the fluorescence ratio of labeled filter paper; the papers used in (A~D) (1-20) were NEO anchoring paper, phosphate buffer incubation paper (control), and paper bound to potassium chloride, calcium chloride, magnesium chloride, ferric chloride, (CH3COO)2Zn, NiCl2, lithium fluoride, barium chloride, Ser, Tyr, Asp, Lys, Cys, Phe, Trp, Glu and Gly.

[0037] Figure 14 In the diagram, (A) represents the potential environmental disturbance to Nin@Eu. 3+ The effect of labeled filter paper fluorescence intensity; (B) the effect of potential environmental interfering substances on Nin@Eu 3+ The effect of the fluorescence ratio of the labeled filter paper; (C) represents the effect of potential environmental interfering substances on Nin@Tb. 3+ The effect of labeled filter paper fluorescence intensity; (D) on the effect of potential environmental interfering substances on Nin@Tb 3+ The effect of the fluorescence ratio of labeled filter paper; the papers used in (A~D) (1-20) are NEO anchoring paper, phosphate buffer incubation paper (control), and paper bound to potassium chloride, calcium chloride, magnesium chloride, ferric chloride, (CH3COO)2Zn, NiCl2, lithium fluoride, barium chloride, Ser, Tyr, Asp, Lys, Cys, Phe, Trp, Glu, and Gly, respectively. The insets in (B, D) are digital photographs of amino acids developed by Nin at 60℃ and 100℃, respectively, from left to right: Gly, Asp, Glu, Phe, Try, Lys, Thr, Cys, Ser, and Tyr;

[0038] Figure 15 In the middle, (A) is ninhydrin-Eu using tap water as a solvent. 3+ (a) Colorimetric charts of NEO fluorescence (top three) / apparent color (bottom three) prepared for the sensing system; (b) Colorimetric charts of ninhydrin-Tb prepared with tap water as solvent. 3+ NEO fluorescence (top three sheets) / apparent color (bottom three sheets) colorimetric cards prepared for the sensing system; (C) is a colorimetric card using river water as solvent and ninhydrin-Eu 3+ NEO fluorescence (top three images) / apparent color (bottom three images) colorimetric cards prepared for the sensing system; (D) is a colorimetric card using river water as solvent and ninhydrin-Tb 3+ NEO fluorescence (top three sheets) / apparent color (bottom three sheets) colorimetric cards prepared for the sensing system; (E) is a colorimetric card using aquaculture pond water as solvent and ninhydrin-Eu 3+ NEO fluorescence (top three sheets) / apparent color (bottom three sheets) colorimetric cards prepared for the sensing system; (F) shows the ninhydrin-Tb colorimetric cards prepared using fishpond water as solvent. 3+ NEO fluorescence (top three sheets) / apparent color (bottom three sheets) colorimetric cards prepared for the sensing system; NEO spiked (1.5 μg / mL); the orange cross arrows in (A~F) represent the color blocks closest to the paper sensors with different sample pretreatments and the corresponding fluorescence / colorimetric doses;

[0039] Figure 16 In the middle, (A) is preloaded NEO filter paper processed with Nin@Eu 3+ (B) Fluorescence spectrum after labeling; 3+ The relationship between the fluorescence intensity ratio after labeling and the NEO concentration; (C) is the relationship between the pre-loaded NEO filter paper and Nin@Tb. 3+ The fluorescence spectrum after labeling; (D) is the fluorescence spectrum of Nin@Tb preloaded NEO filter paper. 3+ The relationship between the fluorescence intensity ratio after labeling and NEO concentration; (A, B) Nin@Eu 3+ I marking the paper 618 / I 696 (C, D)Nin@Tb 3+ I marking the paper 546 / I 621 Among them, the samples used for paper pretreatment were tap water with added NEO (A~D), and the dosage gradient of each sample was 0~10 μg / mL; the inset in (D) is a digital photograph of tap water;

[0040] Figure 17 In the middle, (A) is preloaded NEO filter paper processed with Nin@Eu 3+ (B) Fluorescence spectrum after labeling; 3+The relationship between the fluorescence intensity ratio after labeling and the NEO concentration; (C) is the relationship between the pre-loaded NEO filter paper and Nin@Tb. 3+ The fluorescence spectrum after labeling; (D) is the fluorescence spectrum of Nin@Tb preloaded NEO filter paper. 3+ The relationship between the fluorescence intensity ratio after labeling and NEO concentration; (A, B) Nin@Eu 3+ I marking the paper 618 / I 696 (C, D)Nin@Tb 3+ I marking the paper 546 / I 621 The samples used for paper pretreatment were river water with added NEO (A~D), and the dosage gradient for each sample was 0~10 μg / mL; the inset in (D) is a digital photograph of the river water;

[0041] Figure 18 In the middle, (A) is preloaded NEO filter paper processed with Nin@Eu 3+ (B) Fluorescence spectrum after labeling; 3+ The relationship between the fluorescence intensity ratio after labeling and the NEO concentration; (C) is the relationship between the pre-loaded NEO filter paper and Nin@Tb. 3+ The fluorescence spectrum after labeling; (D) is the fluorescence spectrum of Nin@Tb preloaded NEO filter paper. 3+ The relationship between the fluorescence intensity ratio after labeling and NEO concentration; (A, B) Nin@Eu 3+ I marking the paper 618 / I 696 (C, D)Nin@Tb 3+ I marking the paper 546 / I 621 The samples used for filter paper pretreatment were aquaculture pond water with added NEO (A~D), and the dosage gradient for each sample was 0~10 μg / mL; (D) is an inset of digital photographs of aquaculture pond water;

[0042] Figure 19 In the above, (A) represents ninhydrin-Tb. 3+ The NEO apparent colorimetric card prepared by the sensing system (left of the vertical solid line) and the actual samples of milk, chicken and shrimp meat spiked and analyzed (right of the vertical solid line); (B) is ninhydrin-Tb 3+ NEO fluorescence colorimetric card prepared by the sensing system (left of the vertical solid line) and colorimetric analysis of actual samples of milk, chicken and shrimp (right of the vertical solid line); (C) is ninhydrin-Tb 3+ The AMK apparent colorimetric card prepared by the sensing system (left of the vertical solid line) and the actual samples of milk, chicken and shrimp (right of the vertical solid line); (D) is ninhydrin-Tb 3+The AMK fluorescence colorimetric card prepared by the sensing system (left of the vertical solid line) and the actual samples of milk, chicken and shrimp meat spiked and analyzed colorimetrically (right of the vertical solid line); (E) is ninhydrin-Tb 3+ The apparent colorimetric card of the sensor system (left of the vertical solid line) and the actual samples of milk, chicken and shrimp (right of the vertical solid line) were prepared; (F) is ninhydrin-Tb 3+ The PAR fluorescence colorimetric card prepared by the sensing system (left of the vertical solid line) and the actual samples of milk, chicken and shrimp (right of the vertical solid line) were analyzed by spiked colorimetry. In the actual samples (A~E), 1 were all unspecified samples, 2 were all samples spiked with 0.3 μg / mL of the corresponding antibiotic, and in (A~D), 3 were samples spiked with 1.5 μg / mL of the corresponding antibiotic, and the rest were samples spiked with 0.6 μg / mL of the corresponding antibiotic.

[0043] Figure 20 In the above, (A) represents ninhydrin-Tb. 3+ Fluorescence spectrum of the NEO colorimetric card made from the sensing system; (B) is ninhydrin-Tb 3+ The ratio and concentration relationship of the NEO colorimetric card made by the sensing system; (C) is ninhydrin-Tb 3+ Fluorescence spectrum of the AMK colorimetric card prepared by the sensing system; (D) is ninhydrin-Tb 3+ The ratio and concentration relationship of the AMK colorimetric card made by the sensing system; (E) is ninhydrin-Tb 3+ Fluorescence spectrum of the PAR colorimetric card prepared by the sensing system; for ninhydrin-Tb 3+ The ratio and concentration relationship of the PAR colorimetric card made by the sensing system. The insets in Figures (A, C, E) are digital photographs of commercially available bottled milk, shrimp and chicken samples, respectively.

[0044] Figure 21 In the middle, (A) represents bare filter paper (control group) and filter paper treated with Nin@Tb. 3+ Digital photographs of the fluorescence (top) and apparent color (bottom) of different AGs extracted from filter paper after labeling; (B) is the fluorescence intensity ratio of different AGs (I). 546 / I 621 (A) Comparison of fluorescence intensity and RGB values ​​(apparent color is (R+B) / G, fluorescence is (R+G) / B); (C) Spatial distribution of the five AGs determined based on the ratio of fluorescence intensity to RGB values; (D) Principal component analysis of the five AGs using the ratio of fluorescence intensity to RGB values ​​as the main variable. Detailed Implementation

[0045] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0046] Example 1

[0047] Fabrication of a dual-response paper sensor:

[0048] This embodiment uses neomycin (NEO) as an example to illustrate in detail the process of making a dual-mode colorimetric card.

[0049] S1. Preparation of standard NEO sample solution:

[0050] Accurately weigh NEO standard samples, dissolve and dilute to volume with phosphate buffer (0.1 M, pH 7.2-7.4) to prepare a stock solution with a concentration of 1 mg / mL. Subsequently, perform serial dilutions with PB buffer to obtain NEO solutions with concentrations of 0, 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, and 10 μg / mL. This concentration range covers the national standard limit (e.g., 1.5 μg / mL) and extends to lower and higher concentrations to ensure the applicability of the colorimetric card.

[0051] S2, Antibiotic Adsorption and Color Development:

[0052] Preparation route as follows Figure 1 As shown. First, a bare filter paper disc with a diameter of 6 mm was taken, and its SEM characterization is as follows. Figure 2 A. The filter paper exhibits a typical porous, interwoven cellulose network structure with a large specific surface area, providing favorable conditions for physical adsorption. The filter paper was then washed twice each with ultrapure water and ethanol, 5 min each time, dried in a 60 ℃ oven, and placed in a 96-well microplate. 200 μL of the aforementioned gradient concentration of NEO solution was added to each well, and the plate was incubated at room temperature for 5 min to allow the filter paper to fully adsorb the NEO solution through electrostatic interactions. After incubation, the liquid in the wells was removed with a pipette, and the filter paper was washed twice with PB buffer (pH 7.2-7.4) to remove non-specifically adsorbed impurities. Subsequently, 10 μL of ninhydrin-based solution prepared in Example 3 was added to each filter paper. Colorimetric solution (or ninhydrin-) (Developing solution). Transfer the entire ELISA plate to a 60 °C oven and heat for 10 minutes. During this process, NEO adsorbed on the filter paper reacts with ninhydrin to generate a blue-purple Ruhrmann violet (RP). / The fluorescence is sensitized by the "antenna effect" and then quenched by the internal filtering effect of RP.

[0053] S3. Preparation of the colorimetric card:

[0054] After the reaction was complete, the filter paper was removed and cooled at room temperature. Apparent color images of all filter paper pieces were acquired using a high-resolution scanner (≥300 dpi) under natural light, and fluorescence color images were captured under a 365 nm UV lamp. Using image processing software, the apparent color and fluorescence color images of the filter paper were arranged in two rows, in ascending order of concentration, to create a digital colorimetric template. The corresponding NEO concentration (unit: μg / mL) was clearly labeled below each color patch. Finally, the digital template was printed on high-quality, waterproof, and UV-resistant cardstock, and after cutting, a dual-mode visual colorimetric card for NEO was obtained. The upper half of the colorimetric card represents the fluorescence colorimetric area under UV light, and the lower half represents the apparent colorimetric area under natural light.

[0055] Example 2

[0056] Fluorescence stability test:

[0057] To verify the practicality of the sensing system, ninhydrin- and ninhydrin- The fluorescence stability of the chromogenic solution was systematically evaluated. The optimized Nin@ and Nin@ Bare filter paper was treated with a chromogenic solution and irradiated under a 365 nm UV lamp for 60 min. Fluorescence intensity at the maximum emission wavelength was recorded every 10 min. Results are as follows: Figure 3 As shown in (C), The fluorescence intensity fluctuation at 546 nm does not exceed 9.6%. The fluorescence intensity fluctuation at 618 nm does not exceed 11.2%. This indicates that the lanthanide ion fluorophore used in this invention has excellent photostability and fully meets the requirements of practical detection.

[0058] Example 3

[0059] Investigating the effect of pH changes on color development and fluorescence intensity:

[0060] Phosphate solutions (0.1 M), with pH ranging from 2.5 to 12.5 (adjusted by NaOH and H3PO4), in 1.0 pH increments, were prepared and used as solvents to prepare NEO solutions (1.5 μg / mL). These solutions were then mixed with filter paper (Φ6 mm, 400 μL per sheet) and incubated at room temperature for 30 minutes. Filter paper incubated under the same conditions with the corresponding solvent served as a control. After two washes with the relevant solvents, the resulting filter paper was processed using Nin@Tb. 3+(10 μL per sheet) was subjected to color development at 60 ℃ for 30 minutes. Subsequently, the appearance of the resulting filter paper was scanned, and its fluorescence spectrum was measured using a Synergy H1M microplate reader under 330 nm excitation. Simultaneously, its fluorescence color was recorded using a smartphone under 365 nm UV illumination in a dark chamber. The data were averaged for quantitative analysis, and I... 546 / I 621 The FL intensity ratio was plotted against the system pH value.

[0061] The results are as follows Figure 6 As shown, pH has a certain effect on fluorescence and color development. Without the addition of antibiotics, the apparent color of the test paper showed no significant change after adding the color-developing solution at different pH levels. However, under extreme pH conditions (< 3.5 or > 11.5), the fluorescence intensity decreased. This may be because strong acid and strong alkaline conditions inhibit the reaction of ninhydrin and Tb. 3+ / Eu 3+ Coordination weakens the "antenna effect" and reduces fluorescence. After incubation with antibiotics at room temperature, the filter paper shows a weak ninhydrin color at pH 2.5, with significant color development between pH 3.5 and 9.5, and the strongest color development between pH 3.5 and 6.5. Color development gradually weakens above pH 7.5, and almost no color develops above pH 10.5. The fluorescence change trend is opposite to the apparent color. Figure 6 (E) It can be seen that the fluorescence quenching rate of the filter paper is the highest at pH 4.5, reaching (30.1 ± 2.1)%, at which point the color development is most obvious, the ratio change is the largest, and the internal filtration effect is the strongest. The possible reason for this phenomenon is that acidic conditions favor the ionization of amino groups, leading to an increase in their positive charge, thereby enhancing the electrostatic interaction between the filter paper and AGs. Conversely, under excessively acidic or alkaline conditions, the ionization of carboxylate groups or the protonation of amino groups on the filter paper surface is inhibited, thus inhibiting the binding of the filter paper to antibiotics and resulting in weakened color development. These results indicate that the sensing system has a wide applicable pH range (pH 3.5 ~ 9.5), suitable for the detection of most food samples and environmental water samples, and also demonstrate that the binding mechanism between the filter paper and AGs is electrostatic interaction.

[0062] Example 4

[0063] ninhydrin- Colorimetric solution and ninhydrin - Optimized formulation of the colorimetric reagent:

[0064] Spectroscopic confirmation of the colorimetric reaction of S1, ninhydrin (Nin), and AGs:

[0065] Prepare a 10 mg / mL ninhydrin aqueous solution. Take six 500 μL aliquots of this solution and add NEO, AMK, and PAR standards respectively to achieve final concentrations of 1.5 μg / mL and 5 μg / mL (two concentrations for each AG). Include a control without added AGs. Heat the mixed solution in a 60°C water bath for 30 minutes, cool to room temperature, and then scan the absorption spectrum in the 300-700 nm wavelength range using a UV-Vis spectrophotometer. The results are as follows: Figure 2 As shown in (B), all samples containing AGs exhibited two characteristic absorption peaks near approximately 400 nm and 570 nm. These are the characteristic absorptions of Ruhemann's purple (RP), generated by the reaction of ninhydrin with the primary amine groups in the AGs molecule, confirming the occurrence of the colorimetric reaction. The control sample did not show these absorption peaks.

[0066] S2. The effect of the colorimetric reaction carrier (solution / filter paper) on colorimetric sensitivity:

[0067] Prepare a triketone reaction solution containing 1.5 μg / mL NEO using the method described above, and photograph the solution color after heating. Figure 2 (C) Upper part). Simultaneously, a piece of bare filter paper was immersed in a 1.5 μg / mL NEO solution for 30 minutes for adsorption. After drying, 10 μL of ninhydrin solution of the same concentration was added, and the mixture was heated at 60 ℃ for 30 minutes before photographing. Figure 2 (C) Bottom). As can be seen in the comparison, the solution reaction system is extremely pale in color, making it difficult to distinguish with the naked eye; while the solid-phase reaction system based on filter paper exhibits a bright purplish-red color. This proves that pre-enriching AGs on filter paper before the colorimetric reaction can greatly improve the visual sensitivity of the detection, highlighting the advantages of paper-based sensing.

[0068] S3, Optimization of key components of ninhydrin colorimetric solution:

[0069] 1. Nin concentration optimization: Ninhydrin solutions with concentration gradients of 1, 5, 10, 25, and 50 mg / mL were prepared using 85% ethanol aqueous solution (containing 0.5 M acetic acid / 0.25 M sodium acetate) as solvent. These solutions were then used to treat bare filter paper (background group as shown in the image). Figure 5 A) and filter paper that has adsorbed 1.5 μg / mL NEO (experimental group such as...). Figure 5 (B)). The result is as follows: Figure 5 As shown in (A)-5(B), the background color deepens slightly with increasing Nin concentration. For NEO color development, the optimal concentration of 10 mg / mL is achieved, resulting in the deepest and most uniform color. Further increases in concentration do not significantly improve color depth; instead, background interference increases. Therefore, 10 mg / mL is selected as the optimal Nin concentration.

[0070] 2. Optimization of color development temperature and time: Filter paper adsorbed with NEO was treated with a 10 mg / mL Nin solution. The color development effect of heating at different temperatures (20 ℃, 40 ℃, 60 ℃) for 60 minutes was first investigated. Figure 5 (C) shows that the color development is most rapid and the color is deepest at 60 ℃. Subsequently, the temperature was kept constant at 60 ℃, and the effect of different heating times (0, 1, 3, 5, 10, 20, 30, 60, 90, 120 min) was investigated. Figure 5 (D) The results showed that a stable and significant color development effect could be obtained by heating for 10-30 minutes. Considering the need for rapid detection, the optimal color development condition was determined to be heating at 60 ℃ for 10 minutes.

[0071] 3. Effect of AGs adsorption on colorimetric effect: With a fixed NEO concentration of 1.5 μg / mL, the volume of the adsorption solution was varied (0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mL) to change the total amount of NEO adsorbed by the filter paper. The results after colorimetric development are shown below. Figure 5 As shown in (E), the color depth increases with the increase of NEO adsorption, and tends to saturate after the adsorption amount reaches about 3.0 μg (corresponding to 2.0 mL of solution). This result provides an experimental basis for setting the upper limit of the concentration range when preparing colorimetric cards in the future.

[0072] S4、Nin@ Construction of fluorescence systems and Concentration optimization:

[0073] 1. System Construction and Validation of Carrier Advantages: Three colorimetric solutions were prepared: (a) 10 mg / mL Nin solution; (b) Solution (containing 10 mg / mL Nin and 7.5 mg / mL Nin) (c) Solution (containing 10 mg / mL Nin and 7.5 mg / mL Nin) The solvents used were all 85% aqueous solutions of ethanol containing 0.5 M acetic acid / 0.25 M sodium acetate.

[0074] Solution level: NEO (final concentration 0.05 mg / mL) was added to the above three solutions, and the fluorescence spectrum was measured after heating. Figure 3 A). Nin solution showed no fluorescence; and After the addition of NEO, the solution exhibited different wavelengths at 546 nm. ) and 618 nm ( The characteristic fluorescence emission peak intensity decreased significantly. The inset shows that the solution exhibits weak fluorescence under UV light.

[0075] Paper-based level: Filter paper adsorbed with 0.05 mg / mL NEO was treated with the above three colorimetric solutions, and the fluorescence spectrum was measured. Figure 3 B). Nin-treated paper exhibits no fluorescence; and The treated paper samples exhibited strong characteristic fluorescence, which was significantly quenched upon the addition of NEO. The illustrations clearly show the color change of the paper samples under sunlight and the "on-off" fluorescence response under UV light. This comparison demonstrates that constructing the reaction system on a filter paper substrate effectively avoids the interference of water molecules. The fluorescence quenching effect yields a stronger and more stable fluorescence signal.

[0076] 2. Solvent-based optimization process: First, prepare an aqueous solution containing 70% (v / v) of an organic solvent (ethanol, methanol, 2-methyl-1-propanol, dimethyl sulfoxide, acetonitrile, or dimethylformamide). Then, use this solution as the solvent to prepare six Nin solutions, each with a concentration of 25 mg / mL. Simultaneously, mix six portions of NEO solution (1.5 μg / mL, dissolved in PB) with filter paper (Φ6 mm, 200 μL per sheet) and incubate at room temperature for 30 minutes. After natural drying, add the NEO-extracted filter paper dropwise to the six prepared Nin solutions and incubate at 60 °C for 30 minutes, replenishing the Nin solution every 10 minutes. Subsequently, as... Figure 4 As shown in (A), the obtained filter paper was scanned and its color was recorded. To investigate the effect of ethanol content on the colorimetric reaction, the main steps were the same as described above, but the Nin solution was prepared using aqueous solutions containing different volume fractions of ethanol (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%). Figure 4 (B) It can be seen that the color development effect gradually deepens with the increase of ethanol concentration. Taking into account other components in the color development solution (sodium acetate, ... , To assess the solubility of the colorimetric solution, an aqueous solution of 85% (v / v) ethanol was subsequently used as the solvent for the colorimetric solution.

[0077] 3. and System optimization of concentration: With Nin concentration fixed at 10 mg / mL, preparations were made containing different concentrations or A colorimetric solution with concentrations of (0, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20 mg / mL) was used to treat bare filter paper and filter paper adsorbed with 1.5 μg / mL NEO, and the fluorescence spectra were measured. The fluorescence quenching rate was calculated. ( F represents the fluorescence intensity of the bare filter paper, and F represents the fluorescence intensity of the NEO-adsorbed filter paper.

[0078] like Figure 7 As shown in AF, for In this system, at a concentration of 10 mg / mL, the fluorescence quenching rate reached approximately 46%, and further increasing the concentration had limited contribution to improving the quenching rate. Figure 7 (C)). For The system showed the best quenching rate at a concentration of 2.5 mg / mL, with a quenching rate of approximately 27%. Figure 7 (F)). Figure 8 The visual photographs (A)-8(D) also confirm this conclusion. Therefore, the optimal colorimetric solution formulation is determined as follows:

[0079] 1. Ninhydrin - Colorimetric solution: 10 mg / mL ninhydrin, 10 mg / mL 0.5 M acetic acid and 0.25 M sodium acetate are dissolved in an 85% (v / v) aqueous ethanol solution.

[0080] 2. Ninhydrin - Colorimetric solution: 10 mg / mL ninhydrin, 2.5 mg / mL 0.5 M acetic acid and 0.25 M sodium acetate are dissolved in an 85% (v / v) aqueous ethanol solution. The prepared colorimetric solution is placed in a brown bottle and stored in a refrigerator at 4°C, protected from light, and has a shelf life of at least two weeks.

[0081] Example 5

[0082] Selective testing of the detection system:

[0083] To verify the specific recognition ability of the detection card of this invention for AGs, a total of 17 antibiotics from 3 classes were selected for a control experiment. Experimental group (AGs): NEO (1.5 μg / mL), AMK (1.5 μg / mL), PAR (0.5 μg / mL). Control group (non-AGs): 14 other antibiotics, including chloramphenicol (CAP), oxytetracycline (OTC), cefdinir (CFD), etc., all at a concentration of 10 μg / mL. Blank control: phosphate buffer (PB).

[0084] The bare filter paper discs were immersed in the aforementioned antibiotic solutions or PB, and allowed to adsorb at room temperature for 30 minutes. After removal, they were divided into two groups; one group was treated with the optimized solution. The colorimetric reagent was added dropwise to another set. The colorimetric solution was heated at 60°C for 10 minutes to develop color and fluorescently label it.

[0085] like Figure 10As shown in (A) and 10(B), the filter paper that adsorbed only the three AGs (NEO, AMK, PAR) exhibited a bright purplish-red color, and its green color was observed under a 365 nm UV lamp. ) or red ( Fluorescence was significantly quenched. Filter paper strips adsorbed with other non-AG antibiotics showed similar apparent and fluorescence colors to the blank control (PB), with no significant changes visible to the naked eye. Fluorescence spectral data ( Figure 9 Further quantitative confirmation in (A) and (C) indicates that only the AGs group caused [the condition]. At 546 nm and A significant decrease in the characteristic fluorescence peak at 618 nm. This affects the fluorescence intensity ratio ( system: ; system: ) to perform statistical analysis ( Figure 9 (B), (D)). The fluorescence ratio of the AGs group was significantly lower than that of all other antibiotic groups and the blank group. The above results fully demonstrate that the detection system has high selectivity for aminoglycoside antibiotics and can effectively eliminate interference from other common antibiotics.

[0086] Example 6

[0087] Evaluation of the anti-interference performance of the detection system:

[0088] S1. Interference experiment with high concentrations of food additives:

[0089] Eleven common food additives (DL-malic acid, sodium citrate, fructose syrup, acesulfame potassium, potassium sorbate, sodium benzoate, steviol glycosides, sucralose, sucrose, glucose, and flavorings) were selected and solutions with concentrations as high as 20 mg / mL were prepared (far exceeding the maximum usage limit stipulated by national standards). Bare filter paper was immersed for 30 minutes in each additive solution, a positive control solution containing NEO (1.5 μg / mL), or a PB blank solution. Subsequently, optimized [treatment / treatment] was used. and Treatment with colorimetric solution.

[0090] like Figure 11 and Figure 12 As shown, all filter paper discs containing high concentrations of food additives exhibited colorimetric and fluorescence responses similar to the blank group, but significantly different from the NEO-positive group. Statistical analysis of fluorescence intensity ratios showed that the ratio fluctuations for each interfering group were less than 3.6%. This demonstrates that common food additives do not significantly interfere with the detection card of this invention.

[0091] S2. The impact of potential disruptors in the environmental matrix:

[0092] Eight metal ions that may exist in environmental water bodies were selected. A solution containing 10 amino acids (glycine, aspartic acid, etc.) with a concentration of 0.01 mg / mL was prepared (meeting or exceeding common environmental standards). Interference experiments were conducted using the same method.

[0093] like Figure 13 , 14 As shown, the presence of these metal ions and amino acids has a negligible effect on the detection signal (color and fluorescence) of NEO. Fluorescence ratio analysis indicates that the maximum fluctuation is less than 5.7%. It should be noted that amino acids themselves can react with ninhydrin at high temperatures (e.g., 100 °C) to produce a colorimetric reaction. Figure 14 (B), 14 (D) illustrations), but its color development effect is negligible under the mild conditions (60 °C) used in this invention.

[0094] This detection system has good anti-interference ability against potential interfering substances in complex environmental matrices and is suitable for analysis of actual samples.

[0095] Example 7

[0096] Applications of AGs detection in real-world environmental water samples:

[0097] S1. Preparation of dual-mode colorimetric cards for different water matrices (taking tap water as an example):

[0098] Using tap water as the solvent, a series of NEO standard solutions with concentration gradients of 0, 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, and 10 μg / mL were prepared. Two mL of each concentration solution was immersed in a piece of bare filter paper and allowed to absorb at room temperature for 5 minutes. After removal, half of the filter paper was dripped with... Add the other half of the color developer to the solution. The colorimetric solution was heated at 60°C for 10 minutes. The dried paper discs were arranged in ascending order of concentration to obtain a dual-mode colorimetric card (apparent color - fluorescence color) suitable for this water sample matrix (see example). Figure 15 (A), 15 (B)).

[0099] S2. Actual water sample spiking and visual semi-quantitative analysis:

[0100] Take three types of actual environmental water samples: tap water, river water, and aquaculture pond water. Add 1.5 μg / mL NEO to each sample. Take 2 mL of the spiked water sample and perform adsorption and color development using filter paper as described in "S1" above. Visually compare the developed test paper with a colorimetric card prepared using the same water sample.

[0101] like Figure 15As indicated by samples (1)-(3) in (A)-15(F) and the orange arrow, the apparent color and fluorescence color of the three spiked water samples can be accurately located within the concentration range of 1.0-2.5 μg / mL on the colorimetric card, which is highly consistent with the actual spiked concentration (1.5 μg / mL). This method realizes rapid and reliable semi-quantitative visual analysis of AGs in actual environmental water samples.

[0102] S3. Ratio fluorescence quantitative analysis and spiked recovery verification:

[0103] Fluorescence spectra of the paper pieces used to prepare the colorimetric card in "S1" at each concentration point were scanned. Calculations were performed accordingly. System Ratio and System Ratio. Plot a standard working curve with the ratio on the ordinate (y) and NEO concentration on the abscissa (x). Figure 16 17, 18 (B), (D)). All curves showed good linearity (R² > 0.94). Substituting the fluorescence ratios measured in the spiked samples of “S2” into the corresponding standard curves, the detection concentration and recovery rate of NEO were calculated. Ratio fluorescence mode provides an accurate quantitative method, verifying the reliability of visual colorimetric results and indicating that this method is suitable for the accurate quantitative detection of AGs in actual environmental water samples.

[0104] Example 8

[0105] Applications of AGs detection in actual food samples:

[0106] S1. Food sample pretreatment:

[0107] 1. Milk sample: Take commercially available liquid milk, filter it through a 0.45 μm microporous membrane, and then centrifuge it at 5000 rpm for 30 minutes using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa. Collect the ultrafiltrate to remove large molecular interferences such as proteins and fats.

[0108] 2. Chicken / Shrimp Samples: Weigh 10.0 g of the homogenized sample, add 10 mL of acetonitrile, vortex for 5 minutes, then centrifuge at 6000 rpm for 10 minutes and collect the supernatant. Repeat the extraction with 10 mL of acetonitrile, and combine the two supernatants. Evaporate to dryness under vacuum at 40℃. Redissolve the residue in 10 mL of phosphate buffer (PB, pH 7.4), and filter the solution through a 0.45 μm filter membrane to obtain a clear sample extract.

[0109] S2, based on Preparation and visual inspection of the dual-mode colorimetric card for the system:

[0110] Using PB buffer as the simulated matrix, a series of standard solutions of three AGs, NEO, AMK, and PAR, were prepared (concentration gradient as above). Following the method in Example 5, S1, bare filter paper was used for adsorption... Color development was performed, and two-mode colorimetric cards of "apparent color-fluorescence" for each of the three AGs were prepared. Figure 19 (A)-19(F)).

[0111] Add low, medium, and high concentrations of NEO, AMK, or PAR (e.g., 0.3, 0.6, 1.5 μg / mL) to the processed milk, chicken extract, and shrimp extract, respectively. Take 2 mL of the spiked sample solution and perform filter paper adsorption and color development. Visually compare the test paper with the corresponding colorimetric card. Figure 19 As shown in the right side of the vertical solid line in (A)-19(F), the color and fluorescence of all spiked samples were successfully matched to the corresponding concentration range in the colorimetric card, thus intuitively completing the semi-quantitative detection.

[0112] S3. Quantitative validation and recovery calculation using ratiometric fluorescence method:

[0113] Three AGs were established using fluorescence spectroscopy measurements. Fluorescence ratio under the system ( Standard working curves for concentrations (see example) Figure 20 (B), 20 (D), 20 (F)). Substitute the fluorescence ratios of the spiked samples into the corresponding curves to calculate the detection concentration and spike recovery rate.

[0114] As shown in Table 1, in three complex matrices—milk, chicken, and shrimp—the recoveries of different AGs at various concentration levels ranged from 89.8% to 110.4%, with an RSD of less than 12.9%. This method demonstrates strong resistance to matrix interference, rapid operation, and suitability for on-site rapid detection and accurate quantitative analysis of AG residues in various complex food samples, yielding reliable results.

[0115] Table 1 Ninhydrin - Linear equations and main operating parameters for the detection of NEO, AMK, and PAR using a ratiometric fluorescence sensing system.

[0116]

[0117] Example 9

[0118] Identification of aminoglycoside antibiotics in actual samples:

[0119] S1. Verification of the adsorption and enrichment capacity of various AGs:

[0120] To verify the universal adsorption effect of bare filter paper on AGs with different structures, five common AGs—NEO, AMK, PAR, GEN, and STR—were selected. Standard solutions of these AGs with a concentration of 1.5 μg / mL were prepared using PB buffer as the solvent. Six discs of bare filter paper were immersed in the five AG solutions and one PB buffer solution (blank control), respectively, and allowed to stand at room temperature for 5 minutes for adsorption. After removal, the edge droplets were blotted dry with filter paper, and all the discs were arranged side by side. 10 μL of ninhydrin-terbium (NJT) solution optimized according to Example 2 was added to each disc. The colorimetric solution was placed in a 60 ℃ oven and reacted for 10 minutes.

[0121] After the reaction was complete, the images were observed and photographed under sunlight and a 365 nm ultraviolet lamp. The results are as follows: Figure 21 As shown in (A), all filter paper sheets adsorbing different AGs exhibit distinct purplish-red hues of varying shades (bottom image), and their green fluorescence under UV light is significantly weaker than that of the blank sheet (top image). The blank control filter paper, however, is almost colorless and exhibits the strongest fluorescence. This result directly demonstrates that unmodified filter paper possesses effective adsorption and enrichment capabilities for various AGs, which forms the basis for constructing the subsequent dual-mode detection method.

[0122] S2. Feasibility exploration of distinguishing different AGs based on dual-mode signals:

[0123] right Figure 21 (A) The colorimetric and fluorescence images obtained were digitally analyzed. The apparent color RGB values ​​of each paper piece were extracted, and the (R+B) / G ratio was calculated to quantify the color characteristics; simultaneously, the (R+G) / B ratio was calculated from the fluorescence images to quantify the fluorescence color characteristics. These two ratios were used as two-dimensional coordinates to plot the spatial distribution of the five AGs. Figure 21 (C)). The results show that the coordinate points of different AGs are distributed in different regions of the two-dimensional plane, indicating that their colorimetric and fluorescence response modes differ. Furthermore, the ratios of (R+B) / G, (R+G) / B, and the measured intensity ratios of fluorescence spectra were used to further analyze these relationships. Principal component analysis (PCA) was performed using these as initial variables. Figure 21 As shown in (D), the cumulative variance contribution rate of the first two principal components (PC1 and PC2) exceeds 85%, and the sample points of different AGs show a clear separation trend on the PCA score map. This analysis preliminarily confirms that the "color-fluorescence" dual-mode signal array constructed in this invention has the potential to distinguish and identify different types of AGs.

[0124] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for preparing a dual-mode visual detection card for aminoglycoside antibiotics, characterized in that, Includes the following steps: S1, ninhydrin and Ln 3+ Dissolved in an aqueous ethanol solution containing acetic acid and sodium acetate, a dual-mode labeling reagent for aminoglycoside antibiotics is obtained; S2, prepare standard samples of aminoglycoside antibiotics with concentration gradients, adsorb them with qualitative filter paper, and after adsorption, use the aminoglycoside antibiotic dual-mode labeling reagent obtained in step S1 to label and develop color. Arrange the colored filter paper according to the concentration gradient of aminoglycoside antibiotics to obtain a visual colorimetric card. S3. Place the visual colorimetric card obtained in step S2 into an excitation light source to excite fluorescence, take a picture to record the result, and arrange the filter paper according to the concentration gradient of aminoglycoside antibiotics to obtain the fluorescence colorimetric card.

2. The method for preparing a dual-mode visual detection card for aminoglycoside antibiotics as described in claim 1, characterized in that, In step S1, Ln 3+ For La 3+ Eu 3+ Gd 3+ 、Tb 3+ Any one of the lanthanide metal ions.

3. The method for preparing a dual-mode visual detection card for aminoglycoside antibiotics as described in claim 1, characterized in that, In step S1, the aminoglycoside antibiotic dual-mode labeling reagent contains: ninhydrin 10 mg / mL, acetic acid 0.5 M, sodium acetate 0.25 M, and... 10 mg / mL or 2.5 mg / mL.

4. The method for preparing a dual-mode visual detection card for aminoglycoside antibiotics as described in claim 1, characterized in that, In step S2, the adsorption of the filter paper is either physical adsorption or chemical adsorption.

5. The method for preparing a dual-mode visual detection card for aminoglycoside antibiotics as described in claim 1, characterized in that, In step S2, the visual colorimetric chart includes 10 color blocks for each antibiotic, and each color block corresponds to the concentration of the corresponding aminoglycoside antibiotic.

6. The method for preparing a dual-mode visual detection card for aminoglycoside antibiotics as described in claim 1, characterized in that, In step S2, the concentration gradient of the aminoglycoside antibiotic standard samples is 0, 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, and 10 μg / mL.

7. The method for preparing a dual-mode visual detection card for aminoglycoside antibiotics as described in claim 1, characterized in that, In step S3, the contrast color of each antibiotic in the fluorescent colorimetric card includes 10 color blocks, and each color block corresponds to the concentration of the corresponding aminoglycoside antibiotic.

8. A dual-mode visual detection card for aminoglycoside antibiotics prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the dual-mode visual detection card for aminoglycoside antibiotics as described in claim 8 in the quantitative and semi-quantitative visual detection and identification of aminoglycoside antibiotic content in food, environmental or clinical samples.

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