Molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine and its preparation and application

Abstract

This invention discloses a molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine, its preparation, and its application. The colorimetric sensor (MIP@MIL) is polymerized from a metal-organic framework (MIL-101) using surface molecular imprinting and dual-monomer imprinting techniques. MIP@MIL exhibits a histamine detection limit of 0.77 μM and a detection time of 12.5 min, demonstrating good selectivity. The mechanism involves histamine blocking the imprinted pores of MIP@MIL and acting on MIL-101, thereby weakening the catalytic oxidation process of the chromogenic agent 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS). Furthermore, histamine increases the repulsive force of MIP@MIL against ABTS, thus enabling colorimetric detection of histamine. To further improve practical applicability, MIP@MIL was loaded onto qualitative filter paper and further modified onto microfluidic paper-based chips (μPADs). Using a smartphone, rapid and accurate visual detection of histamine in food was achieved, demonstrating its potential for food freshness detection.

Description

Technical Field

[0001] This invention belongs to the field of food freshness analysis and detection, specifically involving a molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine, its preparation and application. Background Technology

[0002] Early spoilage of food during storage, transportation, and distribution poses a serious threat to food safety and human health. Biogenic amines are important indicators of food freshness, mainly including histamine, putrescine, cadaverine, spermine, spermidine, tyramine, and tryptamine. Among them, histamine is the most toxic; excessive intake can cause headaches, vomiting, and even death. Histamine is also a core indicator of food spoilage, especially in seafood and meat. Currently, histamine detection methods based on chromatography, spectroscopy, electrochemical sensors, and biosensors suffer from drawbacks such as being time-consuming, labor-intensive, cumbersome, costly, and unable to meet the demand for rapid detection. Developing portable, highly selective detection equipment is imperative.

[0003] Metal-organic frameworks (MOFs) are porous crystalline materials composed of metal ions and organic ligands. Due to their high specific surface area, good stability, and diverse functions, MOFs have attracted much attention and have been widely used in energy storage, catalysis, pollutant adsorption, and drug delivery over the past decade. Molecularly imprinted polymers (MIPs) are polymeric materials with the ability to specifically recognize certain molecules (templates). Multi-monomer co-application and surface molecular imprinting technologies have solved the problems of low imprinting efficiency, slow mass transfer, and difficulty in template elution in traditional imprinting methods, significantly improving recognition efficiency and practicality. MIPs can be coated onto MOFs using multi-monomer co-application and surface molecular imprinting technologies to form composite materials with selective adsorption and highly efficient catalytic performance (MIP@MOF).

[0004] Microfluidic paper-based devices (μPADs) utilize paper as a substrate material, relying on capillary action and filtration to guide liquid movement and reaction on the paper through pre-designed microchannels, achieving visualized detection. μPADs, as a simple, integrated, and portable rapid analytical platform, have been widely reported. Paper-based optical, electrochemical, and biosensors can be fabricated by modifying paper with nanomaterials, dyes, printed electrodes, antibodies, nucleic acid probes, and enzymes. However, current optical paper-based sensors heavily rely on the human eye for color discrimination, making it difficult to capture subtle color changes. To resist environmental interference and achieve qualitative and quantitative detection of target substances, the development of smartphone-based paper-based microfluidic platforms is urgently needed. Smartphones offer portable size, excellent data transmission capabilities, and can easily recognize images for rapid detection, enabling personalized applications for portable analyte detection.

[0005] Currently, there are few molecularly imprinted colorimetric sensors used for histamine detection. Several molecularly imprinted sensors have been reported, such as patent CN113092442B, which provides a rapid method for histamine detection. Using a gold-core, silver-shell nanosol as a reinforcing substrate, it applies a molecularly imprinted polymer selectively adsorbing histamine combined with surface-enhanced Raman spectroscopy (SERS) to detect samples. This method shows a significant response to changes in the SERS signal of low concentrations of histamine in the sample and can be used to detect trace amounts of histamine in fish. This method can achieve qualitative and quantitative detection of histamine simply, quickly, and accurately, with good sensitivity and high selectivity. Patent CN111679068B discloses a method for detecting histamine using nanozyme-labeled direct competitive biomimetic immunoassay. This invention uses platinum nanomaterials instead of natural enzymes as markers, combining molecular imprinting technology with nanozymes to achieve highly sensitive detection of histamine. This invention is simple to operate, stable, and highly selective, showing no significant response to other biogenic amines (tyramine, tryptamine), making it suitable for rapid detection of histamine in food. However, both of the above methods require sample pretreatment before detection, which is cumbersome and not user-friendly for non-professionals, thus unsuitable for rapid on-site detection. Patent CN115074111A provides a method for preparing a histamine fluorescent sensing material. This invention synthesizes polymer QDs@COFs@MIP and uses it for the detection of histamine in food. Using QDs with good fluorescence properties as the luminescent element and introducing covalent organic frameworks (COFs), the specific surface area of ​​the entire fluorescent material is increased, improving its adsorption capacity and mass transfer rate. However, this molecularly imprinted sensor uses only a single monomer, and its selectivity for histamine detection needs further improvement. Furthermore, compared to colorimetric detection, fluorescence detection requires external components such as UV lamps, further increasing the detection cost. Therefore, the development of a dual-monomer molecularly imprinted colorimetric sensor, combined with a paper-based microfluidic chip, enables portable and rapid qualitative and quantitative detection of histamine without pretreatment, which is of great significance for on-site detection of food freshness. Summary of the Invention

[0006] To address the shortcomings and deficiencies of the existing technology, the present invention aims to provide a molecularly imprinted paper-based microfluidic colorimetric sensor for visualizing histamine. The molecularly imprinted colorimetric sensor (MIP@MIL) is polymerized from a metal-organic framework (MIL-101) using surface molecular imprinting and dual-monomer imprinting techniques. MIP@MIL exhibits a histamine detection limit of 0.77 μM and a detection time of 12.5 min, demonstrating excellent selectivity. The mechanism involves histamine blocking the imprinted pores of MIP@MIL and acting on MIL-101, thereby weakening the catalytic oxidation process of ABTS. Furthermore, histamine increases the repulsive force of MIP@MIL against ABTS, thus enabling colorimetric detection of histamine. To further improve practical applicability, MIP@MIL was loaded onto microfluidic paper-based chips (μPADs), and the rapid and accurate visual detection of histamine in food was achieved using a smartphone detection platform, demonstrating its potential for food freshness detection.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine, which is obtained by impregnating and loading a molecularly imprinted colorimetric sensor MIP@MIL for detecting histamine onto qualitative filter paper, and then further modifying it onto a paper-based microfluidic chip μPADs, referred to as MIP@MIL@μPADs; MIP@MIL is polymerized from the metal-organic framework MIL-101 using surface molecular imprinting technology and dual monomer imprinting technology; μPADs are designed using PowerPoint, and obtained by laser printing and high-temperature baking on qualitative filter paper before being cut.

[0008] Furthermore, MIP@MIL can detect histamine under the following conditions: Incubate 0.25-4 mg / mL MIP@MIL with histamine aqueous solution for 0-20 min, add buffer solution, 10-100 mM hydrogen peroxide, and 1-50 mM chromogenic agent 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid ABTS. React for 0-180 s, and measure the absorbance at 418 nm using a UV spectrophotometer.

[0009] Furthermore, μPADs consist of two layers: The first layer includes a hydrophobic region, double-sided adhesive within the hydrophobic region, and sensing sites, with the paper-based loaded MIP@MIL adhered to the sensing sites. The second layer consists of a hydrophobic region and a hydrophilic region within the hydrophobic region. The hydrophilic region consists of three vertically arranged circular regions and microchannels connecting the circular regions. The sample droplet area is located in the middle circular region. The second layer is folded and adhered directly to the first layer. The circular area below the hydrophilic region in the second layer overlaps with the sensing site and is placed horizontally. When the sample solution is dropped into the sample drop area, the sample solution will rely on capillary action and filtration to flow through the microchannels of the second layer into the hydrophilic region of the second layer and vertically through the second layer into the sensing site of the first layer.

[0010] This invention also provides a method for preparing the above-mentioned molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine, comprising the following steps: (1) FeCl3 and 1,4-BDC terephthalic acid were dissolved in DMF respectively. Then the two solutions were mixed and sonicated. Acetic acid HAc was added and reacted in a high pressure vessel. After the reaction was completed, the product was washed with DMF and water and dried to obtain metal-organic framework MIL-101. (2) Histamine dihydrochloride was dispersed in ethanol, and monomer 1 methacrylate MAA, monomer 2 p-vinylbenzoic acid 4-VA, MIL-101, ethylene glycol dimethacrylate EGDMA and azobisisobutyronitrile AIBN were added to obtain a mixed solution; the mixed solution was shaken in the dark under N2 protection; ultrasonic elution was performed using a methanol-acetic acid mixture, washed with methanol and dried to obtain MIP@MIL; (3) Immerse the shaped filter paper in the MIP@MIL aqueous solution and shake it. Take it out and let it dry to obtain the paper-based loaded MIP@MIL. (4) The paper-based loaded MIP@MIL is attached to the sensing site of the paper-based microfluidic chip μPADs to obtain the molecularly imprinted paper-based microfluidic colorimetric sensor MIP@MIL@μPADs for detecting histamine.

[0011] Furthermore, the molar volume ratio of histamine dihydrochloride to ethanol is 6.5 mmol: 1 L, and the total amount of monomers added is 0.1-0.5 mmol.

[0012] Furthermore, the molar ratio of monomer 1 to monomer 2 is 4:0-0:4; the amount of MIL-101 added is 10-40 mg.

[0013] Furthermore, the amount of EGDMA added was 0.5-3.5 mmol, the volume ratio of methanol to acetic acid in the methanol-acetic acid mixture was 9:1, the reaction time was 8-24 h, and the elution time was 0-60 min.

[0014] The present invention also provides the application of the above-mentioned molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine in the detection of histamine content in food.

[0015] Furthermore, the food includes shrimp, fruit, or vegetables.

[0016] Furthermore, the aforementioned applications can perform digital analysis using smartphones.

[0017] Furthermore, the detection includes the following steps: (1) Drop the sample solution into the sample drop area of ​​the molecularly imprinted paper-based microfluidic colorimetric sensor MIP@MIL@μPADs for detecting histamine; (2) The sample solution flows through the hydrophilic region of the second layer and flows vertically through the second layer into the sensing site to mix and incubate with the loaded MIP@MIL; (3) Open MIP@MIL@μPADs and add buffer solution, hydrogen peroxide and colorimetric reagent ABTS to the sensing site to carry out the reaction; (4) Take photos with a smartphone and perform digital analysis.

[0018] Furthermore, the concentration of the loaded MIP@MIL was 0.5-4 mg / mL, the incubation time was 2-14 min, the concentration of ABTS was 2.5-30 mM, the concentration of hydrogen peroxide was 0.1-5 M, and the reaction time was 1-7 min.

[0019] The MIP@MIL sensing mechanism of this invention is as follows: First, histamine blocks the pores of the MIP@MIL imprint, directly acting on MIL-101 and inhibiting the catalytic generation of hydroxyl radicals from hydrogen peroxide, thereby weakening the catalytic oxidation process of ABTS. Furthermore, the addition of histamine increases the negative potential of MIP@MIL, increasing the repulsive force against ABTS and weakening its affinity, thus achieving histamine detection through a lighter color development.

[0020] Compared with existing technologies, the key advantages of the present invention, which provides a molecularly imprinted paper-based microfluidic colorimetric sensor for visualizing histamine, include: (1) The sensor core MIP@MIL of the present invention adopts surface molecular imprinting technology and dual monomer imprinting technology, which improves the detection performance of single monomer molecular imprinted polymers.

[0021] (2) The histamine detection limit of the MIP@MIL of the present invention is 0.77 μM, and the detection time is 12.5 min, which shows excellent selectivity. The histamine detection limit of the paper-based microfluidic sensor MIP@MIL@μPADs based on MIP@MIL is 4.61 μM, and the detection time is 15 min.

[0022] (3) The sensing mechanism of this invention has two aspects: First, histamine blocks the pores of the MIP@MIL imprint and acts on MIL-101, thereby weakening the catalytic oxidation process of ABTs; second, histamine increases the repulsive force of MIP@MIL on ABTS. Combining the above two points, the colorimetric detection of histamine is achieved.

[0023] (4) The present invention loads the sensor on a self-designed paper-based microfluidic chip μPADs, which improves the convenience of the sensor and the on-site detection capability.

[0024] (5) The sensor of the present invention uses a smartphone for digital analysis, which can achieve higher sensitivity and accuracy compared with visual recognition analysis, thereby reducing errors related to human observation. Attached Figure Description

[0025] Figure 1 The images are scanning electron microscope (SEM) images (a), transmission electron microscope (TEM) images (b), and SEM images (c) of MIL-101, MIP@MIL, and NIP@MIL before and after loading onto qualitative filter paper in Example 1.

[0026] Figure 2 The infrared spectrum (a), X-ray diffraction pattern (b), and ultraviolet absorption spectrum (cd) of MIL-101, MIP@MIL, and NIP@MIL in Example 1 are shown.

[0027] Figure 3 This is a structural and physical diagram of the paper-based microfluidic chip MIP@MIL@μPADs loaded with MIP@MIL in Example 2.

[0028] Figure 4 This is a statistical chart of UV spectrophotometer measurement results corresponding to the optimized preparation conditions of MIP@MIL in Application Example 1, including: the ratio of the two monomers (a), the total amount of monomers (b), the amount of MIL-101 added (c), the amount of EGDMA added (d), the reaction time (e), and the elution time (f).

[0029] Figure 5 This is a statistical graph of histamine detection imprinting factors for MIL-101, MIP@MIL, and NIP@MIL in Application Example 2.

[0030] Figure 6 This is a statistical chart of UV spectrophotometer measurement results corresponding to the optimized histamine detection conditions of MIP@MIL in Application Example 3, including: material concentration (a), incubation time (b), ABTS concentration (c), hydrogen peroxide concentration (d), and reaction time (e).

[0031] Figure 7 The graphs are: (a) histamine detection dot plots of MIP@MIL and NIP@MIL in Application Example 4; (b) histamine detection linear correlation plots of MIP@MIL and NIP@MIL in Application Example 4.

[0032] Figure 8This is a statistical chart of UV spectrophotometer measurement results for the selectivity experiment of MIP@MIL and NIP@MIL in Application Example 5, including: (a): common amino acids and tyramine, (b): common cations and common anions.

[0033] Figure 9 The application example 6 shows the optimization of histamine detection conditions using MIP@MIL@μPADs and a linear analysis graph of histamine detection, including: material concentration (a), incubation time (b), reaction time (c), ABTS concentration (d), hydrogen peroxide concentration (e), and histamine concentration (f). Detailed Implementation

[0034] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0035] Example 1 A molecularly imprinted colorimetric sensor MIP@MIL for detecting histamine includes the following preparation steps: (1) First, 2.5 mmol FeCl3 was dissolved in 15 mL DMF, and then 2.5 mmol 1,4-BDC was dissolved in 10 mL DMF. The two solutions were mixed and sonicated for 10 minutes, and then 2.5 mL HAc was added. The mixture was reacted in an autoclave at 120 °C for 12 hours. Finally, the product was washed with DMF and water, and dried in an oven at 60 °C for 12 hours to obtain the metal-organic framework MIL-101, which was stored in a desiccator.

[0036] (2) 0.13 mmol histamine dihydrochloride was dispersed in 20 mL of ethanol, and 0.1125 mmol monomer 1 methacrylic acid (MAA), 0.0375 mmol monomer 2 p-vinylbenzoic acid (4-VA), 35 mg MIL-101, 2 mmol ethylene glycol dimethacrylate (EGDMA), and 20 mg azobisisobutyronitrile (AIBN) were added. The mixture was shaken at 60 °C for 16 h in the dark under N2 protection. It was then ultrasonically eluted with a methanol-acetic acid mixture (V:V = 9:1), washed with methanol, and dried in an oven at 60 °C for 12 h to obtain MIP@MIL.

[0037] (3) Following the preparation steps of MIP@MIL, non-molecularly imprinted polymers (NIP@MIL) without the addition of histamine were prepared simultaneously. Molecularly imprinted polymers MAA-MIP@MIL and 4-VA-MIP@MIL were prepared using a single monomer (MAA or 4-VA) for comparison.

[0038] MIL-101, MIP@MIL, and NIP@MIL were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), such as... Figure 1 As shown. Figure 1 In (a), from left to right are scanning electron microscope images of MIL-101, MIP@MIL and NIP@MIL. MIL-101 has an octahedral shape and a particle size of about 800 nm. The surfaces of MIP@MIL and NIP@MIL gradually become rough after imprinting. Figure 1 (b) shows transmission electron microscopy (TEM) images of MIL-101, MIP@MIL, and NIP@MIL, respectively. It can be observed that the imprinted layers of MIP@MIL and NIP@MIL are 60-70 nm thick. Furthermore, MIL-101 is a brown powder, while MIP@MIL and NIP@MIL are light yellow powders.

[0039] MIL-101, MIP@MIL, and NIP@MIL were characterized by infrared spectroscopy, X-ray diffraction, and ultraviolet absorption spectroscopy. The results of the Fourier transform infrared (FT-IR) spectroscopy are as follows: Figure 2 As shown in (a), approximately 555cm -1 The peak at that point should be attributed to the vibration of the Fe-O bond. The CH bending vibration in the benzene ring appears at 748 cm⁻¹. -1 Location. 1593cm -1 and 1390cm -1 Corresponding to the symmetric and asymmetric stretching vibrations of the single bond -COO-. The vibration of C=O occurs at 1647 cm⁻¹. -1 These results demonstrate the presence of abundant functional groups on the surface of MIL-101(Fe). Approximately 1700 cm⁻¹ -1 This is a C=C stretching vibration of a single unit, 3000cm. -1 The nearby structures belong to the stretching vibrations of the monomers methyl and methylene. Because the molecularly imprinted polymers and non-molecularly imprinted polymers are similar in composition to MIL-101 and the synthetic monomers, their spectra exhibit the same characteristics.

[0040] X-ray diffraction (XRD) results are as follows Figure 2As shown in (b), the XRD patterns of MIL-101 at 9.16°, 10.61°, 19.34° and 23.34° correspond to the (2 2 0), (3 1 1), (5 1 1) and (8 5 2) crystal planes, respectively. However, due to the coverage of the imprinted layer, the X-ray diffraction peak intensities of MIP@MIL and NIP are weakened.

[0041] The results of the ultraviolet absorption spectrum are as follows: Figure 2 As shown in (c) and (d), the UV absorption spectrum of MIL-101 is significantly higher than that of MIP@MIL and NIP@MIL, indicating that the successful loading of MIP led to a decrease in absorbance. Furthermore, the UV absorption spectrum of MIP@MIL was found to be higher than that of NIP@MIL, which is attributed to the imprinted cavity of MIP@MIL. After the addition of histamine, a slight increase in the absorption intensity of MIP@MIL in the 250-300 nm range was observed, which is attributed to the recognition of histamine by MIP@MIL.

[0042] Example 2 The molecularly imprinted paper-based microfluidic colorimetric sensor MIP@MIL@μPADs for detecting histamine comprises the following preparation steps: Take the MIP@MIL prepared in Example 1, put a 5mm circular Whatman No.1 filter paper (qualitative filter paper) into a 2mg / mL MIP@MIL aqueous solution, shake at 130rpm for 30min, take it out and let it dry to obtain paper-based loaded MIP@MIL.

[0043] The MIP@MIL loading was characterized before and after qualitative filter paper using scanning electron microscopy (SEM) images. Figure 1 As shown in (c), from left to right are Whatman No1 filter paper and Whatman No1 filter paper loaded with MIP@MIL. The results show that MIP@MIL was successfully loaded onto the qualitative filter paper fibers.

[0044] By attaching the paper-based loaded MIP@MIL to the sensing sites of paper-based microfluidic chip μPADs, MIP@MIL@μPADs can be obtained.

[0045] The paper-based microfluidic chip was designed using PowerPoint, laser-printed using qualitative filter paper, and then cut after baking at 200°C for 3 hours.

[0046] Paper-based microfluidic chip μPADs structure as follows Figure 3As shown in (a), the microfluidic chip consists of two layers. The first layer includes double-sided adhesive, sensing sites, and a black hydrophobic region. The second layer consists of a white hydrophilic region (containing the sample droplet area) and a black hydrophobic region. Each layer of the paper-based microfluidic chip is 3cm × 3cm in size. The double-sided adhesive is a circle with a diameter of 8mm, the sensing sites are circles with a diameter of 5mm, the hydrophilic regions are circles with a diameter of 5mm, the microchannels between the circles are 1mm wide, and the microchannels are 3mm long.

[0047] Figure 3 (b) shows actual images of μPADs and MIP@MIL@μPADs. The left side shows the cut paper-based microfluidic chip μPADs, and the right side shows the MIP@MIL@μPADs folded and placed horizontally after being loaded with MIP@MIL.

[0048] Instructions for use: Fold the second layer directly onto the first layer and place it horizontally. Drop 25 μL of histamine aqueous solution into the sample drop area. The aqueous solution will flow through the hydrophilic region of the second layer and vertically through the second layer into the sensing site. After incubation for 10 min, open the container and drop 7 μL of acetate-sodium acetate buffer solution (pH=3.5), 1 μL of 1M hydrogen peroxide solution, and 2 μL of 10mM ABTS onto the sensing site. After reacting for 5 min, take a picture with a smartphone, extract the RGB values ​​using Photoshop, and construct a standard curve of RGB versus histamine for further analysis of histamine concentration in unknown samples.

[0049] Application Example 1 The synthesis conditions of MIP@MIL in Example 1 were optimized, such as... Figure 4 The parameters shown include: the ratio of the two monomers (a), the total monomer amount (b), the amount of MIL-101 added (c), the amount of EGDMA added (d), the reaction time (e), and the elution time (f). The optimized detection procedure is as follows: Take 50 μL of 3 mg / mL MIP@MIL, add 50 μL of 2 mM histamine aqueous solution or pure water, mix for 10 min, then add 800 μL of 0.2 mM NaAc-HAc (pH=3.5) buffer solution, 50 μL of 75 mM hydrogen peroxide, and 50 μL of 30 mM ABTS. React for 2.5 min, and measure the absorbance at 418 nm using a UV spectrophotometer. Calculate the difference in absorbance between the addition of histamine and the addition of pure water.

[0050] For two monomers (n MAA :n 4-VA Five ratios were set: 4:0, 3:1, 2:2, 1:3, and 0:4. When n MAA :n 4-VAThe absorbance difference before and after histamine addition was largest when the ratio was 3:1. For the total monomer, addition amounts of 0.1 mmol, 0.13 mmol, 0.15 mmol, 0.2 mmol, 0.3 mmol, 0.4 mmol, and 0.5 mmol were set. The largest absorbance difference before and after histamine addition was observed when the total monomer amount was 0.15 mmol. For MIL-101, addition amounts of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, and 40 mg were set. The largest absorbance difference before and after histamine addition was observed when the MIL-101 addition amount was 35 mg. For the crosslinking agent EGDMA, addition amounts of 0.5 mmol, 1 mmol, 1.5 mmol, 2 mmol, 3 mmol, and 3.5 mmol were set. The largest absorbance difference before and after histamine addition was observed when the EGDMA addition amount was 2 mmol. A time gradient of 8h, 12h, 16h, 20h, and 24h was set for the preparation time. The largest difference in absorbance before and after the addition of histamine was observed when the preparation time was 16h. A time gradient of 0min, 7.5min, 15min, 30min, 45min, and 60min was set for the sonication time. The smallest absorbance was observed when the sonication time was 7.5min, indicating that histamine was eluted.

[0051] Application Example 2 Take 50 μL of 3 mg / mL MIP@MIL, NIP@MIL, MAA-MIP@MIL, and 4-VA-MIP@MIL from Example 1, add 50 μL of 2 mM histamine aqueous solution or pure water and mix for 10 min. Then add 800 μL of 0.2 mM NaAc-HAc (pH=3.5) buffer solution, 50 μL of 75 mM hydrogen peroxide, and 50 μL of 30 mM ABTS. React for 2.5 min and measure the absorbance at 418 nm using a UV spectrophotometer.

[0052] The imprinting factor (IF) is calculated as follows: IF = (difference in absorbance between the added histamine and the added pure water in the molecularly imprinted polymer) / (difference in absorbance between the added histamine and the added pure water in the non-molecularly imprinted polymer).

[0053] like Figure 5 As shown, compared with the single-monomer molecularly imprinted polymers MAA-MIP@MIL and 4-VA-MIP@MIL, MIP@MIL exhibits a higher imprinting factor of 2.44, indicating that it has less non-specific adsorption.

[0054] Application Example 3 The histamine detection conditions were optimized using the MIP@MIL method described in Example 1, such as... Figure 6As shown, the parameters include: material concentration (a), incubation time (b), ABTS concentration (c), hydrogen peroxide concentration (d), and reaction time (e). The detection procedure was as follows: 50 μL of 3 mg / mL MIP@MIL was mixed with 50 μL of histamine or pure water for 10 min, followed by 800 μL of 0.2 mM NaAc-HAc (pH=3.5) buffer solution, 50 μL of 75 mM hydrogen peroxide, and 50 μL of 30 mM ABTS. The reaction was allowed to proceed for 2.5 min, and the absorbance at 418 nm was measured using a UV spectrophotometer. The difference in absorbance between the addition of histamine and the addition of pure water was calculated.

[0055] For MIP@MIL, concentration gradients of 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL were set. The largest absorbance difference was observed at a concentration of 2 mg / mL. For incubation time gradients of 0 min, 2.5 min, 5 min, 7.5 min, 10 min, 15 min, and 20 min were set. The largest absorbance difference was observed at a time of 10 min. For ABTS, concentration gradients of 1 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, and 50 mM were set. The largest absorbance difference was observed at a concentration of 30 mM. For hydrogen peroxide, concentration gradients of 10, 20, 40, 50, 75, and 100 mM were set. The largest absorbance difference was observed at a concentration of 75 mM. A time gradient of 0-180s was set for the reaction time. When the time reached 150s, the difference in absorbance value reached its maximum and basically stabilized.

[0056] Application Example 4 Histamine detection was performed using MIP@MIL and NIP@MIL from Example 1. The detection steps were as follows: Take 50 μL of 3 mg / mL MIP@MIL or NIP@MIL, add 50 μL of 30, 60, 120, 180, 300, 600, 900, 1200, 1500, 1800, 2100, or 2400 μM histamine aqueous solution or pure water, and mix for 10 min. Then add 800 μL of 0.2 mM NaAc-HAc (pH=3.5) buffer solution, 50 μL of 75 mM hydrogen peroxide, and 50 μL of 30 mM ABTS. React for 2.5 min, and measure the absorbance at 418 nm using a UV spectrophotometer (detection time is 12.5 min). Calculate the difference in absorbance between the addition of histamine and the addition of pure water.

[0057] Due to volumetric dilution of the reaction, the final histamine concentrations were 1 / 20th of the initial concentrations, specifically 1.5, 3, 6, 9, 15, 30, 45, 60, 75, 90, 105, and 120 μM. Figure 7 As shown in (a), the absorbance difference between MIP@MIL and NIP@MIL gradually increases with increasing histamine concentration. The absorbance difference of MIP@MIL reaches its maximum when the histamine concentration is 75 μM, while the absorbance difference of NIP@MIL reaches its maximum when the histamine concentration is 105 μM. The histamine linear response of MIP@MIL was analyzed, as shown in (a). Figure 7 As shown in (b), MIP@MIL exhibits a linear relationship with histamine in the range of 1.5–75 μM, with the linear equation being ΔA = 0.014C - 0.027, R 2 =0.99, detection limit is 0.77 μM (ΔA is the absorbance difference, C is the histamine concentration). Histamine linear response was analyzed for NIP@MIL, as follows: Figure 7 As shown in (c), NIP@MIL exhibits a linear relationship with histamine in the range of 1.5–105 μM, with the linear equation being ΔA = 0.0043C + 0.018, R 2 =0.97, the linear correlation is worse than that of MIP@MIL, and the difference in absorbance value is smaller than that of MIP@MIL.

[0058] Application Example 5 Selectivity experiments were conducted using MIP@MIL and NIP@MIL from Example 1. The experimental steps are as follows: (1) Common amino acids and tyramine as interfering substances: Take 50 μL of 3 mg / mL MIP@MIL or NIP@MIL, add 50 μL of 1mM histamine, histidine, tyramine, Ala, Phe, Pro, Leu, Val, Ser, Thr, Lys, Arg, Gln, Trp, Cys, Asp, Clu aqueous solution or pure water and mix for 10 min. Then add 800 μL of 0.2 mM NaAc-HAc (pH=3.5) buffer solution, 50 μL of 75 mM hydrogen peroxide, and 50 μL of 30 mM ABTS. React for 2.5 min. Measure the absorbance at 418 nm using a UV spectrophotometer and calculate the difference in absorbance between the added histamine, each interfering substance and the added pure water.

[0059] (2) Common cations and common anions as interfering substances: Add 50 μL of 1mM histamine, KCl, CaCl2, NaCl, MgCl2, ZnCl2, CuCl2, Zn(NO3)2, Na2SO4, Na2SO3, NaHCO3 aqueous solution or pure water, and the rest of the steps are the same as above.

[0060] The absorbance difference of common amino acids and tyramine as interfering substances is as follows: Figure 8 As shown in (a), the difference in absorbance values ​​between common cations and common anions as interfering substances is as follows: Figure 8 As shown in (b), MIP@MIL exhibits good selectivity for common amino acids, cations, anions, and interfering substances such as tyramine, while the non-molecularly imprinted polymer NIP@MIL shows a smaller difference in absorbance, indicating less non-specific adsorption. The selectivity experiment demonstrates the application potential of MIP@MIL in complex food environments.

[0061] Application Example 6 The molecularly imprinted paper-based microfluidic colorimetric sensor MIP@MIL@μPADs used in Example 2 for histamine detection was used for histamine detection condition optimization and histamine linearity analysis. For example... Figure 9 The histamine detection conditions included: the concentration of loaded MIP@MIL (a), incubation time (b), reaction time (c), ABTS concentration (d), and hydrogen peroxide concentration (e); the concentration of histamine used for detection was 2 mM; the detection method was the same as that used in Example 2, and the RGB difference before and after the addition of histamine was calculated.

[0062] A concentration gradient of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL was set for MIP@MIL. The RGB difference was largest at a concentration of 2 mg / mL. A time gradient of 2 min, 4 min, 6 min, 8 min, 10 min, 12 min, and 14 min was set for incubation time. The RGB difference stabilized at an incubation time of 10 min. A time gradient of 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, and 7 min was set for reaction time. The RGB difference reached its maximum at a reaction time of 5 min. The optimal detection time was 15 min. A concentration gradient of 2.5 mM, 5 mM, 10 mM, 20 mM, and 30 mM was set for ABTS. The RGB difference reached its maximum at an ABTS concentration of 10 mM. Hydrogen peroxide concentration gradients of 0.1M, 0.2M, 0.5M, 1M, 2M, and 5M were set. The RGB difference reached its maximum when the hydrogen peroxide concentration was 1M. Since the color value R changed the most during the condition optimization process, the subsequent linear analysis used the difference in R values ​​before and after the addition of histamine as the ordinate.

[0063] Linear detection of histamine (final concentrations of 10, 20, 40, 60, and 80 μM) was performed based on optimized experimental conditions, such as... Figure 9As shown in (f), the R difference and histamine concentration have a good linear relationship in the range of 10-80 μM. The linear equation is: ΔR=0.46C+3.18, R2=0.99, and the detection limit is 4.61 μM (ΔR is the R difference and C is the histamine concentration).

[0064] Application Example 7 To improve the practical application of MIP@MIL@μPADs, a spiked experiment was conducted using shrimp meat. The experimental steps are as follows: Shrimp meat pretreatment: Dissolve 5g of chopped shrimp meat directly in 20mL of water by vortexing for 1min, centrifuge to collect the supernatant, dilute 1000 times, adjust the pH to 7, and use it to prepare histamine gradient solution.

[0065] Histamine detection: Add 25 μL of histamine-spiked sample or water to the sample drop area of ​​MIP@MIL@μPADs, incubate for 10 min, then turn on the device. Add 7 μL of acetate-sodium acetate buffer solution (pH=3.5), 1 μL of 1M hydrogen peroxide solution, and 2 μL of 10mM ABTS to the sensing site. After reacting for 5 min, take a picture with a smartphone, extract the RGB values ​​using Photoshop, and substitute them into the standard curve to obtain the histamine concentration of the sample.

[0066] The results of the shrimp meat spiked experiment using MIP@MIL@μPADs are shown in Table 1. Histamine spike concentrations of 10, 20, 40, 60, and 80 μM were set, with recoveries ranging from 90.43% to 126.52% and RSDs from 1.39% to 3.29%. These results indicate that MIP@MIL@μPADs can accurately assess histamine levels during food spoilage through color changes, thereby evaluating food freshness.

[0067] Table 1. Results of shrimp meat spiked with MIP@MIL@μPADs

Claims

1. A molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine, characterized in that, The MIP@MIL@μPADs are obtained by impregnating and loading a molecularly imprinted colorimetric sensor for detecting histamine onto qualitative filter paper, and then further modifying it onto a paper-based microfluidic chip μPADs. MIP@MIL is polymerized from the metal-organic framework MIL-101 using surface molecular imprinting technology and dual monomer imprinting technology; μPADs are designed using PowerPoint, and obtained by laser printing and high-temperature baking on qualitative filter paper before being cut.

2. The molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine according to claim 1, characterized in that, The MIP@MIL can detect histamine under the following conditions: Incubate 0.25-4 mg / mL MIP@MIL with histamine aqueous solution for 0-20 min, add buffer solution, 10-100 mM hydrogen peroxide, and 1-50 mM colorimetric reagent 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid ABTS. React for 0-180 s, and measure the absorbance at 418 nm using a UV spectrophotometer.

3. The molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine according to claim 1, characterized in that, The μPADs consist of two layers: The first layer includes a hydrophobic region, double-sided adhesive within the hydrophobic region, and sensing sites, with the paper-based loaded MIP@MIL adhered to the sensing sites. The second layer consists of a hydrophobic region and a hydrophilic region within the hydrophobic region. The hydrophilic region consists of three vertically arranged circular regions and microchannels connecting the circular regions. The sample droplet area is located in the middle circular region. The second layer is folded and adhered directly to the first layer. The circular area below the hydrophilic region in the second layer overlaps with the sensing site and is placed horizontally. When the sample solution is dropped into the sample drop area, the sample solution will rely on capillary action and filtration to flow through the microchannels of the second layer into the hydrophilic region of the second layer and vertically through the second layer into the sensing site of the first layer.

4. The method for preparing the molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine as described in claim 1, characterized in that, Includes the following steps: (1) FeCl3 and 1,4-BDC terephthalic acid were dissolved in DMF respectively. The two solutions were then mixed and sonicated. Acetic acid HAc was added and reacted in a high-pressure reactor. After the reaction was completed, the product was washed with DMF and water and dried to obtain metal-organic framework MIL-101. (2) Histamine dihydrochloride was dispersed in ethanol, and monomer 1 methacrylate MAA, monomer 2 p-vinylbenzoic acid 4-VA, MIL-101, ethylene glycol dimethacrylate EGDMA and azobisisobutyronitrile AIBN were added to obtain a mixed solution; the mixed solution was shaken in the dark under N2 protection; ultrasonic elution was performed using a methanol-acetic acid mixture, washed with methanol and dried to obtain MIP@MIL; (3) Immerse the qualitative filter paper in the MIP@MIL aqueous solution and shake it. Take it out and let it dry to obtain the paper-based loaded MIP@MIL. (4) The paper-based loaded MIP@MIL is attached to the sensing site of the paper-based microfluidic chip μPADs to obtain the molecularly imprinted paper-based microfluidic colorimetric sensor MIP@MIL@μPADs for detecting histamine.

5. The preparation method according to claim 4, characterized in that, In step (2), the molar ratio of monomer 1 to monomer 2 is 4:0-0:

4.

6. The preparation method according to claim 4, characterized in that, In step (2), the molar volume ratio of histamine dihydrochloride to ethanol is 6.5 mmol: 1 L, and the total concentration of the added monomer is 5-25 mM.

7. The preparation method according to claim 4, characterized in that, In step (2), the volume ratio of methanol to acetic acid in the methanol-acetic acid mixture is 9:1, the reaction time is 8-24h, and the elution time is 0-60min.

8. The application of the molecularly imprinted paper-based microfluidic colorimetric sensor for detecting histamine as described in claim 1 in the detection of histamine content in food, characterized in that, The food items include shrimp, fruit, or vegetables; the application can perform digital analysis using a smartphone.

9. The application according to claim 8, characterized in that, The detection process includes the following steps: (1) Drop the sample solution into the sample drop area of ​​the molecularly imprinted paper-based microfluidic colorimetric sensor MIP@MIL@μPADs for detecting histamine; (2) The sample solution flows through the hydrophilic region of the second layer and flows vertically through the second layer into the sensing site to mix and incubate with the loaded MIP@MIL; (3) Open MIP@MIL@μPADs and add buffer solution, hydrogen peroxide and colorimetric reagent ABTS to the sensing site to carry out the reaction; (4) Take photos with a smartphone and perform digital analysis.

10. The application according to claim 9, characterized in that, The MIP@MIL loading concentration was 0.5-4 mg / mL, the incubation time was 2-14 min, the ABTS concentration was 2.5-30 mM, the hydrogen peroxide concentration was 0.1-5 M, and the reaction time was 1-7 min.

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