CN114813285A - Liquid-liquid extraction pretreatment method for biological sample - Google Patents
Liquid-liquid extraction pretreatment method for biological sample Download PDFInfo
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- CN114813285A CN114813285A CN202210642581.3A CN202210642581A CN114813285A CN 114813285 A CN114813285 A CN 114813285A CN 202210642581 A CN202210642581 A CN 202210642581A CN 114813285 A CN114813285 A CN 114813285A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
Abstract
The invention relates to a liquid-liquid extraction pretreatment method for a biological sample, which comprises the following steps: adding the biological sample and the extracting agent into a 96-deep-hole plate; vacuum sealing the 96 deep hole plate by using a vacuum packaging machine; carrying out vortex oscillation and centrifugation on the sealed 96 deep hole plate; and transferring out the upper organic phase in the hole of the 96-deep-hole plate, and then carrying out subsequent sample treatment and analysis. The pretreatment method for liquid-liquid extraction of the biological sample skillfully solves the problem that the hole sites of the 96 deep-hole plate and the silica gel pad are difficult to keep tight sealing in the vortex oscillation process during liquid-liquid extraction by the innovative application of a vacuum packaging machine. Vacuum packaging machine can take out the air in the vacuum packaging bag automatically, accomplishes the process of sealing after reaching predetermined vacuum, and vacuum packaging bag can produce powerful package power to 96 deep hole board this moment, and the silica gel pad is pressed fit tightly on 96 deep hole board, and is sealed the effect splendid through the test, can satisfy the requirement of experimental operation such as the long-time vortex oscillation of high rotational speed, low temperature high speed centrifugation.
Description
Technical Field
The invention relates to the technical field of biological analysis, in particular to a liquid-liquid extraction pretreatment method for a biological sample.
Background
Liquid-liquid extraction is a sample pretreatment method commonly used in the field of biological analysis, and is suitable for analyzing non-polar to medium-polar analytes. The method is based on different distribution of target analytes between a biological matrix and an extracting agent, the target analytes are extracted into an organic phase (the extracting agent) through the operation of mixing and oscillating of a biological sample and the extracting agent, the organic phase is dried by blowing, redissolved and the like to obtain purified and diluted analyte solution, and finally, the purified and diluted analyte solution is analyzed by a liquid phase mass spectrometer to determine concentration data.
The mature method in the industry at present is to add a biological sample and an extracting agent into a 96-deep-hole plate, seal the 96-deep-hole plate by using a plate sealing machine, perform vortex oscillation and centrifugation, transfer an upper organic phase to another 96-deep-hole plate by using a pipettor (or a liquid workstation), introduce nitrogen flow into the 96-deep-hole plate by using a nitrogen blowing concentrator until the organic phase is dried, add a mixed solution with a proper proportion for redissolution, complete sample pretreatment, and finally put the sample into a liquid phase mass spectrometer for analysis.
In the method, the key of avoiding pollution among the holes is to ensure that the 96-deep-hole plate can firmly seal the plate during vortex oscillation, and the success or failure of an analysis test result is related. The conventional operation flow (direct sealing plate of the silica gel pad) is used, so that firm sealing cannot be realized, the reason is that the temperature of the extracting agent in the 96 deep hole plate rises and the volatilization amount increases in the high-speed vortex oscillation process, so that the pressure in the hole is increased, the silica gel pad is pushed open when the pressure exceeds a critical value (static friction force between the plug of the silica gel pad and the hole wall of the 96 deep hole plate), and the content leaks to cause the failure of a sample pollution test. In order to solve the problems, the method uses a plate closing machine to seal the 96-deep-hole plate. When the plate is closed, the silica gel pad is replaced by the special hot-melt film, and the plate closing machine fixes the hot-melt film on the deep hole plate in a hot-melt manner in a high-temperature and pressurizing manner, so that the 96 deep hole plate is firmly sealed.
However, the number of types of the plate sealing machines in the current market is large, the sealing effects of instruments of different types are different, the plate sealing machines with sealing performance meeting the requirements of liquid-liquid extraction tests usually depend on import and are expensive. Furthermore, heat-sealing films can interfere with certain analytes, leading to test failures. These factors further limit the use of plate closing machines.
Without a plate sealing machine, the conventional method is to glue and seal the 96-deep-hole plate manually in a tape winding manner. Or carrying out vortex mixing on the sample and the organic reagent in a centrifugal tube, and transferring the organic phases to a 96-deep-hole plate one by one for subsequent preparation and analysis after layering. Both methods are time-consuming and labor-consuming, depend on the operation methods of experimenters, and have low automation degree. Meanwhile, researches show that the vortex can be replaced by repeatedly sucking and releasing the sample and the extracting agent, but the method can be realized only by a liquid workstation, and the liquid workstation is more expensive than a plate sealing machine, so that the cost of performing a biological analysis project and building a laboratory is greatly increased.
Disclosure of Invention
In view of the above, it is necessary to provide a pretreatment method for liquid-liquid extraction of a biological sample, which is easy to use and efficient.
A method of liquid-liquid extraction pretreatment of a biological sample, the method comprising:
adding the biological sample and the extracting agent into a 96-deep-hole plate;
vacuum sealing the 96 deep hole plate by using a vacuum packaging machine;
carrying out vortex oscillation and centrifugation on the sealed 96 deep hole plate;
and transferring out the upper organic phase in the hole of the 96-deep-hole plate, and then carrying out subsequent sample treatment and analysis.
Further, the 96 deep hole plate is a circular hole deep hole plate.
Further, the wells of the 96-deep well plate have a volume of at least 2.0mL, and no more than 0.25mL of sample is added to each well.
Further, the content volume of the wells of the 96-deep well plate does not exceed 1.5 mL.
Further, the sealing of the 96-deep-hole plate by using the vacuum packaging machine previously comprises:
the 96 deep well plate was sealed with a silicone pad.
Further, the performing vortex oscillation includes:
and carrying out vortex oscillation on the 96 deep hole plate by using a multi-test-tube vortex oscillator, wherein the rotating speed is 2500rpm, and the time is 10-20 min.
Furthermore, the vacuum packaging machine has the functions of vacuumizing and vacuum sealing, and the vacuum degree of the vacuum packaging machine after sealing at least reaches-65 kp.
Further, the vacuum packaging bag used by the vacuum packaging machine is made of Polyamide (PA) + Polyethylene (PE) or polyethylene terephthalate (PET) + Polyethylene (PE). The size of the vacuum packaging bag is matched with that of a 96-deep-hole plate, the length of the packaging bag is not more than 25 cm, and the width of the packaging bag is not more than 20 cm.
The pretreatment method for liquid-liquid extraction of the biological sample ingeniously solves the problem that the hole sites of the 96 deep-hole plate and the silica gel pad are difficult to keep tight sealing in the vortex oscillation process through the innovative application of the vacuum packaging machine. Vacuum packaging machine can take out the air in the vacuum packaging bag automatically, accomplishes the process of sealing after reaching predetermined vacuum, and vacuum packaging bag can produce powerful package power to 96 deep hole board this moment, and the silica gel pad is pressed fit tightly on 96 deep hole board, and is sealed the effect splendid through the test, can satisfy the requirement of experimental operation such as the long-time vortex oscillation of high rotational speed, low temperature high speed centrifugation. The vacuum packaging machine has the advantages of low price, low maintenance and consumable material use cost, wide purchase channel and high working efficiency. By adopting the technical scheme, the pretreatment method for liquid-liquid extraction is not limited by a plate sealing machine, a liquid workstation and other expensive experimental equipment, and simultaneously, the labor is greatly saved, and the requirements of high-throughput biological sample analysis are met.
Drawings
FIG. 1 is a flow chart of a pretreatment method for liquid-liquid extraction of a biological sample in one embodiment.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1, in one embodiment, a method for pre-treating a biological sample by liquid-liquid extraction includes the following steps:
step S110, add the biological sample and the extractant to a 96-deep well plate. The common extractant is methyl tert-butyl ether, ethyl acetate, n-hexane or the mixture of two solvents. Internal standards and buffer solutions may also be added as needed prior to the addition of the organic solvent. The function of the internal standard is to aid in analyte quantitation. The buffer solution is used to adjust the matrix PH to aid in the extraction of the analyte from the aqueous phase to the organic phase. The internal standard and buffer solutions and organic solvents need to be selected according to the nature of the analyte.
Wherein 96 deep hole boards have two types of round hole board and square hole board, and the interval between every hole is great on the round hole board, and the hole wall thickness, the firm non-deformable of structure, sealed effectual through the actual measurement round hole board, the square hole board then can't realize sealed. Meanwhile, the total volume of the content in each hole is required to be not more than 1.5mL, so that stable vortex can be generated during vortex oscillation, and the consistency of the extraction rate of the sample in each hole is ensured. Typically, 5 times the sample volume of the extractant ensures efficient extraction, and the sample volume in each well cannot exceed 0.25mL in this method.
Step S120, vacuum sealing the 96 deep-well plate using a vacuum packing machine. After adding the sample and the organic solvent into the 96 deep-hole plate, sealing by using a silica gel pad, and then carrying out vacuum pumping sealing by using a vacuum packaging machine, wherein the size of a vacuum packaging bag needs to be matched with the size of the 96 deep-hole plate as much as possible so as to facilitate subsequent vortex and centrifugation operations.
Through actual measurement, the household vacuum packaging machine has the functions of vacuumizing and vacuum sealing, and the vacuum degree is more than-65 kpa, so that reliable sealing can be realized. Vacuum sealing of a single 96-deep-hole plate can be completed within tens of seconds, the speed is close to that of a plate sealing machine, and compared with manual glue sealing (about 4 minutes is needed for sealing one plate), the method greatly reduces time and labor cost.
And step S130, carrying out vortex oscillation and centrifugation on the sealed 96 deep hole plate. The sealed 96-deep-hole plate is subjected to vortex oscillation by using a multi-tube vortex oscillator, the rotating speed is 2500rpm, the time is 10-20 min (adjusted according to specific conditions), and after the vortex oscillation is finished, a centrifugal machine is used for centrifuging, the centrifugal force is 3200g, and the time is 10 min.
And step S140, transferring the upper organic phase in the hole of the 96 deep-hole plate, and then carrying out subsequent sample treatment and analysis. After centrifugation is finished, liquid in the holes can be observed to be divided into an upper layer and a lower layer, a liquid transfer device is used for transferring an upper layer organic phase to another 96 deep-hole plate for subsequent sample treatment and analysis, the 96 deep-hole plate can be frozen (placed into a dry ice-ethanol bath or a refrigerator with the temperature of-80 ℃) before transfer, and a lower layer water phase is frozen and fixed at the bottom after freezing treatment, so that liquid transfer is convenient. It should be noted that the operation of transferring the organic phase is more preferably performed using a liquid station, where the conditions allow it. And continuously introducing nitrogen flow into the 96-depth plate by using a nitrogen blowing thickener until the transferred organic phase is dried, and then adding the mixed solution in a proper proportion for redissolution. And finally, putting the sample into a liquid phase mass spectrometer for analyzing and determining concentration data.
According to the liquid-liquid extraction pretreatment method for the biological sample, by the innovative application of the vacuum packaging machine, the problem that the silica gel pad and the hole site of the 96 deep hole plate are difficult to keep tight sealing in the vortex oscillation process is solved skillfully, and the laboratory construction cost is greatly reduced while the high-throughput sample analysis is met. The plate sealing machine with reliable sealing performance is generally sold at a price of more than 10 ten thousand yuan RMB, the price is expensive, in addition, the use cost of equipment maintenance, maintenance and consumable materials is also considered, the price of a household vacuum packaging machine is generally below one hundred yuan or even one hundred yuan, the cost of a single vacuum packaging bag is extremely low, the purchase channel is wide, the market stock is sufficient, and the working environment matched with the liquid-liquid extraction method is conveniently and quickly established in a laboratory. From the efficiency of sample processing, the sample processing speed of the method is similar to that of a method for closing the plate by using a plate sealing machine, and compared with the traditional method for manually sealing the plate by glue and whirling the test tube, the method is more suitable for the requirement of high-throughput biological sample analysis, and greatly saves manpower and working time.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A pretreatment method for liquid-liquid extraction of a biological sample, which is characterized by comprising the following steps:
adding the biological sample and the extracting agent into a 96-deep-hole plate;
vacuum sealing the 96 deep hole plate by using a vacuum packaging machine;
carrying out vortex oscillation and centrifugation on the sealed 96 deep hole plate;
and transferring out the upper organic phase in the hole of the 96-deep-hole plate, and then carrying out subsequent sample treatment and analysis.
2. The method for the pretreatment of liquid-liquid extraction of a biological sample according to claim 1, wherein the 96-well plate is a circular-hole deep-well plate.
3. The method according to claim 1, wherein the volume of the wells of the 96-deep well plate is at least 2.0mL, and the volume of the sample added to each well is not more than 0.25 mL.
4. The method according to claim 1, wherein the volume of the contents in the wells of the 96-deep well plate is not more than 1.5 mL.
5. The method of claim 1, wherein the sealing of the 96-deep well plate with a vacuum packaging machine further comprises:
the 96 deep well plate was sealed with a silicone pad.
6. The method according to claim 1, wherein the performing vortex oscillation comprises:
and carrying out vortex oscillation on the 96 deep hole plate by using a multi-test-tube vortex oscillator, wherein the rotating speed is 2500rpm, and the time is 10-20 min.
7. The method for the liquid-liquid extraction pretreatment of a biological sample according to claim 1, wherein the vacuum packaging machine has vacuum pumping and vacuum sealing functions, and the vacuum degree of the vacuum packaging machine after sealing is at least-65 kpa.
8. The method for liquid-liquid extraction pretreatment of a biological sample according to claim 1, wherein the vacuum packaging bag used in the vacuum packaging machine is made of Polyamide (PA) + Polyethylene (PE) or polyethylene terephthalate (PET) + Polyethylene (PE). The size of the vacuum packaging bag is matched with that of a 96-deep-hole plate, the length of the packaging bag is not more than 25 cm, and the width of the packaging bag is not more than 20 cm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210642581.3A CN114813285A (en) | 2022-06-08 | 2022-06-08 | Liquid-liquid extraction pretreatment method for biological sample |
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| CN202210642581.3A CN114813285A (en) | 2022-06-08 | 2022-06-08 | Liquid-liquid extraction pretreatment method for biological sample |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115032413A (en) * | 2022-08-15 | 2022-09-09 | 杭州凯莱谱质造科技有限公司 | Sampling device for sample pretreatment and pretreatment method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115032413A (en) * | 2022-08-15 | 2022-09-09 | 杭州凯莱谱质造科技有限公司 | Sampling device for sample pretreatment and pretreatment method |
| CN115032413B (en) * | 2022-08-15 | 2023-01-03 | 杭州凯莱谱质造科技有限公司 | Sampling device for sample pretreatment and pretreatment method |
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