Preparation method and application of enzymatic D-limonene colorimetric sensor based on MOF stability

Abstract

The invention discloses a preparation method and application of an enzymatic D-limonene colorimetric sensor based on MOF stability. The preparation method comprises the following steps: step 1, preparing an AChE coated Zn-MOF solution; step 2, preparation of a portable test strip based on AChE (at) Zn-MOF: dissolving 0.1-0.2 g of agarose in 10 mL of ultrapure water, and heating until the agarose is completely dissolved; after cooling to 40 DEG C, adding 5 to 10 mL of the AChE coated Zn-MOF solution in the step 1; after fully mixing, uniformly coating 100-200L of the mixed solution on commercial standard precise pH test paper until the pH value is 5.5-9; cooling to form an AChE coated Zn-MOF portable test strip, and storing the test strip in a refrigerator at 4 DEG C for later use; and step 3, establishing a linear regression equation for detecting D-limonene. The method has the characteristics of simplicity and convenience in operation, high portability, high stability, excellent sensitivity and the like, and is particularly suitable for non-professionals such as farmers to use in a field environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of colorimetric sensor detection, and particularly relates to an enzyme-catalyzed colorimetric sensor stabilized by metal-organic framework (MOF), its preparation and its application in portable detection in agricultural environment D - in limonene. Background Art

[0002] Volatile organic compounds (VOCs) released by plants are important indicators for evaluating plant health, quality and environmental stress. D - Limonene is a volatile organic compound widely present in citrus fruits, herbs and spices. Its release amount is closely related to plant health status, fruit quality and environmental stress. Research shows that D - the release amount of limonene will change significantly during plant diseases and insect pests, environmental stress or fruit ripening process, so it can be used as a biomarker for plant health status.

[0003] However, current methods for detecting D - limonene mostly rely on complex instrument equipment such as gas chromatography-mass spectrometry (GC-MS), quartz crystal microbalance (QCM) and electrochemical sensors. These methods have limitations such as complex equipment, long time consumption and the need for professional skills, and are not suitable for on-site rapid detection. Colorimetric sensing technology has become an ideal choice for field detection because of its simple operation, low cost and the need for no complex equipment. However, traditional colorimetric methods often lack sufficient sensitivity and specificity in complex agricultural matrices. Enzyme-catalyzed colorimetric sensing technology has received extensive attention due to its high sensitivity and specificity. However, enzymes have poor stability in complex environments and are easily affected by factors such as temperature and pH value, which limits their application in field environments. To solve this problem, the present invention uses metal-organic framework (MOF) as an enzyme immobilization carrier, which can effectively protect the enzyme activity and improve its stability in complex environments. However, there is no research report on the detection of D - limonene by enzyme-catalyzed colorimetric sensing technology. Summary of the Invention

[0004] The present invention aims to solve the defects in the prior art and provides a preparation method and application of an enzyme-catalyzed D-limonene colorimetric sensor stabilized by MOF. It can realize portable, highly sensitive and specific detection of D - limonene in agricultural environment, and overcome the disadvantages of traditional D - limonene detection technology such as complex operation, long time consumption and easy limitation by environmental factors.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows: An enzyme-catalyzed D- Preparation method of limonene colorimetric sensor, comprising the following steps: Step 1, Preparation of AChE@Zn-MOF solution: Dissolve 5 - 10 mg of acetylcholinesterase (AChE), 150 mg of zinc nitrate, and 20 mg of 2,5-dihydroxyterephthalic acid in 20 mL of Tris-HCl buffer solution (concentration 50 mM, pH 8 - 8.5); after thorough mixing, stir at room temperature for 20 - 30 min; centrifuge at 4000 rpm for 5 min to obtain a precipitate, wash with ultrapure water and disperse in 5 - 10 mL of ultrapure water to obtain AChE@Zn-MOF solution, and store it in a refrigerator at 4°C for later use; Step 2, Preparation of AChE@Zn-MOF-based portable test strip: Dissolve 0.1 - 0.2 g of agarose in 10 mL of ultrapure water and heat until completely dissolved; after cooling to 40°C, add 5 - 10 mL of the AChE@Zn-MOF solution in Step 1; after thorough mixing, evenly coat 100 - 200 µL of the mixture on a commercial standard precision pH test paper with pH 5.5 - 9; after cooling, form an AChE@Zn-MOF portable test strip, and store it in a refrigerator at 4°C for later use; Step 3, Establish a D - Linear regression equation for detecting limonene: Place the AChE@Zn-MOF portable test strip in a 50 mL wide-mouth bottle containing limonene gas with different concentrations, seal and incubate at room temperature for 3 - 5 h; take out the test strip, add 100 µL of acetylcholine solution (concentration 10 mM, pH 8 - 8.5) to each test strip; after incubating at room temperature for 10 - 20 min, take a photo of the still-wet test strip to obtain the RGB value of the test strip; according to the relationship between the G / R value of the detected test strip and the corresponding D - Limonene concentration value, establish a D - Linear regression equation for detecting limonene. D - Limonene.

[0006] In Step 3, use a smartphone to take a photo and obtain the RGB value of the test strip.

[0007] A limonene colorimetric sensor based on MOF-stabilized enzyme catalysis obtained according to the above preparation method, used for detecting D - Limonene gas released by plants: AChE@Zn-MOF on the test strip hydrolyzes acetylcholine to generate acetic acid, thereby causing a change in the pH of the solution, and then triggering a change in the color of the pH test strip; when D - Limonene is present, D - Limonene inhibits the activity of acetylcholinesterase through the dual effects of competitive inhibition and non-competitive inhibition; when acetylcholine is added, D - Limonene inhibits the activity of acetylcholinesterase through the dual effects of competitive inhibition and non-competitive inhibition; when acetylcholine is added, D- The presence of limonene inhibits the hydrolysis of acetylcholine, resulting in a decrease in the amount of acetic acid produced, which in turn causes a color change in the pH test paper, thus achieving D - The rapid and visual detection of limonene.

[0008] The colorimetric sensor is applied to detect the D - Limonene released from citrus fruits infected with Bactrocera dorsalis: Put the infected citrus fruit sample and the prepared AChE@Zn-MOF portable test strip into a sealed bag and incubate at room temperature for 3 - 5 h; after the incubation, take out the test strip and add 100 μL of acetylcholine solution for colorimetric detection; use a smartphone to take a picture of the test strip to obtain the RGB value, and substitute the G / R value of the sample into the D - Limonene linear regression equation to calculate the D - Limonene concentration released from the citrus fruits infected with Bactrocera dorsalis to be measured.

[0009] In the above application, the program of the smartphone automatically calculates the D - Limonene concentration released from the citrus fruits infected with Bactrocera dorsalis to be measured.

[0010] The beneficial technical effects of the present invention are as follows:

[0011] (1) The AChE@Zn-MOF colorimetric sensor prepared by the present invention has excellent stability and can achieve high-sensitivity detection of D - Limonene, and is not interfered by other volatile organic compounds, and the detection results are accurate and reliable.

[0012] (2) By means of the MOF immobilization technology, the stability of AChE in a complex environment is significantly improved, and it can maintain high catalytic activity under different temperature and pH conditions.

[0013] (3) The prepared AChE@Zn-MOF sensor of the present invention is integrated on a commercial standard pH test paper. By combining the adsorption of the hydrogel and the smartphone image processing technology, the D - Real-time and portable detection of limonene in the field environment is realized, with simple operation and no need for complex equipment.

[0014] (4) The present invention first proposes a method for specifically detecting D - Limonene based on colorimetric sensing technology, filling the technical gap in this field. So far, there has been no report on using colorimetric sensing technology to detect D - Limonene. By combining the MOF-stabilized acetylcholinesterase and the pH test strip, the present invention realizes the high-sensitivity and high-specificity detection of D - Limonene, providing a new method for D-The detection of limonene has opened up a new technical approach, which 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

[0015] Figure 1a This is the SEM image of AChE@Zn-MOF in Example 1.

[0016] Figure 1b This is the SEM image of AChE@Zn-MOF in Example 1.

[0017] Figure 2 This is the stability test of AChE@Zn-MOF in Example 1.

[0018] Figure 3 AChE@Zn-MOF portable test strip detection in Example 1 D - Image of the color change of limonene.

[0019] Figure 4 The G / R value of the portable test strip of AChE@Zn-MOF in Example 1 is D -Linear regression curve plotted against limonene concentration values.

[0020] Figure 5 This is the application of the portable test strip of AChE@Zn-MOF in Example 1 on citrus samples infected by Bactrocera dorsalis. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0022] To further illustrate the present invention, a MOF-stabilized enzymatic colorimetric sensor was prepared and used for release of citrus fruits infected by Bactrocera dorsalis. D -Limonene detection is an example, and the specific steps are as follows: Step 1. Preparation of AChE@Zn-MOF: Dissolve 10 mg of acetylcholinesterase, 150 mg of zinc nitrate and 20 mg of 2,5-dihydroxyterephthalic acid in 20 mL of Tris-HCl buffer (50 mM, pH 8). After thorough mixing, stir and react for 20 min. After centrifugation and washing, the resulting mixture was dispersed in 5 mL of ultrapure water and stored in a refrigerator at 4°C for later use. Figure 1a As shown in the figure, the prepared Zn-MOF has a rod-like structure, while after acetylcholinesterase is immobilized, AChE@Zn-MOF exhibits a multi-petal-like structure with a diameter of about 15 µm ( Figure 1b), the significant change in morphology indicates that acetylcholinesterase was successfully immobilized on Zn-MOF. As Figure 2 shown, after using Zn-MOF to immobilize acetylcholinesterase, under harsh conditions such as strong acid, high temperature, proteinase K, and organic solvents, the stability of AChE@Zn-MOF is significantly better than that of free AChE.

[0023] Step 2: Preparation of the portable test strip based on AChE@Zn-MOF: Dissolve 0.2 g of agarose in 10 mL of ultrapure water and heat until completely dissolved. After cooling to 40 °C, add 10 mL of the AChE@Zn-MOF solution from Step 1. After thorough mixing, coat 200 µL of the mixture on a commercial standard precision pH test paper (Maclean, pH 5.5 - 9). After cooling, an AChE@Zn-MOF portable test strip is formed and stored in a 4 °C refrigerator for later use.

[0024] Step 3: Establish the D -limonene linear regression equation: Place the AChE@Zn-MOF portable test strip in a 50 mL wide-mouth bottle containing different D -limonene gases with concentrations of 0, 1, 2, 5, 10, 15, 20, 30, 45, 60, 80, 100 ppm respectively. Seal and incubate at room temperature for 4 h, then take out the test strip, add 100 µL of acetylcholine solution (10 mM, pH 8.5) to each test strip, incubate at room temperature for 10 min, then use a smartphone to take a picture of the still-wet test strip and obtain the RGB value of the test strip. According to the relationship between the G / R value of the detected test strip and the corresponding D -limonene concentration value, establish the D -limonene linear regression equation.

[0025] As Figure 3 shown, AChE@Zn-MOF can hydrolyze acetylcholine to generate acetic acid, thus causing a change in the solution pH, and further triggering a change in the color of the pH test strip. When D -limonene is present, D -limonene inhibits the activity of acetylcholinesterase through the dual effects of competitive inhibition and non-competitive inhibition. When acetylcholine is added, D the presence of -limonene will significantly inhibit the hydrolysis of acetylcholine, resulting in a decrease in the amount of acetic acid generated, and further causing a change in the color of the pH test strip, thus realizing the D rapid and visual detection of -limonene.

[0026] Use a smartphone to obtain the RGB value of the above test strip, and fit the curve according to the change in the G / R value of the test strip and the D -limonene concentration, asFigure 4 As shown, the function corresponding to the curve is: Y=0.014X+0.946, and the correlation coefficient R 2 =0.986, and the linear range is 0.1~80 ppm.

[0027] Step 4: Release of citrus fruits infected with Bactrocera dorsalis D -Limonene detection analysis: Place the infected citrus sample and the AChE@Zn-MOF portable test strip prepared in step 2 in a sealed bag and incubate at room temperature for 4 hours. After the incubation, remove the test strip and add 100 µL of acetylcholine solution (10 mM, pH 8.5) for colorimetric detection. Use a smartphone to obtain the RGB value of the test strip. Figure 5 As shown, the G / R value of the sample is substituted into the G / R value established in step 3. D -Limonene linear regression equation, using a smartphone app to automatically calculate the release of citrus samples infected with Bactrocera dorsalis D- Limonene concentration. Results are as follows Figure 5 As shown, the amount of limonene released by citrus fruits infested with Bactrocera dorsalis was approximately 25.8 ppm.

[0028] 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 in this specification 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; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A MOF-stabilized enzymatic D -A method for preparing a limonene colorimetric sensor, characterized in that: The following steps are involved: Step 1. Preparation of AChE@Zn-MOF solution: 5-10 mg acetylcholinesterase (AChE), 150 mg zinc nitrate and 20 mg 2,5-dihydroxyterephthalic acid were dissolved in 20 mL Tris-HCl buffer (concentration of 50 mM, pH 8-8.5); after thorough mixing, stirred at room temperature for 20-30 min; centrifuged at 4000 rpm for 5 min to obtain a precipitate, washed with ultrapure water and dispersed in 5-10 mL ultrapure water to obtain an AChE@Zn-MOF solution, which was stored in a 4°C refrigerator for later use; Step 2, preparation of portable test strips based on AChE@Zn-MOF: dissolve 0.1~0.2g agarose in 10mL ultrapure water and heat until completely dissolved; after cooling to 40°C, add 5~10mL of the AChE@Zn-MOF solution in step 1; after thorough mixing, evenly apply 100~200µL of the mixture on commercial standard precision pH test paper, pH 5.5~9; after cooling, form the AChE@Zn-MOF portable test strips, and store in a 4°C refrigerator for future use; Step 3: Create a test D -Linear regression equation of limonene: The AChE@Zn-MOF portable test strip was placed in water containing different concentrations D -limonene gas in a 50 mL wide-mouth bottle, sealed and incubated at room temperature for 3-5 h; take out the test strips, add 100 µL of acetylcholine solution (concentration of 10 mM, pH 8-8.5) to each test strip; after incubation at room temperature for 10-20 min, take a picture of the still wet test strips to obtain the RGB value of the test strips; detect the G / R value of the test strips and the corresponding D - The relationship between the concentration values ​​of limonene is established to detect D -Linear regression equation for limonene.

2. The preparation method according to claim 1, characterized in that: In step 3, use a smartphone to capture and obtain the RGB value of the test strip.

3. A MOF-stabilized enzymatic catalyst obtained by the preparation method according to any one of claims 1 to 2 D -Limonene colorimetric sensor, characterized in that For detection of plant release D -Limonene gas: AChE@Zn-MOF on the test strip hydrolyzes acetylcholine to generate acetic acid, which causes a change in the pH of the solution, and then causes a change in the color of the pH test strip; D - When limonene is present, D -Limonene inhibits the activity of acetylcholinesterase through the dual effects of competitive inhibition and non-competitive inhibition; when acetylcholine is added, D -The presence of limonene inhibits the hydrolysis of acetylcholine, resulting in a decrease in the production of acetic acid, which in turn causes the color of the pH test paper to change, thereby achieving D - Rapid, visual detection of limonene.

4. The MOF-stabilized enzymatic reaction according to claim 3 D - Application of a limonene colorimetric sensor, characterized in that The colorimetric sensor is used to detect the release of citrus fruits infected by the oriental fruit fly D -Limonene: Place the infected citrus sample and the prepared AChE@Zn-MOF portable test strip in a sealed bag and incubate at room temperature for 3-5 h. After the incubation, remove the test strip and add 100 µL of acetylcholine solution for colorimetric detection. Use a smartphone to take a picture of the test strip to obtain the RGB value and bring the G / R value of the sample into D -limonene linear regression equation, calculate the release of citrus fruits infected with Bactrocera dorsalis D -Limonene concentration.

5. The use according to claim 4, characterized in that: The smartphone app automatically calculates the number of citrus fruits to be tested for Bactrocera dorsalis infection. D -Limonene concentration.

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