Rapid visual D-limonene detection method and application thereof

Abstract

The invention provides a rapid visual D-limonene detection method and application thereof, and belongs to the technical field of analytical chemistry, the method comprises the following steps: uniformly 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 uniformly stirring at room temperature; after the reaction is completed, washing, centrifuging, drying and calcining an obtained sample to obtain DNCzyme; the method comprises the following steps: adding TMB, H2O2 and DNCzyme into an acetic acid-sodium acetate buffer solution HAc-NaAc, finally adding D-limonene, incubating, and carrying out visual detection on the solution. The method for detecting the D-limonene through colorimetric sensing is rapid, simple, convenient, low in cost, high in sensitivity and good in selectivity, and visual detection is achieved by combining a smart phone.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and relates to a D-limonene rapid visualization detection method and application thereof. Background Art

[0002] Limonene is a natural functional monoterpene that exists in nature in the form of three isomers: right-handed limonene (D-limonene), left-handed limonene (L-limonene) and racemic limonene (DL-limonene). D-limonene has the highest content in natural plants. D-limonene is found in many fruits, vegetables and spices, and can be used as a flavoring and flavor additive in various foods, beverages, medicines and cosmetics. In addition, it has remarkable antioxidant, anti-cancer and anti-inflammatory properties, which can effectively combat a series of metabolic disorders and health problems. Volatile D-limonene is the main component of the aroma of citrus fruits. The change in aroma during storage and mildew often stems from the change in its content. Therefore, the real-time detection of D-limonene content is of great significance to post-harvest agriculture, food science, and medical health.

[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 embodiment of the present invention, the inventor of this patent found at least the following technical problems in the prior art: Both of the above methods have the disadvantages of being time-consuming, costly, requiring expensive equipment, requiring professional personnel, and not being suitable for rapid on-site screening. Therefore, it is necessary to develop a detection method that is lower in cost, easier to operate, and faster.

[0005] Among the many detection methods, the enzyme colorimetric method is based on the relationship between the depth of the solution color and the absorbance, converting the detection signal into a change in the reaction color, which has the characteristic of visualization. Natural biological enzymes have high reaction efficiency and strong specificity and can be used for colorimetric sensing, but they are still limited by their poor stability and high preparation cost. The design and development of artificial synthetic nanozymes with excellent pore performance, adjustable and controllable activity, and stable properties is the key path to achieve specific and efficient detection of D-limonene. There are no reports of synthetic nanozymes for quantitative colorimetric detection of D-limonene in the prior art. In view of this, it is crucial to study and develop a technology that uses artificial synthetic nanozymes to visually detect D-limonene in fruits and agricultural products on site. Summary of the invention

[0006] In view of the content of the background technology, the present invention mainly provides a method for quickly detecting D-limonene using dendritic N-doped meso-macroporous carbon nanozyme (DNCzyme). The present invention first uses phloroglucinol as a nitrogen source to prepare a nitrogen-doped carbon nanozyme with a porous structure. The peroxidase-like activity of the N-doped meso-macroporous carbon nanozyme of the present invention can catalyze a color reaction between hydrogen peroxide (H2O2) and a color developer 3,3',5,5'-tetramethylbenzidine (TMB). When different concentrations of D-limonene are added to the system, the color of the solution changes significantly, thereby realizing a visual rapid detection of D-limonene. The DNCzyme of the present invention is simple to synthesize, and its colorimetric sensing detection method for D-limonene is fast and convenient, and the results are intuitive and clear.

[0007] On the one hand, the present invention provides a method for rapid visual detection of D-limonene, which specifically comprises the following steps: S1: Ethanol and water are mixed evenly to form a solvent system, and templates Pluronic P123 and Pluronic F127, phloroglucinol, 1,3,5-trimethylbenzene and ethylenediamine are added in sequence, and stirred evenly at room temperature; after the reaction is completed, the obtained sample is washed, centrifuged, dried, and calcined to obtain DNCzyme.

[0008] S2: TMB, H2O2, and DNCzyme were added to acetic acid-sodium acetate buffer HAc-NaAc, and finally D-limonene was added. After incubation, the solution was visualized.

[0009] Furthermore, in S1, the volume ratio of ethanol to water is 7:7-7:28, preferably 7:13; the mass ratio of Pluronic P123 to Pluronic F127 is 4:1-1:4, preferably 3:1; the mass ratio of the template to phloroglucinol is 5:1-5:5; the volume ratio of 1,3,5-trimethylbenzene to ethylenediamine is 20:3; and the stirring time at room temperature is 6 h-48 h.

[0010] Furthermore, the mass ratio of the template to phloroglucinol is 5:3, and the stirring time at room temperature is 24 h.

[0011] Furthermore, the concentration of acetic acid-sodium acetate buffer HAc-NaAc in S2 was 0.1 M, pH = 4, the incubation temperature was 25 °C, and the time was 10 min.

[0012] Furthermore, the visual detection in S2 is any one of ultraviolet spectrophotometer detection and gray value extraction detection of red, green and blue components.

[0013] Furthermore, the ultraviolet spectrophotometer detects the absorbance of the solution at 652 nm, and a standard curve and a linear equation are obtained according to the absorbance change of D-limonene at different concentrations.

[0014] Furthermore, the grayscale value extraction of the red, green and blue components includes the following steps: taking pictures of D-limonene with different concentrations added to the mixed system, extracting the grayscale values ​​of the red, green and blue components in each picture, the ordinate of the linear equation is (blue value + green value) / 2×red value, and the abscissa is the concentration of D-limonene. The linear equation is obtained by using the RGB value and different concentrations of D-limonene.

[0015] On the other hand, the present invention also provides an application of a D-limonene rapid visual detection method in the above embodiment for rapid visual detection of D-limonene.

[0016] Furthermore, α-pinene, citric acid, D-sorbitol, myrcene and linalool were added during the detection process for selectivity testing.

[0017] Furthermore, Na was added during the detection process. + , K + 、Al 3+ Mg 2+ , Mn 2+ , Ca 2+ 、Zn 2+ Perform an interference immunity test.

[0018] Furthermore, it is applied to the rapid visual detection of D-limonene in actual samples of green peel, sweet orange, and frankincense.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The N-doped porous carbon nanozyme of the present invention has high peroxidase-like activity and can catalyze hydrogen peroxide to produce hydroxyl radicals. The hydroxyl radicals will oxidize colorless TMB into blue oxidized tetramethylbenzidine (oxTMB). When different concentrations of D-limonene are added to the system, the hydroxyl radicals produced by the catalysis will be consumed, causing the blue color to fade, thereby realizing a visual rapid detection of D-limonene. The present invention establishes a visual detection method for D-limonene, which is intuitive, simple, convenient, and low-cost.

[0020] 2. The detection range of the DNCzyme of the present invention for D-limonene is 0.05-18 μM, the detection limit is 0.049 μM, and it is selective for α-pinene, citric acid, D-sorbitol, myrcene and linalool, and is selective for common ions Al in the solution. 3+ 、Na + , Cl - , K + Mg 2+ 、Zn 2+ , Mn 2+ , Ca 2+ 、CO32- 、SO4 2- It has good anti-interference properties. It is used for the detection of D-limonene in green peel, sweet orange and frankincense. Combined with the color recognition program, it can realize the on-site instant detection of D-limonene.

[0021] 3. The DNCzyme preparation process of the present 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron microscope image of the DNCzyme prepared under the conditions of Example 1 of the present invention; Figure 2 The POD-like activity diagram of the UV-visible absorption spectra of the DNCzyme of the present invention in different TMB-H2O2 systems; Figure 3 The UV-visible spectra of the TMB-DNCzyme-H2O2 system with different concentrations of D-limonene added in the present invention; Figure 4 This is a linear relationship diagram of the DNCzyme detection of D-limonene concentration of the present invention; Figure 5 The results of the present invention using a smartphone color analysis application to extract the grayscale values ​​of the red, green and blue components and process the data of colorimetric photos and standard curves of different D-limonene concentrations. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0024] Example 1: Preparation of DNCzyme 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 of Pluronic P123, 0.25 g of Pluronic F127, 0.6 g of phloroglucinol, 2 mL of 1,3,5-trimethylbenzene and 0.3 mL of ethylenediamine, and stir at room temperature for 24 h.

[0025] 2. The reacted sample was washed three times with a mixed solution of deionized water and ethanol, centrifuged three times, and freeze-dried. The obtained powder was calcined at 800°C for 1 h in a tube furnace to obtain DNCzyme.

[0026] The present invention uses Pluronic P123, Pluronic F127, phloroglucinol, 1,3,5-trimethylbenzene and ethylenediamine as raw materials, self-assembles to form a nanozyme precursor, and then calcines to obtain DNCzyme. Figure 1 The scanning electron microscopy image of the DNCzyme prepared in this example shows that the size of the nanozyme is 450-550 nm, and the dendritic structure can be clearly seen.

[0027] Example 2: Study on the peroxidase-like activity (POD) of DNCzyme Prepare 50 mL of HAc-NaAc buffer solution with an ion concentration of 0.1 M and pH = 4, 4 mL of 50 mM TMB and 4 mL of 30% w / w H2O2; then extract 1880 μL, 100 μL, and 20 μL from each and put them into cuvette No. 1. Then extract 1820 μL of buffer and 100 μL of TMB and put them into cuvette No. 2, and finally add 60 μL of 2 mg / mL N-doped meso-macroporous carbon nanozyme solution before calcination. Cuvette No. 3 is based on cuvette No. 1 and adds 60 μL of 2 mg / mL N-doped porous carbon nanozyme solution. After incubation for 10 min, the absorption spectra of the three cuvettes at a wavelength of 300-800 nm were measured.

[0028] Figure 2 This is a diagram of the POD-like activity of DNCzyme under the conditions of this example. It can be seen that DNCzyme has high POD-like activity, catalyzing H2O2 to produce hydroxyl radicals, and oxidizing the color developer TMB into blue oxTMB.

[0029] Example 3: Testing standard D-limonene solution The DNCzyme-TMB-H2O2 system and different concentrations of D-limonene were added to the HAc-NaAc buffer solution and incubated at 25 °C for 10 min. Then, the relationship between its absorbance at 652 nm and different D-limonene concentrations was studied.

[0030] Figure 3 This is the UV-visible spectrum of the DNCzyme-TMB-H2O2 system with the addition of D-limonene. It can be seen from the figure that with the increase of the concentration of D-limonene, the absorbance at a wavelength of 652 nm gradually decreases, showing a decrease in the degree of blue. Figure 4 This is the linear relationship diagram of DNCzyme detecting D-limonene concentration, and the linear relationship formula is: y = -0.03064x+0.69553(R 2= 0.992), where x is the concentration of D-limonene and 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.

[0031] Example 4: Detection of D-limonene in green peel, sweet orange and frankincense D-limonene extracted from green peel, sweet orange and frankincense was prepared into 2, 10 and 14 μM, and then the samples to be tested were added to the DNCzyme-TMB-H2O2 system. After incubation for 10 min, the absorbance at 652 nm was measured using a UV spectrophotometer. The experimental results are shown in Table 1.

[0032] Table 1

[0033] From the analysis of the test results in Table 1, it can be seen that the detection results of the 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 visual detection of D-limonene by the DNCzyme of the present invention is feasible in actual analysis.

[0034] Example 5: Real-time detection of D-limonene using grayscale value (RGB) color analysis procedure of red (R), green (G) and blue (B) components The DNCzyme-TMB-H2O2 system and D-limonene were added to the HAc-NaAc buffer solution and incubated at 25°C for 10 min. Photos of the reaction were taken with a smartphone. The center of each color-sensitive point was then taken as the region of interest (ROI) using the smartphone, and the grayscale averages of the red, green, and blue components of the ROI area were extracted from the photos using a color analysis program.

[0035] Figure 5 This is the result of RGB extraction and data processing of colorimetric photos of different concentrations of D-limonene and standard curves 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.

[0036] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A rapid visual detection method for D-limonene, characterized in that: The following steps are involved: S1: Ethanol and water are mixed evenly to form a solvent system, and templates Pluronic P123 and Pluronic F127, phloroglucinol, 1,3,5-trimethylbenzene and ethylenediamine are added in sequence, and stirred evenly at room temperature; after the reaction is completed, the obtained sample is washed, centrifuged, dried, and calcined to obtain DNCzyme; S2: TMB, H2O2, and DNCzyme were added to acetic acid-sodium acetate buffer HAc-NaAc, and finally D-limonene was added. After incubation, the solution was visualized.

2. The method according to claim 1, characterized in that The volume ratio of ethanol to water in S1 is 7:7~7:28, the mass ratio of Pluronic P123 to Pluronic F127 is 4:1~1:4, the mass ratio of template to phloroglucinol is 5:1~5:5, the volume ratio of 1,3,5-trimethylbenzene to ethylenediamine is 20:3, and the stirring time at room temperature is 6 h~48 h.

3. The method according to claim 1, characterized in that The concentration of acetic acid-sodium acetate buffer HAc-NaAc in S2 was 0.1 M, pH = 4, the incubation temperature was 25°C, and the time was 10 min.

4. The method according to claim 1, characterized in that: The visual detection in S2 is ultraviolet spectrophotometer detection or gray value extraction detection of red, green and blue components.

5. The method according to claim 4, characterized in that The ultraviolet spectrophotometer detection is to record the absorbance of the solution at 652 nm, and obtain a standard curve and a linear equation according to the absorbance change of D-limonene with different concentrations.

6. The method according to claim 4, characterized in that The grayscale value extraction of the red, green and blue components includes the following steps: taking pictures of D-limonene with different concentrations added to the mixed system, extracting the grayscale values ​​of the red, green and blue components in each picture, the ordinate of the linear equation is (blue value+green value) / 2×red value, and the abscissa is the concentration of D-limonene. The linear equation is obtained by using the RGB value and different concentrations of D-limonene.

7. Use of the method according to any one of claims 1 to 6 for rapid visual detection of D-limonene.

8. The use according to claim 7, characterized in that: During the detection process, α-pinene, citric acid, D-sorbitol, myrcene and linalool were added for selectivity testing.

9. The use according to claim 7, characterized in that: Na was added during the detection process + , K + 、Al 3+ Mg 2+ , Mn 2+ , Ca 2 + 、Zn 2+ Conduct anti-interference test.

10. The use according to claim 7, characterized in that Applied to the rapid visual detection of D-limonene in actual samples of green peel, sweet orange and frankincense.

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