Paper-based sensor, preparation method thereof and application of paper-based sensor in detection of alternariol

Abstract

The invention relates to a paper-based sensor, a preparation method of the paper-based sensor and application of the paper-based sensor in detection of alternariol, and belongs to the technical field of food detection. The paper-based sensor comprises a first protective layer, an immobilization layer, a substrate layer, a color development layer and a second protective layer, the surfaces of the first protective layer and the second protective layer are coated with hydrophobic materials; central areas of the immobilization layer, the substrate layer and the color development layer are coated with acetylcholin esterase, acetylthiocholine and nano-enzyme respectively to form hydrophilic reaction areas, and non-hydrophilic reaction areas are coated with hydrophobic materials to form hydrophobic isolation areas; the first protection layer, the immobilization layer, the substrate layer, the color development layer and the second protection layer are folded according to a preset sequence, and the hydrophilic reaction areas of the immobilization layer, the substrate layer and the color development layer are mutually overlapped after folding. The paper-based sensor is remarkably improved in the aspects of detection sensitivity, stability and convenience.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, and particularly relates to a paper-based sensor, its preparation method, and its application in the detection of cross-linked polysaccharides. Background Technology

[0002] Mycotoxin contamination seriously affects food safety. Alternaria alterniflora (AOH), a secondary metabolite produced by Alternaria fungi, is widely used in grains, fruits and their products. It has attracted much attention due to its significant genotoxicity, cytotoxicity and potential carcinogenicity. Moreover, its physicochemical properties are stable and it is difficult to effectively degrade by conventional food processing. As a result, it can continue to contaminate food through the "farm-to-table" chain. Therefore, it is crucial to establish a rapid and sensitive AOH detection method to protect consumer health and regulate market order.

[0003] Currently, AOH detection mainly relies on instrumental analysis methods such as liquid chromatography-tandem mass spectrometry. Although these methods have high detection accuracy, they have inherent limitations such as expensive equipment, high requirements for professional operation, long analysis cycle, and difficulty in on-site implementation, which cannot meet the urgent needs of large-scale screening and rapid on-site monitoring at the grassroots level. While rapid detection technologies based on immunological principles or nucleic acid aptamers have improved in terms of convenience, their core recognition elements (such as antibodies and aptamers) have high manufacturing costs and poor stability, and are easily interfered with in complex food matrices, affecting the accuracy of detection results and the universality of the methods.

[0004] Paper-based sensors, with their advantages of low cost, portability, easy disposal, and the ability to drive fluids through capillary action, have become one of the ideal platforms for developing rapid on-site detection products. However, existing paper-based sensors (especially colorimetric ones) face two major technical bottlenecks: First, paper is composed of disordered fibers, and its porous and heterogeneous structure leads to severely uneven reagent distribution and colorimetric reactions, resulting in poor signal consistency. Second, result interpretation largely relies on operator visual observation or simple single-point photograph colorimetry, introducing unavoidable human subjective errors, resulting in low quantitative accuracy and poor reproducibility, making it difficult to meet the requirements of precise detection.

[0005] Therefore, there is an urgent need for a sensor for rapid AOH detection that integrates high sensitivity, high accuracy, low cost, and ease of operation. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the difficulty in balancing sensitivity, accuracy, cost and portability in existing AOH detection technologies.

[0007] To address the aforementioned technical problems, this invention provides a paper-based sensor, its preparation method, and its application in the detection of AOH. First, a nanozyme with both high affinity and high efficiency is prepared through heterovalent metal co-doping, overcoming the bottleneck in catalytic performance. Second, based on this nanozyme and AChE, an inhibition-amplification cascade sensing mechanism is constructed to achieve ultrasensitive detection of trace AOH. Finally, utilizing a multilayer spatiotemporally separated sensor structure and an AI quantitative algorithm, a high-performance liquid-phase detection system is successfully and reliably integrated into a portable paper-based platform. This paper-based sensor achieves highly sensitive detection of the target analyte through an efficient signal amplification mechanism. Simultaneously, through spatial isolation structure design and signal analysis strategies, it fundamentally solves the technical problems of uneven color development and subjective interpretation on paper-based platforms, achieving stable and objective accurate quantification, and significantly improving detection sensitivity, stability, and convenience.

[0008] The first objective of this invention is to provide a paper-based sensor, comprising a first protective layer, an immobilization layer, a substrate layer, a color developing layer, and a second protective layer; The first protective layer and the second protective layer are used to protect the immobilization layer, the substrate layer and the color development layer, and the surfaces of the first protective layer and the second protective layer are coated with a hydrophobic material. The central regions of the immobilization layer, substrate layer, and color development layer are coated with acetylcholinesterase (AChE), acetylthiocholine (ATCh), and nanozyme, respectively, to form hydrophilic reaction regions. The non-hydrophilic reaction regions are coated with hydrophobic materials to form hydrophobic isolation regions. The nanozyme is a copper / manganese double-doped cerium dioxide nanozyme (Cu / Mn-CeO2). The first protective layer, the immobilization layer, the substrate layer, the color development layer, and the second protective layer are folded in a preset order, and the hydrophilic reaction zones of the immobilization layer, the substrate layer, and the color development layer overlap after folding.

[0009] In one embodiment of the present invention, the copper-manganese co-doped cerium dioxide nanozyme contains 5.0%-5.5% copper and 1%-2% manganese by mass; the copper-manganese co-doped cerium dioxide nanozyme is used as a core signal probe and has excellent peroxidase-like activity.

[0010] In one embodiment of the present invention, the preparation of the copper-manganese dual-doped cerium dioxide nanozyme includes the following steps: dissolving cerium salt, manganese salt, copper salt and ethylene glycol in water, sealing and reacting in a forced-air drying oven at 175℃-185℃ for 2h-4h, centrifuging, washing and drying to obtain the copper-manganese dual-doped cerium dioxide nanozyme.

[0011] In one embodiment of the present invention, the cerium salt is cerium nitrate; The manganese salt is selected from manganese chloride and / or manganese acetate; The copper salt is copper chloride.

[0012] In one embodiment of the present invention, the hydrophobic material is polydimethylsiloxane (PDMS); a hydrophobic barrier is constructed by the hydrophobic material to achieve spatial physical isolation of different reagents and timing control of the reaction process.

[0013] In one embodiment of the present invention, the loading of the acetylcholinesterase is 0.14 mU-0.16 mU; The loading of acetylthiocholine is 0.09 μmol-0.11 μmol; The loading of the nanozyme is 1.8 μg-2.2 μg.

[0014] In one embodiment of the present invention, the substrate of the paper-based sensor is filter paper.

[0015] A second objective of this invention is to provide a method for preparing the paper-based sensor, comprising the following steps: S1. Coat the substrate with a hydrophobic material solution and cure it to obtain the first protective layer and the second protective layer; Hydrophilic reaction zones were pre-set on the substrate, and hydrophobic materials were coated on the non-hydrophilic reaction zones and cured to form hydrophobic isolation zones. Then, acetylcholinesterase solution, acetylthiocholine solution, and nanozyme solution were coated on each hydrophilic reaction zone and dried to obtain an immobilization layer, a substrate layer, and a color development layer. S2. Fold the first protective layer, immobilization layer, substrate layer, color development layer and second protective layer described in S1 in a preset order, and after folding, the hydrophilic reaction areas of the immobilization layer, substrate layer and color development layer overlap each other to obtain the paper-based sensor.

[0016] In one embodiment of the present invention, in S1, the concentration of the acetylcholinesterase solution is 30 mU / mL; The concentration of the acetylthiocholine solution was 1.8 mmol / L to 2.2 mmol / L; The concentration of the nanozyme solution is 0.36 mg / mL to 0.44 mg / mL.

[0017] A third objective of this invention is to provide an application of the aforementioned paper-based sensor in the detection of cross-linked polysaccharides.

[0018] In one embodiment of the present invention, the application specifically includes the following steps: S1. Add the cross-linked phenol test solution to the hydrophilic reaction zone of the immobilized layer, and incubate to allow the cross-linked phenol in the test solution to fully react with the acetylcholinesterase in the immobilized layer. S2. Folding the first protective layer, immobilization layer, and substrate layer brings the immobilization layer into contact with the substrate layer, triggering a cascade reaction of acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine after incubation; S3. After adding TMB colorimetric solution to the hydrophilic reaction zone of the colorimetric layer, fold all layers completely to carry out the colorimetric reaction. S4. After the colorimetric reaction is complete, digital images are acquired under standard illumination conditions, and the concentration of the test solution of Alternaria solani is calculated by a multi-point sampling color statistical analysis method (AI quantitative algorithm).

[0019] The technical solution of the present invention has the following advantages compared with the prior art: The paper-based sensor described in this invention utilizes cerium dioxide nanozymes doped with heterovalent metals copper and manganese. By introducing these two heterovalent metals to synergistically dope the cerium dioxide lattice, a revolutionary design of the nanozyme's active sites is achieved. Its core mechanism lies in: Cu... 2+ and Mn 2+ As a heterovalent element, it replaces Ce 4 After ⁺, in order to maintain lattice electrical neutrality, it will force the neighboring Ce to... 4 ⁺ Restored to Ce 3+ This process directly induces the formation of a large number of oxygen vacancies. These oxygen vacancies are not random defects, but rather they form a highly active and highly defective interfacial microenvironment through strong electronic cooperative interactions with Cu / Mn sites. This microenvironment not only significantly enhances the adsorption and activation of TMB / H2O2 molecules, but also greatly promotes the generation rate of surface free radicals.

[0020] The paper-based sensor described in this invention constructs a dual-enzyme cascade detection system based on the synergistic effect of AChE and Cu / Mn-CeO2. Its core is a signal switching mechanism: in the absence of Alternaria alternatasol (ALS), AChE hydrolyzes ATCh to generate a strongly reducing TCh, which rapidly quenches the blue ox-TMB produced by nanozyme catalysis, keeping the system in a signal "off" state. In the presence of ALS, it acts as a specific inhibitor of AChE, precisely cutting off this hydrolysis pathway, preventing TCh generation, and allowing Cu / Mn-CeO2 to catalyze TMB color development without interference, driving the signal to the "on" state. Ultimately, thanks to the strong background signal amplification capability of the highly active Cu / Mn-CeO2 and the precise signal cutoff effect of enzyme inhibition, this system achieves a detection limit as low as 0.0173 pg / mL in homogeneous solutions, reaching ultra-trace detection levels.

[0021] The paper-based sensor described in this invention achieves a breakthrough from homogeneous liquid-phase reaction to spatiotemporally separated solid-phase reaction by constructing a "sandwich" type multilayer spatial isolation structure. It utilizes hydrophobic materials to physically isolate AChE, ATCh, and Cu / Mn-CeO2 in different functional layers. Through layering and folding, the liquid flow sequentially triggers the complete time sequence of incubation-reaction-color development along a preset path, accurately reproducing the reaction logic of liquid-phase detection on a solid-phase platform. This spatial isolation structure fundamentally blocks direct contact between AChE, ATCh, and Cu / Mn-CeO2 during storage, effectively solving the problem of background signal increase and reagent failure caused by slow substrate oxidation, and overcoming the long-term stability bottleneck of multi-enzyme test strips. To eliminate the influence of uneven color development on the paper-based substrate and equipment differences on quantitative results, the system further introduces AI algorithms for multi-point sampling and RGB statistical analysis, successfully transforming subjective visual colorimetry into objective and accurate digital image analysis. Ultimately, the paper-based platform achieves a detection limit of 0.0347 ng / mL while maintaining convenience, realizing quantitative accuracy and extremely high sensitivity. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein: Figure 1 This is a schematic diagram of the synthesis of Cu / Mn-CeO2 and a microstructure characterization diagram of the present invention; wherein, A is a schematic diagram of the synthesis, B and C are scanning electron microscope images, D and E are transmission electron microscope images, F is a high-resolution transmission electron microscope image, and G is an enlarged view of the area selected by F. Figure 2 The surface chemical state and oxygen vacancy analysis of Cu / Mn-CeO2 and CeO2 in Test Example 2 of this invention are shown below; where A is the XPS full spectrum scan of Cu / Mn-CeO2, B is the Ce 3d high-resolution XPS spectrum of Cu / Mn-CeO2, C is the Ce 3d high-resolution XPS spectrum of CeO2, and D is the oxygen vacancy-related ESR signal of Cu / Mn-CeO2 and CeO2. Figure 3 The catalytic performance of Cu / Mn-CeO2 in Test Example 3 of this invention is tested; wherein, A is the steady-state kinetics of Cu / Mn-CeO2 with TMB as substrate, and B is the steady-state kinetics of Cu / Mn-CeO2 with H2O2 as substrate; Figure 4 This invention tests the performance of the Cu / Mn-CeO2-AChE dual-enzyme cascade sensing platform in Example 4; where A is a schematic diagram of the AOH detection principle, B is the emission spectrum at different AOH concentrations, and C is 2.50 × 10⁻⁶. -5 Calibration curves for the concentration range of -120 ng / mL; Figure 5 The diagram shows the structure and surface morphology of the paper-based sensor of the present invention; wherein, A is a schematic diagram of the paper-based sensor, C is a physical image of the paper-based sensor, B is a scanning electron microscope image of the hydrophilic reaction region, and D is a scanning electron microscope image of the hydrophobic isolation region. Figure 6 This is a test example 6 of the present invention for the detection and performance evaluation of AOH of the paper-based sensor; wherein, A is the linear relationship between gray value and AOH concentration, B is the specificity of AOH detection, C is the selectivity of AOH detection; D and E are comparisons of gray values ​​of images taken by different smartphones, and F is the stability of the paper-based sensor signal response over 60 days. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0024] In this invention, unless otherwise stated, the chromatography filter paper used in the specification is Whatman No. 5 filter paper.

[0025] In this invention, unless otherwise stated, the PDMS solution used in the specification is purchased from Dow Chemical and is model SYLGARD™ 184. Example 1

[0026] Reference Figure 1 As shown in Figure A, the paper-based sensor and its preparation method of the present invention specifically include the following steps: Preparation of S1 and Cu / Mn-CeO2: 2.714 g of cerium nitrate hexahydrate, 61.3 mg of manganese chloride tetrahydrate, and 74.9 mg of copper nitrate trihydrate were weighed and dissolved sequentially in 1 mL of ultrapure water to form a mixture. 30 mL of ethylene glycol (solvent and structure directing agent) was added to the mixture, and the mixture was vigorously stirred magnetically at room temperature for 30 min to form a homogeneous and transparent solution. The transparent solution was transferred to a 50 mL high-pressure reactor lined with polytetrafluoroethylene, sealed, and placed in a 180 °C drying oven for 3 h. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, centrifuged at 8000 rpm for 10 min to collect the precipitate, and washed three times alternately with anhydrous ethanol and ultrapure water to remove impurities. Finally, the precipitate was dried overnight in a 60 °C vacuum drying oven to obtain Cu / Mn-CeO2.

[0027] S2. Coat 0.5 mL of PDMS solution onto chromatographic filter paper, and then cure in an oven at 60 °C for 2 h to obtain the first protective layer and the second protective layer. Circular hydrophilic reaction zones with a diameter of 8 mm were pre-defined on chromatographic filter paper. 0.5 mL of PDMS solution was then coated onto non-circular hydrophilic reaction zones using a precision spraying method. The zones were then cured in a 60℃ oven for 2 hours to form hydrophobic isolation zones. Next, 5 μL of a 30 mU / mL acetylcholinesterase aqueous solution, 5 μL of a 2 mmol / L acetylthiocholine aqueous solution, and 5 μL of a 0.4 mg / mL Cu / Mn-CeO2 aqueous solution were added dropwise to each hydrophilic reaction zone. The zones were then air-dried at room temperature in the dark to obtain the immobilized layer, substrate layer, and chromogenic layer. S2. Fold the first protective layer, immobilization layer, substrate layer, color development layer, and second protective layer in a preset order, and fix them at one end by physical means to form a multi-layer folded structure. After folding, the hydrophilic reaction areas of the immobilization layer, substrate layer, and color development layer overlap to obtain a paper-based sensor. Figure 5 A and Figure 5 C). Comparative Example 1

[0028] The basic principle is the same as in Example 1, except that CeO2 is not doped. Comparative Example 2

[0029] The basic structure is the same as in Example 1, except that Cu is doped only in CeO2. Comparative Example 3

[0030] The basic structure is the same as in Example 1, except that Mn is doped only in CeO2. Comparative Example 4

[0031] The basic principle is the same as in Example 1, except that Cu / Mn-CeO2 is replaced with natural horseradish peroxidase (HRP). Test Example 1

[0032] The Cu / Mn-CeO2 of Example 1 was characterized using scanning electron microscopy, transmission electron microscopy, and high-resolution transmission electron microscopy. The results are as follows: Figure 1 B- Figure 1 As shown in G. From Figure 1 B- Figure 1 As can be seen from C, the material has a triangular sheet-like structure with a rough surface and clear edges, and the particle size distribution is uniform. Figure 1 D- Figure 1 G further confirmed the lamellar morphology of the material, while showing clear lattice stripes with a crystal plane spacing of about 0.305 nm, corresponding to the (111) crystal plane of CeO2. Test Example 2

[0033] X-ray photoelectron spectroscopy and electron spin resonance spectroscopy were used to characterize Cu / Mn-CeO2 of Example 1 and CeO2 of Comparative Example 1. The results are as follows: Figure 2 As shown. From Figure 2 As can be seen from A, Cu and Mn have been successfully doped; from Figure 2 B- Figure 2 As can be seen from C, Ce exists in Cu / Mn-CeO2. 3+ With Ce 4+ Mixed valence states, and Ce 3+ The relative content was as high as 34.16 at.%, significantly better than the 22.42 at.% of pure CeO2, indicating a higher oxygen vacancy concentration; from Figure 2 As can be seen from D, the intensity of Cu / Mn-CeO2 at g=2.0038 (corresponding to the characteristic signal of unpaired electrons in oxygen vacancies) is much higher than that of CeO2. These results indicate that the introduction of heterovalent metals Cu and Mn produces a strong synergistic effect with the CeO2 support, effectively promoting CeO2 growth. 4+ To Ce 3+ The reduction and induction of a large number of oxygen vacancies, which serve as key active sites, not only significantly enhance the ability of material surface to activate reactant molecules, but also promote the generation of surface free radicals, thereby synergistically driving a significant increase in the catalytic reaction rate. Test Example 3

[0034] 0.2 mol / L sodium acetate buffer (pH=4.0) was prepared, and TMB solutions and H2O2 solutions of various concentrations were prepared. Then, nanozymes (Cu / Mn-CeO2, CeO2, Cu-CeO2, Mn-CeO2, HRP) were dispersed in the above buffer solutions to prepare nanozyme suspensions with a concentration of 0.4 mg / mL. (Steady-state kinetics with TMB as substrate) With a fixed H₂O₂ concentration of 10 mM, TMB solutions of different concentrations were sequentially mixed with nanozyme suspensions and H₂O₂ solutions to construct the reaction system. The absorbance change at 652 nm was continuously monitored using a UV-Vis spectrophotometer, and the initial reaction rate (ΔA / Δt) was recorded. (Steady-state kinetics with H₂O₂ as substrate) With a fixed TMB concentration of 2 mM, H₂O₂ solutions of different concentrations were sequentially mixed with nanozyme suspensions and TMB solutions to construct the reaction system. The absorbance change at 652 nm was continuously monitored using a UV-Vis spectrophotometer, and the initial reaction rate (ΔA / Δt) was recorded. Finally, based on the Michaelis-Menten equation, the apparent properties of the nanozyme for TMB and H₂O₂ were calculated using the Lineweaver-Burk double reciprocal plotting method. K m Value and V max Value, result as Figure 3 As shown in Table 1: Table 1

[0035] from Figure 3 As can be seen from Table 1, Cu / Mn-CeO2 has an apparent effect on TMB. K m The value is 0.25mM. V max 10.33×10 - 8 M·s⁻¹, apparent value of H₂O₂ K m The value is 0.78 mM. V max It is 5.15 × 10 -8 M·s⁻¹; Compared with natural horseradish peroxidase (HRP), single-metal doped and undoped CeO2, Cu / Mn-CeO2 has the highest M·s⁻¹; V max and relatively low K m Value; lower K m The value corresponds to a stronger enzyme-substrate affinity and a higher value. V max This typically represents higher catalytic activity, which fully demonstrates the significant application potential of Cu / Mn-CeO2 in the field of peroxidase simulation. Test Example 4

[0036] 40 μL of 30 mU / mL AChE and 20 μL of 0-120 ng / mL AOH were pre-incubated at 37 °C for 30 min, followed by the addition of 40 μL of 2 mM ATCh and a further reaction for 30 min. The resulting solution was then mixed with 40 μL of 0.4 mg / mL Cu / Mn-CeO2 and 60 μL of TMB colorimetric solution, incubated for 20 min, and the UV-Vis absorption spectrum was measured using a multi-functional microplate reader. The results are as follows: Figure 4 As shown. From Figure 4 It can be seen that the absorbance of the system at 652 nm increases significantly with increasing AOH concentration, where the AOH concentration is between 2.50 × 10⁻⁶. -5 Within the range of ng / mL to 1 ng / mL, the absorbance value showed a good linear relationship with the logarithm of the AOH concentration, and the regression equation was Abs = 0.0064 × lgC. AOH +0.1388 (R) 2 =0.9946), when the concentration is between 1 ng / mL and 120 ng / mL, the absorbance value is directly linearly related to the AOH concentration, and the regression equation is Abs = 0.0031 × lgC. AOH+0.1327 (R) 2 =0.9950), and the detection limit of this method was calculated to be 0.0173 pg / mL according to the formula DL=3σ / k. This is because AOH, as an AChE inhibitor, can prevent the hydrolysis of ATCh. Unhydrolyzed ATCh will not interfere with the oxidation process of TMB catalyzed by Cu / Mn-CeO2, allowing the TMB substrate to produce a normal colorimetric blue signal. However, in the absence of AOH, ATCh will be enzymatically hydrolyzed to generate thiocholine (TCh). TCh, as a reducing agent, can effectively quench the oxidized TMB, thereby inhibiting the colorimetric reaction. Test Example 5

[0037] The hydrophilic reactive region and the hydrophobic isolation region were characterized using scanning electron microscopy, and the results are as follows: Figure 5 B and Figure 5 As shown in D. From Figure 5 B and Figure 5 As can be seen from D, the hydrophilic reaction zone exhibits a porous fibrous structure, while the hydrophobic isolation zone treated with PDMS has a smooth and dense surface. The stark contrast between the two fully demonstrates the effectiveness of the spatial separation design. Test Example 6

[0038] Take 2.5 μL of the pretreated Alternaria sol test solution and add it vertically dropwise into the hydrophilic reaction zone of the paper-based sensor immobilization layer. Incubate the paper-based sensor in a 37°C incubator for 30 min. After removing it, fold the portion containing the first protective layer, immobilization layer, and substrate layer to ensure contact between the immobilization layer and the substrate layer, and continue incubating in a 37°C incubator for another 30 min. Then, add 7.5 μL of the solution to the hydrophilic reaction zone of the chromogenic layer. The TMB colorimetric solution was immediately folded completely and reacted at 45°C for 20 minutes. After the reaction, the paper-based sensor was removed, and a clear image of the colorimetric layer was immediately captured using a smartphone in a standard LED light source box. The captured image was imported into an AI-assisted image processing platform, and the following colorimetric analysis steps were executed sequentially: Image acquisition stage: A series of standard colorimetric images were retrieved to confirm that the colorimetric areas of all images were clearly captured; Data extraction stage: 500 pixels were randomly selected within the preset detection area, and a data table containing their RGB values ​​and corresponding position coordinates was output; Grayscale conversion stage: The standard grayscale conversion formula (grayscale value = 0.299R + 0.587G + 0.114B) was used to convert the extracted RGB data into grayscale values; Model training stage: The calculated grayscale values ​​were fitted with known AOH concentration data to establish a linear calibration curve, and the regression model was learned and stored as the basis for subsequent concentration calculations; Automatic analysis stage: Test sample images captured under the same standardized conditions were retrieved, their RGB values ​​were extracted and converted to grayscale values, and then the corresponding AOH concentration in the sample was calculated and output based on the stored calibration curve. The results are as follows: Figure 6 As shown. From Figure 6 As can be seen from A, within the AOH concentration range of 0.05 ng / mL to 100 ng / mL, the average gray value shows a good linear negative correlation with the concentration, and the linear regression coefficient R0 is [value missing]. 2 The value was 0.9902, and based on a signal-to-noise ratio of 3, the detection limit was 0.0347 ng / mL; from Figure 6 B- Figure 6 As can be seen from C, the interfering substances include common Alternaria toxins (Alternaria monomethyl ether (AME), Alternaria ketoacid (TeA), TEN, Alternaria alternatae (ALT), Alternaria alternatae (ATX)), other mycotoxins that may coexist with AOH (zearalenone (ZEN), ochratoxin A (OTA), fumonisin B1 (FB1)), typical food matrix components (glucose, glutamic acid (Glu), aspartic acid (Asp), proline (Pro), tryptophan (Trp)) and common metal ions (Fe). 2+ Fe 3+ Cu 2+ Zn 2+ Mn 2+ The paper-based sensor exhibits a significantly stronger response to AOH than any of the tested interfering substances, demonstrating excellent specificity. Only when AOH coexists with its structural analogues AME or TeA does it induce a slight synergistic enhancement effect (grayscale increase <9%), which is within a controllable range, indicating good selectivity of the sensor. Figure 6 D- Figure 6 E shows that when using smartphones from different brands such as iPhone, Xiaomi, and Huawei to capture and analyze images under the same conditions, the differences in grayscale values ​​obtained are not statistically significant, demonstrating the strong robustness of the method; from Figure 6 As can be seen from F, after the paper-based sensor is stored under dry and light-protected conditions for 60 days, the gray value change rate of the colorimetric signal for the same concentration of AOH is less than 5%, showing good long-term stability. Test Example 7

[0039] The linear range and detection limit of the paper-based sensor prepared in Example 1 were compared with those of different types of existing sensors for detecting AOH, as shown in Table 2. Table 2

[0040] As can be seen from Table 2, the paper-based sensor of this application has a lower detection limit and a wider linear range. Test Example 8

[0041] Weigh 1.00 g of the homogenized food (wheat, wolfberry, tomato, grape, apple) into a 5 mL centrifuge tube. Add 2.0 mL of 60% methanol aqueous solution as the extraction solvent. Vortex and mix for 30 s. Then, place the tube in a constant temperature shaker at 25±1℃ and shake at 200 rpm for 30 min. After extraction, centrifuge the sample at 8000 rpm for 5 min. Filter the supernatant through a 0.22 μm polytetrafluoroethylene microporous membrane. Discard the initial 0.5 mL filtrate and collect the subsequent filtrate. Accurately transfer 100 μL of this filtrate and dilute it 5 times with 400 μL of phosphate buffer (pH 7.4). Mix well and use this as the test solution. Analyze this test solution according to the method described in Test Example 6. Simultaneously, take parallel samples and analyze them using HPLC-MS as a reference. Repeat the test 3 times under the same conditions. The results are shown in Table 3. Table 3

[0042] As can be seen from Table 3, the results of the method of the present invention are highly consistent with those of the HPLC-MS method, with ideal spike recovery. Furthermore, the statistical t-test showed no significant difference between the two methods (p>0.05), which fully verifies the accuracy and reliability of the method of the present invention in practical applications.

[0043] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A paper-based sensor, characterized in that, It includes a first protective layer, an immobilization layer, a substrate layer, a color developing layer, and a second protective layer; The first protective layer and the second protective layer are used to protect the immobilization layer, the substrate layer and the color development layer, and the surfaces of the first protective layer and the second protective layer are coated with a hydrophobic material. The central regions of the immobilization layer, substrate layer, and color development layer are coated with acetylcholinesterase, acetylthiocholine, and nanozyme, respectively, to form hydrophilic reaction regions, and the non-hydrophilic reaction regions are coated with hydrophobic materials to form hydrophobic isolation regions; the nanozyme is a copper-manganese dual-doped cerium dioxide nanozyme. The first protective layer, the immobilization layer, the substrate layer, the color development layer, and the second protective layer are folded in a preset order, and the hydrophilic reaction zones of the immobilization layer, the substrate layer, and the color development layer overlap after folding.

2. The paper-based sensor according to claim 1, characterized in that, The copper-manganese dual-doped cerium dioxide nanozyme contains 5.0%-5.5% copper and 1%-2% manganese by mass.

3. The paper-based sensor according to claim 1, characterized in that, The preparation of the copper-manganese dual-doped cerium dioxide nanozyme includes the following steps: dissolving cerium salt, manganese salt, copper salt and ethylene glycol in water, sealing and reacting in a forced-air drying oven at 175℃-185℃ for 2h-4h, centrifuging, washing and drying to obtain the copper-manganese dual-doped cerium dioxide nanozyme.

4. The paper-based sensor according to claim 3, characterized in that, The cerium salt is cerium nitrate; The manganese salt is selected from manganese chloride and / or manganese acetate; The copper salt is copper chloride.

5. The paper-based sensor according to claim 1, characterized in that, The hydrophobic material is polydimethylsiloxane.

6. The paper-based sensor according to claim 1, characterized in that, The acetylcholinesterase loading is 0.14 mU-0.16 mU; The loading of acetylthiocholine is 0.09 μmol-0.11 μmol; The loading of the nanozyme is 1.8 μg-2.2 μg.

7. The paper-based sensor according to claim 1, characterized in that, The substrate of the paper-based sensor is filter paper.

8. The method for preparing a paper-based sensor according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Coat the substrate with a hydrophobic material solution and cure it to obtain the first protective layer and the second protective layer; Hydrophilic reaction zones were pre-set on the substrate, and hydrophobic materials were coated on the non-hydrophilic reaction zones and cured to form hydrophobic isolation zones. Then, acetylcholinesterase solution, acetylthiocholine solution, and nanozyme solution were coated on each hydrophilic reaction zone and dried to obtain an immobilization layer, a substrate layer, and a color development layer. S2. Fold the first protective layer, immobilization layer, substrate layer, color development layer and second protective layer described in S1 in a preset order, and after folding, the hydrophilic reaction areas of the immobilization layer, substrate layer and color development layer overlap each other to obtain the paper-based sensor.

9. The method for preparing the paper-based sensor according to claim 8, characterized in that, In S1, the concentration of the acetylcholinesterase solution is 30 mU / mL; The concentration of the acetylthiocholine solution was 1.8 mmol / L to 2.2 mmol / L; The concentration of the nanozyme solution is 0.36 mg / mL to 0.44 mg / mL.

10. The use of the paper-based sensor as described in any one of claims 1-7 in the detection of cross-linked polysaccharides.

Images