WO2020192228A1 - Method for screening compound by means of high-performance thin-layer chromatography combined with bioluminescence method - Google Patents

Abstract

A method for screening a compound by means of a high-performance thin-layer chromatography combined with a bioluminescence method, relating to the technical field of food detection. The high-performance thin-layer chromatography and the bioluminescence method are combined for use. The method comprises: spreading a sample to be detected on a thin-layer plate, then coupling to a luminescent bacterium, performing luminescence imaging, and finally performing scanning by using the thin-layer chromatography. The method for screening the compound by means of the high-performance thin-layer chromatography combined with the bioluminescence method can be used for detecting multiple samples, however, only one sample can be detected by using a high-performance liquid chromatography (HPLC). Compared with an existing HPLC, the present invention can achieve fast screening, simultaneously detect multiple kinds of samples in a single experiment, and achieve high throughput screening; a detection limit can reach 0.5-1 mg/kg; the detection method can achieve the repeatability less than 3.4%, and is economic, fast, simple and convenient.

Description

Method for screening compounds with high-efficiency thin-layer chromatography combined with bioluminescence Technical field

The invention relates to a method for screening compounds by high-efficiency thin-layer chromatography combined with bioluminescence, and belongs to the technical field of food detection.

Background technique

Nifedipine (Nifedipine, NDP) is an antihypertensive chemical drug of the dihydropyridine class. Its efficacy principle is that it is a calcium antagonist, which can selectively inhibit the transmembrane transport of calcium ions into cardiomyocytes and smooth muscle cells, and inhibit Calcium ions are released from the cells without changing the plasma calcium ion concentration, thereby achieving the effect of lowering blood pressure. Nifedipine-type blood pressure lowering chemical drugs have good efficacy and low side effects, and are easily used for adulteration of health tea. In the health care product market, blood pressure-lowering health care products derived from dual-purpose materials for medicine and food occupy a large share. According to the cases disclosed by the media and the Food and Drug Administration, some criminals illegally added the western drug nifedipine to the blood pressure-lowering health care products and illegally mixed them in the blood pressure-lowering health care products in order to achieve the blood pressure and blood sugar lowering effects claimed in the product advertisements. Western medicine phenformin. These behaviors are not only suspected of commercial fraud, but because the addition of chemical drugs has the characteristics of large doses and randomness, consumers will often take long-term use due to temporary and significant effects without knowing it, causing serious toxicity and side effects. In severe cases, it can even be life-threatening. At present, there is no special detection project for the adulteration of illegal chemical drugs in these drugs. Therefore, it is of great significance to establish a screening and detection method for the illegal adulteration of Chinese and Western medicines for blood pressure lowering health products.

At present, the commonly used detection method for nifedipine in food and drugs is high performance liquid chromatography. The sample pretreatment of this method is complicated, most impurities in the sample need to be removed, and the detection time is long. Generally, a sample requires 20 minutes, and the pre-experiment It takes a long time for the system to balance, and the measurement process can only be performed on samples one by one.

N-(trichloromethylthio)-4-cyclohexene-1,2-dicarboximide, belongs to the organic sulfur substance, is one of the broad-spectrum fungicides most widely used for plant protection purposes in agricultural production. Especially for all kinds of fruits and vegetables after harvest, captan has good fresh-keeping performance, which can ensure that the fruits still have a fresh and healthy appearance after storage and processing. Nevertheless, people have noticed the possibility of abuse of captan and its potential threat to public health. Recent studies have shown that in addition to acute poisoning symptoms such as vomiting and conjunctivitis, exposure to captan at low dose levels may also cause damage to the human reproductive system. Therefore, rapid, simple and reliable screening of captan in fruits and vegetables is essential to public food safety. Therefore, it is extremely important to establish a fast and reliable analysis method for rapid screening of captan residues in fruit food.

However, it is worth noting that the characteristics of captan molecules have brought great difficulties to conventional analysis methods. Specifically, captan molecules are not only difficult to ionize under electrospray conditions, but also very unstable at high temperatures and easily decomposed. Due to these limitations, captan is actually neither suitable for HPLC-MS analysis nor GC analysis, although they are the most widely used tools for the confirmation and quantification of hazardous residues in food. In addition, special attention should be paid to that the stability of captan molecules is also very sensitive to pH conditions. It is necessary to avoid the degradation of captan due to pH fluctuations during sample preparation and cleaning. For example, the use of primary-secondary amine reagents as solid phase extraction adsorbents to clean up food samples is a necessary step before column chromatography analysis. However, the addition of this reagent may cause the pH of the extraction solution to rise to neutral or weakly alkaline conditions (pH=7-9), which will eventually lead to the decomposition of captan and give false negative results. It can be seen that the existence of the above-mentioned problems makes it difficult for traditional column chromatography to achieve accurate analysis of captan.

Luminescent bacteria are a class of microorganisms that can emit visible light under normal physiological conditions. The principle of luminescence is that under the action of luciferase catalysis and molecular oxygen, long-chain fatty aldehydes and reduced flavin mononucleotides (FMNH2) are oxidized into long-chain fatty acids and oxidized flavin mononucleotides (FMN), and released at the same time Blue-green light of 450-490nm wavelength. Based on the characteristics of luminescent bacteria, it is often used as the optical sensing element of biological detectors. Compared with common physical and chemical detection methods, the biggest advantage of luminescent bacteria lies in the non-targeting of detection ability: its sensing principle is not based on the recognition of specific chemical structures, but on the degree of bioluminescence inhibition to characterize the toxicity of the target, that is, when When there is interference from toxic substances in the external environment, the physiological process or cellular respiration of the luminescent bacteria is affected, resulting in the inhibition of the luminescence reaction, and the content of toxic substances is related to the degree of weakening of the luminous intensity of the bacteria.

Compared with the toxicity test methods of zebrafish, nematodes and other model organisms with complex preparations and long cycles, the luminescent bacteria method has the advantages of simple operation (use directly after the lyophilized powder of bacteria is recovered) and fast and efficient (within a few minutes at the fastest) Result), so it has a broader application prospect. At present, analysis methods based on luminescent bacteria have played an important role in the EU drinking water safety and early warning system. In my country, luminescent bacteria detection technology has played an important role in water quality emergency assurance in the "5.12 Wenchuan earthquake" disaster area and Taihu Lake water quality monitoring.

Generally, densitometer or microplate reader is the instrument carrier for luminescent bacteria method. The analyst only needs to mix the bacterial solution with the sample, and after a period of reaction, quantitatively detect the change in luminescence intensity. Although this model is simple and efficient, it has the following shortcomings: ①Serious background interference; ②Lack of selectivity for multiple targets.

At present, HPTLC is the only chromatography tool that can be used directly with cell-based biosensors. Unlike the traditional test tube method, the combination with HPTLC can fundamentally solve the problem of strong background interference and poor selectivity in the luminescent bacteria method. HPTLC separation allows the originally mixed targets to be spread out to different positions on the thin-layer plate to form physical isolation according to the difference in molecular structure; subsequently, the luminescent bacteria coupled with the thin-layer plate can be easily diversified in the sample by dipping. Simultaneous detection of targets. Therefore, the analytical method based on the combination of the two has the advantages of high selectivity and good versatility. It has become an emerging hot frontier of analytical chemistry and plays an important role in many fields such as environmental monitoring and natural product analysis.

Summary of the invention

In view of the above problems, the present invention directly uses HPTLC separation so that the originally mixed targets are spread out to different positions on the thin-layer plate to form physical isolation according to the difference in molecular structure; subsequently, the luminescent bacteria coupled with the thin-layer plate are immersed Can quickly realize the simultaneous detection of multiple targets in the sample.

The first object of the present invention is to provide a high-efficiency thin-layer chromatography detection method, which combines high-efficiency thin-layer chromatography and bioluminescence.

In one embodiment of the present invention, the specific steps are: first unfold the sample to be tested on a thin-layer plate, then couple with the luminescent bacteria, then undergo luminescence imaging, and finally scan with thin-layer chromatography.

In one embodiment of the present invention, the thin-layer board includes a silica gel board, a cellulose board, an acid alumina board and an alkaline alumina board.

In an embodiment of the present invention, the imaging exposure time is 20-50s respectively.

The second object of the present invention is to provide an application of the above method in the field of compound detection.

The third object of the present invention is to provide a sample containing nifedipine by using the above method.

In one embodiment of the present invention, the developing agent is a mixed solution of toluene and ethyl acetate, and the volume ratio of toluene and ethyl acetate is 4:6.

In one embodiment of the present invention, the thin layer plate includes a silica gel plate, a cellulose plate, an acid alumina plate, and an alkaline alumina plate. Preferably, the thin layer plate is a silica gel plate and a cellulose plate.

In one embodiment of the present invention, the luminescent bacteria include: (1) Photobacterium psychrophilus CGMCC 1.8932; (2) Photobacterium indica CGMCC 1.8728; (3) Photobacterium hydrofluoride, CGMCC 1.12159, preferably, the luminescent bacteria is Photobacterium psychrophilus CGMCC 1.8932.

In one embodiment of the present invention, the coupling conditions are: luminescent bacteria are cultured in a shake flask at 80-150r/min for 10-16h in an environment of 24-26°C to obtain a bacterial suspension, the dosage is 80-120mL, and the coupling time is 1- 3s, the temperature is 24-26℃, and the pH is 6.5-7.5.

In one embodiment of the present invention, the luminescence imaging conditions are: imaging exposure time is 20-50s, interval 1～3min, and at least 10 photos are taken. Preferably, imaging exposure time is 20-40s.

In an embodiment of the present invention, the thin-layer chromatography scanning condition is: analog scanning of the obtained digital image by videoscan software can realize accurate quantitative analysis of detection results.

In one embodiment of the present invention, the sample to be tested is a health product, food or medicine.

The fourth object of the present invention is to provide a sample for screening captan residues in fruits by using the above method.

In one embodiment of the present invention, the detection method is specifically: preparing the fruit sample to be tested through a simple A-QuEChERS extraction step, then spreading the extract on a thin-layer plate, and then dipping The photoluminescent bacterium suspension is coupled with the chromatographic separation result, the bioluminescence imager is used to record the inhibitory effect of the target on the bacterial bioluminescence, and the detection result is quantitatively analyzed with the help of digital image software analysis.

In one embodiment of the present invention, the mobile phase used for the development is toluene: ethyl acetate=8: 2 (v/v).

In one embodiment of the present invention, the bioluminescence autoradiography detection is based on the detection of Photobacterium luminescens biosensing on high-efficiency thin-layer chromatography, the suspension of Photobacterium luminescens is the liquid required for immersion coupling, and the luminescence bacteria liquid Incubate in a shaker at 25°C and 100r/min for 36h, and then be used for immersion coupling of the expanded thin-layer plate.

In one embodiment of the present invention, the bioluminescence imager is under visualization conditions, and the detection area is the target after chromatographic separation interferes with the physiological metabolism of bacteria in the window area, which causes the inhibition of bioluminescence to form black spots, which provides for visual inspection. Conveniently, the quantitative analysis of imaging results can be realized by using analysis based on digitized image pixel analog scanning.

In one embodiment of the present invention, the sample to be tested is developed by high-efficiency thin-layer chromatography to separate the target substance captan and the sample in the extract interfering with the matrix substance, and then the chromatographic separation result is separated from Photobacterium luminescens by dipping. Biosensing is coupled to quantitatively evaluate the residual concentration of target substances in fruits through bioluminescence imaging combined with digital image analysis.

In one embodiment of the present invention, the fruit samples to be tested include apples, pears and cherries.

In one embodiment of the present invention, add 10g of the homogenate of the fruit sample into a 45-55mL centrifuge tube, continue to add 8-12mL of acetonitrile, 0.1mL of formic acid, shake and mix, and ultrasonic water bath for 8-12min; then add 4g of anhydrous Magnesium sulfate and 1g anhydrous sodium acetate, shake and mix well, centrifuge at 3500-4500r/min for 4-6min, take 2-4mL of the upper acetonitrile phase, and pass through a 0.45μm filter membrane to obtain a liquid sample that can be directly used for analysis.

In an embodiment of the present invention, the expanded and dried thin-layer plate is subjected to bioluminescence autoradiography detection, and then the detection result is recorded by the bioluminescence imaging device, and a very intuitive semi-quantitative result can be obtained by visual inspection.

In an embodiment of the present invention, the videoscan software is used to perform analog scanning on the obtained digital image, which can realize accurate quantitative analysis of the detection result.

In one embodiment of the present invention, the thin-layer plate obtained after chromatographic expansion and bioluminescence auto-imaging is placed on a bioluminescence imager to take pictures, and combined with digital image software for quantitative analysis, by drawing a standard curve, the calculation of the fruit sample The content of target captan. According to the detection results obtained by scanning quantification, a standard curve is obtained: y=163.46x+1336.1, and the content of captan in the fruit sample is calculated; where y is the chromatographic peak area in AU; x is the content of captan in ng.

The beneficial effects of the present invention:

(1) The present invention establishes a high-efficiency thin-layer chromatography combined with bioluminescence method for screening nifedipine. The method of the present invention can detect multiple samples, up to 20 samples can be detected at a time, and the duration of a single experiment is 30 minutes. However, the HPLC method can only detect one sample at a time, and the duration of a single experiment is 20 minutes. Therefore, compared with the existing high-efficiency liquid phase method, the method of the present invention can realize rapid screening, and simultaneously detect multiple samples in a single experiment, realizing high-throughput screening; the recovery rate of the method of the present invention is 96.8%-98.7%, The detection limit can reach 0.5-1mg/kg, and the detection method can achieve repeatability RSD<2.3%, which has the advantages of economy, speed and simplicity.

(2) The present invention also establishes a high-efficiency thin-layer chromatography-bioluminescence auto-imaging combination method for rapid and quantitative screening of captan residues in fruits. A single run detects multiple samples simultaneously, realizing high-throughput screening Requirements. The detection limit can reach 0.5～1mg/kg, the detection method can achieve repeatability RSD<3.4%, and the recovery rate of standard addition is 78.4%-97.7%. This method has the advantages of fast, accurate and economical; it is also based on high-efficiency thin-layer chromatography and The establishment of the combined detection method of bioluminescence and autography provides a method example for the rapid and reliable screening of harmful residues in food with unobvious spectral characteristics.

Description of the drawings

Figure 1 shows the linear relationship of different doses of nifedipine on the inhibition of bioluminescence in Example 2.

Figure 2 shows the coupling effect of thin-layer plates of different materials and luminescent bacteria in Example 3. The spotting tracks: (1) silica gel plate; (2) cellulose plate; (3) acid alumina plate; (4) alkali Alumina board.

Figure 3 shows the coupling effect of different luminescent bacteria and thin-layer plates in Example 3, where the spot track: 1 is Photobacterium psychrophilus CGMCC 1.8932; 2 is Photobacterium indica CGMCC 1.8728; 3 is Photobacterium hydrophobe, CGMCC 1.12159.

4 is a photographing effect diagram of different exposure times in Embodiment 3.

Figure 5 is the bioluminescence image of the sample and the standard product after development in Example 4. The spotting track: 1 is health care product 1, 2 is health care product 1 + nifedipine standard solution, and 3 to 4 are nifedipine standards Solution, 5 is health care product two, 6 is health care product two + nifedipine standard solution.

Figure 6 shows the expanded view and linear relationship of the standard solution of gtandan in Example 5.

Figure 7 is an expanded view of the matrix effect of Example 5 with a black and white background.

Fig. 8 is a scanning image of the matrix effect of Example 5, with a color background.

Figure 9 shows the thin-layer plates of different materials in Example 6 and their influence on the test results; among them, (1) silica gel plates; (2) cellulose plates; (3) acid alumina plates; (4) alkaline alumina plates.

Figure 10 shows the effects of different luminescent bacteria on the test results in Example 6; among them, (1) Photobacterium luminescens ATCC 11040; (2) Photobacterium psychrophilus CGMCC 1.8932; (3) Photobacterium indica CGMCC 1.8728; (4) water Photobacterium photobacterium, CGMCC 1.12159.

Figure 11 shows the influence of different developing agents on the test results in Example 6; among them, (1) is toluene: ethyl acetate = 6: 4 (v/v); (2) toluene: ethyl acetate = 7: 3 ( v/v); (3) Toluene: ethyl acetate = 8: 2 (v/v).

Figure 12 is an expanded view of the recovery rate of standard addition in Example 7; among them, 1: apple; 2: pear; 3: captan standard solution; 4: cherry.

detailed description

The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention, and are not intended to limit the present invention.

The bacteria used in the following examples (Photobacterium psychrophilus CGMCC 1.8932; Photobacterium indica CGMCC 1.8728; Photobacterium aquaticum, CGMCC 1.12159; Photobacterium luminescenti ATCC 11040; Photobacterium aquaticum, CGMCC 1.12159;) are all purchased from the Microbial Culture Collection .

Example 1: Preparation of working luminescence suspension of high performance thin layer chromatography combined with bioluminescence method

The preparation process of the working luminescent suspension is as follows:

(1) Preparation of simulated seawater liquid:

Prepare simulated seawater liquid culture medium according to the following formula: 30g/L NaCl, 5g/L Na ₂ HPO ₄ , 5g/L KH ₂ PO ₄ , 3mL/L glycerol, 5g/L peptone and 5g/L yeast extract; add 1L After the ultrapure water is stirred and dissolved, the pH value of the prepared liquid medium is adjusted to 7 with a 1mol/L sodium hydroxide aqueous solution, and then sterilized in an autoclave at 121°C for 15 minutes, and the prepared liquid medium is sealed The device is kept in the refrigerator for later use, and can be stored for 7 days at 4°C when not in use;

(2) Cultivation and preservation of luminescent bacteria: inoculate the luminescent bacteria frozen and preserved with glycerin into a triangular flask containing the 100mL liquid culture medium prepared in step (1); the bottle mouth is wrapped with a sterile four-layer folded foil paper To ensure that outside oxygen can enter the bottle during the culture process, shake the flask at 100r/min at 25°C to obtain the bacterial mother liquor; then add an equal volume of fresh liquid medium to the mature bacterial mother liquor to obtain a working luminous suspension When the working luminescent suspension is not in use, it can be stored at 4°C for 3 days.

Example 2: Drawing of nifedipine standard curve based on high performance thin layer chromatography combined with bioluminescence method

(1) Preparation of standard solution: accurately weigh 50mg of nifedipine standard with an electronic balance, place it in a 10mL volumetric flask, and dilute with methanol to obtain a standard stock solution with a concentration of 5mg/mL; keep the standard stock solution away from light at ordinary times Place it in a 4℃ environment. When the analysis is about to start, take out 1mL and place it in a 10mL volumetric flask, dilute with methanol to a constant volume to obtain a standard working solution with a concentration of 0.5mg/mL; the standard working solution must be reconfigured before each test;

(2) Use the prepared standard solution directly for HPTLC spotting;

(3) Chromatographic separation: first use a 100μL spotting needle to manually draw the solution to be tested, and quantitatively purge it to a position 10mm away from the bottom of the thin layer plate with the assistance of 0.5MPa nitrogen flow by a semi-automatic thin layer spotting instrument, The scanning speed is 100nL/s, the pre-arrangement volume is 0.2μL, the strip width is 6mm, and the distance from both sides is at least 15mm; each sample prepared by steps (1) and (2) is spotted, and one sample is spotted manually after finishing Take out the spotting needle and wash it with methanol three times before spotting the next sample; after spotting all samples, take out the thin-layer plate and dry it with a hair dryer for 1 min. The purpose is to volatilize the residual methanol in the spotting origin. Does not affect the subsequent steps;

The chromatographic separation is carried out in a fully automatic thin-layer developing instrument, the mobile phase is a mixture of toluene/ethyl acetate, the volume ratio is 4/6, v/v, and the expansion distance is 60mm; chromatographic conditions: the expansion cylinder is controlled by bubbling saturated magnesium chloride solution Relative humidity, last for 3 minutes, adjust the relative humidity to 35%, and pre-equilibrate the thin layer plate for 10 minutes; when the mobile phase front reaches the predetermined height, the system automatically ends, take the thin layer plate out, and place it on the thin layer heater at 80°C for 5 minutes , In order to volatilize and remove the residual organic solvent on the thin layer;

(4) Bioluminescence imaging detection: Use an automatic dipping device to immerse the unfolded and dried thin-layer plate in the working luminescent suspension, the immersion speed is 1mm/s, the residence time is 2s, and then the thin-layer plate immersed in the working luminescent suspension is placed Into the bioluminescence imager for imaging detection, the imaging exposure time is 40s, the interval between each photo is 2min, and 15 photos are taken continuously;

(5) Analysis: Save the photo taken by the bioluminescence imager, then open it with Videoscan software, digitally analyze the pixel polarity in the picture to obtain a processable chromatogram, and then set the integration parameters and conditions for quantitative analysis.

The linear relationship between different doses of nifedipine on the inhibition of bioluminescence is shown in Figure 1. When the concentration of nifedipine is 0.05～0.30mg/g, the concentration of nifedipine has a linear relationship with the chromatographic peak area, and the linear equation is y=39.127 x-479.432, R ² =0.99752, the detection limit is 0.5-1μg/kg.

Example 3: Optimizing the conditions for detecting nifedipine by high performance thin layer chromatography combined with bioluminescence

1. The influence of different silica gel plates on the test results

The detection principle of the method is that the luminescent strain is specifically a type of microorganism that can emit visible light under normal physiological conditions. The luminescence principle of this type of bacteria is that long-chain fatty aldehydes and reduced flavin mononucleotides (FMNH2) are oxidized to long-chain fatty acids and oxidized flavin mononucleotides (FMN) under the catalysis of luciferase and molecular oxygen. At the same time, blue and green light of 450-490nm wavelength is emitted. Based on this feature, luminescent bacteria are often used as optical sensing elements of biological detectors. Compared with common physical and chemical detection methods, the biggest advantage of luminescent bacteria biosensing lies in the non-targeting of detection ability: its sensing principle is not based on the recognition of specific chemical structures, but on the degree of bioluminescence inhibition to characterize the toxicity of the target .

The experiment process is as follows:

(1) Prepare extracts of blood pressure-lowering health care products separately: Weigh 1.0g of blood-lowering health care product powder, add 10mL methanol, extract in an ultrasonic water bath at 25°C for 30 minutes, take it out and centrifuge at 5000 rpm for 10 minutes, after centrifugation, take the upper 5mL clear solution and pass 0.45 Filtration with a μm nylon filter membrane, and the resulting sample clear liquid is directly used for HPTLC spotting;

(2) Chromatographic separation: first use a 100μL spotting needle to manually draw the solution to be tested, and quantitatively purge it to a position 10mm away from the bottom end of the thin layer plate with the assistance of 0.5MPa nitrogen flow by a semi-automatic thin layer spotting instrument, The scanning speed is 100nL/s, the pre-arrangement volume is 0.2μL, the strip width is 6mm, and the distance from both sides is at least 15mm; each sample prepared by steps (1) and (2) is spotted, and one sample is spotted manually after finishing Take out the spotting needle and wash it with methanol three times before spotting the next sample; after spotting all samples, take out the thin-layer plate and dry it with a hair dryer for 1 min. The purpose is to volatilize the residual methanol in the spotting origin. Does not affect the subsequent steps; the thin-layer board material includes (1) silica gel board; (2) cellulose board; (3) acid alumina board; (4) alkaline alumina board;

The chromatographic separation is carried out in a fully automatic thin-layer developing instrument, the mobile phase is a mixture of toluene/ethyl acetate, the volume ratio is 4/6, v/v, and the spreading distance is 60mm; the chromatographic conditions: the spreading cylinder is controlled by bubbling a saturated magnesium chloride solution Relative humidity, last 3 minutes, adjust the relative humidity to 35%, and pre-equilibrate the thin layer plate for 10 minutes; when the mobile phase front reaches the predetermined height, the system will automatically end, take the thin layer plate out, and place it on the thin layer heater at 80°C for 5 minutes , In order to volatilize and remove the residual organic solvent on the thin layer;

(3) Bioluminescence imaging detection: Use an automatic dipping device to immerse the unfolded and dried thin-layer plate into the working luminescent suspension, the immersion speed is 1mm/s, the residence time is 2s, and then the thin-layer plate immersed in the working luminescent suspension is placed Enter the bioluminescence imager for imaging detection, the imaging exposure time is 40s, the interval between each photo is 2min, and the continuous shooting is 15; the luminescent bacteria selects Photobacterium psychrophilus CGMCC 1.8932;

(4) Analysis: Save the photo taken by the bioluminescence imager, then open it with Videoscan software, digitally analyze the pixel polarity in the picture to obtain a processable chromatogram, and then set the integration parameters and conditions for quantitative analysis.

Figure 2 shows the coupling effect diagram of the thin-layer plates of four different materials and the photobacterium Photobacterium psychrophilus. It can be seen from Figure 2 that after the thin-layer plate made of cellulose, acidic alumina and alkaline alumina material is coupled with the luminescent bacteria, the band imaging is not clear. Only the thin-layer plate made of silica gel material shows clear bands, indicating the most The stationary phase suitable for screening nifedipine is a silica gel plate.

2. The influence of different luminescent bacteria on the test results

Using the above-mentioned preferred thin-layer plate, the difference is that: the luminescent bacteria include: (1) Photobacterium psychrophilus CGMCC 1.8932; (2) Photobacterium india CGMCC 1.8728; (3) Photobacterium hydrofluoride, CGMCC 1.12159, other steps and parameters As in the first part of Example 3, the coupling effect diagram of the luminescent bacteria of several different strains and the silica gel plate is shown in Figure 3.

It can be seen from Figure 3 that the band imaging is not clear after the photobacteria of Photobacterium indica and Photobacterium hydrophobes are coupled with the thin-layer plate, and only the photobacterium of Photobacterium psychrophilus shows clear bands, indicating that it is most suitable for screening nitrobenzene The luminescent bacteria of Diping is Photobacterium psychrophilus.

3. The influence of different exposure conditions

Using the above-mentioned preferred thin-layer plate and luminescent bacteria, the difference is that: the imaging exposure time is respectively 50s, 40s, 30s, 20s, and the other steps and parameters are the same as the first part of Example 3. By selecting exposure times under different conditions for imaging, the results are shown in Figure 4, indicating that the optimal exposure time for nifedipine screening is 40s.

Example 4 Performance test of high performance thin layer chromatography combined with bioluminescence method to detect nifedipine

The sample test steps are as follows:

(1) Repeatable experiment

Using the above-mentioned preferred detection method, respectively detect 50mg/kg, 70mg/kg, 90mg/kg and 110mg/kg samples, repeat 10 times for each concentration, calculate the standard deviation (SD), relative standard deviation (RSD) and coefficient of variation (CV). The results are shown in Table 1. The CV of the measurement results is less than 2.3%, indicating that the method has good repeatability in the linear range.

Table 1 Repeatability experiment

(2) Standard addition recovery experiment

Add different concentrations of nifedipine standard to the sample, and use the above-mentioned preferred high-performance liquid thin-layer chromatography combined with biological method for detection. The expanded view of the sample and standard is shown in Figure 5, and each concentration is measured 5 times in parallel , Take the average value and compare it with the HPLC method. The measured recovery rate of standard addition is shown in Table 2. The average recovery rate is 98%, and the overall test results are consistent with the HPLC results, indicating that the test strip has a good Accuracy. Among them, the specific operation method of HPLC is: octadecyl-bonded silica gel is used as a chromatographic column, methanol-water is used as the mobile phase for gradient elution, the detection wavelength is 235nm, and the column temperature is 30°C.

Table 2 Standard recovery rate

In addition, the method of the present invention can detect multiple samples, up to 20 samples at a time, and the duration of a single experiment is 30 minutes, while the HPLC method can only detect one sample at a time, and the duration of a single experiment is 20 minutes. When the accuracy of the two methods is equivalent, the method of the present invention can realize rapid screening, and a single experiment can detect 20 samples at the same time, realizing high-throughput screening, and having the advantages of economy, speed, and simplicity.

Example 5: Drawing of Captan standard curve based on high performance thin layer chromatography combined with bioluminescence method

(1) Preparation of captan standard solution: use ethyl acetate as a solvent to prepare a standard solution of captan with a concentration of 0.01 mg/mL, and then to establish a standard curve, the standard solution must be diluted to 0.001 and 0.0005 mg/mL ；

(2) High-performance thin-layer chromatography: 4-8μL of captan standard, apple, pear and cherry samples are accurately spotted with Linomat 5, and the developing solution (toluene: ethyl acetate = 8: 2(v/v)) Unfold, the upward unfolding distance is 60mm, after unfolding, take out the silica gel plate and place it on a flat heater at 100℃ to fully dry it for 5min;

(3) Bioluminescence imager imaging: the step (2) high-efficiency thin-layer chromatography plate is dipped to couple the photoluminescent bacillus suspension with the chromatographic separation result, and then placed in the bioluminescence imager for imaging;

(4) Quantitative analysis of digital image software: The thin-layer plate obtained after chromatographic expansion and bioluminescence auto-development is placed on a bioluminescence imager to take pictures, and then quantitative analysis is combined with digital image software. After the scan, the peak area of the scan chromatogram is taken as the y-axis, and the content of captan on the x-axis is used to prepare a standard curve.

The expansion diagram and linear relationship of captan standard solution are shown in Figure 6, the matrix effect expansion diagram is shown in Figure 7, and the matrix effect scanning diagram is shown in Figure 8. Calculate the content of captan in the sample by the standard curve.

Example 6: Optimizing the conditions for detecting captan by high performance thin layer chromatography combined with bioluminescence

1. The influence of different silica gel plates on the test results

The detection principle of the method is that the luminescent strain is specifically a type of microorganism that can emit visible light under normal physiological conditions. The luminescence principle of this type of bacteria is that long-chain fatty aldehydes and reduced flavin mononucleotides (FMNH2) are oxidized to long-chain fatty acids and oxidized flavin mononucleotides (FMN) under the catalysis of luciferase and molecular oxygen. At the same time, blue and green light of 450-490nm wavelength is emitted. Based on this characteristic, luminescent bacteria are often used as optical sensing elements of biological detectors. Compared with other physical and chemical detection methods, the biggest advantage of luminescent bacteria biosensing lies in the non-targeting of detection ability: its sensing principle is not based on the recognition of specific chemical structures, but on the degree of bioluminescence inhibition to characterize the toxicity of the target High and low.

The experiment process is as follows:

(1) Prepare the fruit sample extract separately: add 10g of the homogenate of the fruit sample into a 50mL centrifuge tube, continue to add 10mL of acetonitrile, 0.1mL of formic acid, shake and mix, ultrasonic bath for 10min; then add 4g of anhydrous magnesium sulfate and 1g of anhydrous Sodium acetate, shake and mix well, centrifuge at 4000r/min for 5min, take 2-4mL of the upper acetonitrile phase, and pass through a 0.45 micron filter membrane to obtain a liquid sample that can be directly used for analysis, and refrigerate it at 4°C;

(2) Chromatographic separation: first use a 100μL spotting needle to manually draw the solution to be tested, and quantitatively purge it to a position 8mm away from the bottom of the thin layer plate with the help of 0.5MPa nitrogen flow by a semi-automatic thin layer spotting instrument, The scanning speed is 100nL/s, the pre-arrangement volume is 0.2μL, the strip width is 6mm, and the distance between the two sides is at least 12mm; spot each sample prepared in step (1), and manually take out the spot after one sample is finished. After the needle is cleaned three times with methanol, the next sample is spotted; after all samples are spotted, take out the thin-layer plate and dry it with a hair dryer for 1 min. The purpose is to volatilize the residual methanol in the spotting origin so that it will not affect the follow-up Step; The thin-layer board material includes (1) silica gel board; (2) cellulose board; (3) acid alumina board; (4) alkaline alumina board;

The chromatographic separation was carried out in a fully automatic thin-layer developing instrument. The mobile phase was optimized and finally determined to be a toluene/ethyl acetate mixture with a volume ratio of 8/2, v/v, and an expansion distance of 60mm; chromatographic conditions: passing through a saturated magnesium chloride solution drum The bubble controls the relative humidity in the unfolding cylinder for 3 minutes, adjust the relative humidity to 35%, and the thin layer plate is pre-balanced for 10 minutes; when the mobile phase front reaches the predetermined height, the system automatically ends, take the thin layer plate out and place it on the thin layer heater Bake at 100°C for 5 minutes to volatilize and remove residual organic solvents on the thin layer;

(3) Bioluminescence imaging detection: Use an automatic dipping device to immerse the unfolded and dried thin-layer plate into the working luminescent suspension, the immersion speed is 1mm/s, the residence time is 2s, and then the thin-layer plate immersed in the working luminescent suspension is placed Into the bioluminescence imager for imaging detection, the imaging exposure time is 40s, the interval between each photo is 2min, and 15 pictures are taken continuously; after the optimization of the luminescent bacteria, the photoluminescent bacteria ATCC 11040 is selected;

(4) Analysis: Save the photo taken by the bioluminescence imager, then open it with Videoscan software, digitally analyze the pixel polarity in the picture to obtain a processable chromatogram, and then set the integration parameters and conditions for quantitative analysis.

Figure 9 shows the coupling effect diagram of the thin-layer plate with four different materials and Photobacterium luminescens. It can be seen from Figure 9 that after the thin-layer plate made of cellulose, acidic alumina and alkaline alumina material is coupled with the luminescent bacteria, the image of the band is not clear. Only the thin-layer plate made of silica gel material shows clear bands, indicating the most The stationary phase suitable for screening captan is a silica gel plate.

2. The influence of different luminescent bacteria on the test results

Using the above-mentioned preferred thin-layer plate, the difference is: the luminescent bacteria include: (1) Photobacterium luminescens ATCC 11040; (2) Photobacterium psychrophilus CGMCC 1.8932; (3) Photobacterium indica CGMCC 1.8728; (4) Hydroluminescence Bacillus, CGMCC 1.12159; other steps and parameters are the same as in the first part of Example 6, and the coupling effect diagram of luminescent bacteria of several different strains and silica gel plate is shown in Figure 10.

It can be seen from Fig. 10 that the photobacteria of Photobacterium indica and Photobacterium hydrophobe are unclearly imaged after coupling with the thin-layer plate, while the photobacteria of Photobacterium psychrophilus and Photobacterium luminescenti show clearer bands. Below, the band of Photobacterium luminescens is the clearest, indicating that the luminescence bacterium most suitable for screening captan is Photobacterium luminescens.

3. The influence of the ratio of different developing agents on the test results

Using the above-mentioned preferred thin-layer plate and luminescent bacteria, the difference is that the ratio of the developing agent is different: for the test of the mobile phase, first select toluene: ethyl acetate=6:4 (v/v) for the test, because of the selected mobile phase The polarity is too strong, causing the target strip to be too close to the bottom of the plate, and the effect is not good, as shown in Figure 11(1), so by reducing the amount of ethyl acetate and increasing the amount of toluene, the polarity of the developing agent is reduced; The test selects toluene: ethyl acetate=7:3(v/v). Because the polarity of the selected mobile phase is reduced, the expansion distance is higher than before, but it is still lower and has tailing phenomenon, as shown in Figure 11(2); For the three tests, toluene: ethyl acetate=8:2(v/v) was selected, as shown in Figure 11(3), the expansion effect was the best, which solved the expansion distance and tailing phenomenon, and finally selected toluene: ethyl acetate=8:2( v/v) as the developing mobile phase. Other steps and parameters are the same as the first part of Example 6. By selecting different proportions of the developing agent, the results are shown in Figure 11, indicating that the best mobile phase suitable for screening captan is toluene: ethyl acetate = 8: 2 (v/v).

Example 7 Performance test of high performance thin layer chromatography combined with bioluminescence method to detect nifedipine

Test for apples, pears and cherries and different spiked amounts:

Pretreatment of fruits: apples, pears and cherries: add 10g of the homogenate of the fruit sample into a 50mL centrifuge tube, continue to add 10mL of acetonitrile, 0.1mL of formic acid, shake and mix, ultrasonic water bath for 10min; then add 4g of anhydrous magnesium sulfate and 1g anhydrous sodium acetate, shake and mix well, centrifuge at 4000r/min for 5min, take 2-4mL of the upper acetonitrile phase, and pass through a 0.45 micron filter membrane to obtain a liquid sample that can be directly used for analysis, and refrigerate it at 4°C.

Pretreatment of spiked recovery sample solution: add 10g of fruit sample homogenate to a 50mL centrifuge tube, continue to add 10mL of acetonitrile, 0.1mL of formic acid, shake and mix, ultrasonic bath for 10min; then add 4g of anhydrous magnesium sulfate and 1g of anhydrous Sodium acetate, shake and mix well, centrifuge at 4000r/min for 5min, take 2-4mL of the upper acetonitrile phase, and pass through a 0.45 micron filter membrane to obtain a liquid sample that can be directly used for analysis. Add 15μL of 0.01mg/mL captan in the apple extract, 10μL of 0.01mg/mL captan in the pear extract, and add 10μL of 0.01mg/mL captan in the cherry extract, and put it at 4℃ Medium refrigeration.

Using 0.5MPa nitrogen as the carrier, use a 100μL syringe (CAMAG) to accurately spot the sample and captan standard with Linomat5 on a 20×10cm thin-layer plate. The spotted strip is 6mm long and the strip is 8mm from the bottom. , 12mm from the left end, with a strip spacing of 1.7mm. After spotting is completed, use the ADC-2 (CAMAG) expander to expand. Before expanding, inject 10 mL of mobile phase into another tank to make the cylinder saturated. Take 10 mL of the optimized developing solution (toluene: ethyl acetate = 8: 2 (v/v)), expand it upward for 60 mm, take it out, and place it on a plate heater at 100° C. to fully dry it for 5 minutes. The suspension of Photobacterium luminescens is then coupled with the chromatographic separation result by dipping.

After that, it was placed on a bioluminescence imager, and an image of the silica gel plate was acquired under 40-second exposure conditions, and then digital image software was used for quantitative analysis on a computer. Artificial addition of different content of captan can obtain clear imaging. The results in Figure 12 show that the standard curve of the recovery rate of different samples of captan can be calculated based on the detection results of 1-4, and the chromatographic peaks of apples, pears and cherries are substituted into According to the standard curve, the content of captan in apple is 734.22, the content of captan in pear is 455.21, the content of captan in cherry is 461.23, the recovery rate of captan in apple is 97%, and that in pear The spiked recovery rate of jundan was 91%, and that of captan in cherries was 92.2%, which was basically consistent with the HPLC detection results.

Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention should be defined by the claims.

Claims (21)

1. A high-efficiency thin-layer chromatography detection method, which is characterized in that the method is a combination of high-efficiency thin-layer chromatography and bioluminescence; the specific method is: first unfold the sample to be tested on a thin-layer plate, and then interact with luminescent bacteria Coupled, go through luminescence imaging, and finally scan with thin layer chromatography.
2. The method according to claim 1, wherein the thin layer plate comprises a silica gel plate, a cellulose plate, an acid alumina plate and an alkaline alumina plate.
3. Application of the method of claim 1 or 2 in the field of compound detection.
4. A method for screening nifedipine, characterized in that the method of claim 1 is used to detect a sample to be tested containing nifedipine.
5. The method according to claim 4, wherein the unfolding is to use a mixed solution of toluene and ethyl acetate as a developing agent.
6. The method of claim 5, wherein the volume ratio of toluene and ethyl acetate is 4:6.
7. The method according to claim 4, characterized in that the coupling conditions are: luminescent bacteria are cultured in a shake flask at 80-150r/min for 10-16h in an environment of 24～26℃ to obtain a bacterial suspension, and the dosage is 80～120mL. The time is 1 to 3s, the temperature is 24 to 26°C, and the pH is 6.5 to 7.5.
8. The method according to claim 4, wherein the luminescence imaging conditions are: the imaging exposure time is 20-50s, the interval is 1-3min, and at least 10 photos are taken.
9. The method according to claim 4, wherein the sample to be tested is a health product, food or medicine.
10. A method for screening captan residues in fruits, which is characterized in that the method according to claim 1 is used to detect a test sample containing captan.
11. The method according to claim 10, wherein the specific screening method is: first sample preparation and sample cleaning of the fruit homogenate to be tested by solid-liquid extraction, and then the extract is developed by high-performance thin-layer chromatography to make the target Separation of the substrate material interfered with the sample of Wu Kejundan and the extract, and then coupled the chromatographic separation result with the photoluminescent bacterium biosensor by dipping, and quantitatively evaluate the target substance in the fruit through bioluminescence autography combined with digital image analysis The residual concentration of bactam.
12. The method according to claim 11, wherein the Photobacterium luminescens is specifically: Photobacterium luminescens ATCC 11040.
13. The method according to claim 11, wherein the bioluminescence autoradiography detection is a detection based on photoluminescent bacterium luminescens biosensing on high efficiency thin layer chromatography.
14. The method according to claim 11, wherein the fruits to be tested are apples, pears and cherries.
15. The method according to claim 11, wherein the sample preparation and sample cleaning are specifically: adding 10 g of the fruit sample homogenate into a 45-55 mL centrifuge tube, continuing to add 8-12 mL of acetonitrile, 0.1 mL of formic acid, shaking and mixing Then add 4g anhydrous magnesium sulfate and 1g anhydrous sodium acetate, shake and mix well, centrifuge at 3500-4500r/min for 4-6min, take 2-4mL of the upper acetonitrile phase, pass through a 0.45μm filter membrane, That is, a liquid sample that can be directly used for analysis is obtained.
16. The method according to claim 11, characterized in that the unfolding adopts a mixed solution of toluene and ethyl acetate as the developing agent.
17. The method according to claim 16, characterized in that the volume ratio of toluene and ethyl acetate is 8:2.
18. The method according to claim 11, wherein the expansion is specifically: spotting 1-10 μL of a liquid sample for analysis, and after spotting, toluene:ethyl acetate=8:2 is used as the mobile phase. Unfold, stop after the upward unfolding distance of 60mm. After unfolding, place the thin-layer board on a flat plate heater at 100°C and fully dry for 5 minutes.
19. 11. The method of claim 11, wherein the expanded and dried thin-layer plate is subjected to bioluminescence autoradiography detection, and then the detection result is recorded by a bioluminescence imaging device, and an intuitive semi-quantitative result is obtained by visual inspection.
20. The method according to claim 11, characterized in that the thin-layer plate obtained after chromatographic expansion and bioluminescence auto-imaging is placed in a bioluminescence imager to take a picture, and combined with digital image software for quantitative analysis, and calculating by drawing a standard curve The content of target captan in fruit samples.
21. The method according to claim 11, wherein the standard curve is obtained according to the detection result: y=163.46x+1336.1, calculating the content of captan in the fruit sample; wherein y is the chromatographic peak area, in units of AU; x is Captan content, unit ng.

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