A method for rapid detection of dibutyl phthalate based on CdSe / ZnS quantum dots

Abstract

This invention discloses a rapid method for detecting dibutyl phthalate (DBP) using CdSe / ZnS quantum dots as a fluorescent probe, belonging to the field of food testing technology. The method includes: adding the sample to be tested to an aqueous dispersion containing CdSe quantum dots; measuring the fluorescence intensity of the CdSe quantum dots after the reaction; and then substituting the fluorescence intensity value into a standard curve to calculate the DBP content in the sample. This invention uses CdSe / ZnS quantum dots as a fluorescent probe and establishes a rapid fluorescence detection method for DBP based on the fluorescence enhancement effect of DBP on CdSe / ZnS quantum dots. This method can be used to determine the DBP content in baijiu (Chinese liquor). The accuracy of this method is comparable to that of the traditional GC-MS method, and it has a lower detection limit.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a method for rapid detection of dibutyl phthalate based on fluorescent probe CdSe / ZnS quantum dots. Background Technology

[0002] Dibutyl phthalate (DBP) is an excellent plasticizer, currently the most widely produced and used plasticizer. It exhibits good solubility in various resins and is characterized by its light color, low toxicity, good electrical properties, low volatility, mild odor, and low-temperature resistance. However, DBP has irritant and mild sensitizing effects on skin and mucous membranes. Exposure can cause polyneuritis, spinal neuritis, and cerebral polyneuritis, affecting physiological regulation and possessing carcinogenic and teratogenic effects. DBP can also damage the liver, causing changes in liver tissue morphology, seriously endangering human health. With the rapid development of the plastics industry and the widespread use of plastic products, DBP has entered the environment in large quantities, commonly found in soil, sediment, water, organisms, air, and atmospheric dust, becoming one of the most prevalent pollutants globally.

[0003] In an era where food safety issues are receiving increasing attention and environmental pollution continues to worsen, the hazards of plasticizers, represented by dibutyl phthalate (Dbutyl phthalate), have become increasingly prominent, making the development of rapid, simple, accurate, and sensitive detection methods particularly important. Existing methods mainly include ultraviolet spectrophotometry, gas chromatography, high-performance liquid chromatography (HPLC), solid-phase extraction-near-infrared spectroscopy, electrochemical methods, and fluorescence analysis. However, some of these methods have drawbacks such as requiring expensive instruments, cumbersome operation, and long detection cycles. Fluorescence analysis, on the other hand, is a more efficient and convenient analytical method.

[0004] Quantum dots are nanomaterials with a size of approximately 1–10 nm, and are widely used in fluorescence analysis and detection due to their unique and excellent fluorescence properties. Common semiconductor quantum dots such as CdSe have attracted much attention due to their narrow emission spectrum, high stability, and controllable size. However, Cd atoms have certain biological toxicity, and CdSe quantum dots themselves have surface defects. ZnS and CdS are often used as shell materials for quantum dots due to their high energy bandwidth. To avoid the introduction of toxic Cd, CdSe quantum dots are coated with ZnS to form core-shell structured quantum dots. This not only compensates for the surface defects of CdSe quantum dots and reduces the toxicity caused by Cd atoms, but also enhances their fluorescence and significantly improves the fluorescence quantum yield (Preparation of CdSe quantum dots and ZnS core-shell structured quantum dots in aqueous phase. Progress in Materials, 2017, 36(5): 389-394.). CdSe / ZnS quantum dots have been widely used in the detection of toxic and hazardous substances, such as mercury ions (Fluorescence enhancement of CdSe / ZnS quantum dots induced by mercury ions and its applications to the on-site sensitive detection of mercury ions. Microchimica Acta, 2021, 188(6): 655-661.), aflatoxin B1 (A CdSe / ZnS core / shell competitive quantum dot-based fluorescence-linked immunosorbent assay for the sensitive and accurate detection of aflatoxin B1 in corn sample. Journal of Food Measurement and Characterization, 2022, 16(1): 857-866.), and hydrogen peroxide (Development of Ratiometric Fluorescence Sensors Based on CdSe / ZnS Quantum Dots for the Detection of Hydrogen) Peroxide.Sensors, 2019, 19(22): 4977-4995.), and demonstrated high detection sensitivity and convenience.

[0005] Currently, there are no reports on using CdSe / ZnS quantum dots as fluorescent probes for the determination of dibutyl phthalate. Summary of the Invention

[0006] The purpose of this invention is to provide a fluorescence detection method that can rapidly determine dibutyl phthalate (DBP) with high sensitivity and accuracy.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides the application of CdSe / ZnS quantum dots as fluorescent probes in the preparation of a dibutyl phthalate detection kit.

[0009] This invention has found that dibutyl phthalate enhances the fluorescence response signal of CdSe / ZnS quantum dots, and the fluorescence signal is linearly related to the logarithm of the dibutyl phthalate concentration. Therefore, by measuring the fluorescence intensity of the quantum dots after the reaction of dibutyl phthalate with CdSe / ZnS quantum dots, the concentration of dibutyl phthalate can be calculated.

[0010] Specifically, the CdSe / ZnS quantum dots are prepared by a hydrothermal method, and the CdSe quantum dots are formed by coating CdSe quantum dots with ZnS to form core-shell structure quantum dots.

[0011] Preferably, the preparation method of the CdSe / ZnS quantum dots includes: firstly, dispersing cadmium acetate dihydrate and sodium oleate in ethanol to obtain an ethanol dispersion, dispersing oleic acid and selenium powder in water to obtain an aqueous dispersion, mixing the two and heating at 40-100℃ for 0.5-3h in a non-oxygen atmosphere, and then reacting at 100-300℃ for 0.5-3h to obtain CdSe quantum dots; then, alternately adding zinc acetate and sodium sulfide solution to the CdSe quantum dot solution, and reacting at 60-100℃ for 0.5-2h to obtain CdSe / ZnS core-shell structured quantum dots.

[0012] More preferably, the ethanol dispersion and the water dispersion are mixed and heated at 40°C for 2 hours under nitrogen protection, then transferred to a reaction vessel and reacted at 180°C for 3 hours to obtain CdSe quantum dots.

[0013] The maximum excitation wavelength of the CdSe / ZnS core-shell quantum dots prepared by this invention is 270 nm, and the maximum emission wavelength is 540 nm.

[0014] This invention provides a method for rapid detection of dibutyl phthalate, comprising: adding the sample to be tested to an aqueous dispersion containing CdSe quantum dots, measuring the fluorescence intensity value of the CdSe quantum dots after the reaction, and then substituting the fluorescence intensity value into the standard curve calculation formula to calculate the content of dibutyl phthalate in the sample to be tested.

[0015] In the above method, neutral water is used as the reaction solvent. Studies have shown that the fluorescence response of CdSe quantum dots is the largest and most stable when the pH of the QDs-DBP system is 7.0, and the ionic strength has no significant effect on the fluorescence response.

[0016] Preferably, the concentration of CdSe quantum dots in the reaction system is 0.01-0.1 μmol / L. More preferably, the concentration of CdSe quantum dots is 0.01 μmol / L.

[0017] Preferably, the reaction conditions are 15-20 min at room temperature. After the QDs-DBP system is maintained for 15 min, the fluorescence signal remains essentially unchanged.

[0018] After the reaction was completed, the fluorescence response signal was measured. The excitation wavelength was set to 270 nm, and the fluorescence intensity value at a wavelength of 540 nm was recorded.

[0019] Preferred fluorescence measurement conditions: excitation and emission slit width: 5 nm, PMT voltage: 500 V, scan rate: 1200 nm / min, scan range: 280-800 nm, response time: 0.002 s.

[0020] The method for constructing the standard curve includes: using CdSe / ZnS quantum dots as fluorescent labels to detect a series of dibutyl phthalate solutions with concentrations ranging from 0.3 to 20 μg / L, with a fluorescence excitation wavelength of 270 nm, recording the fluorescence intensity value at a wavelength of 540 nm, and linearly fitting the fluorescence intensity value with the logarithm of the dibutyl phthalate concentration to obtain the standard curve.

[0021] The above method can rapidly and accurately quantify dibutyl phthalate (DBP) in food. Furthermore, the sample to be tested is baijiu (Chinese liquor). This invention demonstrates that the above method can achieve specific quantitative detection of DBP in baijiu samples, unaffected by interference from diethyl phthalate (DEP), diisobutyl phthalate (DIBP), dioctyl phthalate (DOP), or similar substances.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention utilizes CdSe / ZnS quantum dots as fluorescent probes and establishes a rapid fluorescence detection method for DBP based on the fluorescence enhancement effect of dibutyl phthalate (DBP) on CdSe / ZnS quantum dots. DBP enhances the fluorescence signal of CdSe / ZnS quantum dots, exhibiting a linear relationship with the logarithm of DBP concentration in the range of 0.3-20.0 μg / L. The detection limit is 0.1 μg / L, and the correlation coefficient R0 is [value missing]. 2The limit of detection is 0.99. This method can be used to determine the DBP content in baijiu (Chinese liquor). The accuracy of this method is comparable to that of the traditional GC-MS method, and it has a lower detection limit. Attached Figure Description

[0024] Figure 1 The images show transmission electron microscopy (TEM) images (A) (morphology and energy dispersive spectroscopy elemental analysis) and fluorescence response maps (B) (QDs concentration of 0.1 μmol / L) of CdSe / ZnS quantum dots. In Figure A, the first row from left to right shows the TEM morphology, high-resolution elemental distribution maps of Cd and Se, and the second row from left to right shows the elemental distribution maps of quantum dots, Zn, and S.

[0025] Figure 2 The effect of reaction time on fluorescence intensity in the QDs-DBP system.

[0026] Figure 3 Figure A shows the fluorescence response of CdSe / ZnS to DBP, and Figure B shows the fluorescence intensity versus DBP concentration curve. In Figure A, a to k represent DBP concentrations of 0, 0.3, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, and 200.0 μg / L, respectively. In Figure B, the inset plot shows the logarithmic linear relationship between fluorescence intensity and DBP concentration in the concentration range of 0.3–20.0 μg / L.

[0027] Figure 4 This is an interference experiment (the effect of DEP, DIBP, and DOP on the fluorescence response of DBP).

[0028] Figure 5 To determine DBP in a baijiu sample by GC-MS. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] F-7000 fluorescence spectrometer (Hitachi, Japan); Agilent 7000D-8890B gas chromatograph-mass spectrometer; IKAVORTEX 1 circular oscillator (IKA, Germany); DK-S28 constant temperature water bath (Shanghai Jinghong); PHS-3C pH meter (Shanghai Leici); 200kV field emission transmission electron microscope (Talos F200X, Thermo Fisher Scientific); Millipore ultrapure water system (Merck Millipore, Germany); YQ-120 ultrasonic cleaner (Shanghai Yiqing); drying oven (Hangzhou Biaodun); centrifuge (Shanghai Anting), etc.

[0032] Dibutyl phthalate (DBP) (99%, AR), sodium sulfide nonahydrate (99%, AR), cadmium acetate dihydrate (99%, AR), and sodium oleate were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Zinc acetate (98%, AR), potassium dihydrogen phosphate, and disodium hydrogen phosphate (98%, AR, used for preparing PBS) were purchased from China National Pharmaceutical Group Co., Ltd.

[0033] Baijiu sample (DBP characteristic value 4.3 mg / kg, standard deviation of capacity evaluation 0.3 mg / kg, characteristic value 3.7-4.9 mg / kg, China Academy of Inspection and Quarantine Science Testing and Evaluation Center).

[0034] The water used in the experiment was all ultrapure water (resistivity 18.2 MΩ·cm).

[0035] Example 1

[0036] 1. Preparation of CdSe / ZnS quantum dots

[0037] First, 1 mM cadmium acetate dihydrate and 1.2 g sodium oleate were dispersed in 10 mL of ethanol. Then, 2 mL of oleic acid and 1 mmol of selenium powder were added to 20 mL of water. The ethanol dispersion and the water dispersion were mixed evenly and heated at 40 °C for 2 hours under N2 protection. The mixture was then transferred to a 100 mL reactor and reacted at 180 °C for 3 hours to obtain CdSe quantum dots.

[0038] 1 mmol zinc acetate and 1 mmol sodium sulfide solution were added dropwise to a CdSe quantum dot solution while stirring. The mixture was reacted at 100 °C for 2 hours. Unreacted raw materials and impurities were removed by filtration. The solution was then centrifuged at high speed and dried in a vacuum oven at 80 °C to obtain CdSe / ZnS core-shell quantum dots.

[0039] Weigh out a certain amount of quantum dots and redisperse them in water to obtain a 1.0 μmol / L quantum dot solution, which is then refrigerated for later use.

[0040] 2. Characterization of CdSe / ZnS quantum dots

[0041] 2.1 The size, morphology and elemental composition of the prepared quantum dots were analyzed using field emission transmission electron microscopy.

[0042] The results are as follows Figure 1 As shown in (A), the internal morphology and elemental composition of the prepared water-soluble CdSe / ZnS quantum dots were determined using high-resolution transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS). It was clearly observed that the prepared quantum dots were uniform in size, with a particle size between 10-20 nm, and were approximately spherical with slightly irregular outer edges. This is because it is difficult to achieve a completely uniform thickness of the ZnS shell when coating the CdSe quantum dot core. The EDS spectrum showed that Cd and Se elements were coated by Zn and S elements, demonstrating the successful preparation of CdSe / ZnS core-shell quantum dots.

[0043] 2.2 Fluorescence response analysis was performed using a fluorescence spectrometer. Fluorescence measurement conditions: excitation and emission slit width: 5 nm, PMT voltage: 500 V, scan rate: 1200 nm / min, scan range: 280-800 nm, response time: 0.002 s.

[0044] To analyze the fluorescence response of CdSe / ZnS quantum dots, a quantum dot solution of a specific concentration (0.1 μmol / L) was selected for fluorescence measurement. The results are as follows: Figure 1 As shown in (B), the maximum excitation wavelength of the quantum dot is 270 nm, and the maximum emission wavelength is 540 nm.

[0045] 2.3 Effect of dibutyl phthalate (DBP) on the fluorescence response of CdSe / ZnS quantum dots. To preliminarily test the effect of DBP on the fluorescence response of CdSe / ZnS quantum dots, a mixed solution containing 0.1 μmol / L quantum dots and the same concentration of DBP was prepared.

[0046] A precise amount of dibutyl phthalate standard was dissolved in a small amount of ethanol, then diluted with water to prepare the required concentration. CdSe / ZnS quantum dots were used as fluorescent probes to react with the dibutyl phthalate solution. A 300 μL sample was taken and fluorescence was measured according to the aforementioned instrument parameters, revealing a significant enhancement in the fluorescence signal.

[0047] Example 2

[0048] 1. Optimization of fluorescence determination conditions for dibutyl phthalate

[0049] 1.1 Based on the preliminary tests in Example 1, to further investigate the effect of DBP on the fluorescence response of quantum dots and the effect of different conditions, the fluorescence intensity of quantum dots at different concentrations (probe concentrations of 0.005, 0.01, and 0.1 μmol / L) was further tested. It was found that 0.01 μmol / L of quantum dots produced a suitable fluorescence intensity, which is convenient for analysis. Therefore, 0.01 μmol / L was selected as the working concentration of CdSe / ZnS quantum dots.

[0050] 1.2 To investigate the effect of pH on the fluorescence signal of the QDs-DBP system, PBS solutions (0.1 mol / L) with different pH values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) were prepared as solvents, and the fluorescence signals were measured. The results showed that the response was the largest and most stable at pH 7.0.

[0051] 1.3 To test the effect of ions on the fluorescence response of quantum dots, water and 0.1 mol / L PBS (pH 7.0) were used as solvents for fluorescence measurement. The results showed no significant difference in fluorescence intensity. Therefore, neutral water was chosen as the solvent in this experiment.

[0052] 1.4 To investigate the effect of reaction time of DBP in quantum dot solution on fluorescence response, the DBP concentration was selected as 0.1 mg / L as the reaction concentration, and the reaction times at room temperature were 1 min, 4 min, 7 min, 10 min, 13 min, 15 min, 16 min, 17 min, and 18 min.

[0053] The results are as follows Figure 2 As shown, the fluorescence signal remained essentially unchanged after 15 minutes in the QDs-DBP system. Therefore, 15 minutes was selected as the optimal reaction time, i.e., the waiting time, for DBP to enhance the fluorescence signal of QDs.

[0054] 2. Working Curve

[0055] Under optimal experimental conditions, CdSe / ZnS quantum dots were used as fluorescent labels to detect a series of DBP concentrations. Dibutyl phthalate solutions with concentrations of 0, 0.3, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, and 200.0 μg / L were measured, and standard curves were plotted.

[0056] The results are as follows Figure 3 The results showed that within the low concentration range of 0.3-20 μg / L, the fluorescence intensity exhibited a linear relationship with the logarithm of the concentration, with the linear equation being I = 226.0lg C. DBP +1399.3, correlation coefficient R 2=0.99, and the detection limit is 0.1 μg / L (S / N = 3). However, the fluorescence intensity decreases slightly when the concentration is higher than 20.0 μg / L. Therefore, this method is more suitable for the detection of low concentrations of DBP.

[0057] The mechanism by which the addition of DBP enhances the fluorescence signal of CdSe / ZnS quantum dots may be that the rigid conjugated structure within the DBP molecule maintains a certain distance between the quantum dots, preventing them from agglomerating. At the same time, the conjugated electrons form an electron cloud with the acid radical ions remaining on the surface during the quantum dot preparation process, reducing the external attachment of acid radical ions and thus covering the spatial defects on the quantum dot surface, thereby enhancing the fluorescence.

[0058] 3. Interference Experiment

[0059] To test the applicability of the constructed detection method in the presence of interfering substances, diethyl phthalate (DEP), diisobutyl phthalate (DIBP), and dioctyl phthalate (DOP), which have similar structures to DBP, were selected as interfering substances. They were added to a 0.01 mg / L solution of DBP at the same concentration (0.01 mg / L), and then reacted with CdSe / ZnS quantum dots under the same reaction conditions. The fluorescence response was then measured. The results are as follows. Figure 4 As shown, the interfering substances did not have a significant impact on the detection of DBP (maximum standard deviation of 5.0%).

[0060] Application Example 1

[0061] 1. Actual sample testing

[0062] To further verify the applicability of the constructed detection method, it was applied to the determination of DBP in purchased baijiu samples. Under the aforementioned detection conditions, 10 μL of baijiu sample was diluted and mixed with CdSe / ZnS quantum dots to prepare test solutions with concentrations of 4.0-5.5 μg / L and 20.0-22.0 μg / L, respectively, and fluorescence detection was performed. The results obtained were 5.2 μg / L and 16.1 μg / L, with relative standard deviations (RSDs) of 4.0% and 23.0%, respectively (see Table 1). This shows that the method is more accurate at low concentrations, while the deviation is larger at high concentrations.

[0063] 2. Gas chromatography / mass spectrometry (GC-MS)

[0064] GC-MS is one of the conventional methods for DBP detection. In this experiment, four liquor samples with DBP concentrations (50.0, 100.0, 200.0, and 500.0 μg / mL) were selected as the detection objects, and GC-MS detection and accuracy evaluation were performed on them.

[0065] 2.1 Sample pretreatment for gas chromatography / mass spectrometry (GC-MS) detection

[0066] Accurately weigh 1.0 g (accurate to 0.0001 g) of the sample into a 25 mL stoppered ground glass centrifuge tube, add 2-5 mL of distilled water, vortex to mix, then accurately add 10 mL of n-hexane, vortex for 1 min, shake vigorously for 1 min, sonicate for 30 min, centrifuge at 1000 r / min for 5 min, and take the supernatant for GC-MS analysis.

[0067] 2.2 GC-MS Detection Conditions

[0068] Test method: GB 5009.271-2016 Method II.

[0069] Chromatographic conditions: Injector temperature: 250℃; Temperature program: Initial column temperature 60℃, hold for 1 min, increase to 220℃ at 20℃ / min, hold for 1 min, then increase to 280℃ at 5℃ / min, hold for 5 min; Carrier gas: Helium, flow rate 1 mL / min, splitless injection; Injection volume: 1 μL.

[0070] Mass spectrometry conditions: Chromatography-mass spectrometry interface temperature: 280℃; Electron impact source: Ion scan mode selected, Ionization energy: 70 eV; Solvent delay: 5 min.

[0071] The diagram is as follows Figure 5 As shown in the figure (taking a sample with a DBP concentration of 50.0 μg / mL as an example, with a retention time of 9.87 minutes), detailed results are shown in Table 1. The standard curve of this method is I = 128514.3C - 2184.2, and the correlation coefficient is R. 2 =0.99, and the limit of quantitation is 0.33 μg / mL. This is significantly higher than the detection limit of the fluorescence detection method constructed in this invention, demonstrating high accuracy at higher concentrations.

[0072] Table 1. Determination of DBP in Baijiu Samples by CdSe / ZnS QDs Fluorescence and GC-MS Methods

[0073]

[0074]

[0075] Conclusion: In the above embodiments, a core-shell CdSe / ZnS quantum dot was first prepared, and its morphology and structure were characterized and confirmed by transmission electron microscopy. The prepared quantum dots were then used to determine DBP, and the results showed that DBP enhanced the fluorescence signal of the quantum dots. Under optimal conditions (neutral aqueous solution, reaction time 15 minutes), the fluorescence signal of the quantum dots showed an increasing trend at DBP concentrations of 0.3-20 μg / L, exhibiting a linear relationship with the logarithm of the DBP concentration. The accuracy of this method is comparable to that of the traditional GC-MS method, with a lower detection limit. DBP in liquor samples was also measured in the experiment, and satisfactory results were obtained.

Claims

1. A method for rapid detection of dibutyl phthalate, characterized in that, include: The sample to be tested was added to an aqueous dispersion containing CdSe / ZnS quantum dots. The concentration of CdSe / ZnS quantum dots in the reaction system was 0.01-0.1 μmol / L. The reaction was carried out at room temperature for 15-20 min. After the reaction, the fluorescence intensity of CdSe / ZnS quantum dots was measured. The maximum excitation wavelength of CdSe / ZnS quantum dots was 270 nm, and the maximum emission wavelength was 540 nm. The fluorescence intensity value was then substituted into the standard curve to calculate the content of dibutyl phthalate in the sample to be tested. The sample to be tested was baijiu (Chinese liquor). Dibutyl phthalate enhances the fluorescence response signal of CdSe / ZnS quantum dots; The preparation method of the CdSe / ZnS quantum dots includes: firstly, dispersing cadmium acetate dihydrate and sodium oleate in ethanol to obtain an ethanol dispersion, and dispersing oleic acid and selenium powder in water to obtain an aqueous dispersion. After mixing the two, heating at 40-100℃ for 0.5-3 h in a non-oxygen atmosphere, and then reacting at 100-300℃ for 0.5-3 h to obtain CdSe quantum dots; then alternately adding zinc acetate and sodium sulfide solution to the CdSe quantum dot solution, and reacting at 60-100℃ for 0.5-2 h to obtain CdSe / ZnS core-shell structured quantum dots.

2. The method as described in claim 1, characterized in that, The concentration of CdSe / ZnS quantum dots in the reaction system was 0.01 μmol / L.

3. The method as described in claim 1, characterized in that, The reaction conditions were 15 min at room temperature.

4. The method as described in claim 1, characterized in that, Fluorescence measurement conditions: excitation and emission slit width: 5 nm, PMT voltage: 500 V, scan rate: 1200 nm / min, scan range: 280 - 800 nm, response time: 0.002 s.

5. The method as described in claim 1, characterized in that, The method for constructing the standard curve includes: using CdSe / ZnS quantum dots as fluorescent labels to detect a series of dibutyl phthalate solutions with concentrations ranging from 0.3 to 20 μg / L, with a fluorescence excitation wavelength of 270 nm, recording the fluorescence intensity value at a wavelength of 540 nm, and linearly fitting the fluorescence intensity value with the logarithm of the dibutyl phthalate concentration to obtain the standard curve.

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