Preparation method and application of linalool colorimetric sensor based on MIP-coated His-MOF nano enzyme

Abstract

The invention discloses a preparation method and application of a linalool colorimetric sensor based on an MIP-coated His-MOF nano enzyme. The preparation method comprises the following steps: 1, dissolving histidine and terephthalic acid in a DMF (Dimethyl Formamide) solution by adopting a solvothermal method, and adding ferric chloride hexahydrate under ultrasonic stirring; then transferring the mixture into a high-pressure kettle, and reacting at 110 DEG C for 20 hours; washing and drying to obtain a His-MOF material; step 2, dispersing the His-MOF in acetonitrile, and adding linalool and methacrylic acid; pre-assembling for 30 minutes under the protection of nitrogen, adding ethylene glycol dimethacrylate and azodiisobutyronitrile, and polymerizing for 12 hours at 60 DEG C; washing the obtained product with ethanol to thoroughly remove linalool molecules, and drying for 12 hours under a vacuum condition to obtain an MIP-coated His-MOF nano enzyme; and cutting and dipping the qualitative filter paper, and airing at room temperature to obtain the portable test strip. Establishing a linear regression equation for detecting linalool and then detecting tea leaves. The method provided by the invention solves the problems of complex operation, long time consumption, poor environmental adaptability and the like in the existing linalool detection technology.

Description

Technical Field

[0001] The present invention belongs to the field of nanozyme probes and agricultural detection technology, and specifically relates to a linalool colorimetric sensing platform based on molecularly imprinted metal-organic framework (MIP@His-MOF) nanozymes, and its application in agricultural product quality assessment. Background Art

[0002] Linalool, a monoterpenoid volatile organic compound (VOC) widely found in plants, plays a crucial role in plant physiological regulation, environmental adaptation, and agricultural product quality assessment. Linalool release can increase significantly (by 5-10 times) when plants are exposed to biotic stresses, such as pests and diseases, making it a valuable biomarker for plant health monitoring. Furthermore, the linalool content in agricultural products such as tea and fruit directly influences their aroma quality and is therefore a key indicator for evaluating product grade.

[0003] However, the detection of linalool currently faces the following technical bottlenecks: the traditional GC-MS method relies on laboratory equipment, is complex to operate, and is difficult to apply on-site; existing sensors (such as metal oxide semiconductors, QCMs) are easily interfered by humidity and lack selectivity; colorimetric sensors based on nanomaterials (such as gold nanoparticles, metal-organic frameworks, etc.) have attracted attention due to their advantages such as simple operation and visual detection, but traditional colorimetric materials have problems such as poor stability (such as easy aggregation of nanoparticles) and easy inactivation of biological enzymes, which limit their practical application.

[0004] To address these challenges, the present invention innovatively combines the catalytic advantages of MOF nanozymes with the selectivity of molecular imprinting to develop a novel biomimetic colorimetric sensing platform. This technology mimics the active center of natural enzymes through histidine modification, while leveraging molecular imprinting for specific recognition and integrating smartphone detection technology. This provides a breakthrough solution for plant health monitoring and agricultural product quality assessment. Summary of the Invention

[0005] This paper addresses the challenges of existing linalool detection technologies, such as complex operations, time-consuming processes, and poor environmental adaptability. By adopting a "bionic recognition-catalytic coupling" strategy, this paper proposes a method for preparing and applying a linalool colorimetric sensor based on the MIP@His-MOF nanozyme. By organically combining a molecularly imprinted MOF nanozyme (MIP@His-MOF) with smartphone colorimetric analysis, this paper constructs a novel colorimetric sensing platform that enables simple, rapid, intuitive, and highly sensitive detection of linalool.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: Step 1. Preparation of His-MOF: Using a solvothermal method, 0.62 mmol of histidine and 1.25 mmol of terephthalic acid were dissolved in 15 mL of DMF solution. 2.5 mmol of ferric chloride hexahydrate was added under ultrasonic stirring. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and reacted at 110°C for 20 hours. The resulting product was thoroughly washed with DMF and ethanol (three times each) and then dried at 60°C to obtain the His-MOF material. Step 2. Preparation of MIP@His-MOF nanozyme and portable test strip: 80-100 mg of His-MOF was dispersed in 30 mL of acetonitrile, and 15-20 μL of linalool and 40-50 μL of methacrylic acid were added; after pre-assembly under nitrogen protection for 30 minutes, 450-500 μL of ethylene glycol dimethacrylate and 5-10 mg of azobisisobutyronitrile were added, and polymerized at 60°C for 12 hours; the resulting product was washed 9 times with ethanol to completely remove the linalool molecules, and dried under vacuum conditions for 12 hours to obtain MIP@His-MOF nanozyme; Whatman qualitative filter paper was cut into 2×2 cm squares; it was immersed in 200 μL of MIP@His-MOF nanozyme solution and dried at room temperature to obtain MIP@His-MOF portable test strip.

[0007] An application of a linalool colorimetric sensor based on MIP@His-MOF nanozyme comprises the following steps: Step 1. Establish a standard curve for linalool detection: Place the MIP@His-MOF portable test strips in a 50 mL wide-mouth bottle containing different linalool gases, seal the bottle, and incubate at room temperature for 2 hours. Remove the test strips and add 25 μL of 20 mM TMB, 25 μL of 160 mM H2O2, and 50 μL of pH 4 NaAc-HAc buffer to each test strip. After color development at room temperature for 5 minutes, use a smartphone to photograph the test strips and use Color Picker software to obtain the grayscale value. Based on the relationship between the grayscale value of the test strips and the corresponding linalool concentration value, establish a linear regression equation for linalool detection. Step 2. Determination of linalool in the actual sample: Place 0.1 g of the actual sample in a 50 mL wide-mouth bottle containing the MIP@His-MOF portable test strip, and react according to the steps described. Obtain the grayscale value of the MIP@His-MOF portable test strip in the actual sample, substitute it into the standard curve, and calculate the linalool content in the actual sample.

[0008] Preferably, the actual sample described in step 2 is tea.

[0009] Beneficial technical effects of the present invention:

[0010] (1) This invention innovatively combines the catalytic activity of histidine-modified MOF with the selectivity of molecular imprinting to achieve the dual functions of "recognition-catalysis" and has excellent enzyme-like catalytic activity (K m =1.33 mM, V max =17.71×10⁻ 8 M / s).

[0011] (2) The prepared MIP@His-MOF sensor was integrated on filter paper and combined with smartphone image processing technology to achieve real-time, portable, highly selective and highly sensitive detection of linalool in field environments, with a minimum detection limit of 0.62 ppm.

[0012] (3) This invention proposes for the first time a method for the specific detection of linalool based on colorimetric sensing technology, filling the technical gap in this field. After linalool is bound to the specific molecular imprinting cavity of the MIP@His-MOF nanozyme, it binds to histidine through hydrogen bonding and π-π stacking, and utilizes the steric hindrance effect to hinder the H2O2 / TMB substrate from approaching the catalytic site, thereby inhibiting the TMB oxidation color development reaction and producing a concentration-dependent color gradient change (blue weakening), thereby achieving rapid and visual detection of linalool. To date, there has been no report on the detection of linalool using colorimetric sensing technology. This invention has opened up a new technical approach for the detection of linalool and has important scientific research value and practical application potential. Compared with the existing technology, the present invention has the characteristics of simple operation, strong portability, high stability and excellent sensitivity, and is particularly suitable for use by non-professionals such as farmers in field environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the SEM images of His-MOF and MIP@His-MOF in Example 1.

[0014] Figure 2 This is the steady-state kinetic curve of MIP@His-MOF to TMB in Example 1.

[0015] Figure 3 This is a comparative graph showing the inhibition of linalool on the catalytic activity of His-MOF and MIP@His-MOF in Example 1.

[0016] Figure 4 This is a comparison chart of the selectivity of MIP@His-MOF for linalool in Example 1.

[0017] Figure 5 This is the color response of the portable test strip of MIP@His-MOF in Example 2 to linalool.

[0018] Figure 6This is the standard curve equation for plotting the grayscale value of the portable test strip of MIP@His-MOF in Example 2 against the linalool concentration value. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the scope of protection of the present invention.

[0020] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, those skilled in the art, based on their understanding of the prior art and the description of the present invention, can also use any methods, equipment and materials in the prior art that are similar or equivalent to the methods, equipment and materials described in the embodiments of the present invention to implement the present invention.

[0021] Example 1: Preparation of MIP@His-MOF nanozyme

[0022] Step 1. Preparation of His-MOF: Dissolve 0.62 mmol of histidine and 1.25 mmol of terephthalic acid in 15 mL of DMF solution. Then, add 2.5 mmol of ferric chloride hexahydrate under ultrasonic stirring. The mixture is then transferred to a Teflon-lined autoclave and reacted at 110°C for 20 hours. The resulting product is thoroughly washed with DMF and ethanol (three times each) and then dried at 60°C to obtain the His-MOF material, as shown in FIG. Figure 1 As shown in part a, the prepared His-MOF has a hexagonal bipyramid structure, a particle size of about 1 μm, and a smooth surface.

[0023] Step 2. Preparation of MIP@His-MOF nanozyme and portable test strips: 80 mg of His-MOF was dispersed in 30 mL of acetonitrile, followed by the addition of 15 μL of linalool and 40 μL of methacrylic acid. After pre-assembly under nitrogen protection for 30 minutes, 450 μL of ethylene glycol dimethacrylate and 5 mg of azobisisobutyronitrile were added, and polymerization was carried out at 60°C for 12 hours; the resulting material was washed 9 times with ethanol to completely remove the linalool molecules, and dried under vacuum conditions for 12 hours to obtain MIP@His-MOF nanozyme. The prepared MIP@His-MOF nanozyme has a rough surface and has an imprinting layer of about 120 nm thick compared to His-MOF, with a specific recognition cavity, which proves the successful synthesis of MIP@His-MOF ( Figure 1 Part b).

[0024] The catalytic kinetic curve of MIP@His-MOF was further studied. Figure 2 It can be seen that with the increase of TMB concentration, the initial reaction rate increases; when the TMB concentration increases to a certain concentration, the initial reaction rate tends to the maximum value, which is consistent with the typical Michaelis equation. Through curve simulation, it can be seen that when TMB is used as the substrate, the K m =1.33 mM, V max =17.71×10⁻ 8 M / s, with excellent peroxidase-like activity.

[0025] After the His-MOF surface was modified with a molecular imprinting layer, the inhibitory effect of linalool on the catalytic activity of the synthesized nanozyme was significantly enhanced ( Figure 3 ), this is because linalool can specifically bind to the molecularly imprinted cavity of the nanozyme, on the one hand, hindering the H2O2 / TMB substrate from approaching the catalytic site through the steric effect, and on the other hand, it can interact with the histidine-modified active center, affecting its catalytic performance, and ultimately inhibiting the TMB oxidation color reaction, resulting in a concentration-dependent color gradient change (blue weakening). And, as Figure 4 As shown in Figure 2, due to the presence of specific recognition sites on the molecular imprinting, the MIP@His-MOF nanozyme has excellent selectivity for linalool. Subsequently, Whatman qualitative filter paper was cut into 2×2 cm squares, impregnated with 200 μL of the MIP@His-MOF nanozyme solution, and dried at room temperature to obtain the MIP@His-MOF portable test strip.

[0026] Example 2: Practical application of MIP@His-MOF nanozyme test strips in black tea samples of different qualities

[0027] Step 1. Establish a standard curve for the detection of linalool: Place the MIP@His-MOF portable test strips in a 50 mL wide-mouth bottle containing different linalool gas concentrations of 0, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 80, and 120 ppm, seal, and incubate at room temperature for 2 hours. Remove the test strips and add 25 μL of 20 mM TMB, 25 μL of 160 mM H2O2, and 50 μL of pH 4 NaAc-HAc buffer to each test strip. Figure 5As shown in the figure, after 5 minutes of reaction at room temperature, the test strip showed a significant concentration gradient color change, gradually changing from dark blue (0 ppm) to light blue (120 ppm). This is because linalool inhibited the catalytic activity of the MIP@His-MOF nanozyme. The grayscale value of the test strip was obtained using Color Picker software, and a curve was fitted based on the grayscale value of the test strip and the concentration change of linalool. Due to the wide concentration range of linalool (0.5–120 ppm), the grayscale value response of the test strip showed a saturation inhibition trend, as shown in the figure. Figure 6 As shown in the figure, the concentration of linalool and the gray value of the test strip show a typical double exponential decay trend, and the curve equation is y=-4.91*exp(-x / 2.55)-19.89*exp(-x / 44.51)+209.89, R 2 =0.995. The fast initial response phase (τ1 = 2.55 ppm) indicates the high affinity binding of the molecularly imprinted cavity to linalool, and the subsequent slower phase (τ2 = 44.51 ppm) reflects the saturation behavior of the signal, achieving a sensitive detection of 0.62 ppm.

[0028] Step 2: Detection and Analysis of Linalool Released from Black Tea Samples of Different Quality: 0.1 g of each of three black tea samples (Jinhao, Jinmaohao, and Jinyinghong) were placed in a 50 mL headspace vial with a MIP@His-MOF portable test strip and allowed to react at room temperature for 2 hours. Subsequently, 25 μL of 20 mM TMB, 25 μL of 160 mM H₂O₂, and 50 μL of pH 4 NaAc-HAc buffer were added. After a 5-minute reaction, the color change was recorded using a smartphone, and the grayscale value of the test strip was obtained. This grayscale value was then applied to the linalool standard curve established in Step 1 to calculate the linalool concentrations released by the different quality black tea samples. The results are shown in Table 1. The premium-grade Jinhao tea sample had the highest linalool content (49.21 ± 1.88 ppm), followed by the first-grade Jinmaohao (40.18 ± 1.73 ppm), and the second-grade Jinyinghong had the lowest (23.37 ± 1.65 ppm), fully consistent with the actual quality grades of the tea.

[0029] Table 1. Analysis of linalool content in black tea samples of different qualities

[0030] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for preparing a linalool colorimetric sensor based on MIP@His-MOF nanozyme, characterized in that: The following steps are involved: Step 1. Preparation of His-MOF: Using a solvothermal method, 0.62 mmol of histidine and 1.25 mmol of terephthalic acid were dissolved in 15 mL of DMF solution. 2.5 mmol of ferric chloride hexahydrate was added under ultrasonic stirring. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and reacted at 110°C for 20 hours. The resulting product was thoroughly washed with DMF and ethanol and then dried at 60°C to obtain the His-MOF material. Step 2. Preparation of MIP@His-MOF nanozyme and portable test strip: 80-100 mg of His-MOF was dispersed in 30 mL of acetonitrile, and 15-20 μL of linalool and 40-50 μL of methacrylic acid were added; after pre-assembly under nitrogen protection for 30 minutes, 450-500 μL of ethylene glycol dimethacrylate and 5-10 mg of azobisisobutyronitrile were added, and polymerized at 60°C for 12 hours; the resulting product was washed 9 times with ethanol to completely remove the linalool molecules, and dried under vacuum conditions for 12 hours to obtain MIP@His-MOF nanozyme; Whatman qualitative filter paper was cut into 2×2 cm squares; it was immersed in 200 μL of MIP@His-MOF nanozyme solution and dried at room temperature to obtain MIP@His-MOF portable test strip.

2. An application of a linalool colorimetric sensor based on MIP@His-MOF nanozyme, characterized in that: The method according to claim 1 is used to prepare the product, and the application comprises the following steps: Step 1) Establish a standard curve for linalool detection: Place the MIP@His-MOF portable test strips in a 50 mL wide-mouth bottle containing different linalool gases, seal the bottle, and incubate at room temperature for 2 hours. Remove the test strips and add 25 μL of 20 mM TMB, 25 μL of 160 mM H2O2, and 50 μL of pH 4 NaAc-HAc buffer to each test strip. After color development at room temperature for 5 minutes, use a smartphone to photograph the test strips and use Color Picker software to obtain the grayscale value. Based on the relationship between the grayscale value of the test strips and the corresponding linalool concentration, a linear regression equation for linalool detection was established. Step 2) Determination of linalool in the actual sample: Place 0.1 g of the actual sample in a 50 mL wide-mouth bottle containing the MIP@His-MOF portable test strip for reaction. Obtain the grayscale value of the MIP@His-MOF portable test strip in the actual sample and substitute it into the standard curve to calculate the linalool content in the actual sample.

3. The use of the linalool colorimetric sensor based on MIP@His-MOF nanozyme according to claim 2, characterized in that The actual sample is tea.

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