A solid phase extraction method and pipetting module

Abstract

The application discloses a solid phase extraction method and a pipetting module, and the solid phase extraction method comprises the following steps: step one, placing a sample to be separated and a corresponding solvent in a microwell plate with a porous membrane to prepare a solid-liquid phase mixed solution; step two, removing a pipetting head on the pipetting module and driving the pipetting module to move downward to above the microwell plate; step three, driving the pipetting module to move downward until the pipetting head adapter is pressed on the microwell plate; and step four, generating a positive pressure acting on each hole of the microwell plate by the pipetting module, so that the liquid phase in the solid-liquid phase mixed solution is separated from the solid-liquid phase mixed solution through the porous membrane. Compared with the prior art, the application can simplify the structure of the automatic liquid workstation and solve the technical problem that the liquid in other holes cannot be completely discharged due to the air communication of part of the holes of the microwell plate in the prior art solid phase extraction method by using the pipetting module of the automatic liquid workstation to perform solid phase extraction without the need of using special equipment for solid phase extraction.

Description

Technical Field

[0001] This invention relates to the field of separation and extraction technology, and in particular to a solid-phase extraction method and a pipetting module. Background Technology

[0002] In automated liquid workstations used in pharmaceuticals, food, environment, commodity inspection, and chemical industries, pipetting modules are essential functional modules. Meanwhile, solid-liquid separation technologies (such as protein precipitation plates) and solid-phase extraction (SPE) are sample pretreatment techniques that have emerged in recent years. These techniques combine liquid-solid extraction columns and liquid chromatography, and are primarily used for sample separation, purification, and concentration. Compared to traditional liquid-liquid extraction methods, SPE can improve analyte recovery rates, more effectively separate analytes from interfering components, and reduce sample pretreatment steps.

[0003] Solid-liquid separation (SPE) technology can be used for the purification of small molecules (hormones, metabolites, vitamins, etc.) or large molecules (nucleic acids, proteins), the separation of large particles and liquids, and the separation of proteins and small molecules (e.g., protein precipitation plates). Microplates with embedded membrane materials of specific pore sizes are commonly used as SPE media, or microplates with embedded surface-modified fillers with specific functional groups are used as SPE media (hereinafter collectively referred to as membranes). Currently, the common method involves applying positive or negative pressure gas to a single tube or microplate. Under pressure, the liquid in each pore of the tube or microplate flows through the membrane layer to the outside of the pore. A drawback of this method is that since the same pressure source applies to all pores of the microplate, once one pore becomes connected to air, the pressure in the other pores can no longer be maintained, leading to incomplete drainage of liquid from other pores. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing a solid-phase extraction method that utilizes the pipetting module of an automated liquid workstation for solid-phase extraction, eliminating the need for additional dedicated equipment. This simplifies the structure of the automated liquid workstation and solves the technical problem in existing solid-phase extraction methods where some pores of the microplate are connected to air, preventing the complete drainage of liquid from other pores.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a solid-phase extraction method, comprising the following steps:

[0007] The sample to be separated is placed in a microplate with a porous membrane and a solvent is added by a pipetting module or manually by the operator to prepare a solid-liquid mixture.

[0008] Remove the pipette tip from the pipette module and drive the pipette module to move down above the microplate. The pipette module includes an aspiration chamber with multiple air chambers penetrating its upper and lower surfaces. The hole spacing of the microplate is the same as or an integer multiple of the air chamber spacing. Each air chamber has a piston that can move up and down along its axial direction. The piston is driven by a drive mechanism. A pipette tip adapter is also connected below the aspiration chamber. The pipette tip adapter has multiple through holes that correspond one-to-one with the air chambers.

[0009] Drive the pipetting module downwards until the pipetting tip adapter is pressed against the microplate;

[0010] The control drive mechanism drives the piston to move downward. The downward movement of the piston generates positive pressure in the gas chamber and the through hole. Under the action of the positive pressure, the liquid phase in the solid-liquid mixture is separated from the solid-liquid mixture through the pore membrane.

[0011] Preferably, during the preparation of the solid-liquid mixture, the liquid pipetting module is used to blow the liquid to accelerate the dissolution rate of the sample to be separated in the solvent.

[0012] Preferably, mechanical oscillation is used during the preparation of the solid-liquid mixture to accelerate the dissolution rate of the sample to be separated in the solvent.

[0013] Preferably, the sample to be separated is paraffin-embedded tissue, and the step of placing the sample to be separated in a microplate with a porous membrane and adding solvent to prepare a solid-liquid mixture includes:

[0014] The paraffin-embedded sample was placed in a microplate with a porous membrane, and the appropriate solvent was added by means of a pipetting module or by the operator manually.

[0015] The microplate is heated so that the paraffin, biological tissue and molecules in the paraffin-embedded sample dissolve into the solvent to form a mixture;

[0016] The mixture is cooled until the paraffin wax precipitates out of the mixture, solidifies into flakes, and floats on the aqueous phase of the mixture, thereby producing the solid-liquid phase mixture.

[0017] Preferably, the sample to be separated is a solid environmental sample extracted by forensic experts from the crime scene. The step of placing the sample to be separated in a microplate with a porous membrane and adding a solvent to prepare a solid-liquid mixture includes:

[0018] The solid sample of the environmental scenario is placed in a microplate with a porous membrane, and the corresponding solvent is added by means of a pipetting module or by the operator manually.

[0019] The microplate is heated so that nucleic acid substances or other soluble molecules in the solid sample of the environmental scene dissolve into the solvent, thereby preparing the solid-liquid mixture.

[0020] In a second aspect, the present invention provides a pipetting module for the solid-phase extraction method described in the first aspect above, comprising:

[0021] The liquid suction chamber has several air chambers that penetrate its upper and lower surfaces. Each air chamber has a piston that can move up and down along its axial direction. A sealing connection is provided between the side wall of the piston and the side wall of the air chamber.

[0022] A pipette tip adapter is fixedly connected to the lower side of the aspiration chamber. The pipette tip adapter has several through holes that correspond one-to-one with the air chamber. Each through hole is connected to its corresponding air chamber to form a pipetting channel. A sealing element is provided between the pipette tip adapter and the aspiration chamber. A pipette tip can be mounted at the bottom of the through hole.

[0023] A drive mechanism is provided for driving the piston to move up and down along the axial direction of the air chamber.

[0024] Preferably, the number of air chambers in the liquid absorption cavity is one of 1, 2, 4, 8, 12, 16, 24, 32, 48 and 96.

[0025] Preferably, the spacing between the air chambers is 0.1 mm to 100 mm.

[0026] Preferably, the spacing between the air chambers is one of 2.25 mm, 4.5 mm, 9 mm, and 18 mm.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The solid-phase extraction method and pipetting module provided by this invention, on the one hand, utilize the pipetting module of an automated liquid workstation for solid-phase extraction, eliminating the need for separate dedicated equipment, thus simplifying the structure of the automated liquid workstation and reducing its manufacturing cost; on the other hand, since each air chamber and through-hole on the pipetting module is independent of each other, the pressure source of each hole in the microplate during solid-phase extraction is generated by the downward movement of the piston in its corresponding air chamber. The independent pressure source of each hole effectively avoids the problem of liquid not being completely discharged from other holes due to some holes of the microplate being connected to air during solid-phase extraction. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the specific embodiments will be briefly described below. It should be noted that in all the drawings, the elements or parts are not necessarily drawn to actual scale.

[0030] Figure 1 This is a schematic diagram of the overall structure of the pipetting module in one embodiment of the present invention;

[0031] Figure 2 This is a schematic diagram of the overall structure of the suction chamber of the pipetting module in one embodiment of the present invention;

[0032] Figure 3 This is a cross-sectional view of the overall structure of the suction chamber of the pipetting module in one embodiment of the present invention;

[0033] Figure 4 This is a schematic diagram of the overall structure of the pipette tip adapter of the pipetting module in one embodiment of the present invention.

[0034] In the picture:

[0035] 1. Aspiration chamber; 11. Air chamber; 12. Piston; 2. Pipette tip adapter; 21. Through hole; 3. Drive mechanism. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the system or component referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," etc., used to define components are merely for the convenience of distinguishing the aforementioned components. Unless otherwise stated, these terms have no special meaning and should not be construed as indicating or implying relative importance.

[0038] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0039] Most existing automated liquid workstations require dedicated equipment for solid-phase extraction (SPE). This increases the complexity and manufacturing cost of the automated liquid workstation. Furthermore, during SPE in microplates, the pressure in each well is supplied by the same pressure source. If some wells become open to air during SPE, the pressure in the remaining wells cannot be maintained, leading to incomplete drainage of liquid from those wells. Therefore, this invention provides a SPE method and pipetting module that utilizes the pipetting module of an automated liquid workstation for SPE, eliminating the need for dedicated equipment. This simplifies the structure of the automated liquid workstation and solves the technical problem in existing SPE methods where some wells become open to air, resulting in incomplete drainage of liquid from other wells.

[0040] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0041] Example 1

[0042] This invention provides a solid-phase extraction method, comprising the following steps:

[0043] Step 1: Place the sample to be separated into a microplate with a porous membrane and prepare a solid-liquid mixture by adding solvent through a pipetting module or manually by the operator.

[0044] Step 2: Remove the pipette tip from the pipetting module and move the pipetting module down to above the microplate;

[0045] Specifically, such as Figures 1-4As shown, the pipetting module includes a suction chamber 1, which has multiple air chambers 11 extending through its upper and lower surfaces. The spacing between the microplate holes is the same as or an integer multiple of the spacing between the air chambers 11. Each air chamber 11 contains a piston 12 that can move up and down along its axial direction. The piston 12 is driven by a drive mechanism 3. A pipette tip adapter 2 is also connected below the suction chamber 1. The pipette tip adapter 2 has multiple through holes 21 that correspond one-to-one with the air chambers 11. The drive mechanism 3 includes general-purpose devices such as a through-type stepper motor.

[0046] Step 3: Drive the pipetting module downwards until the pipetting tip adapter 2 is pressed against the microplate;

[0047] Specifically, in the implementation of step three, a sealing gasket can be placed between the pipette tip adapter 2 and the microplate, or other methods can be used to keep the contact point between the pipette tip adapter 2 and the microplate sealed from the outside.

[0048] Step 4: Control the drive mechanism 3 to drive the piston 12 to move downward. The downward movement of the piston 12 generates positive pressure in the gas chamber 11 and the through hole 21. Under the action of positive pressure, the liquid phase in the solid-liquid mixture is separated from the solid-liquid mixture through the pore membrane.

[0049] Furthermore, in the solid-liquid mixture preparation process of this embodiment, the liquid pipetting module is used to blow the liquid to accelerate the dissolution rate of the sample to be separated.

[0050] Specifically, in this embodiment, when preparing a solid-liquid mixture, after adding the sample to be separated and the corresponding solvent, the microplate is placed below the pipetting module and the pipetting module is driven to move downwards until the pipetting tip adapter 2 is close to the microplate. Then, the piston 12 in each air chamber 11 is driven by the driving mechanism 3 to move up and down reciprocally. During the up and down reciprocating movement of the piston 12, an airflow is generated in the air chamber 11 and the through hole 21 to blow the microplate. The airflow can blow the sample to be separated and the solvent in the microplate, thereby accelerating the dissolution rate of the sample to be separated in the solvent.

[0051] It is understood that in other embodiments of the present invention, the dissolution rate of the sample to be separated in the solvent can also be accelerated by other means such as mechanically oscillating the microplate.

[0052] Example 2

[0053] This embodiment provides a pipetting module that can be used in the solid-phase extraction method as described in Embodiment 1, including:

[0054] The liquid suction chamber has several air chambers that penetrate its upper and lower surfaces. Each air chamber has a piston that can move up and down along its axial direction. A sealing connection is provided between the side wall of the piston and the side wall of the air chamber.

[0055] A pipette tip adapter is fixedly connected to the lower side of the aspiration chamber. The pipette tip adapter has several through holes that correspond one-to-one with the air chambers. Each through hole is connected to its corresponding air chamber to form a pipetting channel. There is a sealing element between the pipette tip adapter and the aspiration chamber. A pipette tip can be loaded at the bottom of the through hole.

[0056] The drive mechanism is used to drive the piston to move up and down along the axial direction of the air chamber.

[0057] It should be noted that the number of air chambers 11 on the aspiration chamber 1 of the pipetting module in this embodiment can be set according to actual needs. For example, the number of air chambers 11 on the aspiration chamber 1 can be 1, 2, 4, 8, 12, 16, 24, 32, 48 and 96.

[0058] Preferably, in this embodiment, the number of air chambers 11 on the liquid aspiration chamber 1 of the pipetting module is 96.

[0059] Furthermore, in this embodiment, the spacing between the gas chambers 11 on the pipetting module is 0.1 mm to 100 mm.

[0060] Of course, the spacing of the gas chambers 11 on the pipetting module of the present invention is not limited to the above range, and can be set accordingly in other specific embodiments according to actual needs.

[0061] Preferably, the spacing between the gas chambers 11 on the pipetting module in this embodiment is one of 2.25 mm, 4.5 mm, 9 mm and 18 mm.

[0062] Example 3

[0063] This embodiment uses the solid-phase extraction method provided in Embodiment 1 to perform solid-phase extraction on paraffin-embedded tissue to achieve dewaxing, including the following steps:

[0064] Step 1: Place the paraffin-embedded tissue sample in a microplate with a porous membrane and prepare a solid-liquid mixture by adding appropriate solvents such as water, lysis buffer or other reagents through a pipetting module or manually by the operator.

[0065] The paraffin-embedded tissue samples ranged from 1 to 10 pieces, with a thickness of 1 μm to 1000 μm; the solvent volume was 10 μL.

[0066] ~1000μL.

[0067] Step 2: Remove the pipette tip from the pipetting module and move the pipetting module down to above the microplate;

[0068] Step 3: Drive the pipetting module downwards until the pipetting tip adapter 2 is pressed against the microplate;

[0069] Step 4: Control the drive mechanism 3 to drive the piston 12 to move downward. The downward movement of the piston 12 generates positive pressure in the gas chamber 11 and the through hole 21. Under the action of positive pressure, the liquid phase in the solid-liquid mixture is separated from the solid-liquid mixture through the pore membrane, thereby leaving the paraffin layer on the pore membrane and achieving the purpose of dewaxing.

[0070] Specifically, the preparation of the solid-liquid mixture in this embodiment includes the following steps:

[0071] (1) Place the paraffin-embedded sample in a microplate with a porous membrane and add appropriate solvents such as water, lysis buffer or other reagents;

[0072] (2) The microplate is heated so that the paraffin, biological tissue and molecules in the paraffin-embedded sample dissolve into the solvent to form a mixture;

[0073] (3) Cool the mixture until the paraffin wax precipitates out of the mixture, solidifies into flakes, and floats on the aqueous phase of the mixture, thus producing a solid-liquid mixture.

[0074] Example 4

[0075] This embodiment uses the solid-phase extraction method provided in Embodiment 1 to perform solid-phase extraction on environmental scene solid samples (such as bloodstains on a wall) extracted by forensic experts from a crime scene to extract soluble molecules (such as nucleic acids) from the environmental scene samples, including the following steps:

[0076] Step 1: Place the environmental scene sample in a microplate with a porous membrane and prepare a solid-liquid mixture by adding appropriate solvents such as water, lysis buffer or other reagents through a pipetting module or manually by the operator.

[0077] The environmental scene sample mass ranges from 0.1g to 10g, and the solvent volume ranges from 0.01mL to 10mL.

[0078] Step 2: Remove the pipette tip from the pipetting module and move the pipetting module down to above the microplate;

[0079] Step 3: Drive the pipetting module downwards until the pipetting tip adapter 2 is pressed against the microplate;

[0080] Step 4: Control the drive mechanism 3 to drive the piston 12 to move downward. The downward movement of the piston 12 generates positive pressure in the gas chamber 11 and the through hole 21. Under the action of positive pressure, the liquid phase in the solid-liquid mixture is separated from the solid-liquid mixture through the pore membrane, so that the liquid phase containing dissolved soluble small molecules passes through the membrane layer while the solid particles remain on the pore membrane, thereby realizing the extraction of soluble molecules (such as nucleic acids) in environmental scene samples.

[0081] Specifically, the preparation of the solid-liquid mixture in this embodiment includes the following steps:

[0082] (1) Place the solid sample of the environmental scene in a microplate with a porous membrane and add appropriate solvents such as water, lysis buffer or other reagents;

[0083] (2) The microplate is heated so that the nucleic acid or other soluble molecules in the solid sample of the environmental scene dissolve into the solvent, thereby making a solid-liquid mixture.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A solid-phase extraction method, characterized in that, For use in a pipetting module, the pipetting module includes: The liquid suction chamber has several air chambers that penetrate its upper and lower surfaces. Each air chamber has a piston that can move up and down along its axial direction. A sealing connection is provided between the side wall of the piston and the side wall of the air chamber. A pipette tip adapter is fixedly connected to the lower side of the aspiration chamber. The pipette tip adapter has several through holes that correspond one-to-one with the air chamber. Each through hole is connected to its corresponding air chamber to form a pipetting channel. A sealing element is provided between the pipette tip adapter and the aspiration chamber. A pipette tip can be mounted at the bottom of the through hole. A drive mechanism is used to drive the piston to move up and down along the axial direction of the air chamber; A sealing gasket is placed between the pipette tip adapter and the microplate to keep the contact point between the pipette tip adapter and the microplate sealed from the outside environment. The solid-phase extraction method includes the following steps: The sample to be separated is placed in a microplate with a porous membrane and a solvent is added by a pipetting module or manually by the operator to prepare a solid-liquid mixture. Remove the pipette tip from the pipette module and drive the pipette module to move down above the microplate. The pipette module includes an aspiration chamber with multiple air chambers penetrating its upper and lower surfaces. The hole spacing of the microplate is the same as or an integer multiple of the air chamber spacing. Each air chamber has a piston that can move up and down along its axial direction. The piston is driven by a drive mechanism. A pipette tip adapter is also connected below the aspiration chamber. The pipette tip adapter has multiple through holes that correspond one-to-one with the air chambers. Drive the pipetting module downwards until the pipetting tip adapter is pressed against the microplate; The control drive mechanism drives the piston to move downward. The downward movement of the piston generates positive pressure in the air chamber and the through hole. Under the action of the positive pressure, the liquid phase in the solid-liquid mixture is separated from the solid-liquid mixture through the pore membrane, so that the liquid phase containing dissolved soluble small molecules passes through the membrane layer while the solid particles remain on the pore membrane, thereby realizing the extraction of soluble molecules in environmental scene samples. During the preparation of the solid-liquid mixture, the liquid transfer module is used to blow the liquid to accelerate the dissolution rate of the sample to be separated in the solvent. The drive mechanism drives the piston in each air chamber to move up and down reciprocally. During the up and down reciprocating movement of the piston, an airflow is generated in the air chamber and through hole to blow the microplate. The airflow can blow the sample to be separated and the solvent in the microplate, thereby accelerating the dissolution rate of the sample to be separated in the solvent.

2. The solid-phase extraction method as described in claim 1, characterized in that, During the preparation of the solid-liquid mixture, mechanical oscillation is used to accelerate the dissolution rate of the sample to be separated in the solvent.

3. The solid-phase extraction method as described in claim 1, characterized in that, The sample to be separated is paraffin-embedded tissue. The sample is placed in a microplate with a porous membrane and a solvent is added to prepare a solid-liquid mixture, comprising: The paraffin-embedded tissue was placed in a microplate with a porous membrane, and the appropriate solvent was added by means of a pipetting module or by manual operation. The microplate is heated so that the paraffin, biological tissue, and molecules in the paraffin-embedded tissue dissolve into the solvent to form a mixture; The mixture is cooled until the paraffin wax precipitates out of the mixture, solidifies into flakes, and floats on the aqueous phase of the mixture, thereby producing the solid-liquid phase mixture.

4. The solid-phase extraction method as described in claim 1, characterized in that, The sample to be separated is a solid environmental sample extracted by forensic experts from the crime scene. The sample is placed in a microplate with a porous membrane and a solvent is added to prepare a solid-liquid mixture, including: The solid sample of the environmental scene is placed in a microplate with a porous membrane, and the corresponding solvent is added by means of a pipetting module or by the operator manually. The microplate is heated so that nucleic acid substances or other soluble molecules in the solid sample of the environmental scene dissolve into the solvent, thereby preparing the solid-liquid mixture.

5. The solid-phase extraction method as described in claim 1, characterized in that, The number of air chambers in the liquid absorption cavity is one of 1, 2, 4, 8, 12, 16, 24, 32, 48, and 96.

6. The solid-phase extraction method as described in claim 1, characterized in that, The spacing between the air chambers is 0.1mm to 100mm.

7. The solid-phase extraction method as described in claim 6, characterized in that, The spacing between the air chambers is one of 2.25 mm, 4.5 mm, 9 mm, and 18 mm.

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