CN111189819A - Method for detecting content of Chinese and western vitamin in food by liquid-phase microextraction combined with thread microfluid colorimetric method - Google Patents
Method for detecting content of Chinese and western vitamin in food by liquid-phase microextraction combined with thread microfluid colorimetric method Download PDFInfo
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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
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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
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Abstract
The invention discloses a method for detecting the content of the Chinese and western vitamin in food by combining liquid-phase microextraction with a thread microfluidic colorimetry. The method comprises the following steps: 1) treating cotton thread to be neutral, fixing, wetting and adding diazonium salt; 2) crushing a sample to be detected, extracting to obtain an extracting solution, and filtering by using a membrane; 3) sequentially adding carbonate, an extracting agent and citric acid into the extracting solution, and centrifuging to obtain an upper organic phase; 4) dropping the organic phase onto cotton thread; photographing the cotton threads, and calculating the RGB intensity according to the formula 1); 5) preparing carbaryl standard substance solutions with different concentrations, and obtaining RGB (red, green and blue) intensities corresponding to the carbaryl standard substance solutions according to the steps 3) and 4); taking the concentration of the carbaryl standard as an abscissa and the RGB intensity as an ordinate to make a standard curve; and (4) obtaining the concentration of carbaryl in the sample to be detected according to the standard curve and the RGB intensity measured in the step 4). The reagent used in the method is environment-friendly, the operation process is simple and quick, the experimental result is accurate and reliable, and the remote and on-site quick detection can be realized.
Description
Technical Field
The invention relates to a method for detecting the content of Chinese and western vitamin in food by a liquid-phase microextraction combined thread microfluid colorimetric method, belonging to the field of food safety detection.
Background
Crop pest and disease prevention is an important aspect of agricultural management. In recent years, a number of pesticides have been developed to control pests, greatly increasing the overall yield of crops. Carbaryl is a carbamate broad-spectrum pesticide, which has high insecticidal activity and low bioaccumulation capacity, and thus is widely applied to grains, grease, vegetables and other crops to prevent the influence of insect pests. However, the unreasonable use of carbaryl can cause carbaryl to remain in food, and further enter human body through food chain to cause carbaryl enrichment in the human body, thus generating toxic action on the human body. Research shows that carbaryl can obviously inhibit the activity of acetylcholinesterase (AChE) in human body and neuron nicotinic acetylcholine receptor. This leads to increased acetylcholine in the synaptic cleft, undesirable effects such as anorexia, memory loss, paralysis, increased risk of Alzheimer's disease and, in extreme cases, death. Therefore, the method has important significance in detecting the content of the carbaryl in the food.
To date, many methods for determining the content of the western and western vitamins in food have been developed, such as High Performance Liquid Chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), Gas Chromatography (GC), Surface Enhanced Raman Spectroscopy (SERS), enzyme-linked immunosorbent assay (ELISA), electrochemical biosensor method, and the like. Although the detection method has sensitive and accurate results, the detection method still has room for improvement in detection equipment, sample preparation methods and the like. Therefore, an analysis method which has accurate detection results, simple and portable instruments and high efficiency and environmental protection of a sample preparation method is needed to be established for determining the residual quantity of the western and Chinese vitamins in the food.
Disclosure of Invention
The invention aims to provide a method for detecting the content of the Chinese and western vitamin in food by a digital image colorimetric method, and particularly relates to a method for detecting the content of the Chinese and western vitamin in food by combining an effervescent auxiliary convertible solvent solidification-based liquid phase microextraction (EA-SSHS-LPME) pretreatment method with a thread microfluid (mu TAD) digital image colorimetric method (DIC).
The food of the present invention may be rice, wheat, soybean, Chinese cabbage, etc.
Specifically, the method for detecting the content of the Chinese and western vitamin in the food by the digital image colorimetric method provided by the invention comprises the following steps:
(1) treating cotton thread to be neutral, fixing the cotton thread on a plate, and adding diazonium salt after wetting;
(2) crushing a sample to be detected, extracting to obtain an extracting solution, and filtering by using a membrane;
(3) sequentially adding carbonate, an extracting agent and citric acid into the extracting solution, and taking an upper organic phase after centrifugal treatment;
(4) dropwise adding the organic phase onto the cotton threads for reaction; photographing the cotton threads, and calculating the RGB intensity according to the formula (1);
I＝255–G (1)
wherein, I represents RGB intensity, G represents the value of green channel in image RGB mode;
(5) preparing carbaryl standard substance solutions with different concentrations, and obtaining RGB (red, green and blue) intensities corresponding to the carbaryl standard substance solutions according to the step (3) and the step (4); taking the concentration of the carbaryl standard as an abscissa and the RGB intensity (I) as an ordinate to prepare a standard curve; and (5) obtaining the concentration of carbaryl in the sample to be detected according to the standard curve and the RGB intensity measured in the step (4).
In the method, in the step (1), the cotton threads are processed to be neutral according to the following steps: placing the cotton thread on Na2CO3Washing in boiling solution;
the concentration of the sodium carbonate boiling solution is 10mg mL-1The washing time is 5 min;
rinsing the cotton threads with distilled water after washing until the pH value of rinsing liquid is neutral, drying at room temperature, and storing in a dryer for later use;
fixing the cotton thread on the board by using double-sided adhesive tape, wherein the board can be a white plastic board;
the cotton threads were wetted with an aqueous solution having a pH of 7.
In the above method, in the step (1), the diazonium salt is 4-methoxybenzene diazonium tetrafluoroborate or p-nitrobenzene tetrafluoroborate diazonium salt;
adding the diazonium salt as a solution of the diazonium salt;
the concentration of the solution of the diazonium salt is 6-10 mmol L-1；
The amount of the solution of the diazonium salt used was 7. mu.L relative to the cotton thread having a length of 1. + -. 0.1cm (the diameter of the cotton thread was 20. + -.3 mm);
and adding the diazonium salt, naturally airing, and sealing and storing in a dryer.
In the method, acetonitrile is adopted for vortex extraction in the step (2);
the dosage of the acetonitrile is as follows: 1 to 5mL of-1Sample to be tested, preferably 1mL g-1A sample to be tested;
the vortex extraction time is 3-10 min, preferably 3 min;
the diameter of the filter membrane is 0.22 mu m;
and crushing the sample to be detected and then extracting.
In the above method, in step (3), the carbonate may be sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, or the like;
the addition amount of the carbonate is 35-50 mg g-1A sample to be tested;
the carbonate adjusting solution is alkaline so that carbaryl is hydrolyzed into 1-naphthol;
the extractant is caprylic acid or pelargonic acid, preferably caprylic acid;
the caprylic acid or the pelargonic acid is converted into hydrophilic caprylate or pelargonic acid salt under alkaline conditions, and the caprylate or pelargonic acid salt is dissolved into an aqueous phase;
the dosage of the extracting agent is 60-160 mu L g-1Sample to be tested, preferably 80. mu. L g-1A sample to be tested;
the dosage of the citric acid is 60-80 mg g-1Sample to be tested, preferably 70mg g-1A sample to be tested;
after the citric acid is added, the caprylate or pelargonate is converted into a hydrophobic solvent under an acidic condition, the caprylic acid extracting agent or pelargonic acid extracting agent is generated in situ, the caprylic acid extracting agent or pelargonic acid extracting agent is fully contacted with a water phase, and bubbles generated in an effervescence reaction assist the caprylic acid or pelargonic acid to complete extraction.
In the above method, in the step (3), the centrifugation conditions are as follows: 5000r/min, 3 min;
after the centrifugation, the organic phase was solidified by ice bath.
In the method, in the step (4), the dropping amount of the organic phase is 6-8 mu L g-1Sample to be tested, preferably 7. mu.Lg-1A sample to be tested;
1-naphthol in the organic phase and diazonium salt on the cotton thread are subjected to coupling reaction to generate an orange azo compound;
the reaction time was 20 min.
In the above method, in step (4), the photographing is performed in the following photographing apparatus:
the periphery and the top are provided with reflectors, the bottom is a light softening plate, and the top is provided with a light source so as to ensure that the photographing conditions are consistent each time;
after photographing, selecting an RGB mode by using Adobe Photoshop CC software, reading red (R), green (G) and blue (B) channel values of an effective part, and calculating according to a formula (1).
In the method, in the step (5), the concentration of the carbaryl standard substance solution is 0.30-30.00 mg kg-1。
The method of the invention has the following beneficial effects:
as carbaryl is easy to hydrolyze under alkaline conditions to generate 1-naphthol (figure 2), the 1-naphthol can generate a coupling reaction with diazonium salt (figure 3) to form a water-soluble azo compound which is combined with cotton threads for colorimetric detection. The invention uses natural medium-chain fatty acid with convertible property as an extracting agent, the extraction process can be completed only by adjusting the pH value of the solution, and bubbles are generated in the process to assist the extracting agent in completing dispersion and extraction. The extraction process is rapid and efficient, environment-friendly and green, low in energy consumption and less in generated waste. The μ TAD uses a more hydrophilic cotton thread, and the sample flows along the threads under capillary force. 1-naphthol and diazonium salt deposited on cotton threads are subjected to coupling reaction to generate color change, the color change is photographed by a smart phone, effective data is read in an RGB mode, and the intensity (I) is calculated to obtain an experimental result.
The method provided by the invention has the advantages that the adopted detection equipment is simple, the required amount of samples is less, expensive precision instruments (such as mass spectrum or high performance liquid chromatography) are not needed, the common electronic equipment of a smart phone is used, and the experimental result can be accurately analyzed by utilizing an image processing system.
The reagent used in the method is environment-friendly, the operation process is simple and quick, the experimental result is accurate and reliable, and the remote and on-site quick detection can be realized.
Drawings
FIG. 1 is a flow chart of the detection method of the present invention.
FIG. 2 shows the mechanism of carbaryl hydrolysis.
FIG. 3 is a coupling reaction mechanism.
FIG. 4 is a graph showing the effect of extractant type on recovery.
Figure 5 is a graph of the effect of sodium carbonate usage on recovery.
FIG. 6 is a graph showing the effect of extractant dosage on recovery.
FIG. 7 shows the effect of acid type on recovery.
FIG. 8 is a graph showing the effect of citric acid dosage on recovery.
FIG. 9 is a graph of the effect of adjusting the pH of a solution on the intensity (I) of a line test.
FIG. 10 is a graph of the effect of diazonium salt concentration on thread detection intensity (I).
FIG. 11 is a graph showing the effect of extract dosage on thread detection intensity (I).
FIG. 12 is a graph of the effect of reaction time on thread detection intensity (I).
FIG. 13 is a graph of the effect of different storage modes on thread detection strength (I).
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.
The flow chart of the detection method of the present invention is shown in FIG. 1.
Example 1 pretreatment of Cotton yarn
The cotton thread was kept at 10mg mL-1Na2CO3Washing in boiling solution for 5 min. Then, the cotton threads were rinsed with distilled water until the pH of the rinsing solution was neutral, dried at room temperature, and stored in a desiccator for later use.
Example 2 effervescent assisted liquid phase microextraction condition optimization based on convertible solvent solidification
A. Kind of extractant
(1) The experimental method comprises the following steps: 1g of the pulverized sample was weighed, and 100. mu.L of carbaryl (10mmol L) was added-1Methanol) and 1mL acetonitrile solution, vortex for 3 min. The extract was filtered through a membrane (0.22 μm), and 600. mu.L of the sample filtrate was taken and added to 4.4mL of water in a 10mL centrifuge tube. Adding 40mg sodium carbonate to adjust the pH of the solution to alkalinity, hydrolyzing carbaryl to 1-naphthol, adding 80. mu.L of a hydrophobic extractant (caprylic acid, pelargonic acid, capric acid), converting the extractant into a water-soluble ionic form under an alkaline condition, and dispersing the water-soluble ionic form into a sample solution. Then 70mg of citric acid is added, the extractant is generated in situ for extraction, and the bubbles generated by the effervescence reaction cause a larger surface area to be formed between the extraction solvent and the water phase to assist in completing the extraction. Centrifuging at 5000r/min for 3min to obtain organic phase in the upper layer of the centrifuge tube. The centrifuge tube was ice-cooled to solidify the organic phase and remove the lower aqueous phase. And (3) after the organic phase is melted, taking out 7 mu L of 1-naphthol extracting solution, adding the extracting solution into the thread microfluidic detection equipment, reacting for twenty minutes, photographing to calculate the RGB intensity (I), and calculating the recovery rate.
(2) The results of the experiment are shown in FIG. 4.
(3) And (4) analyzing results: the extractant plays a decisive role in the extraction process.
Under the same extraction conditions, the influence of caprylic acid, pelargonic acid and capric acid on the extraction effect is examined. The results show that the extraction of caprylic acid is the best. The freezing point of the decanoic acid is high, and the decanoic acid is easily influenced by the change of the room temperature, so that the recovery volume of the extractant is unstable. Caprylic acid was therefore chosen as the extractant.
B. Amount of sodium carbonate
(1) The experimental method comprises the following steps: the same as (1) in A, except that different amounts of sodium carbonate (5mg, 15mg, 25mg, 35mg, 40mg, 45mg, 50mg) were added, respectively.
(2) The results of the experiment are shown in FIG. 5.
(3) And (4) analyzing results: the main effects of sodium carbonate in this experiment are three: firstly, regulating the solution to be alkaline so as to hydrolyze carbaryl into 1-naphthol; second, inducing the conversion of hydrophobic caprylic acid to hydrophilic caprylate; thirdly, the sodium carbonate can be neutralized with acid added in the subsequent process to generate bubbles, so that the extraction efficiency is improved, and therefore, the sodium carbonate is important for the whole extraction process. The results show that: the recovery rate of the sodium carbonate is increased from 5mg to 40mg, and the recovery rate is stable from 40mg to 50 mg. The reason is that in the process of increasing the using amount of sodium carbonate, the alkalinity of the solution is gradually enhanced, the conversion rate of hydrolyzing carbaryl into 1-naphthol is increased, the efficiency of converting caprylic acid into caprylate is improved, the amount of bubbles generated by effervescence is increased, the auxiliary extraction efficiency is improved, and therefore the recovery rate is gradually increased along with the using amount of sodium carbonate. Finally 40mg of sodium carbonate was used.
C. Amount of extractant used
(1) The experimental method comprises the following steps: the same as in (1) in A, except that different amounts of caprylic acid extractant (40. mu.L, 50. mu.L, 60. mu.L, 80. mu.L, 100. mu.L, 120. mu.L, 160. mu.L) were added, respectively.
(2) The results of the experiment are shown in FIG. 6.
(3) And (4) analyzing results: the dosage of the extractant is in the range of 40-80 mu L, the recovery rate is in an ascending trend, and the recovery rate is in a gentle trend within the range of 80-160 mu L. This is because the sample cannot be completely recovered when the amount of the extractant used is small, and the recovery rate gradually increases as the amount of the extractant used increases and the target analyte is completely extracted. The dosage of the extracting agent is further increased, the recovery rate is not obviously changed, and 80 mu L of caprylic acid extracting agent is finally selected.
D. Kind of acid
(1) The experimental method comprises the following steps: the same as (1) in A, except that different acids (oxalic acid, malic acid, citric acid) were added.
(2) The results of the experiment are shown in FIG. 7.
(3) And (4) analyzing results: the acid can play three roles in this experiment: firstly, reducing the pH value of a system, converting hydrophilic caprylate into a hydrophobic organic phase, and generating an extracting agent in situ; secondly, complete extraction of 1-naphthol (pKa 9.34, 25 ℃) is ensured under acidic conditions; third, the air bubbles generated in the effervescent aid can accelerate the extraction efficiency of caprylic acid. The invention researches the influence of oxalic acid, malic acid and citric acid on the recovery efficiency. The experimental results show that: the recovery rate of the citric acid is highest, and the extraction efficiency and the extraction speed of the caprylic acid are improved because the amount of bubbles generated by the citric acid is large and the speed of generating the bubbles is high. Therefore, citric acid is finally selected.
E. The amount of citric acid
(1) The experimental method comprises the following steps: the same as (1) in A, except that citric acid (10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg) was added in different amounts, respectively.
(2) The results of the experiment are shown in FIG. 8.
(3) And (4) analyzing results: the recovery rate is gradually increased along with the increase of the dosage of the citric acid within the range of 10mg to 70mg, because the pH value of the solution is gradually reduced along with the increase of the dosage of the citric acid, the hydrophilic caprylate is favorably converted into the hydrophobic caprylic acid to promote the phase separation speed, the 1-naphthol is ensured to exist in a molecular form, the quantity of generated bubbles is gradually increased, and the extraction efficiency is improved. The decrease in recovery with increasing citric acid dosage in the range of 70mg to 100mg may be due to the inability of 1-naphthol to be completely extracted into octanoic acid due to the fast phase separation, the shortened dispersion time, and the viscous drag. Therefore, 70mg of citric acid was finally used.
Example 3 threaded microfluidic detection part optimization
a. pH optimization of the adjusted solution
(1) The experimental method comprises the following steps: the treated cotton thread is cut into small sections with the length of 1 plus or minus 0.1cm and is adhered to a white plastic plate by double-sided adhesive tape. Preparing aqueous solutions with different pH values (pH 1, pH 2, pH 3, pH 4, pH 5, pH 6, pH),pH 7, pH 8, pH 9, pH 10, pH 11, pH 12, pH 13, pH 14, pH adjusted with HCl in the range of 1 to 6, pH adjusted with NaOH in the range of 7 to 14). Adding 5 μ L of water solution with different pH to 1cm cotton thread, wetting, adding 5 μ L of 4-methoxybenzene diazo tetrafluoroborate (10mmol L)-1) Air drying at room temperature, and adding 7 μ L1-naphthol octanoic acid solution (concentration is 1mmol L)-1) After twenty minutes of reaction, the RGB intensity (I) was calculated by photographing.
(2) The results of the experiment are shown in FIG. 9.
(3) And (4) analyzing results: pH is an important reaction condition in the coupling reaction. The results show that: the line color of the cotton thread with pH values of 13 and 14 is yellow in the drying process, and probably because the diazo salt is changed in a strong alkali environment, only the caprylic acid solution of 1-naphthol is added into thread microfluid equipment with the pH value within the range of 1-12. Pictures were taken after twenty minutes of reaction and RGB intensity (I) was calculated. The results are shown in FIG. 9, which indicates that strong acid and strong base conditions are unfavorable for the coupling reaction. An aqueous solution with a pH of 7 was finally selected for addition to the threaded microfluidic detection apparatus.
b. Optimisation of diazonium salt concentration
(1) The experimental method comprises the following steps: the treated cotton thread is cut into small sections with the length of 1 plus or minus 0.1cm and is adhered to a white plastic plate by double-sided adhesive tape. Preparing different concentrations (5 mu mol L)-1、50μmol L-1、500μmol L-1、1mmol L-1、2mmol L-1、4mmol L-1、6mmol L-1、8mmol L-1、10mmol L-1、12mmol L-1、14mmol L-1) And 4-methoxybenzene diazo tetrafluoroborate aqueous solution. mu.L of an aqueous solution having a pH of 7 was added to 1cm of cotton thread, and after the thread was wetted, 5. mu.L of different concentrations of diazonium salts were added and the thread was dried at room temperature. Finally, 7. mu.L of a 1-naphthol octanoic acid solution (concentration: 1mmol L) was added-1) After twenty minutes of reaction, the RGB intensity (I) was calculated by photographing.
(2) The results of the experiment are shown in FIG. 10.
(3) And (4) analyzing results: the diazo salt and 1-naphthol are subjected to coupling reaction and are important reaction substrates. The results show that: diazonium salt concentrationAt 5. mu. mol L-1～8mmol L-1With increasing diazonium salt concentration the intensity (I) increases, 8mmol L-1～14mmol L-1There was no significant difference in the concentration range. Thus, the final selected concentration was 10mmol L-14-methoxybenzene diazotetrafluoroborate.
c. Optimization of the amount of the extract
(1) The experimental method comprises the following steps: the treated cotton thread is cut into small sections with the length of 1 plus or minus 0.1cm and is adhered to a white plastic plate by double-sided adhesive tape. Adding 5 μ L of pH 7 water solution onto 1cm cotton thread, wetting, adding 5 μ L of 10mmol L-1And (4) naturally drying the 4-methoxybenzene diazo tetrafluoroborate. Finally, different volumes (3. mu.L, 4. mu.L, 5. mu.L, 6. mu.L, 7. mu.L, 8. mu.L) of 1-naphthol octanoic acid solution (concentration of 1mmol L) were added-1) After twenty minutes of reaction, the RGB intensity (I) was calculated by photographing.
(2) The results of the experiment are shown in FIG. 11.
(3) And (4) analyzing results: the intensity (I) of the extract gradually increases within the range of 3-6 μ L. The internal strength (I) of 6-8 mu L reaches a stable state. When the volume of the extracting solution is small, the reaction products are few and cannot be uniformly distributed on a line, and the accuracy of the detection result is influenced. Along with the increase of the volume of the sample extracting solution, the reaction products gradually increase and are uniformly distributed on the line along with the flow of the liquid, so that the accuracy and the sensitivity of the detection result are improved. When more than 8 μ L of the extraction solution is added, the thread cannot absorb an excessive volume of the solution, resulting in a portion of the solution overflowing the cotton thread to be detected, affecting the detection result. Thus, the optimal volume of the sample extract was determined to be 7. mu.L.
d. Reaction time optimization
(1) The experimental method comprises the following steps: the treated cotton thread is cut into small sections with the length of 1 plus or minus 0.1cm and is adhered to a white plastic plate by double-sided adhesive tape. Add 7. mu.L of pH 7 solution (adjusted with NaOH) to the wire, wait for the wire to be fully wetted, and add 5. mu.L of 10mmol L-1The diazonium salt of (a). After the line had dried naturally, 7. mu.L of a solution of 1-naphthol in octanoic acid (concentration 1mmol L) was added-1) After twenty minutes of reaction, the RGB intensity (I) was calculated by photographing.
(2) The results of the experiment are shown in FIG. 12.
(3) And (4) analyzing results: the intensity (I) gradually increased with the reaction time, and there was no significant difference in intensity (I) after the reaction time reached 20 min. Thus, the optimal reaction time for this experiment was determined to be 20 min.
Example 4 preparation of a sevin Standard Curve
(1) The experimental method comprises the following steps: weighing 1g of pulverized sample (rice, wheat, soybean, Chinese cabbage; the addition concentration of the sample is 0.30mg kg-1、1.00mg kg-1、5.00mg kg-1、15.00mg kg-1、30.00mg kg-1、50.00mg kg-1) 1mL of acetonitrile was added and vortex extracted for 3 min. The extract was filtered through a 0.22 μm filter, and 600 μ L of sample filtrate was added to 4.4mL of water in a 10mL centrifuge tube. Adding 40mg sodium carbonate to adjust the pH of the solution to alkalinity so that carbaryl is hydrolyzed into 1-naphthol, adding 80. mu.L caprylic acid extractant, and converting caprylic acid into hydrophilic salt under the alkaline condition. Then 70mg citric acid is added, the caprylate salt generates in situ a hydrophobic caprylic acid extractant and the effervescence generates bubbles that assist in completing the extraction. Centrifuging for 3min under the condition of 5000r/min, wherein the organic phase of the 1-naphthol octanoic acid is positioned at the upper layer of a centrifugal tube. The centrifuge tube was ice-cooled to solidify the organic phase and remove the lower aqueous phase. After the organic phase is melted, 7 mu L of 1-naphthol caprylic acid extracting solution is taken out and added to the cotton thread after the diazo salt is dripped, and after twenty minutes of reaction, the RGB intensity (I) is calculated by photographing. And (3) plotting the concentration of the carbaryl as an abscissa and the concentration of the carbaryl as an ordinate, wherein I is 255-G, so as to obtain the linear detection range of different matrixes. The standard deviation of each substrate was calculated for 10 blanks according to the formula for limit of quantitation (LOQ): LOQ 10 σ/K and limit of detection (LOD): the LOD is 3 sigma/K (sigma is the standard deviation of blank sample; the slope of K working curve) to calculate the limit of quantification and detection under the optimized condition.
(2) The results of the experiment are shown in table 1.
TABLE 1 Linear equation for food samples
(3) And (4) analyzing results: the content of carbaryl is 0.30mg kg-1～30mg kg-1There is a good linear relationship within the concentration range of (c). R20.9929-0.9982, and a limit of detection (LOD) of 0.012mg kg-1～0.016mg kg-1The limit of quantitation (LOQ) is 0.040mg kg-1～0.054mg kg-1. The experimental results show that the method has higher sensitivity and accuracy.
Example 5 detection of actual samples
(1) The experimental method comprises the following steps: weighing 1g of pulverized sample (rice, wheat, soybean, Chinese cabbage; the addition concentration of the sample is 1.00mg kg-1、15.00mg kg-1、30mg kg-1) 1mL of acetonitrile was added and vortex extracted for 3 min. The extract was filtered through a 0.22 μm filter, and 600 μ L of sample filtrate was added to 4.4mL of water in a 10mL centrifuge tube. Adding 40mg sodium carbonate to adjust the pH of the solution to alkalinity so that carbaryl is hydrolyzed into 1-naphthol, adding 80. mu.L caprylic acid extractant, and converting caprylic acid into hydrophilic salt under the alkaline condition. Then 70mg of citric acid was added, the caprylate salt converted to hydrophobic caprylic acid and the effervescence generated bubbles assisted the completion of the extraction. Centrifuging for 3min under the condition of 5000r/min, wherein the organic phase of the 1-naphthol octanoic acid is positioned at the upper layer of a centrifugal tube. The centrifuge tube was ice-cooled to solidify the organic phase and remove the lower aqueous phase. After the organic phase is melted, 7 mu L of 1-naphthol caprylic acid extracting solution is taken out and added into a white plastic plate on a cotton thread (in the white plastic plate) containing the diazonium salt, and after twenty minutes of reaction, the mixture is photographed to calculate the RGB intensity (I). The recovery was calculated according to the following formula:
Cfound: represents the total concentration of the analyte obtained after adding a standard substance with a known concentration to the actual sample; crealInitial concentration of the object to be measured in the actual sample; cadded: the concentration of the known analyte is added.
(2) The results of the experiment are shown in table 2.
Table 2 additive recovery of food samples
(3) And (4) analyzing results: the sample recovery rate is 92.3% -105.9%, and the RSD is less than 4.65% (n is 3). The result shows that the test result is accurate and reliable, the precision is high, and the method can be widely used for rapidly detecting the carbaryl content of food samples on site.
Example 6 study of the stability of threaded microfluidic devices
(1) The experimental method comprises the following steps: the treated cotton thread is cut into small sections with the length of 1 plus or minus 0.1cm and is adhered to a white plastic plate by double-sided adhesive tape. Add 7. mu.L of pH 7 solution (adjusted with NaOH) to the wire, wait for the wire to be fully wetted, and add 5. mu.L of 10mmol L-1The diazonium salt of (a). After the strands were naturally dried, they were placed under two different storage conditions: a: storing the μ TAD in a refrigerator; b: the μ TAD was stored in a desiccator. At the same time point each day, the same time point was taken out of the storage conditions and 7. mu.L of 1-naphthol octanoic acid solution (concentration: 1mmol L) was added-1) After twenty minutes of reaction, the RGB intensity (I) was calculated by photographing. The stability of μ TAD under two different storage conditions for 10 days was examined.
(2) The results of the experiment are shown in FIG. 13 (dryer on the left and refrigerator on the right).
(3) And (4) analyzing results: the detection result of the detection device under two different storage modes has no obvious change within 10 days, which shows that the device has good stability and can be used for detecting actual samples.
Claims (9)
1. A method for detecting the content of the Chinese and western vitamin in food by a digital image colorimetric method comprises the following steps:
(1) treating cotton thread to be neutral, fixing the cotton thread on a plate, and adding diazonium salt after wetting;
(2) crushing a sample to be detected, extracting to obtain an extracting solution, and filtering by using a membrane;
(3) sequentially adding carbonate, an extracting agent and citric acid into the extracting solution, and taking an upper organic phase after centrifugal treatment;
(4) dropwise adding the organic phase onto the cotton threads for reaction; photographing the cotton threads, and calculating the RGB intensity according to the formula (1);
I＝255–G (1)
wherein, I represents RGB intensity, G represents the value of green channel in image RGB mode;
(5) preparing carbaryl standard substance solutions with different concentrations, and obtaining RGB (red, green and blue) intensities corresponding to the carbaryl standard substance solutions according to the step (3) and the step (4); taking the concentration of the carbaryl standard as an abscissa and the RGB intensity as an ordinate to prepare a standard curve; and (5) obtaining the concentration of carbaryl in the sample to be detected according to the standard curve and the RGB intensity measured in the step (4).
2. The method of claim 1, wherein: in the step (1), the cotton threads are processed to be neutral according to the following steps: placing the cotton thread on Na2CO3Washing in boiling solution;
the concentration of the sodium carbonate boiling solution is 10mg mL-1The washing time is 5 min;
fixing the cotton thread on the board by using double-sided adhesive tape;
the cotton threads were wetted with an aqueous solution having a pH of 7.
3. The method according to claim 1 or 2, characterized in that: in the step (1), the diazonium salt is 4-methoxybenzene diazonium tetrafluoroborate or p-nitrobenzene diazonium tetrafluoroborate;
adding the diazonium salt as a solution of the diazonium salt;
the concentration of the solution of the diazonium salt is 6-10 mmol L-1；
The amount of the solution of the diazonium salt used was 7. mu.L relative to the cotton thread having a length of 1. + -. 0.1 cm.
4. The method according to any one of claims 1-3, wherein: in the step (2), performing vortex extraction by using acetonitrile;
the dosage of the acetonitrile is as follows: 1 to 5mL of-1A sample to be tested;
the vortex extraction time is 3-10 min;
the diameter of the filter membrane is 0.22 μm.
5. The method according to any one of claims 1-4, wherein: in the step (3), the adding amount of the sodium carbonate is 35-50 mg g-1A sample to be tested;
the extractant is caprylic acid or pelargonic acid;
the dosage of the extracting agent is 60-160 mu L g-1A sample to be tested;
the dosage of the citric acid is 60-80 mg g-1And (5) testing the sample to be tested.
6. The method according to any one of claims 1-5, wherein: in the step (3), the centrifugation conditions are as follows: the rotating speed is 5000r/min, and the time is 3 min;
after the centrifugation, the organic phase was solidified by ice bath.
7. The method according to any one of claims 1-6, wherein: in the step (4), the dropping amount of the organic phase is 6-8 mu L g-1A sample to be tested;
the reaction time was 20 min.
8. The method according to any one of claims 1-7, wherein: in the step (4), photographing is carried out in the following photographing devices:
the periphery and the top are all reflectors, the bottom is a light softening plate, and the top is provided with a light source.
9. The method according to any one of claims 1-8, wherein: in the step (5), the concentration of the carbaryl standard substance solution is 0.30-30.00 mg kg-1。
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