CN114136957B - Instrument-free visual traceability method for dendrobium nobile producing area - Google Patents
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- G—PHYSICS
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
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Abstract
The invention discloses a novel instrument-free dendrobium origin tracing method, which is based on the protection effect of dendrobium extract on nano gold and effectively inhibits metal ions (Cu 2+ ) The resulting aggregation effect of the nano-gold; by utilizing the aggregation degree difference and color change of the nano gold caused by the action of the dendrobium extract and the metal ions in different producing areas and combining a chemometrics method, the excellent traceability of the dendrobium in different producing areas is realized. Compared with the traditional detection means such as chromatography, the method has the advantages of no instrument, low cost, short time consumption, simple operation and the like, and has important application value in the field of analysis and detection.
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to an instrument-free visual traceability method for dendrobium nobile producing areas.
Background
Dendrobium nobile is a traditional Chinese medicine in China, and is derived from stems of various plants of Dendrobium of Orchidaceae, sweet and light in taste and slightly cold in nature; has effects of promoting gastric secretion, clearing heat and nourishing yin, and can be used for treating fever, dry mouth, polydipsia, asthenic fever after illness, yin injury, and dim eyesight. Dendrobium nobile contained in 2015 edition pharmacopoeia comprises common varieties of dendrobium nobile, dendrobium chrysotoxum, dendrobium fimbriatum and the like, and dendrobium huoshanense is also contained in 2020 edition pharmacopoeia. Modern pharmacological researches show that dendrobium contains saccharides, alkaloids, dibenzyls, phenanthrenes, coumarins, fluorenes, steroid saponins, alocasia saponins, other glycosides, diterpenes and other compounds, and has various medicinal values of immunoregulation, anti-tumor, gastrointestinal tract effect, antioxidation, anti-aging, anti-fatigue, blood sugar reduction, blood fat reduction, blood pressure reduction and the like. The total of 76 varieties of dendrobium nobile in China comprises two varieties, wherein more than 40 medicinal dendrobium nobile are mainly distributed in Yunnan, guizhou, sichuan, anhui, guangxi and Guangdong. Areas such as Hainan and the like, fujian, hunan, hubei, henan, jiangxi and the like are also distributed. Because of the complex sources, the long-term knowledge of the variety, quality evaluation, efficacy and the like has more problems.
At present, the methods for detecting different sources of dendrobium nobile at home and abroad mainly depend on large-scale instruments, such as high performance liquid chromatography, meteorological chromatography and the like, and the methods have the advantages of high detection accuracy, reliable results and the like, but have high cost, operators need to have professional knowledge skills, and the defects of complex sample pretreatment, long time consumption and the like still greatly limit the application of the methods in rapid detection of foods and medicines. Therefore, a simple, quick, low-cost and short-time dendrobium origin tracing method needs to be further established to adapt to market demands.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel method for tracing the dendrobium nobile producing area without instruments, which utilizes the interaction of various components in the dendrobium nobile extracting solution and the nano gold to form protection, so as to lead to the difference of aggregation degree and color of the nano gold protected by the dendrobium nobile extracting solution in different producing areas based on metal ion induction and realize the rapid and accurate identification of the dendrobium nobile producing area; the method has important significance for protecting the health and benefits of consumers, detecting and identifying the related departments and maintaining good market environment.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an instrument-free dendrobium origin tracing method comprises the following steps:
1) Extracting a dendrobium sample to obtain dendrobium extract;
2) Sequentially introducing nano gold, dendrobium extract and copper salt, uniformly mixing, and performing room temperature reaction; obtaining a color response picture of the obtained reaction system;
3) According to the method in the steps 1) to 2), color response pictures of the known dendrobium samples at the production places are obtained, color information in the color response pictures is extracted, and quick tracing of the unknown dendrobium samples at the production places is achieved according to the relation between the color information and the dendrobium production places.
In the above scheme, the dendrobium producing area comprises Anhui, zhejiang, yunnan or Guizhou province and the like.
In the above scheme, the color information is RGB values.
In the scheme, a chemometric method is adopted to establish the relation between the color information in the step 3) and the dendrobium origin, so that the dendrobium origin can be rapidly traced.
In the above embodiment, the chemometric method is Partial Least Squares Discriminant (PLSDA) or Linear Discriminant Analysis (LDA) analysis means.
In the above scheme, the extracting step adopts an ultrasonic extracting process; the extractant is methanol water solution with the concentration of 25-50 vol% and the feed liquid ratio of 50mg (20-30) mL.
In the scheme, the reaction temperature in the step 2) is 25-30 ℃ at room temperature, and the reaction time is 5-10 min.
In the scheme, the spherical nano gold in the step 2) is synthesized by taking chloroauric acid as a main raw material and trisodium citrate as a reducing agent by adopting a microwave method.
In the scheme, the molar ratio of the chloroauric acid to the trisodium citrate is 1 (10-20).
In the above scheme, the microwave process parameters include: the power is 600-800W and the time is 3-5 min.
In the scheme, the content of the dendrobium component in the mixed solution obtained in the step 2) is 0.5-1 mg/ml based on the crude drug introduced into the dendrobium sample in the step 1).
In the scheme, the concentration of the nano gold introduced into the mixed solution obtained in the step 2) is 3.0-4.0 nmol/L, and the concentration of the copper salt is 2-3 multiplied by 10 -3 mol/ml。
In the above scheme, the copper salt can be copper chloride or copper sulfate.
Preferably, the reaction in the step 2) is placed in a 96-well plate, the colors are recorded through mobile phone photographing, and pattern recognition is performed after RGB value extraction, so that the rapid instrument-free and batch traceability of dendrobium nobile places is realized.
Based on the instrument-free color change response capability of different dendrobium samples, the invention utilizes the aggregation effect of copper ions on nano gold (the dendrobium extract can protect the nano gold from aggregation caused by copper ions) and the aggregation degree difference of the nano gold caused by the action of the dendrobium extract and the copper ions in different producing areas, so that different dendrobium samples show different color change responses; then recording color change by means of mobile phone photographing and the like, and extracting R, G and B values by means of Photoshop software and the like; further processing is carried out by combining chemometric pattern recognition, so that the method can show excellent identification performance for tracing dendrobium nobile in different producing areas.
The principle of the invention is as follows:
according to the invention, based on the characteristic that copper ions can cause nano gold to be aggregated, by introducing the dendrobe extracting solution into a copper ion-nano gold solution system, various components in the dendrobe extracting solution can be fixed on the surface of the nano gold to form protection through interaction of surface charge, hydrogen bond or Van der Waals force and the like with the surface of the nano gold, so that the nano gold aggregation phenomenon caused by the copper ions is reduced, and different color changes are further caused by the nano gold aggregation of different degrees; and combining the difference of the chemical component contents in different dendrobium samples to generate different aggregation degrees and color changes, further forming characteristic color information of each dendrobium sample, and finally combining pattern recognition to realize quick visual recognition of dendrobium production places.
Compared with the prior art, the invention has the beneficial effects that:
1) The invention utilizes the aggregation effect of copper ions on the nano gold and the aggregation degree and color change response difference of the nano gold caused by the action of the extracting solution of the dendrobium nobile in different producing areas and the copper ions, and combines the mode identification means of chemometrics, thereby realizing the excellent identification performance of the dendrobium nobile in different producing areas;
2) The detection method has the advantages of no instrument detection, high precision, quick response, visualization and the like, can rapidly realize the accurate discrimination of the dendrobium production place, and effectively solves the problems of high cost, long time consumption, complex operation and the like of the existing detection method.
Drawings
Fig. 1 is a schematic diagram of a detection flow of the instrument-free dendrobe origin tracing method.
FIG. 2 is a TEM characterization of different states of nanogold; wherein a is the original nano gold condition, and b is nano gold+Cu 2+ Under the condition that c is nano gold+Cu 2+ Conditions (fold down to aggregation state at 20 nm); d is nano gold, dendrobium extract and Cu 2+ Conditions.
Fig. 3 shows the color change response test results of 4 different dendrobium nobile lindl sources based on the nano gold aggregation effect.
FIG. 4 is a Partial Least Squares Discriminant (PLSDA) analysis of 4 dendrobium nobile producing areas by an instrument-free visual sensor based on nano-effects.
Fig. 5 is a graph showing the result of Linear Discriminant Analysis (LDA) analysis of 4 kinds of dendrobium nobile in origin by using a nano-effect based instrument-free visual sensor.
FIG. 6 shows the color change response test results of the nano-gold system obtained under the action conditions of different metal ions and different dendrobium nobile extracts in different places.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the following examples, the preparation method of the nano gold solution adopted comprises the following steps:
taking 2mL of HAuCl with mass concentration of 1% 4 The solution was added to 98mL of boiled ultra-pure water, and 12mL of trisodium citrate dihydrate (Na) at a concentration of 50mmol/L was added 3 C 6 H 5 O 7 ·2H 2 O) heating the solution in a microwave high heat with the power of 800W for 3min until the color becomes red; the resulting product was purified by high speed centrifugation at 12000r/min for 10 minutes to remove unreacted chemicals and stored at 4 ℃. The concentration of the AuNPs solution (nano-gold solution) obtained by calculation according to the lambertian law is about 11.8nmol/L.
Example 1
An instrument-free dendrobium origin tracing method comprises the following steps:
1) respectively taking 10 dendrobium samples of Anhui (f 01), zhejiang (f 02), yunnan (f 03) and Guizhou province (f 04), extracting by using a 25vol% methanol aqueous solution, wherein the adopted feed-liquid ratio is 50mg:20mL, and carrying out ultrasonic treatment for 30min at 25 ℃; respectively obtaining dendrobe extracting solutions (20 mL) of dendrobe samples of different producing areas;
2) Adding 700 mu L of ultrapure water into 300 mu L of nano gold solution to fix the volume to 1mL to obtain a blank solution; mu.L of the gold nanoparticle solution was taken and 250. Mu.L of copper chloride solution (1X 10) -2 mol/L) and 450 mu L of ultrapure water are added at the same time to fix the volume to 1mL to be used as a control solution; 300. Mu.L of the gold nanoparticle solution, 350. Mu.L of the dendrobe extract and 250. Mu.L of the copper chloride solution (1X 10) were sequentially added -3 mol/L) and adding 100 mu L of water to a constant volume of 1mL to serve as an experimental group, wherein each dendrobium nobile has 10 parallel samples, the reaction time is 10min, the reacted samples are placed in a 96-well plate, and a camera is used for photographing and recording the color change response picture of each sample;
3) 10×10 pixels are extracted through Photoshop 2021 software, R, G and B values are extracted through MATLAB R2017a platform, and finally pattern recognition is carried out on 4 dendrobium nobile in the producing area based on a data matrix of RGB values, wherein the pattern recognition mainly comprises Linear Discriminant Analysis (LDA) and Partial Least Squares Discriminant Analysis (PLSDA).
FIG. 2 is a TEM characterization of different states of a nanogold solution; it can be seen that the introduction of copper ions into the nanogold solution can promote aggregation of nanogold; however, when the dendrobe extract is introduced in advance, the nano gold can be protected from aggregation caused by metal ions.
Fig. 3 is a color-change response picture based on dendrobe in different places of origin in Anhui, zhejiang, yunnan and Guizhou provinces, and it can be seen that under the same other conditions, different color-change responses can be obtained after the effect of the dendrobe in different places of origin.
Step 3) the partial least square discrimination results are shown in table 1, table 2 and fig. 4, wherein the table 1 shows that the number of training set samples is larger than the number of prediction set samples in PLSDA discrimination of 4 producing places dendrobe based on RGB values, and the table 2 shows that when state=2, the best hidden variable (Lvm) is selected to be 3, and the discrimination accuracy of the training set and the prediction set can reach 100%; table 3 and FIG. 5 show the linear discrimination results of 4 dendrobium nobile in the producing areas based on RGB values, and the LDA discrimination results show that the dendrobium nobile in the 4 producing areas can achieve 100% of correct discrimination, and the accuracy of the knife-cut cross-validation is 73%. The result shows that the mode identification accuracy of the 4 dendrobium nobile lindl of origin based on RGB values is high, and the dendrobium nobile lindl of different origins can be identified rapidly and effectively.
Table 1 4 partitioning results of the training set and the prediction set of RGB values PLSDA of Dendrobium nobile in different places
Table 2 4 different places of dendrobe RGB values PLSDA discrimination results (state=2)
Table 3 4 dendrobe RGB value LDA discrimination results for different producing areas
Specificity study
300. Mu.L of the gold nanoparticle solution, 350. Mu.L of the dendrobe extract and 250. Mu.L of the different metal ion solution (concentration of (1X 10) -2 mol/L, metal ions are Mg respectively 2+ 、Zn 2+ 、Mn 2+ 、K + 、Fe 3+ 、Sn 2+ 、Cu 2+ 、Ag + ) And adding 100 mu L of water to a constant volume of 1mL to serve as an experimental group, wherein 1 sample is selected as the most representative for each production place, the reaction time is 10min, the reacted samples are placed in a 96-well plate, and a camera is used for photographing and recording the color change response picture of each sample, and the specific view is shown in fig. 6.
The result shows that the dendrobium nobile extract in different producing areas can cause obvious difference of aggregation degree and color change response of the nano gold only after the dendrobium nobile extract in different producing areas is acted with copper ions, thereby realizing effective identification of dendrobium nobile producing areas.
The above examples are presented for clarity of illustration only and are not limiting of the embodiments. Other variations and modifications of the above description will be apparent to those of ordinary skill in the art, and it is not necessary or exhaustive of all embodiments, and thus all obvious variations or modifications that come within the scope of the invention are desired to be protected.
Claims (4)
1. An instrument-free visual traceability method for dendrobium nobile production places is characterized by comprising the following steps:
1) Extracting a dendrobium sample to obtain dendrobium extract;
2) Sequentially introducing nano gold, dendrobium extract and copper salt, uniformly mixing, and performing room temperature reaction; obtaining a color response picture of the obtained reaction system;
3) Obtaining color response pictures of known dendrobium nobile samples at the production places according to the method in the steps 1) to 2), extracting color information in the color response pictures, and realizing quick tracing of the production places of the unknown dendrobium nobile samples according to the relation between the color information and the dendrobium nobile production places;
the color information is RGB value;
adopting a chemometric method to establish the relationship between the color information in the step 3) and the dendrobium nobile producing area; the chemometrics method is partial least square discrimination or linear discrimination analysis;
step 1) adopting an ultrasonic extraction process; the extracting agent is a methanol aqueous solution with the concentration of 25-50vol% and the feed liquid ratio of 50mg (20-30) mL;
the nano-gold in the step 2) is spherical nano-gold; the method is characterized in that chloroauric acid is used as a main raw material, trisodium citrate is used as a reducing agent, and a microwave method is adopted for synthesis; the technological parameters of the microwave method comprise: the power is 600-800W, and the time is 3-5 min;
the concentration of the nano gold introduced into the mixed solution obtained in the step 2) is 3.0-4.0 nmol/L, and the copper salt concentration is 2-3 multiplied by 10 -3 mol/ml。
2. The method of claim 1, wherein the dendrobe producing area comprises an Anhui, zhejiang, yunnan or Guizhou province.
3. The method according to claim 1, wherein the room temperature reaction temperature in step 2) is 25-30 ℃ and the reaction time is 5-10 min.
4. The method of claim 1, wherein the content of the dendrobe component in the mixed solution obtained in step 2) is 0.5-1 mg/ml in terms of the crude drug introduced into the dendrobe sample in step 1).
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