High-throughput screening method of thujopsene sample
By using high-throughput screening method of DPPH solution and Rohan Benene samples in 96-well plates, the problems of small samples and long detection time in traditional methods are solved, and fast and accurate Rohan Benene concentration detection is achieved, suitable for Large-scale screening and application development.
Patent Information
- Application Number
- CN202411258998.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-10
AI Technical Summary
Traditional methods have problems with small samples and long detection time when detecting Luohan Bernen samples, which are difficult to apply to high-throughput screening. Compounds with similar structures are prone to errors and peak superposition, affecting component distinction and precise quantification.
Using a high-throughput screening method based on DPPH solution, the absorbance is detected using a microplate reader, and the concentration is calculated by combining the standard curve. Dodecane is preferred as solvent and automatic pipetting station Ensure consistent fading rate, the detection time takes only 3 minutes.
It realizes efficient detection of 96 samples in a short time, with small errors, and is suitable for high-throughput screening of Luohan Berene samples, improving detection efficiency and accuracy, and is especially suitable for large-scale screening and application development.
Smart Images
Figure CN119104506A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of active compound screening, and particularly relates to a high-throughput screening method for thujopsis samples based on DPPH solution. Background Art
[0002] Terpenoids can be regarded as a class of natural compounds formed by isoprene or isopentane linked in various ways. The sources of terpenoids include flowers, fruits, leaves, stems and roots of various plants. According to the number of isoprene / isopentane, terpenoids can be divided into monoterpenes, sesquiterpenes, diterpenes, triterpenes and tetraterpenes; according to the different "connection" methods, terpenoids can be divided into chain terpenes and cyclic terpenes. Terpenoids are widely present in nature and have a variety of physiological activities. Therefore, terpenoids are often used in medicine, such as clinical applications and new drug development. The chemical structure of terpenoids is diverse, resulting in more than 40,000 species. At the same time, they have attracted much attention due to their great potential in medicine. Sesquiterpenoids are a type of terpenoids with a molecular formula of C15H24. They are the most diverse type of terpenoids. Rudorphelen is a type of sesquiterpenoid with a wide range of application value. It can be used to prepare various organic compounds, such as turpentine, spices, and drugs. Rudhoacene is widely used. It has biological activity and physiological effects including anti-inflammatory, anti-nociceptive, immunomodulatory and antibacterial. This series of effects has made people aware of its potential in the development of new therapies and its potential targets for medical purposes. In addition, Rudhoacene can also be used to prepare polymer materials, coatings, rubber, etc. Rudhoacene has been widely used in industrial production due to its high chemical and thermal stability.
[0003] The traditional analysis method for terpenes is mainly gas chromatography-mass spectrometry. However, this method may have errors when facing compounds with highly similar structures, because it is likely to retrieve multiple close matching results in the mass spectrum library, which increases the difficulty of identification. At the same time, if the retention times of the compounds are similar, they will be eluted from the gas chromatography column almost at the same time, resulting in overlapping peaks in the mass spectrum, which affects the clear distinction and precise quantification of the various components. Finally, because this method can detect a small number of samples at the same time and takes a long time to detect a single sample, it is not advantageous in large-scale screening and testing.
[0004] In summary, the potential value of ruthenium is high and has good economic benefits. However, the method for detecting ruthenium concentration has the disadvantages of small number of samples detected at the same time and long detection time, which is not suitable for high-throughput screening. Therefore, establishing an effective and cost-effective high-throughput screening method for ruthenium samples is of great significance for the research and development of this type of new drugs. Summary of the invention
[0005] The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a high-throughput screening method for thujopsis samples.
[0006] To achieve the above object, the present invention provides a high-throughput screening method for thujopsis samples based on DPPH solution, aiming to solve the problems of small number of detection samples and long detection time in the prior art. By using DPPH as a free radical scavenger, the method of the present invention can detect multiple thujopsis samples in a short time, and is suitable for high-throughput screening.
[0007] The following steps are involved:
[0008] (1) preparing a 0.1 mM DPPH solution, wherein the solvent of the solution is dodecane;
[0009] (2) Add 100 μl of DPPH solution into each well of a 96-well plate;
[0010] (3) Add 100 μl of the test sample of ruthenium to each well of a 96-well plate, and detect the absorbance in each well using an ELISA reader;
[0011] (4) Calculate the concentration of the cypermethrin sample by substituting it into the standard curve.
[0012] The concentration of the DPPH solution can be selected between 0.05 mM and 0.2 mM, but the preferred concentration is 0.1 mM.
[0013] The solvent of the DPPH solution is dodecane, and dodecane is selected as the best solvent in the experiment.
[0014] The DPPH solution was transferred into the 96-well plate in batches by an automatic pipetting station to ensure that the natural fading rate of the DPPH solution in each well was consistent.
[0015] The optimal detection time of the microplate reader is the third minute of the reaction, and the obtained absorbance data is used to calculate the concentration of the thujopsisene sample.
[0016] The rubotryne sample was derived from the fermentation broth and obtained by extraction with dodecane.
[0017] This method is suitable for detecting samples of thujopsis in a concentration range of 0.04 g / L to 13 g / L.
[0018] The beneficial effects of the present invention are as follows: the method can process 96 samples in a short time, and the detection time only takes 3 minutes, which greatly improves the detection efficiency. In addition, the error of the method is small, and it is suitable for high-throughput screening of thujopsis samples. The present invention provides a fast and efficient high-throughput screening method for thujopsis samples. The method is based on the fading mechanism of DPPH solution, and the concentration of thujopsis is calculated by measuring the absorbance of the sample by an enzyme marker. Compared with the traditional detection method, the method has higher detection efficiency and smaller error, and is particularly suitable for large-scale screening of thujopsis samples and its application development. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Attached Figure 1 : Diagram of the mechanism of fading of DPPH solution due to free radical loss.
[0020] Attached Figure 2 : Schematic diagram of the structure of a 96-well plate.
[0021] Attached Figure 3 : Schematic diagram of 96-well plate operation steps. DETAILED DESCRIPTION
[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the implementation scope of the present invention is not limited thereto.
[0023] like Figures 1 to 3 As shown, the term "high-throughput screening" in the present invention means that the number of detections in a single experiment is greater than 96, and the screening method can simultaneously detect the concentrations of 96 samples each time it is applied.
[0024] The fading mechanism of the DPPH developer used in the present invention is as follows Figure 1 As shown, from Figure 1 It can be seen that the cause of DPPH fading is the loss of free radicals.
[0025] Currently, there are few methods for screening ruthenium samples, and they are mainly gas chromatography-mass spectrometry, which has disadvantages such as long processing time and small number of processed samples, and is not suitable for high-throughput screening. Based on the free radical loss mechanism of 2,2-diphenyl-1-pyridine hydrazide (DPPH), the present invention successfully constructed a screening method for ruthenium samples. This method is relatively low in cost, short in time, and suitable for high-throughput screening.
[0026] Embodiment 1:
[0027] A high-throughput screening method for thujopsis samples comprises the following steps:
[0028] (1) preparing a 0.1 mM DPPH solution, wherein the solvent of the solution is dodecane;
[0029] (2) Add 100 μl of DPPH solution into each well of a 96-well plate;
[0030] (3) Add 100 μl of the test sample of ruthenium to each well of a 96-well plate, and detect the absorbance in each well using an ELISA reader;
[0031] (4) Calculate the concentration of the cypermethrin sample by substituting it into the standard curve.
[0032] Specifically, a DPPH solution is prepared as a screening tool, and the solvent of the solution is dodecane. After comparing the actual effects of dodecane with ethanol, methanol, and tetradecane, the dissolution effect of dodecane is the best; after adding 100 μl of DPPH solution to each well of a 96-well plate, the absorbance in each well is detected by an enzyme marker. Taking the specific removal of DPPH free radicals by rubotrys as an example, the screening principle is explained: the screening tool DPPH designed by the present invention is a very stable nitrogen-centered free radical. When there is a free radical scavenger, such as rubotrys, its absorption gradually disappears due to pairing with its single electron, and its fading degree is quantitatively related to the number of electrons it accepts, so a rapid quantitative analysis can be performed using an enzyme marker.
[0033] Preferably: in step (1), the solution may be tetradecane, dodecane, ethanol, methanol, which are well known in the art; in the preferred embodiment, it is dodecane. Since the sample of thujopsis to be tested is extracted from the fermentation broth, dodecane is the preferred solvent for extracting hydrophobic compounds in living cultures due to its low toxicity and good phase separation. Therefore, dodecane is selected as the preferred solvent in this invention.
[0034] Preferably: in the above step (3), after the reaction is completed, the concentration of ruthenium can be determined by absorbance detection, such as ultraviolet spectrophotometer, microplate reader, etc., and those skilled in the art can make a conventional selection of the detection means based on factors such as cost; in the implementation method of detecting by microplate reader, the detection steps are as follows: first set the microplate reader running program, shake the well plate for ten seconds before the formal measurement to ensure sufficient mixing, the time interval for each absorbance detection is half a minute, and after the reaction is carried out for five minutes, the reaction can be considered to be completed, the program is closed, and the data is stored.
[0035] Preferably, the concentration of the DPPH solution can be selected between 0.05 mM and 0.2 mM, but the preferred concentration is 0.1 mM.
[0036] Preferably, the solvent of the DPPH solution is dodecane, and dodecane is selected as the best solvent in the experiment.
[0037] Preferably, the DPPH solution is transferred in batches to the 96-well plate by an automatic pipetting station to ensure that the natural fading rate of the DPPH solution in each well is consistent.
[0038] Preferably, the optimal detection time of the microplate reader is the third minute of the reaction, and the obtained absorbance data is used to calculate the concentration of the thujopsisene sample.
[0039] Preferably, the rubotryne sample is derived from a fermentation broth and is obtained by extraction with dodecane.
[0040] Preferably, the method is suitable for detecting a concentration range of thujopsisene samples of 0.04 g / L to 13 g / L.
[0041] This method can process 96 samples in a short time, and the detection time only takes 3 minutes, which greatly improves the detection efficiency. In addition, the error of this method is small, which is suitable for high-throughput screening of thujopsis samples.
[0042] Among the engineered bacteria producing thujoides, each mutant strain produces different thujoides yields. The above screening method provided by the present invention screens thujoides mutant engineered bacteria with different yields. In order to further screen the thujoides engineered bacteria with the highest yield, the screening method also includes the following steps: obtaining target gene fragments with different base sequences through random mutation, constructing a recombinant plasmid, allowing it to express thujoides synthase in Saccharomyces cerevisiae, culturing on a shaking table, and extracting to obtain a product thujoides sample; applying the prepared DPPH solution to each thujoides sample, and screening by the above screening method, thereby confirming the thujoides mutant strain with the highest yield.
[0043] The screening method provided by the present invention uses the rubotrysene extracted from the yeast fermentation broth as a sample for screening, which is closer to the actual application scenario, and the screening result has a higher credibility. In addition, the screening method can achieve the effect of simultaneous detection of multiple samples based on conventional detection means such as an ELISA instrument, and is a low-cost high-throughput screening method.
[0044] In this embodiment, the rutosiderine engineering bacteria that have been subjected to drug mutagenesis treatment are selected, and the specific steps are as follows:
[0045] The mutagenized plasmid was introduced into the yeast strain and cultured on YPD solid medium.
[0046] 100 ml of YPD liquid culture medium was prepared for strain expansion culture, and dodecane was added to extract thujopsisene.
[0047] The rubotryne samples obtained after the culture were added to a 96-well plate, with 100 μl of the sample added to each well.
[0048] DPPH solution was added, and the absorbance was measured using an enzyme reader to obtain the concentration data of thujopsisene.
[0049] The experimental results show that this method has a small detection error and is suitable for high-throughput screening of cypermethrin samples.
[0050] Embodiment 2:
[0051] Step 1: Prepare a DPPH solution of appropriate concentration, wherein the solvent is dodecane:
[0052] The alternative concentrations of DPPH are 0.05mM, 0.1mM, and 0.2mM, and 2g / L, 4g / L, 6g / L, 8g / L, and 10g / L of rubotrysene standards are prepared respectively. Figure 2 As shown, various concentrations of rutinene standards were added to a 96-well plate, 100 μl was added to each well, and then 0.05 mM DPPH solution was added simultaneously through an automatic pipetting station, and the absorbance after fading every 30 seconds was measured by a microplate reader, and the regression line R2 of the standard curve was 0.933. Similarly, the same operation was performed, but the concentration of the DPPH solution was changed to 0.1 mM and 0.2 mM, and the R2 obtained was 0.99212 and 0.9724, respectively. Therefore, the optimal concentration of the DPPH solution for this method is 0.1 mM, and the standard curve is y=-0.0011x+0.126, where x is the concentration of the analyte and y is the absorbance measured by the microplate reader.
[0053] Step 2: Mix the sample to be tested with the DPPH solution and place it in the microplate reader for determination:
[0054] This method has an upper detection limit and a lower detection limit, that is, the range in which the concentration of the analyte can be detected. First, determine the lower detection limit, prepare a 0.1 mM DPPH solution, prepare 0.02 g / L, 0.04 g / L, 0.06 g / L, 0.08 g / L, and 0.1 g / L rubotrysene standard solutions, and perform the operation as in step 1. Mix the standard solution and the DPPH solution and place them in an enzyme reader to detect the absorbance. The absorbance shows that the standard curve of the 0.04 g / L to 0.1 g / L standard solution presents a linear relationship, R2 is 0.9462, and the standard curve is y=-0.1533x+0.1704, where x is the concentration of the analyte and y is the absorbance measured by the enzyme reader. Continue to find the detection upper limit as in the above steps, prepare 10g / L, 11g / L, 12g / L, 13g / L, 14g / L, 15g / L rubotrysene standard solutions, step 1, mix the standard solution and DPPH solution and put them into the microplate reader to detect the absorbance. The absorbance shows that the standard curve of the 10g / L to 13g / L standard solution presents a linear relationship, R2 is 0.9086, and the standard curve is y=-0.0003x+0.1358, where x is the concentration of the analyte and y is the absorbance measured by the microplate reader. Same as the above steps, replace the analyte with 0.1-1g / L rubotrysene standard sample, and obtain the standard curve of y=-0.0198x+0.1694, R 2=0.968. Therefore, the lowest concentration of thujopsis that can be detected by this method is 0.04 g / L, and the highest concentration of thujopsis that can be detected is 13 g / L. In the actual operation, 100 μl of each of several analytes was added to the wells of a 96-well plate, and then the DPPH solution was added simultaneously through an automatic pipetting station, and then quickly placed in an ELISA instrument to detect the absorbance.
[0055] Step 3: Substitute the standard curve to obtain the concentration of the substance to be tested:
[0056] In this operation step, the best detection time is the third minute of the reaction, so after obtaining the absorbance data of the third minute, the different absorbance ranges are substituted into a suitable standard curve to obtain the concentration value of the analyte.
[0057] This embodiment achieves the experimental purpose of rapid, accurate and high-throughput. The currently commonly used detection method is gas chromatography, and the detection time of a single sample is 20-30 minutes. This method can detect 96 samples at the same time and the detection time is 3 minutes, and the detection efficiency is greatly improved.
[0058] Embodiment 3:
[0059] The thujopsis engineering bacteria used in this embodiment is obtained by random mutation of the original thujopsis engineering bacteria, and the thujopsis production is increased or decreased. Therefore, this method is used for detection, and the specific steps are as follows:
[0060] (1) The drug-mutated plasmid was introduced into the yeast substrate, spread on a ball plate, and cultured in a constant temperature incubator at 28-30°C on a YPD solid medium plate.
[0061] (2) Prepare 100 ml of YPD liquid culture medium, with the ingredients of 1 g yeast extract powder, 2 g peptone, and 2 g glucose. Sterilize at 116°C for 25 minutes and dispense into 20 50 ml sterile centrifuge tubes. Add 5 ml YPD culture medium to each sterile centrifuge tube. Pick a single colony grown on the plate and place it in a test tube. When the OD600 value of the bacterial solution in the sterile centrifuge tube is about 0.8-1.2, inoculate it into a sterilized and cooled shake flask for expansion culture. After 24 hours of shake flask culture, add 5 ml of sterilized dodecane and continue fermentation culture for 80 hours. The culture conditions of the shake flask are shaker at 30°C and 200 rpm.
[0062] (3) After 80 h of shaking flask culture, the upper organic phase was taken and added to a 96-well plate. A total of 20 samples were added to the 96-well plate. Figure 3 shown.
[0063] (4) Prepare 0.1mM DPPH solution and add it to the 96-well plate with samples added in advance through an automatic pipetting station. After the well plate is prepared, put it into the microplate reader. The microplate reader program settings are as follows: shake for 10 seconds before testing, the temperature is 25°C, the detection time is 30 seconds per test, and the detection automatically stops after five minutes;
[0064] (5) After the test is completed, the absorbance value is derived and substituted into the appropriate standard curve to calculate the sample concentration. The absorbance obtained in this experiment is shown in Table 1 below, and the sample concentration value is obtained by substituting into the standard curve:
[0065] Sample name <![CDATA[Absorbance (OD 600 )]]> Determination concentration (g / L) No. 1 shake bottle 0.164133 0.265993266 Shake bottle No. 2 0.163721 0.286818182 No. 3 shake bottle 0.168344 0.053333333 No. 4 shake bottle 0.164850 0.22979798 No. 5 shake bottle 0.166469 0.148030303 No. 6 shake bottle 0.164642 0.24030303 No. 7 shake bottle 0.167234 0.109393939 No. 8 shake bottle 0.166453 0.148838384 No. 9 shake bottle 0.168224 0.059393939 No. 10 shake bottle 0.165673 0.188232323 No. 11 Shaker Bottle 0.167376 0.102222222 No. 12 shake bottle 0.167902 0.075656566 No. 13 Shaker Bottle 0.167204 0.110909091 No. 14 shake bottle 0.166717 0.135505051 No. 15 shake bottle 0.165668 0.188484848 No. 16 shake bottle 0.165143 0.215 No. 17 Shaker Bottle 0.165946 0.174444444 No. 18 shake bottle 0.163947 0.27540404 No. 19 Shaker Bottle 0.168269 0.057121212 No. 20 shake bottle 0.166986 0.121919192
[0066] (6) Take 20 μl of the upper organic phase of each shake flask and put it into a gas phase vial, then add 180 μl of tetradecane internal standard, and put it into a gas chromatograph for detection to verify the accuracy of the invention. The accurate concentration and error are shown in Table 2 below:
[0067]
[0068]
[0069] (7) It can be seen from the final experimental results that the error of this method is generally small and is within an acceptable range in high-throughput screening. Therefore, this method can be applied to the high-throughput screening of thujopsis samples. It can be seen from the above embodiments that compared with the method of detecting the concentration of thujopsis by gas chromatograph, the DPPH solution is more efficient in the screening method and has a smaller error, which is acceptable in the experiment. Such results are based on the correct judgment of the upper and lower limits of detection, which improves the detection efficiency in practical applications. The DPPH color developer method is selected to achieve high-speed and accurate detection of the concentration of thujopsis samples, avoid the long detection time of a single sample, resulting in a slowdown in the experimental progress, and reduce the frequency of experimental detection. Detecting the concentration of thujopsis samples by DPPH is an efficient and accurate detection method.
[0070] In order to verify the accuracy of this method, some samples were tested by gas chromatography. The results showed that the error between the concentration of thujopsisene detected by this method and the result of gas chromatography was within an acceptable range, which further verified the effectiveness and reliability of this method.
[0071] The present invention provides a rapid and efficient high-throughput screening method for thujopsis samples. The method is based on the fading mechanism of DPPH solution, and the concentration of thujopsis is calculated by measuring the absorbance of the sample with an enzyme marker. Compared with traditional detection methods, the method has higher detection efficiency and smaller error, and is particularly suitable for large-scale screening of thujopsis samples and its application development.
[0072] It should be understood that although the present invention has been described through specific embodiments, those skilled in the relevant art may make various modifications and changes to the present invention without departing from the spirit and scope of the present invention, and these modifications and changes should also be regarded as the scope of protection of the present invention.
Claims
1. A high-throughput screening method for thujopsis samples, characterized in that: The following steps are involved: (1) preparing a 0.1 mM DPPH solution, wherein the solvent of the solution is dodecane; (2) Add 100 μl of DPPH solution into each well of a 96-well plate; (3) Add 100 μl of the test sample of ruthenium to each well of a 96-well plate, and detect the absorbance in each well using an ELISA reader; (4) Calculate the concentration of the cypermethrin sample by substituting it into the standard curve.
2. The high-throughput screening method for a thujopsis sample according to claim 1, characterized in that: The concentration of the DPPH solution can be selected between 0.05 mM and 0.2 mM, but the preferred concentration is 0.1 mM.
3. The high-throughput screening method for thujopsis samples according to claim 1, characterized in that: The solvent of the DPPH solution is dodecane, and dodecane is selected as the best solvent in the experiment.
4. The high-throughput screening method for thujopsis samples according to claim 1, characterized in that: The DPPH solution was transferred into the 96-well plate in batches by an automatic pipetting station to ensure that the natural fading rate of the DPPH solution in each well was consistent.
5. The high-throughput screening method for thujopsis samples according to claim 1, characterized in that: The optimal detection time of the microplate reader is the third minute of the reaction, and the obtained absorbance data is used to calculate the concentration of the thujopsisene sample.
6. The high-throughput screening method for thujopsis samples according to claim 1, characterized in that: The rubotryne sample was derived from the fermentation broth and obtained by extraction with dodecane.
7. The high-throughput screening method for thujopsis samples according to claim 1, characterized in that: This method is suitable for detecting samples of thujopsis in a concentration range of 0.04 g / L to 13 g / L.