A rapid visual detection method for d-limonene and application thereof

Abstract

This invention provides a rapid and visual detection method for D-limonene and its application, belonging to the field of analytical chemistry. The method includes: mixing ethanol and water to form a solvent system, sequentially adding template agents Pluronic P123 and Pluronic F127, phloroglucinol, 1,3,5-trimethylbenzene, and ethylenediamine, and stirring at room temperature until homogeneous; after the reaction is complete, washing, centrifuging, drying, and calcining to obtain DNCzyme; adding TMB, H2O2, and DNCzyme to an acetate-sodium acetate buffer solution HAc-NaAc, and finally adding D-limonene, incubating, and then visually detecting the solution. The colorimetric sensing method for detecting D-limonene provided by this invention is rapid, simple, low-cost, highly sensitive, and selective, and can be visualized using a smartphone.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry technology, and specifically to a rapid visual detection method for D-limonene and its application. Background Technology

[0002] Limonene is a natural functional monoterpene that exists in nature in three isomers: dextrorotatory limonene (D-limonene), levorotatory limonene (L-limonene), and racemic limonene (DL-limonene), with D-limonene being the most abundant in natural plants. D-limonene is found in many fruits, vegetables, and spices and can be used as a flavoring and fragrance additive in various foods, beverages, pharmaceuticals, and cosmetics. Furthermore, it possesses remarkable antioxidant, anticancer, and anti-inflammatory properties, effectively combating a range of metabolic disorders and health problems. Volatile D-limonene is a major component of the aroma of citrus fruits, and changes in aroma during storage and mold growth often stem from variations in its content. Therefore, real-time monitoring of D-limonene content is of great significance for postharvest agriculture, food science, and pharmaceuticals.

[0003] Currently, the commonly used methods for detecting D-limonene are gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC).

[0004] In the process of implementing the technical method of the embodiments of this invention, the inventors of this patent have discovered at least the following technical problems in the prior art:

[0005] Both of the above methods have drawbacks such as being time-consuming, costly, requiring expensive equipment, needing professional personnel, and unsuitable for rapid on-site screening. Therefore, it is necessary to develop and research a detection method that is lower in cost, easier to operate, and faster.

[0006] Among numerous detection methods, enzymatic colorimetry, based on the relationship between solution color intensity and absorbance, converts the detection signal into a change in reaction color, offering a visual advantage. Natural biological enzymes, with their high reaction efficiency and specificity, can be used for colorimetric sensing, but they are still limited by poor stability and high preparation costs. Designing and developing synthetic nanozymes with excellent pore performance, tunable and controllable activity, and stable properties is a key pathway to achieve specific and efficient detection of D-limonene. Currently, there are no reports on the quantitative colorimetric detection of D-limonene using synthetic nanozymes. Therefore, researching and developing a technology for the visual on-site detection of D-limonene in fruits and agricultural products using synthetic nanozymes is crucial. Summary of the Invention

[0007] In response to the background art, this invention mainly provides a method for rapid detection of D-limonene using dendritic nitrogen-doped meso-macroporous carbon nanozymes (DNCzyme). This invention first prepares a nitrogen-doped carbon nanozyme with a porous structure using phloroglucinol as the nitrogen source. The peroxidase-like activity of the nitrogen-doped meso-macroporous carbon nanozyme of this invention can catalyze a colorimetric reaction between hydrogen peroxide (H₂O₂) and the chromogenic agent 3,3',5,5'-tetramethylbenzidine (TMB). When different concentrations of D-limonene are added to the system, the solution color changes significantly, thus achieving rapid and visual detection of D-limonene. The DNCzyme of this invention is simple to synthesize, and its colorimetric sensing detection method for D-limonene is rapid, convenient, and provides intuitive and clear results.

[0008] This invention provides a rapid and visual method for detecting D-limonene, specifically comprising the following steps:

[0009] S1: Ethanol and water were mixed evenly to form a solvent system. Template agents Pluronic P123 and Pluronic F127, phloroglucinol, 1,3,5-trimethylbenzene and ethylenediamine were added in sequence and stirred evenly at room temperature. After the reaction was completed, the resulting sample was washed, centrifuged and dried, and calcined to obtain DNCzyme.

[0010] S2: Add TMB, H2O2, and DNCzyme to the acetate-sodium acetate buffer HAc-NaAc, and finally add D-limonene. After incubation, perform visual detection of the solution.

[0011] Further, in S1, the volume ratio of ethanol to water is 7:7 to 7:28, preferably 7:13; the mass ratio of Pluronic P123 to Pluronic F127 is 4:1 to 1:4, preferably 3:1; the mass ratio of template agent to phloroglucinol is 5:1 to 5:5; the volume ratio of 1,3,5-trimethylbenzene to ethylenediamine is 20:3; and the stirring time at room temperature is 6 h to 48 h.

[0012] Furthermore, the mass ratio of the template agent to phloroglucinol was 5:3, and the stirring time at room temperature was 24 hours.

[0013] Furthermore, in S2, the concentration of the acetate-sodium acetate buffer HAc-NaAc was 0.1 M, the pH was 4, the incubation temperature was 25℃, and the time was 10 min.

[0014] Furthermore, in S2, the visualization detection can be either ultraviolet spectrophotometer detection or grayscale value extraction detection of the red, green and blue components.

[0015] Furthermore, the ultraviolet spectrophotometer detects and records the absorbance of the solution at 652 nm, and a standard curve and linear equation are derived based on the absorbance changes of different concentrations of D-limonene.

[0016] Furthermore, the extraction of grayscale values ​​of the red, green, and blue components includes the following steps: taking pictures of D-limonene added to the mixing system at different concentrations, extracting the grayscale values ​​of the red, green, and blue components in each picture, and using (blue value + green value) / 2 × red value as the vertical axis of the linear equation and the concentration of D-limonene as the horizontal axis. The linear equation is obtained by using the RGB values ​​and different concentrations of D-limonene.

[0017] On the other hand, the present invention also provides an application of the rapid visual detection method for D-limonene in the above embodiments for rapid visual detection of D-limonene.

[0018] Furthermore, α-pinene, citric acid, D-sorbitol, myrcene, and linalool were added during the detection process for selective testing.

[0019] Furthermore, Na is added during the detection process. + K + Al 3+ Mg 2+ Mn 2+ Ca 2+ Zn 2+ Conduct anti-interference tests.

[0020] Furthermore, it can be applied to the rapid and visual detection of D-limonene in actual samples of green peel, sweet orange, and frankincense.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. The N-doped porous carbon nanozyme of the present invention has high peroxidase-like activity and can catalyze the generation of hydroxyl radicals from hydrogen peroxide. The hydroxyl radicals oxidize colorless TMB to blue oxidized tetramethylbenzidine (oxTMB). When different concentrations of D-limonene are added to the system, the hydroxyl radicals generated by catalysis are consumed, causing the blue color to fade, thereby achieving rapid and visual detection of D-limonene. The present invention establishes a visual detection method for D-limonene, which is intuitive, simple, convenient, and low in cost.

[0023] 2. The DNCzyme of this invention has a detection range of 0.05-18 μM for D-limonene and a detection limit of 0.049 μM. It also exhibits selectivity for α-pinene, citric acid, D-sorbitol, myrcene, and linalool, and is effective against common Al ions in solution. 3+ Na + Cl - K +Mg 2+ Zn 2+ Mn 2+ Ca 2+ CO3 2- SO4 2- It exhibits good anti-interference properties. When used for the detection of D-limonene in green peel, sweet orange, and frankincense, combined with a color recognition program, it can achieve on-site, real-time detection of D-limonene.

[0024] 3. The DNCzyme preparation process of this invention has low cost and simple synthesis conditions. Compared with some traditional large-scale instrument detection methods, the detection of D-limonene has the advantages of being fast, convenient, low cost, and on-site visualization. Attached Figure Description

[0025] Figure 1 Here is a scanning electron microscope image of DNCzyme prepared under the conditions of Example 1 of this invention;

[0026] Figure 2 This is a POD-like activity diagram of the UV-Vis absorption spectra of DNCzyme in different TMB-H2O2 systems of the present invention;

[0027] Figure 3 UV-Vis spectra of the TMB-DNCzyme-H2O2 system with different concentrations of D-limonene added in this invention;

[0028] Figure 4 This is a linear relationship graph of the DNCzyme detection of D-limonene concentration according to the present invention;

[0029] Figure 5 This is a result of the extraction and data processing of grayscale values ​​of red, green and blue components from colorimetric photographs and standard curves of different D-limonene concentrations using a smartphone color analysis application, as described in this invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] Example 1: Preparation of DNCzyme

[0032] 1. Add 65 mL of ethanol and 35 mL of deionized water to a clean 250 mL three-necked flask, mix thoroughly, then add 0.75 g Pluronic P123, 0.25 g Pluronic F127, 0.6 g phloroglucinol, 2 mL of 1,3,5-trimethylbenzene and 0.3 mL of ethylenediamine, and stir at room temperature for 24 h.

[0033] 2. The sample after reaction was washed three times with a mixture of deionized water and ethanol, centrifuged three times, freeze-dried, and the resulting powder was calcined in a tube furnace at 800℃ for 1 h to obtain DNCzyme.

[0034] This invention uses Pluronic P123, Pluronic F127, phloroglucinol, 1,3,5-trimethylbenzene, and ethylenediamine as raw materials to self-assemble a nanozyme precursor, which is then calcined to obtain DNCzyme. Figure 1 The scanning electron microscope image of the DNCzyme prepared in this embodiment shows that the nanozyme has a size of 450-550 nm and a clear dendritic structure.

[0035] Example 2: Study on the peroxidase-like activity (POD) of DNCzyme

[0036] Prepare 50 mL of HAc-NaAc buffer solution (0.1 M, pH 4), 4 mL of 50 mM TMB, and 4 mL of 30% w / w H2O2. Then, draw 1880 μL, 100 μL, and 20 μL from each buffer and place them into cuvette #1. Next, draw 1820 μL of buffer solution and 100 μL of TMB and place them into cuvette #2. Finally, add 60 μL of 2 mg / mL N-doped mesoporous carbon nanozyme solution before calcination. Cuvette #3 is prepared by adding another 60 μL of 2 mg / mL N-doped porous carbon nanozyme solution to cuvette #1. After incubating for 10 min, measure the absorption spectra of all three cuvettes at wavelengths of 300–800 nm.

[0037] Figure 2 The diagram shows the activity of DNCzyme-like POD under the conditions of this embodiment. It can be seen that DNCzyme has high POD-like activity, catalyzing the generation of hydroxyl radicals from H2O2, which oxidizes the colorimetric reagent TMB into blue oxTMB.

[0038] Example 3: Detection standard D-limonene solution

[0039] The DNCzyme-TMB-H2O2 system and different concentrations of D-limonene were added to HAc-NaAc buffer solution and incubated at 25°C for 10 minutes. Then, the relationship between the absorbance at 652 nm and different D-limonene concentrations was studied.

[0040] Figure 3 The image shows the UV-Vis spectrum of the DNCzyme-TMB-H2O2 system with added D-limonene. As can be seen from the image, the absorbance at 652 nm gradually decreases with increasing D-limonene concentration, indicating a decrease in the intensity of blue. Figure 4 The linear relationship between DNCzyme detection of D-limonene concentration was plotted, and the linear relationship was obtained as: y = -0.03064x + 0.69553 (R 2 = 0.992), where x is the concentration of D-limonene, y is the absorbance of the solution, the detection range is 0.25-18 μM, and the limit of detection (LOD) is 0.049 μM.

[0041] Example 4: Detection of D-limonene in green peel, sweet orange, and frankincense

[0042] D-limonene extracted from green peel, sweet orange, and frankincense was prepared into concentrations of 2, 10, and 14 μM. The samples to be tested were then added to the DNCzyme-TMB-H2O2 system and incubated for 10 min. The absorbance at 652 nm was then measured using a UV spectrophotometer. The experimental results are shown in Table 1.

[0043] Table 1

[0044]

[0045] As can be seen from the analysis of the test results in Table 1, the results of the detection of D-limonene content in green peel, sweet orange, and frankincense by this method are similar to those of the standard large-scale instrument, indicating that the visualization detection of D-limonene by the DNCzyme of this invention is feasible in actual analysis.

[0046] Example 5: Real-time detection of D-limonene using a color analysis program based on the grayscale values ​​(RGB) of the red (R), green (G), and blue (B) components.

[0047] The DNCzyme-TMB-H2O2 system and D-limonene were added to the HAc-NaAc buffer solution and incubated at 25°C for 10 minutes. Photos of the reaction were taken with a smartphone. The center of each color-sensitive point was then used as the region of interest (ROI) and the grayscale average of the red, green and blue components of the ROI was extracted from the photos using a color analysis program.

[0048] Figure 5The image shows the results of RGB extraction and data processing of colorimetric photographs and standard curves of different concentrations of D-limonene using a smartphone. The linear relationship is as follows: y = -0.05875x + 2.0075 (R 2 =0.9916), and the LOD was calculated to be 0.626 μM.

[0049] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they are not intended to limit the present invention. It should be noted that various changes and modifications can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rapid visual detection method for D-limonene, characterized in that, A rapid and visual detection method for D-limonene in actual samples of green peel, sweet orange, and frankincense is applied. This method, combined with a smartphone color recognition app, enables real-time on-site detection of D-limonene, and includes the following steps: S1: Ethanol and water were mixed evenly to form a solvent system. Template agents Pluronic P123 and Pluronic F127, phloroglucinol, 1,3,5-trimethylbenzene and ethylenediamine were added in sequence and stirred evenly at room temperature. After the reaction was completed, the resulting sample was washed, centrifuged and dried, and calcined to obtain DNCzyme. S2: Add TMB, H2O2, and DNCzyme to the acetate-sodium acetate buffer solution, and finally add D-limonene. After incubation, perform visual detection of the solution. Among them, DNCzyme has high peroxidase-like activity, which catalyzes the generation of hydroxyl radicals from H2O2. The hydroxyl radicals oxidize colorless TMB to blue oxidized tetramethylbenzidine oxTMB. When different concentrations of D-limonene are added to the system, the hydroxyl radicals generated by the catalysis are consumed, causing the blue color to fade, thereby achieving rapid and visual detection of D-limonene. DNCzyme exhibits a detection range of 0.05–18 μM for D-limonene, with a detection limit of 0.049 μM. It also demonstrates selectivity for α-pinene, citric acid, D-sorbitol, myrcene, and linalool, and is effective against common Al ions in solution. 3+ Na + Cl - K + Mg 2+ Zn 2+ Mn 2+ Ca 2+ CO3 2- SO4 2- It has good anti-interference properties; In S1, the volume ratio of ethanol to water is 7:7 to 7:28, the mass ratio of Pluronic P123 to Pluronic F127 is 4:1 to 1:4, the mass ratio of template agent to phloroglucinol is 5:1 to 5:5, the volume ratio of 1,3,5-trimethylbenzene to ethylenediamine is 20:3, and the stirring time at room temperature is 6 h to 48 h.

2. The method according to claim 1, characterized in that, The concentration of the acetate-sodium acetate buffer in S2 was 0.1 M, pH=4, the incubation temperature was 25℃, and the time was 10 min.

3. The method according to claim 1, characterized in that, In S2, the visual detection is performed by extracting the grayscale values ​​of the red, green, and blue components.

4. The method according to claim 3, characterized in that, Gray value extraction of the red, green and blue components The process includes the following steps: taking pictures of D-limonene at different concentrations added to the mixing system, extracting the grayscale values ​​of the red, green and blue components in each picture, and obtaining a linear equation using the RGB values ​​and different concentrations of D-limonene. The vertical axis of the linear equation is (blue value + green value) / 2 × red value, and the horizontal axis is the concentration of D-limonene.

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