Method for detecting lycopene content by paper-based surface enhanced Raman

Abstract

The invention belongs to the technical field of food safety and quality detection, and provides a method for detecting lycopene content by paper-based surface enhanced Raman. The method comprises the following steps: reacting chloroauric acid with sodium citrate to obtain a gold nano solution, and carrying out centrifugal concentration on the gold nano solution to obtain a concentrated gold nano solution; mixing a lycopene-containing solution to be detected with the concentrated gold nano solution to obtain a sample solution; dipping the paper tip substrate in a sample solution, sequentially performing drying and lycopene Raman signal detection after dipping, and obtaining the content of lycopene in the to-be-detected solution according to a Raman signal detection result, the preparation method of the paper tip substrate comprises the following steps: sequentially cutting filter paper and sealing the filter paper with wax oil to obtain the paper tip substrate. The method not only can give full play to the advantages of the SERS technology, but also has the characteristics of high detection precision, high detection speed, good detection stability and repeatability and low detection cost, and is beneficial to wide application in the field of rapid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety and quality detection, and in particular to a method for detecting lycopene content by surface-enhanced Raman analysis of paper-based materials. Background Art

[0002] Lycopene, a natural carotenoid, is widely used for its potent antioxidant properties, cancer prevention, cardiovascular protection, and immune-boosting benefits. Accurate quantitative detection of lycopene is crucial in the food industry, especially in beverages and other foods. Currently, there are three common quantitative detection methods: Spectrophotometry, based on the selective absorption of light by a substance, quantifies its content by measuring absorbance at specific wavelengths. However, spectrophotometry's limitations become apparent when dealing with samples with complex matrices, such as fruit and vegetable juices containing other pigments or carotenoids, or when high-precision quantitative analysis is required. Thin-layer chromatography (TLC), a separation and analysis technique based on the principle of adsorption chromatography, exploits the differences in adsorption capacity of different components on the same adsorbent to separate and detect components in a mixture. However, this method's separation efficiency is affected by various factors, resulting in poor repeatability and reproducibility, and limited quantitative analysis capabilities, making it difficult to meet the requirements of high-precision detection. High-performance liquid chromatography (HPLC), based on chromatographic separation technology and light absorption characteristics, is also used. Although HPLC has high sensitivity and high separation efficiency, in actual application, the sample pretreatment process is cumbersome, the purity of the mobile phase and the performance of the chromatographic column are extremely strict, the analysis time is long, and the cost of the instrument equipment is high. These factors have limited its widespread application in the field of rapid detection.

[0003] Therefore, there is an urgent need to provide a detection method for lycopene in complex matrix samples with high detection accuracy, fast detection speed, good detection stability and repeatability, and low detection cost. Summary of the Invention

[0004] In view of this, the present invention provides a paper-based surface-enhanced Raman method for detecting lycopene content to solve the problem that existing lycopene detection methods cannot simultaneously take into account detection accuracy, speed, stability, repeatability and cost.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for detecting lycopene content using paper-based surface-enhanced Raman spectroscopy, comprising the following steps:

[0007] 1) reacting tetrachloroauric acid with sodium citrate to obtain a gold nanoparticle solution, and then centrifuging and concentrating the gold nanoparticle solution to obtain a concentrated gold nanoparticle solution;

[0008] 2) mixing the test solution containing lycopene with the concentrated gold nanoparticle solution to obtain a sample solution;

[0009] 3) Immersing the paper tip substrate in the sample solution, followed by drying and Raman signal detection of lycopene, and determining the lycopene content in the sample solution based on the Raman signal detection results;

[0010] The method for preparing the paper tip base comprises the following steps: cutting filter paper and sealing with wax oil in sequence to obtain the paper tip base.

[0011] Preferably, the tip angle of the paper tip base is 10-20°; the wax oil seal is performed at a distance of ≥9 mm from the tip of the paper tip base; and the pore size of the filter paper in the paper tip base is 10-12 μm.

[0012] Preferably, the molar concentration of the concentrated gold nanoparticle solution in step 1) is 6.14×10 -10 ~7.20×10 -10 mol / mL; the average particle size of the gold nanoparticles in the concentrated gold nanosolution is 60 to 70 nm.

[0013] Preferably, the volume ratio of the test solution containing lycopene to the concentrated gold nanoparticle solution in step 2) is 1:1-10.

[0014] Preferably, the mass concentration of lycopene in the test solution containing lycopene in step 2) is 100-500 μg / mL.

[0015] Preferably, the drying in step 3) is specifically performed by placing the impregnated paper tip substrate on a heating plate for drying; wherein the surface of the heating plate is arranged parallel to the ground, and the surface of the impregnated paper tip substrate is arranged perpendicular to the surface of the heating plate; the angle between the oblique edge of the paper tip substrate close to the surface of the heating plate after the impregnation is 25 to 60 degrees and the surface of the heating plate.

[0016] Preferably, the drying temperature is 70-80° C. and the drying time is 3-5 minutes.

[0017] Preferably, the immersion time in step 3) is 15 to 30 seconds.

[0018] Preferably, the parameters for the Raman signal detection of lycopene in step 3) are: excitation light wavelength: 633 nm, acquisition time: 5-10 s, cumulative number of times: 1-2 times.

[0019] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention uses surface-enhanced Raman spectroscopy (SERS) to quickly detect the Raman signal of lycopene. The detection principle is based on the surface plasmon resonance (SPR) effect of gold nanostructures. By generating a "hot spot" area with high electric field intensity on the surface of the nanostructure, the Raman scattering signal of the adsorbed molecules is significantly enhanced, thereby achieving high-sensitivity detection of low-concentration or weakly Raman-active molecules. In addition, the present invention utilizes the capillary force and retention effect of the paper tip substrate to promote the accumulation of dispersed gold nanoparticles in the cellulose pores of the filter paper, forming abundant optically active detection points, thereby providing a good SERS enhancement effect. The detection method of the present invention combines the paper tip substrate with surface-enhanced Raman spectroscopy, which can not only give full play to the advantages of SERS technology, but also has the characteristics of high detection accuracy, fast detection speed, good detection stability and repeatability, and low detection cost. It is expected to become a very promising on-site detection platform, especially suitable for environments with limited resources or scenarios requiring immediate detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0022] Figure 1 This is a photo of the paper tip substrate prepared in Example 1;

[0023] Figure 2 Schematic diagram of drying the paper tip substrate on a heating plate after impregnation in Example 1;

[0024] Figure 3 The UV-visible spectrum of the gold nanoparticle solution prepared in Example 1 is within the wavelength range of 400 to 800 nm;

[0025] Figure 4 This is the UV-visible spectrum of the concentrated gold nanoparticle solution prepared in Example 1 within a wavelength range of 400 to 800 nm. DETAILED DESCRIPTION

[0026] The present invention provides a method for detecting lycopene content using paper-based surface-enhanced Raman spectroscopy, comprising the following steps:

[0027] 1) reacting tetrachloroauric acid with sodium citrate to obtain a gold nanoparticle solution, and then centrifuging and concentrating the gold nanoparticle solution to obtain a concentrated gold nanoparticle solution;

[0028] 2) mixing the test solution containing lycopene with the concentrated gold nanoparticle solution to obtain a sample solution;

[0029] 3) Immersing the paper tip substrate in the sample solution, followed by drying and Raman signal detection of lycopene, and determining the lycopene content in the sample solution based on the Raman signal detection results;

[0030] The method for preparing the paper tip base comprises the following steps: cutting filter paper and sealing with wax oil in sequence to obtain the paper tip base.

[0031] In the present invention, the filter paper is preferably qualitative filter paper.

[0032] In the present invention, the tip angle of the paper tip base is 10-20°, preferably 11-18°, more preferably 12-17°, and more preferably 13-16°, which facilitates subsequent operations. The wax oil seal is performed at a distance of ≥9 mm from the tip of the paper tip base, preferably 9.2-10.5 mm from the tip of the paper tip base, more preferably 9.5-10.3 mm from the tip of the paper tip base, and more preferably 9.6-10 mm from the tip of the paper tip base, so that the gold nanoparticles and lycopene are quickly aggregated at the tip of the paper tip base, preventing diffusion in the opposite direction toward the tip due to the coffee ring effect.

[0033] In the present invention, the pore size of the filter paper in the paper tip substrate is 10 to 12 μm, preferably 10.2 to 11.8 μm, more preferably 10.5 to 11.5 μm, and even more preferably 11 μm.

[0034] In the present invention, the molar concentration of the concentrated gold nanoparticle solution in step 1) is 6.14×10 -10 ~7.20×10 -10 mol / mL, preferably 6.15×10 -10 ~7.10×10 -10 mol / mL, more preferably 6.20×10 -10 ~7.00×10 -10 mol / mL, more preferably 6.35×10 -10 ~6.80×10 -10 mol / mL.

[0035] In the present invention, the rotation speed of the centrifugal concentration in step 1) is preferably 3000-4000 rpm, more preferably one of 3000 rpm, 3200 rpm, 3500 rpm, 3800 rpm, and 4000 rpm; the time of the centrifugal concentration is preferably 5-10 min, more preferably one of 5 min, 6 min, 7 min, 8 min, 9 min, and 10 min.

[0036] In the present invention, the average particle size of the gold nanoparticles in the concentrated gold nanoparticle solution in step 1) is 60 to 70 nm, preferably 62 to 68 nm, more preferably 63 to 67 nm, and even more preferably 64 nm.

[0037] In the present invention, the volume ratio of the test solution containing lycopene to the concentrated gold nanoparticle solution in step 2) is 1:1-10, preferably 1:2-8, more preferably 1:3-6, and even more preferably 1:5.

[0038] In the present invention, the mixing in step 2) is preferably ultrasonic oscillation mixing; the power of the ultrasonic oscillation mixing is preferably 400 W, the time is preferably 5 min, the temperature is preferably 4° C., and the environmental conditions are preferably dark-proof.

[0039] In the present invention, the mass concentration of lycopene in the test solution containing lycopene in step 2) is 100-500 μg / mL, preferably 150-450 μg / mL, more preferably 200-400 μg / mL, and even more preferably 250-300 μg / mL.

[0040] In the present invention, the test liquid containing lycopene preferably comprises a lycopene liquid drink.

[0041] In the present invention, the drying in step 3) is specifically performed by placing the impregnated paper tip substrate on a heating plate for drying, and utilizing gravity and the coffee ring effect to cause the sample (lycopene and gold nanoparticles) to gather at the tip of the paper tip substrate; wherein the surface of the heating plate is arranged parallel to the ground, and the surface of the impregnated paper tip substrate is arranged perpendicular to the surface of the heating plate; and the angle between the hypotenuse of the impregnated paper tip substrate close to the surface of the heating plate and the surface of the heating plate is 25-60°, preferably 26-50°, more preferably 28-40°, and even more preferably 30°.

[0042] In the present invention, the drying temperature in step 3) is 70-80°C, preferably one of 70°C, 72°C, 75°C, 76°C, 78°C, and 80°C; the drying time is 3-5 min, preferably 3.2-4.5 min, more preferably 3.5-4.2 min, and more preferably 4 min.

[0043] In the present invention, the drying temperature in step 3) can ensure that the temperature gradient self-assembly effect of the gold nanoparticles at the tip of the paper tip substrate is optimal and the SERS signal is strongest.

[0044] In the present invention, the immersion time in step 3) is 15 to 30 seconds, preferably 18 to 28 seconds, more preferably 20 to 26 seconds, and even more preferably 22 to 25 seconds, so as to load the sample on the paper tip substrate.

[0045] In the present invention, the parameters for the Raman signal detection of lycopene in step 3) are as follows: excitation light wavelength: 633 nm, acquisition time: 5-10 s, cumulative number of times: 1-2 times.

[0046] In the present invention, the acquisition time is preferably 6 to 8 seconds, more preferably 7 seconds; the cumulative number of times is preferably 2 times.

[0047] In the present invention, the instrument for detecting the Raman signal of lycopene in step 3) is preferably a laser confocal Raman detection instrument.

[0048] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] 1) 1.5 mL of 1 wt% chloroauric acid (HAuCl4) solution was dissolved in 148.5 mL of deionized water (RO H2O) and mixed. The mixture was heated to boiling in an oil bath. During the waiting time, 0.1 g of sodium citrate was weighed and mixed with 10 mL of deionized water to prepare a 1 wt% sodium citrate solution (prepared immediately after use). After 150 mL of HAuCl4 solution was boiled, 0.9 mL of the prepared sodium citrate solution was added. The reaction system was kept boiling for 40 min while ensuring continuous stirring. Finally, the generated gold nanoparticles (AuNPs) solution was naturally cooled to room temperature and stored in a refrigerator at 4 ° C in the dark for later use. Then, 1 mL of the gold nanoparticle solution was centrifuged and concentrated at 4000 rpm for 10 min. 700 μL of the supernatant was discarded, leaving a precipitate layer to obtain a concentrated gold nanoparticle solution. The average particle size of the gold nanoparticles in the concentrated gold nanoparticle solution was 64 nm and the molar concentration was 6.19×10 -10 mol / mL;

[0051] 2) Lycopene liquid drink (lycopene concentration of 500 μg / mL) and concentrated gold nanoparticle solution were mixed at a volume ratio of 1:10 and ultrasonically vibrated at a power of 400 W, a duration of 5 minutes, a temperature of 4°C, and in the dark to obtain a sample solution;

[0052] 3) Cut the qualitative filter paper with a pore size of 11 μm into a paper tip with a tip angle of 11.25° and seal it with wax oil at a position 9 mm away from the paper tip to obtain a paper tip base (the actual picture of the paper tip base is as follows Figure 1 The paper tip substrate is then immersed in the sample solution in step 2) for 30 seconds. After the immersion is completed, the paper tip substrate is taken out and fixed on a clamp above a heating plate at a temperature of 70°C (the surface of the heating plate is set parallel to the ground), and the surface of the paper tip substrate is perpendicular to the surface of the heating plate. The angle between the oblique edge of the paper tip substrate close to the surface of the heating plate and the surface of the heating plate is 30° (schematic diagram as shown). Figure 2 The sample was dried for 3 minutes using a confocal laser Raman detector with parameters set to an excitation wavelength of 633 nm, an acquisition time of 5 seconds, and two cumulative times. The Raman signal of lycopene at the tip of the paper tip base (the filter paper fibers protruding from the tip) was detected. Based on the Raman signal detection results, the lycopene content in the test solution was determined to be 500 μg / mL.

[0053] The gold nanoparticles solution prepared in this example and the concentrated gold nanoparticles solution were quantified using NanoDrop TM One instrument measures UV absorbance in the wavelength range of 400 to 800 nm. The results are as follows Figures 3-4 As shown. Among them, from Figure 3 From the UV-visible spectrum of the gold nanoparticle solution, it can be seen that the surface plasmon resonance peak (SPR) is at 540 nm, which indicates that the diameter of the gold nanoparticles is 66 nm. The molar concentration of the gold nanoparticle solution is calculated to be 5.68×10 - 10 mol / mL; from Figure 4 From the UV-visible spectrum of the concentrated gold nanoparticle solution, it can be seen that the surface plasmon resonance peak (SPR) is at 538 nm, which indicates that the diameter of the gold nanoparticles is 64 nm. The molar concentration of the gold nanoparticle solution is calculated to be 6.19×10 -10 mol / mL.

[0054] Example 2

[0055] 1) 1.5 mL of 1 wt% chloroauric acid (HAuCl4) solution was dissolved in 148.5 mL of deionized water (RO H2O) and mixed. The mixture was heated to boiling in an oil bath. During the waiting time, 0.1 g of sodium citrate was weighed and mixed with 10 mL of deionized water to prepare a sodium citrate solution with a mass concentration of 1 wt% (prepared immediately after use). After 150 mL of HAuCl4 solution was boiled, 0.9 mL of the freshly prepared sodium citrate solution was added. While ensuring continuous stirring, the reaction system was kept boiling for 40 min. Finally, the generated gold nanoparticles (AuNPs) solution was naturally cooled to room temperature and stored in a refrigerator at 4 ° C in the dark for later use. Then, 1 mL of the gold nanoparticle solution was centrifuged and concentrated at a speed of 3000 rpm for 5 min. 700 μL of the supernatant was discarded, leaving a precipitate layer to obtain a concentrated gold nanoparticle solution. The average particle size of the gold nanoparticles in the concentrated gold nanoparticle solution was 61 nm and the molar concentration was 7.20 × 10 -10 mol / mL;

[0056] 2) Lycopene liquid drink (lycopene concentration of 100 μg / mL) and concentrated gold nanoparticle solution were mixed in a 1:1 volume ratio and ultrasonically vibrated at a power of 400 W, a duration of 5 minutes, a temperature of 4°C, and in the dark to obtain a sample solution;

[0057] 3) Cut a 12 μm pore size qualitative filter paper into a tip with a 10° tip angle and seal it with wax at a position 11 mm from the tip to obtain a tip base, which is then set aside. The tip base is then immersed in the sample solution from step 2) for 15 seconds. After immersion, the tip base is removed and secured to a clamp above an 80°C hot plate (the hot plate surface is parallel to the ground), with the surface of the tip base perpendicular to the hot plate surface. The angle between the hypotenuse of the tip base, closest to the hot plate surface, and the hot plate surface is 25°. Dry the paper. After drying for 5 minutes, use a laser confocal Raman spectrometer with the following parameters: excitation wavelength: 633 nm, acquisition time: 10 seconds, and cumulative count: 1. The Raman signal of lycopene at the tip of the tip base (the filter paper fibers protruding from the tip) is detected. Based on the Raman signal detection results, the lycopene content in the test solution is determined to be 100 μg / ml.

[0058] Example 3

[0059] 1) 1.5 mL of 1 wt% chloroauric acid (HAuCl4) solution was dissolved in 148.5 mL of deionized water (RO H2O) and mixed. The mixture was heated to boiling in an oil bath. During the waiting time, 0.1 g of sodium citrate was weighed and mixed with 10 mL of deionized water to prepare a sodium citrate solution with a mass concentration of 1 wt% (prepared immediately after use). After 150 mL of HAuCl4 solution was boiled, 0.9 mL of the freshly prepared sodium citrate solution was added. While ensuring continuous stirring, the reaction system was kept boiling for 40 min. Finally, the generated gold nanoparticles (AuNPs) solution was naturally cooled to room temperature and stored in a refrigerator at 4 ° C in the dark for later use. Then, 1 mL of the gold nanoparticle solution was centrifuged and concentrated at a speed of 3800 rpm for 8 min. 700 μL of the supernatant was discarded, leaving a precipitate layer to obtain a concentrated gold nanoparticle solution. The average particle size of the gold nanoparticles in the concentrated gold nanoparticle solution was 62 nm and the molar concentration was 6.80 × 10 -10 mol / mL;

[0060] 2) Lycopene liquid drink (lycopene concentration of 250 μg / mL) and concentrated gold nanoparticle solution were mixed at a volume ratio of 1:5 and ultrasonically vibrated at a power of 400 W, a duration of 5 minutes, a temperature of 4°C, and in the dark to obtain a sample solution;

[0061] 3) Cut a qualitative filter paper with a pore size of 10 μm into a paper tip with a tip angle of 15°. Seal the tip with wax at a position 10 mm from the tip to obtain a paper tip base, which is then set aside. Then, immerse the paper tip base in the sample solution prepared in step 2) for 20 seconds. After immersion, remove the paper tip base and secure it to a clamp above a 75°C hot plate (the surface of the hot plate is parallel to the ground), with the surface of the paper tip base perpendicular to the surface of the hot plate. The angle between the hypotenuse of the paper tip base, closest to the hot plate surface, and the hot plate surface is 60°. Dry the paper tip base for 4 minutes. Using a laser confocal Raman detector, with parameters set to excitation wavelength: 633 nm, acquisition time: 7 seconds, and cumulative count: 1, detect the Raman signal of lycopene at the tip of the paper tip base (the filter paper fibers protruding from the tip). Based on the Raman signal detection results, the lycopene content in the test solution is determined to be 250 μg / mL.

[0062] Example 4

[0063] The only difference between Example 4 and Example 1 is that in step 3), the paper tip substrate is not dried using a heating plate. Instead, the impregnated paper tip substrate is directly tested using a laser confocal Raman spectrometer. The testing process is the same as in Example 1, and the lycopene content in the test solution is determined based on the Raman signal detection results.

[0064] It can be seen from this embodiment that the method of the present invention is also applicable to rapid detection of lycopene on-site where it is inconvenient to use heating equipment.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate, characterized in that: The steps include: 1) reacting tetrachloroauric acid with sodium citrate to obtain a gold nanoparticle solution, and then centrifuging and concentrating the gold nanoparticle solution to obtain a concentrated gold nanoparticle solution; 2) mixing the test solution containing lycopene with the concentrated gold nanoparticle solution to obtain a sample solution; 3) Immersing the paper tip substrate in the sample solution, followed by drying and Raman signal detection of lycopene, and determining the lycopene content in the sample solution based on the Raman signal detection results; The method for preparing the paper tip base comprises the following steps: cutting filter paper and sealing with wax oil in sequence to obtain the paper tip base.

2. The method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate according to claim 1, characterized in that: The tip angle of the paper tip base is 10 to 20 degrees; The wax oil seal is sealed at a distance of ≥9 mm from the tip of the paper tip base; The pore size of the filter paper in the paper tip substrate is 10 to 12 μm.

3. The method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate according to claim 2, characterized in that: The molar concentration of the concentrated gold nanoparticle solution in step 1) is 6.14×10 -10 ~7.20×10 -10 mol / mL; The average particle size of the gold nanoparticles in the concentrated gold nanoparticle solution is 60-70 nm.

4. The method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate according to any one of claims 1 to 3, characterized in that: The volume ratio of the test solution containing lycopene to the concentrated gold nanoparticle solution in step 2) is 1:1-10.

5. The method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate according to claim 4, characterized in that: The mass concentration of lycopene in the test solution containing lycopene in step 2) is 100-500 μg / mL.

6. The method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate according to claim 5, characterized in that: The drying in step 3) is specifically to place the paper tip substrate after impregnation on a heating plate for drying; wherein the surface of the heating plate is arranged parallel to the ground, and the surface of the paper tip base after the impregnation is completed is arranged perpendicular to the surface of the heating plate; After the impregnation is completed, the angle between the oblique edge of the paper tip base close to the surface of the heating plate and the surface of the heating plate is 25 to 60 degrees; The drying temperature is 70-80° C. and the drying time is 3-5 minutes.

7. The method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate according to claim 6, characterized in that: The immersion time in step 3) is 15 to 30 seconds.

8. The method for detecting lycopene content by surface-enhanced Raman spectroscopy on a paper substrate according to claim 7, characterized in that: The parameters for the Raman signal detection of lycopene in step 3) are as follows: excitation light wavelength: 633 nm, acquisition time: 5-10 s, cumulative number of times: 1-2 times.

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