CN114646628A - Method for detecting chlorogenic acid substances in plants based on SERS (surface enhanced Raman scattering) - Google Patents
Method for detecting chlorogenic acid substances in plants based on SERS (surface enhanced Raman scattering) Download PDFInfo
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- CN114646628A CN114646628A CN202011532963.8A CN202011532963A CN114646628A CN 114646628 A CN114646628 A CN 114646628A CN 202011532963 A CN202011532963 A CN 202011532963A CN 114646628 A CN114646628 A CN 114646628A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- B82Y40/00—Manufacture or treatment of nanostructures
Abstract
The invention discloses a method for detecting chlorogenic acid substances in plants based on SERS. The method comprises the following steps: s1, crushing a sample to be detected, extracting to obtain an extracting solution, and taking a filtrate obtained after the filtrate passes through a microfiltration membrane as a liquid to be detected; s2, dropwise adding the diluted solution to be detected onto a surface enhanced Raman substrate for Raman spectrum detection to obtain a Raman characteristic peak signal of chlorogenic acid substances in the solution to be detected; s3, preparing a standard solution of chlorogenic acid substances, then dropwise adding the standard solution onto a surface enhanced Raman substrate, and performing Raman spectrum detection to obtain a signal of a Raman characteristic peak of the chlorogenic acid substances in the standard solution, so as to obtain a standard curve between the concentration of the chlorogenic acid substances in the standard solution and the signal of the Raman characteristic peak; and detecting the chlorogenic acid substances in the sample to be detected according to the standard curve and the signal of the Raman characteristic peak of the chlorogenic acid substances in the liquid to be detected. The invention is suitable for detecting the content of chlorogenic acid substances in eucommia ulmoides, stevia rebaudiana, honeysuckle and other plants.
Description
Technical Field
The invention relates to a method for detecting chlorogenic acid substances in plants, in particular to a method for detecting the chlorogenic acid substances in the plants based on SERS.
Background
Chlorogenic acids (CGAs) have various biological properties including antibacterial, antiviral, anticancer, antiinflammatory, antioxidant, blood sugar lowering and blood lipid reducing effects. In recent years, CGAs have been widely used, particularly in the fields of pharmacology, chemical industry, food industry, cosmetic industry, and the like. CGAs have high content in eucommia ulmoides, honeysuckle, stevia rebaudiana and other plants, and the conventional detection methods comprise high performance liquid chromatography, reverse phase chromatography, liquid chromatography-mass spectrometry, electrochemical method, spectrophotometry and the like. The high performance liquid chromatography requires various standards of CGAs to perform qualitative or quantitative analysis, and the electrochemical method and the spectrophotometric method require specific reaction conditions to perform qualitative or quantitative analysis. Generally, these methods are relatively time-consuming and prone to secondary contamination, and thus it is desirable to establish an economical, rapid and sensitive detection method.
The Surface Enhanced Raman Scattering (SERS) can measure the fingerprint of a target substance, has excellent accuracy and sensitivity, and can be used for qualitative and quantitative detection of the target substance. However, no report of enhanced Raman spectroscopy for detecting the content of CGAs exists at present. Therefore, the establishment of the enhanced Raman technology for simply, rapidly and accurately detecting the CGAs is an important research direction.
Disclosure of Invention
The invention aims to provide a method for detecting chlorogenic acid substances in plants based on SERS, which is suitable for detecting the chlorogenic acid substances in plants such as eucommia ulmoides, stevia rebaudiana and honeysuckle.
The method for detecting the content of chlorogenic acid substances in plants based on SERS provided by the invention comprises the following steps:
s1, crushing a sample to be detected, extracting to obtain an extracting solution, and taking a filtrate obtained after the filtrate passes through a microfiltration membrane as a liquid to be detected;
s2, dropwise adding the diluted solution to be detected onto a surface enhanced Raman substrate, and performing Raman spectrum detection to obtain a signal of a Raman characteristic peak of a chlorogenic acid substance in the solution to be detected;
s3, preparing at least 3 standard solutions of the chlorogenic acids with different concentrations, dripping the standard solutions onto the surface enhanced Raman substrate, and performing Raman spectrum detection under the same detection conditions as those in the step S2 to obtain Raman characteristic peak signals of the chlorogenic acids in the different standard solutions, so as to obtain a standard curve between the concentration of the chlorogenic acids in the standard solutions and the Raman characteristic peak signals;
s4, detecting the content of the chlorogenic acid substances in the sample to be detected according to the Raman characteristic peak signal of the chlorogenic acid substances in the liquid to be detected obtained in the step S2 and the standard curve obtained in the step S3.
The method is suitable for detecting chlorogenic acid substances in plant samples, particularly for plants with homology of medicine and food, namely the plants which are food and medicine, and can be used as plant products of medicine, health-care products, food and the like, such as eucommia ulmoides, stevia rebaudiana or honeysuckle and the like.
The chlorogenic acid substance in the invention refers to depside natural phenolic compounds generated from Caffeic acid (Caffeic acid) and Quinic acid (Quinic acid, 1-hydroxyhexahydro gallic acid), and comprises the following components: 3-caffeoylquinic acid (3-caffeoylquinic acid, 3-CQA), 4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-dicaffeoylquinic acid, 3,4-dicQA), 3,5-dicaffeoylquinic acid (3,5-dicaffeoylquinic acid, 3,5-dicQA), 4, 5-dicaffeoylquinic acid (4,5-dicQA) and the like, wherein the content of the total chlorogenic acid substances in the sample to be detected can be detected by adopting any standard substance of the chlorogenic acid substances, and 3-CQA with higher content and low price is preferably selected as the standard substance.
In the method, in step S1, a methanol aqueous solution with a volume concentration of 40-70% is used for ultrasonic extraction of the sample to be detected, preferably a 50% methanol aqueous solution is used for ultrasonic extraction, and the dosage of the methanol aqueous solution is 100-200 mL/g of the sample to be detected;
the ultrasonic extraction time is 20-30 min;
the aperture of the microfiltration membrane is 0.45 mu m.
In the above method, the surface-enhanced raman substrate may be Cu2An O-Ag composite substrate, the Cu being prepared by the method2O-Ag compositeSubstrate:
mixing Cu2Adding the O sheet into a silver nitrate aqueous solution to react to obtain the product;
growing the Cu on a copper sheet or glass substrate by a wet chemical method, an electrochemical method or a high-temperature annealing method2And (3) O thin plates.
In the above method, in step S3, the concentration of the standard solution is 0.5 to 50. mu.g/mL;
the detection limit of the CGAs detected by the detection method is 0.13 mu g/mL.
The signal of the Raman characteristic peak refers to the peak area or intensity of the Raman characteristic peak.
In the method, a light source with the wavelength of 532nm, 633nm or 785nm is used as an excitation light source for Raman spectrum detection.
In the method, the Raman characteristic peak comprises a shift of 1615 +/-5 cm-1、1573±5cm-1、1498±5cm-1、1330±5cm-1、1254±5cm-1、1162±5cm-1、1114±5cm-1And 975. + -.5 cm-1The absorption peak at (c).
The method utilizes the surface enhanced Raman technology to detect the liquid to be detected, can accurately detect the spectrum of the CGAs Raman signal fingerprint area in the liquid to be detected, has small deviation of the detection result, and accurately quantifies the content of the chlorogenic acid compound in the sample according to the fingerprint spectrum. According to the embodiment of the invention, the total chlorogenic acid compounds in the sample to be detected can be accurately quantitatively/qualitatively analyzed without using various standard substances, the detection process is simple and quick, the result accuracy is high, and the method sensitivity is high.
Compared with the prior art, the invention is based on Cu2The O-Ag substrate has good sensitivity to CGAs, has good linear relation in the range of 0.5-50 mu g/mL, and R2The detection limit is 0.99, and the detection limit is 0.13 mu g/mL, and the method is applied to measuring the content of CGAs in plant samples; the method can meet the requirement on detection portability, is a brand-new and efficient CGAs detection method, and has the advantages of simple and rapid detection process and low cost.
Drawings
FIG. 1 shows Cu prepared in example 1 of the present invention2Scanning electron micrographs of O-Ag substrates.
FIG. 2 is a schematic diagram of a standard curve linear fit constructed in example 1 of the present invention.
FIG. 3 shows the extraction of stevia rebaudiana Bertoni in Cu of example 1 of the present invention2O-Ag substrate, 10. mu.g/mL 3-CQA solution in Cu2O-Ag and 10. mu.g/mL 3-CQA solution in Cu2Comparison of raman spectra on O-substrate.
FIG. 4 is an enhanced Raman spectrum of various chlorogenic acids.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 detection of content of chlorogenic acids in stevia rebaudiana
1)Cu2Preparation of O-Ag SERS substrate
Polishing a copper sheet (the thickness of which is about 0.3mm) by 7000-mesh polishing paper, cutting the copper sheet into blocks of 1.5cm multiplied by 1.5cm, sequentially performing ultrasonic cleaning on acetone, ethanol and water for 10min respectively, taking out the copper sheet, putting the copper sheet into a 50mL centrifuge tube, adding etching solution (9mL of water, 4mL of 10mol/L NaOH and 1mL of 1mol/L ammonium persulfate), reacting at room temperature for about 120min to generate copper oxide (CuO), taking out the copper sheet, cleaning the copper sheet for 4 times by using deionized water and ethanol, drying the copper sheet, putting the copper sheet into a tube furnace, and adding N2Sintering at 550 ℃ for 5h in atmosphere to generate cuprous oxide (Cu)2O), cooling to room temperature and taking out. Mixing Cu2O into a 50mL centrifuge tube, 20mL 5X 10 was added-4Aqueous solution of silver nitrate (containing 2.5X 10 mol/L)-4mol/L nitric acid) for 60s, while shaking constantly to generate Cu2And taking out the O-Ag, washing with deionized water and ethanol for 4 times, putting in a vacuum drying oven, drying at 50 ℃ for 6 hours in vacuum, and taking out for later use.
Cu prepared in this example2The scanning electron micrograph of the O-Ag substrate is shown in FIG. 1, and it can be seen that Ag nanoparticles are distributed in Cu2On the surface of O, the size of Ag nano particles is about 90nm, and Cu2Size of O particlesAbout 900 nm.
2) Construction of a Standard Curve
The 3-CQA was prepared as a 1mg/mL stock solution with 50% aqueous methanol and diluted with 50% aqueous methanol to a series of standard solutions (0.5, 1, 5, 10, 25 and 50. mu.g/mL). Dropping 2 microliter of standard solution with different concentrations in Cu2And carrying out Raman spectrum collection on the O-Ag substrate.
Measurement conditions were as follows: a laser light source: 785nm, laser power: 25mW, integration time: 1s, objective lens: 10X.
Using the 1573cm in Raman spectrum-1And constructing a linear model by using the peak intensity of the characteristic peak and the concentration of the 3-CQA. FIG. 2(A) is a Raman spectrum of 3-CQA at different concentrations, and FIG. 2(B) is a characteristic peak of 1573cm-1The peak intensity of the part (strong peak intensity) and the concentration of 3-CQA are used for constructing a linear model, the fitting equation y is 1339.5x +5631.8, the correlation coefficient R2 is 0.9909, and y is 1573cm-1The peak intensity is strong, x is the concentration of 3-CQA, the unit is mu g/mL, the linear range is 0.5-50 mu g/mL, the detection limit obtained by calculation is 0.13 mu g/mL, and the constructed concentration linear model has a better linear relation and a lower detection limit.
3) Detection of actual samples
After the stevia sample is crushed, 0.1g of the sample is weighed, 10mL of 50% methanol aqueous solution is added, ultrasonic extraction is carried out for 30min, the mixture passes through a 0.45 mu m membrane and is diluted by 10 times, 2 mu L of solution to be measured is taken, and the solution is dripped on a substrate for Raman spectrum measurement.
Measurement conditions were as follows: a laser light source: 785nm, laser power: 25mW, integration time: 1s, objective lens: 10X.
The obtained Raman spectrum is shown in figure 3, and the content of chlorogenic acid substances in the stevia rebaudiana is obtained according to the linear model obtained in the step 2), wherein the content of the chlorogenic acid substances in the solution to be tested is 19.56 mu g/mL, and the content of the chlorogenic acid substances in the stevia rebaudiana is 19.56 g/kg.
Because stevia contains a plurality of different CGAs isomers, the SERS spectra of 6 CGAs different isomers are detected, 50% methanol aqueous solution is adopted to prepare 10 microgram/mL solution which is dripped in Cu2Raman spectroscopy on O-Ag substratesThe results are shown in FIG. 4, in which curves a to f in FIG. 4 represent Raman peak intensity spectra of 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA), respectively. As can be seen from the graph, the SERS spectra of the isomers of the 6 CGAs have no obvious difference, so that the method detects the content of the total CGAs in the sample.
The invention also compares Cu2O-Ag composite substrate and Cu2SERS performance on O-substrate. Under the above measurement conditions, 3-CQA (10. mu.g/mL) in Cu2O-Ag composite substrate and Cu2The Raman spectrum on the O substrate is shown in FIG. 3, and it can be seen that 3-CQA is in Cu2No Raman signal on O substrate, and on Cu2Obvious Raman signals are generated on the O-Ag composite substrate, which indicates that Cu is generated2The O-Ag composite substrate has excellent SERS performance
The invention compares the SERS method and the result of HPLC detection of different plant samples.
The sample pretreatment method was the same as in example 1. The conditions for HPLC detection were as follows:
a chromatographic column: c18A column with a length of 250nm, an inner diameter of 4.6mm and a particle size of 5 μm;
column temperature: 30 ℃;
detection wavelength: 327 nm;
mobile phase: a: acetonitrile, B: 0.1% formic acid-water solution, elution gradient is shown in table 1;
flow rate: 1.0 mL/min;
sample introduction amount: 10 μ L.
TABLE 1 high Performance liquid chromatography elution gradient time
A stevia sample was treated in the same manner as in example 1, and then subjected to Raman spectrum detection under the same conditions, and concentration conversion was performed according to the standard curve shown in FIG. 2.
The test results are shown in Table 2, wherein samples 1 to 6 are stevia rebaudiana samples from Shaanxi, Xinjiang, Hubei, Hebei, Gansu and Jiangsu, respectively.
As can be seen from the test results in Table 2, the relative error of the SERS result based on the HPLC method is less than 3%, and the SERS result has better consistency, which indicates that the SERS method of the invention has better accuracy and is comparable to the detection result of the HPLC method.
TABLE 2 SERS and HPLC detection results comparing CGAs content in stevia rebaudiana sample
The Cu with excellent SERS performance is obtained by preparation according to the embodiment of the invention2The O-Ag substrate realizes the rapid and portable detection of chlorogenic acid substances. And the method has the advantages of high sensitivity, simple and quick operation, low cost and the like.
Claims (9)
1. A method for detecting chlorogenic acid substances in plants based on SERS comprises the following steps:
s1, crushing a sample to be detected, extracting to obtain an extracting solution, and taking a filtrate obtained after the filtrate passes through a microfiltration membrane as a liquid to be detected;
s2, dropwise adding the diluted solution to be detected onto a surface enhanced Raman substrate, and performing Raman spectrum detection to obtain a signal of a Raman characteristic peak of a chlorogenic acid substance in the solution to be detected;
s3, preparing at least 3 standard solutions of the chlorogenic acids with different concentrations, dripping the standard solutions onto the surface enhanced Raman substrate, and performing Raman spectrum detection under the same detection conditions as those in the step S2 to obtain Raman characteristic peak signals of the chlorogenic acids in the different standard solutions, so as to obtain a standard curve between the concentration of the chlorogenic acids in the standard solutions and the Raman characteristic peak signals;
s4, detecting the chlorogenic acid substances in the sample to be detected according to the Raman characteristic peak signal of the chlorogenic acid substances in the liquid to be detected obtained in the step S2 and the standard curve obtained in the step S3.
2. The method of claim 1, wherein: the sample to be detected is a plant sample, in particular to a plant with homology of medicine and food.
3. The method according to claim 1 or 2, characterized in that: the sample to be detected is eucommia ulmoides, stevia rebaudiana or honeysuckle.
4. The method according to any one of claims 1-3, wherein: in the step S1, a methanol water solution with the volume concentration of 40-70% is adopted for ultrasonically extracting the sample to be detected, and the dosage of the sample to be detected is 100-200 mL/g;
the ultrasonic extraction time is 20-30 min;
the aperture of the microfiltration membrane is 0.45 mu m.
5. The method according to any one of claims 1-4, wherein: the chlorogenic acid substances comprise 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4, 5-dicaffeoylquinic acid.
6. The method according to any one of claims 1-5, wherein: the surface enhanced Raman substrate is Cu2And (3) an O-Ag composite substrate.
7. The method of claim 6, wherein: the Cu was prepared as follows2O-Ag composite substrate:
mixing Cu2Adding the O sheet into a silver nitrate aqueous solution to react to obtain the product;
growing the Cu on a copper sheet or glass substrate by a wet chemical method, an electrochemical method or a high-temperature annealing method2And (7) O thin plate.
8. The method according to any one of claims 1-7, wherein: a light source with the wavelength of 532nm, 633nm or 785nm is adopted as an excitation light source for Raman spectrum detection.
9. The method according to any one of claims 1-8, wherein: the Raman characteristic peak comprises a displacement of 1615 +/-5 cm-1、1573±5cm-1、1498±5cm-1、1330±5cm-1、1254±5cm-1、1162±5cm-1、1114±5cm-1And 975. + -. 5cm-1The absorption peak at (b).
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| CN116106452A (en) * | 2023-02-27 | 2023-05-12 | 浙江景岳堂药业有限公司 | Construction method of stevia rebaudiana formula granule characteristic spectrum, characteristic spectrum constructed by method and application of characteristic spectrum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116106452A (en) * | 2023-02-27 | 2023-05-12 | 浙江景岳堂药业有限公司 | Construction method of stevia rebaudiana formula granule characteristic spectrum, characteristic spectrum constructed by method and application of characteristic spectrum |
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