Composite material DexTracker for dexamethasone detection and application thereof

Abstract

The invention discloses a composite material DexTracker for dexamethasone detection and application of the composite material DexTracker, and belongs to the technical field of new materials. According to the present invention, the aptamer having the ssDNA sequence is combined with the iron-based organic framework (MOF) material to prepare the composite material DexTracker nano-enzyme, and the DexTracker nano-enzyme has characteristics of excellent peroxidase-like activity and excellent dexamethasone (DEX) specific recognition ability. Under optimized conditions, the designed sensor can effectively separate, capture and detect DEX, and shows good recovery rate in complex samples such as milk, honey and the like. Finally, a three-dimensional paper-based (3D-[mu] PAD) device based on DexTracker is developed, and a new theoretical solution is provided for real-time detection of hormones in food samples.

Description

Technical Field

[0001] The present invention relates to the technical field of new materials, and particularly to a composite material DexTracker for dexamethasone detection and its application. Background Art

[0002] With the exacerbation of food safety problems and the increasing severity of drug residue problems, especially the potential health risks brought by drug pollutants in food, the development of efficient and sensitive detection methods has become a key focus in environmental chemistry and food safety research. Dexamethasone (DEX) is a widely used synthetic steroid drug, mainly used widely due to its anti-inflammatory and immunosuppressive properties. However, long-term intake of DEX poses a significant threat to human health. Traditional detection techniques, such as liquid chromatography and mass spectrometry, provide high accuracy and sensitivity. However, these methods are costly, require complex operating procedures, and are not suitable for rapid on-site detection. Therefore, there is an urgent need to develop simple, efficient, and portable DEX detection methods. Summary of the Invention

[0003] The purpose of the present invention is to provide a composite material DexTracker for dexamethasone detection and its application to solve the problems existing in the above-mentioned prior art.

[0004] To achieve the above purpose, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a composite material DexTracker for dexamethasone detection, which is formed by combining an iron-based MOF material and a dexamethasone aptamer.

[0006] Another technical solution of the present invention is a preparation method of the composite material DexTracker, including the following steps:

[0007] (1) Add FeCl3·6H2O and 2-aminobenzoic acid to N,N-dimethylformamide, continuously stir for 2 hours, and then transfer it to an autoclave for high-temperature reaction;

[0008] (2) Add the dexamethasone aptamer and react for 2 h to allow it to grow naturally on the surface of the product, and centrifuge to collect the product.

[0009] Another technical solution of the present invention is the application of the composite material DexTracker in dexamethasone detection.

[0010] Another technical solution of the present invention is the application of the composite material DexTracker in the preparation of products for detecting dexamethasone.

[0011] Another technical solution of the present invention is a product for detecting dexamethasone, including the composite material DexTracker.

[0012] Based on the above technical solutions, the present invention has the following technical effects:

[0013] The present invention prepared a composite material DexTracker nanozyme, which has excellent peroxidase-like activity and specific recognition ability for dexamethasone (DEX). The binding of DEX to NH2-MIL-88B reduces the electron density of the active site of DexTracker and inhibits the ability of the material to effectively catalyze H2O2 to generate reactive oxygen species (ROS). In addition, the incorporation of aptamers improves the selectivity and capture efficiency of the material for DEX. These characteristics endow DexTracker with enhanced detection and adsorption capabilities for DEX. Under optimized conditions, the designed sensor can effectively separate and capture DexTracker for DEX detection, showing good recovery rates in complex samples such as milk and honey. Finally, a three-dimensional μPAD device based on DexTracker was developed, providing new theoretical insights for the real-time detection of hormones in food samples. Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0015] Figure 1 SEM image (A), 50 nm TEM image (B), 10 nm TEM image (C), elemental mapping image (D), UV image (E), XPS image (F), XRD pattern (G) of DexTracker.

[0016] Figure 2 Schematic diagram for DEX detection using a DexTracker-based biosensor (A), steady-state kinetic analysis of DexTracker: TMB (B), H2O2 (C), UV spectra of DEX in the concentration range of 0.01 - 50 μmol (D) and linear analysis of absorbance at 650 nm versus DEX concentration (E), UV spectra (F) and response values of DEX and its structural analogs at a concentration of 3 μmol to the sensing system (G). In (G), a: cortisone, b: dexamethasone, c: prednisone, d: estradiol, e; erythromycin, f: enrofloxacin, g: chloramphenicol, h: metronidazole, I: potassium chloride j: calcium chloride.

[0017] Figure 3Design drawings and physical diagrams of the 3D paper-based device (A), SEM image of the hydrophobic region (B), SEM image of the detection region (C), linear relationship diagram between DEX concentration in the range of 0.01 - 3 μm and the R / G ratio of the colorimetric detection region, and diagrammatic paper-based diagram (D).

[0018] Figure 4 Adsorption capacity of DEX at different concentrations and Langmuir isotherm diagram (A); Freundlich isotherm diagram (B); Pseudo-first-order adsorption kinetics fitting (C) and Pseudo-second-order adsorption kinetics fitting (D).

[0019] Figure 5 Effects of pH value (A), aptamer concentration (B), incubation time of aptamer (C), and capture reaction time of DEX (C) on the absorbance of DexTracker at 650 nm in the sensing system.

[0020] Figure 6 High-performance liquid chromatography diagram of DEX in the concentration range of 0.5 - 50 μM (A), and linear relationship between the peak area under the curve and DEX concentration (B). Detailed implementation mode

[0021] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of this application are merely exemplary.

[0025] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0026] The technical solutions of the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0027] An embodiment of the present invention provides a composite material DexTracker for dexamethasone detection, which is formed by combining an iron-based MOF material and a dexamethasone aptamer.

[0028] In some specific embodiments, the nucleotide sequence of the dexamethasone aptamer is as shown in SEQ ID NO.1.

[0029] In some specific embodiments, the 5′ end of the dexamethasone aptamer is modified with FAM.

[0030] An embodiment of the present invention also provides a preparation method of the composite material DexTracker, including the following steps:

[0031] (1) Add FeCl3·6H2O and 2-aminobenzoic acid to N,N-dimethylformamide, continuously stir for 2 hours, then transfer it to an autoclave for high-temperature reaction;

[0032] (2) Add the dexamethasone aptamer and react for 2 h to allow it to grow naturally on the surface of the product, and centrifuge to collect the product.

[0033] In some specific embodiments, the molar ratio of FeCl3·6H2O, 2-aminobenzoic acid, and the dexamethasone aptamer is 60:60:1;

[0034] The conditions for the high-temperature reaction are: heat at 120 °C for 24 hours.

[0035] An embodiment of the present invention also provides the application of the composite material DexTracker in dexamethasone detection.

[0036] An embodiment of the present invention also provides the application of the composite material DexTracker in the preparation of a product for detecting dexamethasone.

[0037] An embodiment of the present invention also provides a product for detecting dexamethasone, including the composite material DexTracker.

[0038] In some specific embodiments, the product includes reagents, reagent kits, and 3D-μPAD devices.

[0039] To achieve rapid on-site detection of dexamethasone (DEX) in food, the present invention prepared a three-dimensional paper-based (μPAD) device based on the composite material DexTracker. DEX specifically binds to DexTracker through hydrogen bonds via its functional groups (hydroxyl group, carbonyl group), which changes the ligand field effect in DexTracker and reduces the electron density of the catalytic center, thereby inhibiting the peroxidase activity of DexTracker. Under optimized conditions, the absorbance linear response is closely related to the DEX concentration range (10 - 3000 nM), and the limit of detection (LOD) is 6.1 nM. The spiked recoveries in milk and honey are 88.2% - 107.6%. It provides a portable and efficient platform for food safety monitoring and drug residue detection.

[0040] Metal-organic frameworks (MOFs) have become a research hotspot in the fields of sensing, adsorption, and catalysis due to their unique porous structure, large specific surface area, and tunable functional properties. By selecting different metal ions and organic ligands, the structural diversity and high porosity of MOF materials can be precisely controlled. This enables them to effectively capture and concentrate target molecules (e.g., tetracycline, coumarin, etc.), facilitating their effective separation from complex sample matrices. The present invention designed a novel composite material DexTracker colorimetric sensor for the detection of DEX in food. The detection mechanism is as Figure 2 shown in A. In the absence of DEX, DexTracker catalyzes the oxidation of colorless TMB to blue oxTMB in the presence of H2O2. When DEX is present, it is specifically adsorbed by DexTracker through chemical bonds, inhibiting its peroxidase-like activity and inhibiting the Fenton reaction. The DexTracker colorimetric sensor has a low limit of detection (LOD) and can effectively separate and identify DEX in complex food samples. To further improve its practicality, a three-dimensional paper-based device (μPAD) based on the composite material DexTracker was constructed. This innovative combination of portability and simplicity paves the way for a revolutionary advancement in DEX detection methods.

[0041] Example 1

[0042] 1 Reagents and Instruments

[0043] The base sequence of the DEX aptamer and its FAM-modified sequence are respectively: (SEQ ID NO.1) 5′-CGGGGGACGTTGCCAACGGTAACGTCGTTGGAT-3′.

[0044] These sequences were provided by Shanghai Sangon Biotech Co., Ltd. Iron(III) chloride hexahydrate (FeCl3·6H2O), 2-aminoterephthalic acid (NH2-BDC), and N,N-dimethylformamide (DMF) were purchased from Aladdin Biochemical Technology Co., Ltd. Methanol, sodium acetate (NaAc), acetic acid (HAc), and dimethyl sulfoxide (DMSO) were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. All chemical reagents were of analytical grade; 3,3',5,5'-tetramethylbenzidine (TMB), dexamethasone, cortisone, prednisone, estrogen, chloramphenicol, metronidazole, and erythromycin were purchased from Shanghai Macklin Biochemical Co., Ltd. Potassium chloride and calcium chloride were purchased from Tianjin Beilian Fine Chemical Development Co., Ltd. Hydrophilic-lipophilic balance columns (HLB columns) were purchased from Waters (Massachusetts, USA). Milk and honey samples were purchased from the local market. All experimental procedures were carried out using Wahaha purified water (Hangzhou Wahaha Group Co., Ltd.).

[0045] The morphologies of DexTracker and the paper-based device were investigated using a scanning electron microscope (SEM) (ZEISS Sigma 300, Germany). Their microstructures were detected by transmission electron microscopy (TEM) (JEOL JEM-F200, Japan). Cu Kα radiation was used. Powder X-ray diffraction (XRD) measurements were carried out on a diffractometer (Rigaku SmartLab SE, Japan). X-ray photoelectron spectroscopy (XPS) was performed on a PHI5000 Versaprobe III XPS (ULVAC-PHI, Inc., Japan) to analyze the elements and chemical states using an Al Kα X-ray source. Fourier transform infrared spectroscopy (FTIR) was recorded with a Nicolet IS10 spectrometer. The Zeta potential of the samples was analyzed using a ZetaSizer Pro nanoparticle size and potential analyzer (Malvern Instruments Ltd.). The specific surface area and pore size of the samples were measured by N2 adsorption-desorption isotherms at 77 K using a Quantachrome Autosorb (ASAP 2460, Micromeritics, USA). Before the measurement, the samples were degassed in vacuo at 120 °C for 6 h. The ultraviolet absorption spectra of various substances were recorded using an ultraviolet-visible spectrophotometer (UV2700, Shimadzu, Japan). The recovery rate of DEX in food was analyzed by high performance liquid chromatography (1220Infinity LC, Agilent Technologies Co., Ltd.).

[0046] Analysis of the adsorption process

[0047] Preparation of the composite material DexTracker

[0048] 1.44 mmol of FeCl3·6H2O and 1.44 mmol of 2-aminobenzoic acid (NH2-BDC) were added to 30 mL of N,N-dimethylformamide (DMF) and continuously stirred for 2 h. The dissolved mixture was placed in a 50 mL autoclave and then heated at 120 °C for 24 h. After the reaction was completed, the product was cooled to room temperature, which was the iron-based MOF material.

[0049] 150 nM concentration of DEX aptamer was added and reacted for 2 h to allow it to grow naturally on the surface of the product. Finally, the product was collected by centrifugation, which was the composite material DexTracker.

[0050] The adsorption kinetics of DEX by DexTracker is as follows: In this experiment, 5 mg of DexTracker was added to a reactor containing 100 mL of DEX solution (30 mg / L). The reactor was placed in the dark, and the suspension was homogenized using magnetic stirring (800 rpm). The test lasted for 60 minutes. Samples of 1.2 mL were collected from each reactor every 10 minutes, placed in 2 mL centrifuge tubes, centrifuged at 8000 rpm for 15 minutes, and filtered using a 0.22 μm PTFE syringe filter. The residual concentration of tetracycline in the centrifuged solution was determined using a UV-visible spectrophotometer. The maximum adsorption wavelength of DexTracker was 240 nm. The adsorption capacity and adsorption efficiency were calculated as follows:

[0051]

[0052] where C0 (mg / L) is the initial concentration of the solute in the solution before adsorption, Ce (mg / L) is the equilibrium concentration of the solute after adsorption reaches equilibrium. V (L) represents the volume of the solution, and m (g) represents the mass of the adsorbent used.

[0053] Study the adsorption kinetics of the composite material DexTracker for DEX: Add 100 mL of DEX with different concentrations (8, 10, 15, 20, 25, 30, 35 mg / L) into 250 mL conical flasks respectively. Add 5 mg / ml of the composite material DexTracker and mix well. Subsequently, shake the mixed solution horizontally for 1 hour and centrifuge at 8000 rpm for 10 minutes. Finally, take the supernatant and determine the residual concentration of DEX in each solution using a UV-visible spectrophotometer. Calculate the amount of DEX adsorbed per unit mass of the adsorbent.

[0054] 3 Analysis of the pseudo-enzyme activity of the material

[0055] Perform enzyme kinetics analysis on the composite material DexTracker. Add 200 μL (pH = 4.0), 100 μL of H2O2 solutions with different concentrations (0.02, 0.06, 0.12, 0.18, 0.3, 0.6, 0.8, 1.1 mM) and 100 μL of TMB solutions with different concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mM) to 100 μL of the composite material DexTracker solution. Add the mixed solution to a UV-Vis spectrophotometer and record the change in absorbance at a wavelength of 650 nm. And perform kinetic analysis on the enzyme-catalyzed reaction.

[0056]

[0057] In the formula, Vmax is the maximum initial velocity, [S] is the concentration of TMB, and Km is the Michaelis constant.

[0058] Detection of DEX by 4 colorimetric sensors

[0059] The typical colorimetric analysis process is as follows: Add 50 μL of 1 mg / mL DexTracker solution into an EP tube, and then add dexamethasone solutions with different concentrations (0.01 - 50 μm). Vortex for 15 seconds and incubate for 20 minutes. Finally, add 200 μL of 0.1 M sodium acetate buffer solution with pH 4, 100 μL of 3 mM TMB solution 2, and 100 μL of 10 μM H2O2 solution into the mixed solution, and incubate for 15 minutes. Observe and record the color change and spectrum of the mixed solution. Measure the absorbance value at 650 nm and construct a standard curve with dexamethasone concentration. Determine the absorbance value at 650 nm and construct a standard curve of dexamethasone concentration vs. A at 650 nm.

[0060] 5 Sample pretreatment

[0061] The pretreatment methods for different methods are as follows: First, according to the composite material DexTracker colorimetric method, add 10.0 mL of acetonitrile aqueous solution into 2 mL of milk and honey respectively. Then ultrasonicate the mixture for 10 minutes and let it stand for 20 minutes. Finally, centrifuge the mixture at 12000 rpm for 15 minutes. The supernatant is used for the next determination. For column separation method, transfer the supernatant to a hydrophilic-lipophilic balance (HLB) column. Collect the filtrate and dry it under nitrogen at 40°C. Collect the residue, dissolve it in 2 ml of pure water, and then dry it under nitrogen at 40°C. Finally, redissolve the residue in 2 ml of pure water for colorimetric method determination. For HPLC method, inject the supernatant into the HPLC system for further analysis. The chromatographic conditions are: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase is methanol-aqueous solution (v:v; 7:3), detection wavelength is 240 nm, flow rate is 1.0 mL / min, column temperature is room temperature, and injection volume is 20 μL.

[0062] 6 Preparation of 3D-μPAD device

[0063] The paper-based 3D-μPAD was prepared using Whatman No. 1 filter paper. Initially, the desired paper pattern was designed using Adobe Illustrator software. The 3D-μPAD consists of four parts: (1) the feeding layer, (2) the functional layer, (3) the colorimetric layer, and (4) the anti-pollution layer. The functional zones 1, 2, and 3 consist of hydrophilic circular areas with a diameter of 15 mm, and the remaining areas are hydrophobic circular areas, which are sealed with PDMS glue. Then, the sealed filter paper was heated in an oven at 60 °C for 8 hours to cure the PDMS and form hydrophobic regions. Subsequently, 10 μL of 1 mg / mL DexTracker nanoprobe solution was added to the functional zones of the 3D μPAD. Another 10 μL of the mixed solution containing 3 mM TMB and 10 μM H2O2 was added to the colorimetric layer, and the paper was air-dried for DEX detection.

[0064] 7 Results and Discussion

[0065] 7.1 Characterization of DexTracker

[0066] The DexTracker nanoparticles used in this example are in the shape of spindle-shaped crystals, with a length of approximately 3.00 μm ( Figure 1 A-C in). In addition, DexTracker has excellent crystallinity and mesoporous properties ( Figure 1 G in), with an average pore diameter of 2.21 nm and a specific surface area of 381.73 m 2 / g. The high specific surface area of the material is beneficial for adsorption. High-resolution XPS spectra show distinct signals of Fe, oxygen, carbon, and nitrogen ( Figure 1 F in), and these elements are evenly distributed within the material ( Figure 1 D in). As Figure 1 shown in E, the UV-Vis absorption spectrum of DexTracker shows absorption peaks in the range of 350 - 450 nm, which can be attributed to the d-d transitions of Fe 3+ ions within the Fe-based MOF. These transitions occur when the electrons in the d orbitals of iron ions are electronically excited, resulting in light absorption in this wavelength range. These findings indicate that DexTracker has strong light absorption characteristics. DexTracker exhibits excellent peroxidase-like activity (H2O2 Km = 0.053 mM, TMB Km = 0.129 mM) ( Figure 2 B-C and Table 1 in). It effectively catalyzes the oxidation of the TMB substrate in the presence of H2O2 to produce the blue product oxTMB ( Figure 2 A in).

[0067] Table 1

[0068]

[0069]

[0070] 7.2 Characteristics of DexTracker Adsorbent and Its Interaction with DEX

[0071] The pore size and framework structure of DexTracker are very suitable for adsorbing dexamethasone (DEX). As Figure 1 shown, in the presence of DEX, the UV absorption peak of DexTracker at 350 nm is significantly weakened ( Figure 1 F in [reference]), indicating an electronic interaction between the two, and DEX affects the electronic transition of DexTracker. This indicates a coordination bond and charge transfer between DEX and DexTracker. To analyze the adsorption isotherm, the Langmuir and Freundlich models were applied to the adsorption data using the fast Fourier transform (FFT) ( Figure 4 A - B in [reference]). The experimental data of DEX adsorption conforms to the Langmuir model, indicating that the adsorption occurs in a monolayer on the surface of the adsorbent, and the binding energy of all sites is uniform. In contrast, the Freundlich model, which is usually used to describe heterogeneous adsorption systems, provides a poorer fit (Table 2). In addition, the pseudo-first-order and pseudo-second-order models were used to analyze the adsorption kinetics of DEX on DexTracker ( Figure 4 C - D in [reference] and Table 3). The experimental data conforms to both models. The pseudo-second-order model can more accurately characterize the DEX adsorption process. These findings indicate that the adsorption of DEX on DexTracker follows a chemisorption mechanism that promotes electron sharing or exchange.

[0072] Table 2 Calculation of Adsorption Equilibrium Constants

[0073]

[0074] Table 3 Kinetic Parameters for Adsorbing DEX

[0075]

[0076] 7.3 Detection of DEX by DexTracker Colorimetry

[0077] To further improve the detection performance of the sensor, the sensing conditions were studied. First, the effect of pH on the catalytic activity was studied, and the results are as Figure 5 shown in A of [reference]. The catalytic activity of DexTracker gradually increases under acidic conditions and is optimal at a pH of 4.0. Subsequently, the concentration and incubation time of the added aptamer were further studied. The absorbance response is maximum when the aptamer concentration of the composite DexTracker is 150 nM ( Figure 5 B in [reference]), and the whole process takes about 15 minutes (Figure 5 C). Finally, the reaction time between the composite material DexTracker and DEX was investigated. It was found that the whole reaction was completed within 20 minutes, which also reflected the fast kinetics between them. Figure 5 D). Under the optimized conditions, the detection performance of the DEX aptasensor was studied. It showed a good linear relationship in the concentration range of 0.01 - 3 μM, and the detection limit was 6.7 nM. Figure 2 D - E). The ability of the aptamer to specifically capture DEX and enhance the enzyme activity of DexTracker endows this colorimetric aptasensor with excellent DEX detection performance. To further evaluate the anti - interference ability of the aptasensor, common DEX analogues in food (including prednisone, hydrocortisone, estrogen), antibiotics (chloramphenicol, metronidazole, erythromycin, enrofloxacin) and metal ions (K + , Ca 2+ ) were determined. As shown in Figure 2 F - G, only DEX could cause a significant change in the absorbance and color of the sensing system, while the cross - reaction signals of other structurally similar compounds were as low as 10%. This indicates that the sensor has excellent selectivity for DEX and has the potential to detect DEX in real samples.

[0078] 7.4 Detection of DEX in real samples

[0079] To further verify the applicability and feasibility of the sensor for detecting DEX in real samples, DEX solutions with different concentrations were added to milk and honey samples respectively. After removing the proteins in the food with acetonitrile, the spiked recovery was calculated using the constructed biosensor. At the same time, it was compared with the previously reported pre - treatment method of column separation. The results are shown in Table 1. The recovery rate was 88.2% - 107.6%, which had no significant difference from the column separation method (86.8 - 103.6%). This is due to the strong DEX adsorption performance of the composite material DexTracker, which enables the effective capture and separation of DEX in real samples. This also greatly simplifies the sample pre - treatment process. In addition, these samples were detected by the standard HPLC - based method, and the recovery rate was 98.4 - 102.5% (Table 4 and Figure 6 ), which was similar to the detection results of the aptasensor. Compared with the biosensors reported in recent years (Table 5), the colorimetric sensor constructed in this invention also showed excellent detection performance and has the potential for on - site detection of DEX in food.

[0080] Table 4 Results of detecting DEX in food by different methods

[0081]

[0082]

[0083] Comparison of dexamethasone determination methods

[0084]

[0085] 7.5 3D μPAD device based on DexTracker

[0086] To promote the application of this sensor in the on-site detection of DEX, the present invention constructs a three-dimensional vertical μPAD that integrates biorecognition, nanomaterial adsorption, and colorimetric model output ( Figure 3 as shown in A - C in the figure). After the sample is injected into the sample layer, it will flow vertically downward along the hydrophilic channel. In the presence of DEX, it is separated and captured from the layer DexTracker, and then the ternary mixture acts on TMB in layer III. Since the peroxidase activity is inhibited after DexTracker binds to DEX, only slight color change occurs in layer III. When the sample does not contain DEX, DexTracker will directly react with TMB in layer III, producing a large amount of blue products. Finally, the color change in the third layer is used as the detection signal. Combined with a smartphone, the detection signal is further converted into an RGB value (G / R) for the quantitative analysis of DEX. When the DEX concentration is in the range of 0.01 - 3 μM, the G / R value shows a good linear relationship with the DEX concentration ( Figure 3 as shown in D in the figure), and the equation is y = 0.1322x + 0.696 (R 2 = 0.976). It shows that the constructed 3D μPAD device can realize the visual analysis and semi-quantitative detection of DEX, and has important application potential in the POCT detection of DEX in food.

[0087] In summary, the present invention prepares a composite material DexTracker nanozyme, which has excellent peroxidase-like activity and specific recognition ability for dexamethasone (DEX). The binding of DEX to DexTracker reduces the electron density of the active sites of the metal-organic framework (MOF), and the surface hydroxylation of the Fe center inhibits the ability of the MOF to effectively catalyze H2O2 to generate reactive oxygen species (ROS). These characteristics endow DexTracker with enhanced detection and adsorption capabilities. Under optimized conditions, the designed sensor can effectively separate, capture, and detect DEX, showing good recovery rates in complex samples such as milk and honey. Finally, a three-dimensional μPAD device based on DexTracker is developed, providing new theoretical insights for the real-time detection of hormones in food samples.

[0088] Obviously, the above embodiments of the present invention are merely examples given to clearly illustrate the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A composite material DexTracker for dexamethasone detection, characterized in that, It is formed by combining an iron-based MOF material and a dexamethasone aptamer.

2. The composite material DexTracker according to claim 1, wherein, The nucleotide sequence of the dexamethasone aptamer is shown as SEQ ID NO.

1.

3. The composite material DexTracker according to claim 2, wherein The 5′ end of the dexamethasone aptamer is modified with FAM.

4. The preparation method of the composite material DexTracker according to any one of claims 1-3, characterized in that, It includes the following steps: (1) Add FeCl3·6H2O and 2-aminobenzoic acid into N,N-dimethylformamide, continuously stir for 2 hours, then transfer it into an autoclave for high-temperature reaction; (2) Add the dexamethasone aptamer and react for 2 h to make it grow naturally on the surface of the product, and centrifuge to collect the product.

5. The preparation method according to claim 4, wherein The molar ratio of FeCl3·6H2O, 2-aminobenzoic acid and the dexamethasone aptamer is 60:60:1; The conditions of the high-temperature reaction are: heating at 120 °C for 24 hours.

6. Use of the composite material DexTracker according to any one of claims 1-3 in dexamethasone detection.

7. Use of the composite material DexTracker according to any one of claims 1-3 in the preparation of a product for detecting dexamethasone.

8. A product for detecting dexamethasone, characterized in that, It includes the composite material DexTracker according to any one of claims 1-3.

9. The product according to claim 8, wherein The product includes reagents, reagent kits and 3D-μPAD devices.

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