WO2023216591A1

WO2023216591A1 - Purification separation column for clinical detection pretreatment, pretreatment apparatus, pretreatment method and application

Info

Publication number: WO2023216591A1
Application number: PCT/CN2022/139665
Authority: WO
Inventors: 贺笑非; 冯梦雪
Original assignee: 美羅醫藥科技有限公司
Priority date: 2022-05-11
Filing date: 2022-12-16
Publication date: 2023-11-16
Also published as: CN116407871A; CN114949929A

Abstract

A purification separation column for clinical detection pretreatment, a pretreatment apparatus, a pretreatment method, and an application. The purification separation column comprises a column tube (1) open at at least one end, the column tube (1) having disposed therein a filtering assembly, and a filtrate to be filtered entering through the opening from the outside of the column tube (1), passing through the filtering assembly and then entering an inner cavity of the column tube (1); the filtering assembly comprises, sequentially arranged from bottom to top, a first sieve plate (6), a water blocking layer (4), a filler layer (3) and a second sieve plate (2). The purification separation column is primarily used for carrying out pretreatment on a clinically detected sample to be detected; conventional solid-phase extraction requires undergoing a plurality of operations such as activation, adsorption, washing and elution, wherein the involved solution reagents differ from one another; the present purification separation column achieves one-step pretreatment of a sample to be detected.

Description

A purification separation column, pretreatment device, pretreatment method and application for clinical detection pretreatment

Technical field

This application belongs to the field of clinical detection technology, and specifically relates to a purification separation column used for pre-treatment of clinical detection, a pre-treatment device, a pre-treatment method and an application.

Background technique

Solid-phase extraction (SPE) technology is a sample pretreatment method based on chromatography theory. It uses selective adsorption and selective elution to enrich, separate and purify samples. Solid-phase extraction uses solid adsorbents to adsorb target substances in liquid samples to separate the target substances from the matrix and interfering compounds of the sample, and then uses eluent to elute or heat to mediate adsorption to achieve the purpose of separating and enriching the target substances. .

Compared with traditional liquid-liquid extraction, solid-phase extraction does not require a large amount of mutually immiscible solvents, and no emulsification will occur during the treatment process; due to the use of highly selective adsorbents (stationary phases), solid-phase extraction can significantly reduce solvent emissions. Dosage; The pretreatment process of solid phase extraction is relatively simple, has the advantages of strong selectivity, high recovery rate and easy automation. It is widely used in fields such as water quality testing, pharmaceuticals, environmental analysis, food analysis and tobacco analysis. It is a very common pretreatment method for testing organic compounds (including clinical applications), including vitamin D, aldosterone, steroid hormones, bile acids, etc.

The solid-phase extraction operation can be divided into four steps: column pretreatment, sample loading, column washing, and analyte elution, as shown in the figure below. Whether each step is properly designed will have an impact on the recovery rate of the experiment, leading to premature penetration of solutes, incomplete washing of interferences, loss of target substances, incomplete elution, etc. At this time, it is generally difficult to recover the analytes. accomplish.

CN212974266U discloses a solid phase extraction rapid separation device and a solid phase extraction tube, including a pressurizing structure, a solid phase extraction tube and a filter head. The pressurizing structure includes a foot-operated inflatable cylinder and a buffer airbag. The foot-operated The air outlet of the inflatable cylinder is connected to the connector through a pipeline, the buffer air bag is arranged on the pipeline, and the filter head is inserted into the opening of the liquid outlet pipe of the solid phase extraction tube. When the adsorbent material completely adsorbs the sample liquid in the solid phase extraction tube body, use a foot-operated inflatable cylinder to continuously pressurize the solid phase extraction tube body to expel the processed sample fluid in the solid phase extraction tube body. , flows out of the solid phase extraction tube body through the filter head.

CN212039361U discloses a centrifugal and pneumatic dual-purpose solid phase extraction and separation device, which includes two separate upper and lower components. The upper part is the solid phase extraction and separation component, and the lower part is the collection component. Solid-phase extraction and separation components include a column, a sealing cover, a sieve plate and a packing. The upper end of the column is connected to the sealing cover, and the lower part of the column can be connected to the lower components through a spiral; the sealing cover has an air inlet The air inlet hole can be connected with an external pressurizing device to increase the air pressure in the column; the sieve plate is located in the middle of the column, and two or more sieve plates are fixed at intervals. The collection component is a collection tube, the upper end of which is connected to the sealing cover; there is an air hole in the middle and upper part of the collection tube to ensure communication between the inside of the tube and the outside world.

CN202161857U discloses a solid-phase extraction pre-treatment filter, which is used in the pre-treatment process of test samples. The filter is a funnel-shaped device made of glass. The upper part has a trapezoidal structure and the lower part has a cylindrical structure. The outer diameter of the cylindrical part is consistent with the inner diameter of the solid phase extraction column. When used, filter paper is built into the filter, and then the cylinder is Part of it is placed over the solid phase extraction column.

When performing solid-phase extraction, it usually takes several steps to purify the pre-treatment solution to obtain the sample, including preliminary sample treatment, packing activation, sample adsorption, one to three washings and elution, etc. Since the pores of the solid-phase extraction cartridge packing are small, there are special requirements for the purity of the sample. There must be no suspended matter or solid particles, otherwise the cartridge will be blocked and subsequent operations will be difficult to perform; due to activation, adsorption, washing, elution, etc. Several different solutions are involved in this step. When performing pre-treatment operations, you need to use a positive pressure or negative pressure device to roughly drain the solution in the solid-phase extraction cartridge before adding the next step solution. However, do not over-dry and maintain the consistency of the filler. The wettability ensures its separation performance. Therefore, during the processing process, the degree of dryness of the filler surface, the groove path formed by the residual liquid, and the resistance of the sample liquid are difficult to maintain consistent, thus affecting the intra-batch and inter-batch consistency of the sample.

Contents of the invention

This application provides a purification separation column, pre-treatment device, pre-treatment method and application for pre-processing of clinical testing. It is mainly used for pre-processing samples to be tested for clinical testing. Conventional clinical testing uses solid-phase extraction. Pretreatment requires multiple steps such as activation, adsorption, washing and elution, and the solution reagents involved are also different. In order to simplify the pretreatment steps, this application provides a purification separation column to achieve the purpose of testing. The one-step pre-processing of the sample greatly reduces the pre-processing operation process, and does not require careful operation during the process, lowering the operating threshold.

In a first aspect, this application provides a purification and separation column for pre-treatment of clinical testing. The purification and separation column includes a column tube with at least one end open. A filter component is provided in the column tube. The liquid to be filtered is discharged from the outside of the column tube. Enter through the opening, pass through the filter assembly and then enter the inner cavity of the column tube;

Wherein, the filter assembly includes a first screen plate, a water-blocking layer, a filler layer and a second screen plate arranged in sequence from bottom to top.

The purification separation column provided in this application is mainly used for pre-processing the samples to be tested for clinical testing. Conventional clinical testing uses solid-phase extraction for pre-processing, which requires multi-step operations such as activation, adsorption, washing and elution, which involves The solution reagents obtained are also different. In order to simplify the pre-treatment steps, this application provides a purification separation column, which realizes one-step pre-treatment of the sample to be tested, greatly simplifying the pre-treatment operation process and reducing the amount of reagents. , improve sample processing efficiency; and no need to be careful during the process, lowering the operating threshold. When used, the sample to be tested passes through the filter assembly from the bottom of the purification separation column and then enters the main chamber of the purification separation column. In the process of passing through the filter assembly, the impurities in the sample to be tested are initially separated through the first sieve plate, blocking The polymer compounds in the water layer allow the organic phase to pass smoothly, and expand rapidly when encountering the water phase. While blocking the water, the organic phase and the aqueous phase are separated at the same time. The water phase and protein precipitation in the liquid to be filtered are blocked in the water blocking layer. Under the layer, the organic phase then continues to pass through the packing layer. The packing layer is filled with adsorption materials with specific adsorption functions. Through the selective adsorption of the adsorption materials, specific substances in the organic phase are adsorbed, so that the extraction liquid is purified. The second sieve plate can filter some layered impurities and granular impurities missed in the previous layers to avoid clogging the chromatographic column.

As a preferred technical solution of the present application, the filter assembly is located at the bottom of the inner cavity of the column tube, and the filtrate passes through the filter assembly from the bottom of the column tube and then enters the inner cavity of the column tube.

Preferably, the filler layer is filled with purification materials, and the purification materials include specific adsorption materials for adsorbing polypeptides and phospholipid compounds.

Preferably, the purification materials include silica gel, octadecyl bonded silica gel and its derivatives, octadecyl bonded silica gel and its derivatives, diatomaceous earth, activated carbon, N-propylethylenediamine, quartz sand, Any one or a combination of at least two of molecular sieves, hydrophilic lipophilic polymeric fillers, strong cation exchange resins, weak cation exchange resins, strong anion exchange resins or weak anion exchange resins.

It should be noted that considering that blood samples mainly contain proteins, peptides, fats (mainly phospholipid compounds), inorganic salts, etc., they need to be separated from the target substances to be measured so as not to affect subsequent detection, and try to avoid large matrix effects. Therefore, during the extraction process using acetonitrile, the proteins in the sample first precipitate, and the inorganic salts in the sample still remain in the aqueous phase due to their strong water solubility. Therefore, the purification filler used in this application is preferably one that can adsorb polypeptides and phospholipid compounds. Specific adsorption materials to further remove impurities from the sample.

Preferably, the particle size of the purification material is 5 to 2000 μm, for example, it can be 5 μm, 50 μm, 100 μm, 300 μm, 500 μm, 700 μm, 900 μm, 1000 μm, 1200 μm, 1400 μm, 1600 μm, 1800 μm or 2000 μm, but is not limited to those listed. value, other unlisted values within this value range are also applicable.

Preferably, the filling mass of the purification material is 10 to 100 mg, for example, it can be 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg, but is not limited to For the listed values, other unlisted values within the range are also applicable.

As a preferred technical solution of the present application, the pore diameter of the first sieve plate is the same as or different from the pore diameter of the second sieve plate.

Preferably, the pore diameter of the first sieve plate is 5-50 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, but is not limited to the listed values. The same applies to other values within the range not listed.

Preferably, the pore diameter of the second sieve plate is 5-50 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, but is not limited to the listed values. The same applies to other values within the range not listed.

Preferably, the water-blocking layer is a water-blocking film, and the pore diameter of the water-blocking film is 5 to 50 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, but not Not limited to the listed values, other unlisted values within this range are also applicable.

It should be noted that the water-blocking layer defined in this application can adopt a sieve plate structure with a certain pore size. The inner surface of the pores is coated with a polymer compound. The organic phase can pass through the water-blocking layer, and the polymer compound absorbs water and expands when exposed to water. , thereby achieving the effect of hindering the passage of the water phase, and finally achieving the separation of the water phase and the organic phase.

As a preferred technical solution of this application, a filter layer is also provided between the first screen plate and the water-blocking layer.

During the pretreatment process of the sample to be tested using the purification separation column provided by this application, it is necessary to add an extraction agent, such as acetonitrile, etc. For blood samples, it mainly contains proteins and peptides. After adding the extraction agent, the protein in the blood sample Precipitation will form. By setting a filter layer between the first sieve plate and the water-blocking layer, the protein precipitation can be filtered and intercepted to avoid clogging the water-blocking layer at the rear end.

Preferably, the filter layer includes at least one filter membrane.

Preferably, the filter layer includes at least two layers of filter membranes, and the filter membranes have the same or different pore sizes.

Preferably, the filter layer includes at least two layers of filter membranes with different pore sizes, and the pore sizes of the filter membranes decrease sequentially from bottom to top.

Preferably, the filtration layer includes a first filter membrane, a second filter membrane and a third filter membrane stacked sequentially from bottom to top, and the pore size of the first filter membrane ≥ the pore size of the second filter membrane ≥ the third filter membrane aperture.

Preferably, the pore size of the first filter membrane is 0.5-2.5 μm, for example, it can be 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.2 μm, 1.5 μm, 2.0 μm, 2.5 μm, but are not limited to the listed values, and other unlisted values within this numerical range are also applicable.

Preferably, the pore size of the second filter membrane is 0.2-0.8 μm, for example, it can be 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7μm, 0.75μm or 0.8μm, but are not limited to the listed values, and other unlisted values within this numerical range are also applicable.

Preferably, the pore size of the third filter membrane is 0.1-0.5 μm, for example, it can be 0.1 μm, 0.15 μm, 0.2 μm, 0.22 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm or 0.5 μm, However, it is not limited to the listed values, and other unlisted values within the range of values are also applicable.

In this application, when the pore sizes of the first filter membrane, the second filter membrane and the third filter membrane are different, they decrease in sequence from bottom to top, so that the purification separation column can adapt to the filtration of different liquids to be filtered, thus expanding the capabilities of the purification separation column. Scope of application; at the same time, the pore sizes of each layer of filter membrane are different, which makes the separation process of the liquid to be filtered progressive layer by layer and enhances the separation effect of the liquid to be filtered.

In a second aspect, the present application provides a pre-treatment device for clinical testing, which includes a liquid storage tube and a purification separation column for pre-processing of clinical testing described in the first aspect.

The liquid storage tube is used in conjunction with the purification separation column. The extraction reagent is pre-distributed in the liquid storage tube. The sample to be tested is added to the liquid storage tube. The sample to be tested is mixed with the extraction reagent and then left to stratify. The purification and separation column is pressed into the liquid storage tube, and the upper organic phase passes through the filter assembly from the bottom of the purification and separation column and enters the inner cavity of the column tube of the purification and separation column.

As a preferred technical solution of the present application, the inner cavity of the column tube of the purification separation column is divided into a first liquid storage chamber and a second liquid storage chamber from bottom to top.

Preferably, the diameter of the outer wall of the column tube corresponding to the first liquid storage chamber is smaller than the diameter of the outer wall of the column tube corresponding to the second liquid storage chamber, so that an annular shape is formed at the outer wall of the column tube between the first liquid storage chamber and the second liquid storage chamber. Step surface; after the purification separation column is pressed into the liquid storage tube, the open end surface of the liquid storage tube contacts the annular step surface.

As a preferred technical solution of the present application, a sealing ring is provided along the circumferential direction on the outer wall of the column tube at a corresponding position of the filter assembly.

In this application, the outer wall of the column tube is provided with a sealing ring along the circumferential direction. After the purification separation column is pushed into the liquid storage tube, the sealing ring can close the gap between the purification separation column and the liquid storage tube to prevent the sample to be tested from purifying. The gap between the separation column and the liquid storage tube flows out, ensuring that the sample to be measured stored in the liquid storage tube can only enter through the bottom opening of the purification separation column.

Preferably, a cutout is provided at the top opening of the column tube of the purification separation column. When the purification separation column is pressed down toward the liquid storage pipe, the air in the liquid storage pipe is discharged through the cutout.

In the third aspect, this application provides a pre-processing method for clinical detection, which uses the pre-processing device for clinical detection described in the second aspect to pre-process the sample to be tested. The pre-processing method includes:

Add the sample to be tested into the liquid storage tube, mix the sample to be tested with the pre-placed extraction reagent and let it stand. After the aqueous phase and organic phase are initially separated, press the purification separation column into the liquid storage tube, and the organic phase will pass through the purification separation column. The bottom passes through the filter component and enters the inner cavity of the column tube of the purification separation column. The aqueous phase is isolated by the filter component. The organic phase entering the inner cavity is the sample to be tested after pretreatment. The sample to be tested after pretreatment is put on the machine for detection or nitrogen blowing. Then reconstitute and test on the machine.

In addition to redesigning the structure of the purification separation column, this application also improves the operation process of the pretreatment device. Compared with traditional dispersive solid phase extraction and solid phase extraction, the operation of the pretreatment method provided by this application is It is simple and has good purification effect, realizing one-step purification treatment. in particular:

(1) The conventional operation process of the traditional dispersive solid phase extraction method includes: adding extraction reagents to the sample to be tested to form a mixed solution, adding a separation material package to the mixed solution, followed by vortex mixing, and then performing the first centrifugation. After the aqueous phase and the organic phase are separated, transfer the upper organic phase to another container, then add the purification agent and vortex for a second time to mix, let it stand or centrifuge, and then use a filter membrane to filter the organic phase and transfer it to another container. In a container, the organic phase is finally taken and tested on the machine or the organic phase is reconstituted by nitrogen blowing and then tested on the machine. It can be seen from this that the dispersive solid phase extraction method requires multiple transfers of the organic phase, and also involves complex operations such as two centrifugations and two vortex mixings. In addition, the purification agent used in the dispersive solid phase extraction method only relies on its adsorption for purification. In this application, the purification agent packing is designed as a bed structure and filled into the column tube. When the extract liquid passes through the packing layer, it uses the adsorption of the purification agent. While purifying, it can also intercept some impurities, thereby flowing out the purified supernatant.

(2) The traditional solid-phase extraction method has a complicated process. Most samples need to be pre-treated before using the solid-phase extraction method, such as adding regulators, diluents or precipitating agents to the samples to be tested to pre-precipitate proteins to ensure no clogging. Column. In addition, the solid-phase extraction cartridge needs to complete a two-step activation, multiple rinses and elution processes under a positive pressure or negative pressure device. During the process, the degree of drying needs to be controlled to ensure that the filler is moist and does not affect the column efficiency. At the same time, a large amount of Waste liquid, during elution, additional collection plates need to be replaced to collect the eluate, which places higher requirements on operators. Using the pretreatment device provided by this application, you only need to press the purification separation column into the liquid storage tube, which saves the complex operations such as activation, washing, elution and replacement of the collection plate required by the traditional solid phase extraction method, which greatly improves the efficiency of the process. The operation difficulty is reduced and the operation time is saved. At the same time, the method of the present application basically produces no waste liquid and is more environmentally friendly than the solid-phase extraction method.

As a preferred technical solution of this application, the extraction reagent includes salts and extraction agents.

Preferably, the salts include any one or a combination of at least two of magnesium sulfate, sodium sulfate, sodium chloride or potassium chloride.

Preferably, the extraction agent includes acetonitrile, a mixture of acetonitrile and methanol, acidified acetonitrile or a mixture of acidified acetonitrile and methanol.

Preferably, the extraction reagent further includes a purifying agent.

Preferably, the purification agent includes any one or a combination of at least two of N-propylethylenediamine, activated carbon or C18.

In a fourth aspect, the present application provides an application of the pre-processing device described in the second aspect, where the pre-processing device is used to pre-process a sample to be tested before clinical testing.

The numerical range described in this application not only includes the point values listed above, but also includes any point value between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, this application will not exhaustively list the ranges. Specific point values included.

Compared with the existing technology, the beneficial effects of this application are:

The purification separation column provided in this application is mainly used for pre-processing the samples to be tested for clinical testing. Conventional clinical testing uses solid-phase extraction for pre-processing, which requires multi-step operations such as activation, adsorption, washing and elution, which involves The solution reagents obtained are also different. In order to simplify the pre-treatment steps, this application provides a purification separation column, which realizes one-step pre-treatment of the sample to be tested, greatly simplifying the pre-treatment operation process and reducing the amount of reagents. , improve sample processing efficiency; no need to operate carefully during the process, lowering the operating threshold; basically no waste liquid is produced, and it is environmentally friendly. When used, the sample to be tested passes through the filter assembly from the bottom of the purification separation column and then enters the main chamber of the purification separation column. In the process of passing through the filter assembly, the impurities in the sample to be tested are initially separated through the first sieve plate, blocking The polymer compounds in the water layer allow the organic phase to pass smoothly, and expand rapidly when encountering the water phase. While blocking the water, the organic phase and the aqueous phase are separated at the same time. The water phase and protein precipitation in the liquid to be filtered are blocked in the water blocking layer. Under the layer, the organic phase then continues to pass through the packing layer. The packing layer is filled with adsorption materials with specific adsorption functions. Through the selective adsorption of the adsorption materials, specific substances in the organic phase are adsorbed, so that the extraction liquid is purified. The second sieve plate can filter some layered impurities and granular impurities missed in the previous layers to avoid clogging the chromatographic column.

Description of the drawings

Figure 1 is a schematic structural diagram of a purification separation column provided by a specific embodiment of the present application;

Figure 2 is a schematic structural diagram of a pre-processing device provided by a specific embodiment of the present application;

Among them, 1-column tube; 2-second sieve plate; 3-packing layer; 4-water blocking layer; 5-filtration layer; 51-first filter membrane; 52-second filter membrane; 53-third filter membrane ; 6-First sieve plate; 7-Sealing ring; 8-Notch; 9-Liquid storage tube; 10-Organic phase; 11-Aqueous phase.

Detailed ways

It should be understood that in the description of this application, the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "back", "left", "right", " The orientations or positional relationships indicated by "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present application and The simplified description is not intended to indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be construed as a limitation on the present application. Furthermore, the terms “first”, “second”, etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, features defined by "first," "second," etc. may explicitly or implicitly include one or more of such features. In the description of this application, unless otherwise stated, "plurality" means two or more.

It should be noted that in the description of this application, unless otherwise clearly stated and limited, the terms "set", "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection or a detachable connection. Connection, or integrated connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be an internal connection between two components. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood through specific circumstances.

The technical solutions of the present application will be further described below through specific implementations.

In a specific embodiment, the present application provides a purification and separation column for pre-treatment of clinical detection. As shown in Figure 1, the purification and separation column includes a column tube 1 with at least one end open. A filter component is provided, and the liquid to be filtered enters from the outside of the column tube 1 through the opening, and then enters the inner cavity of the column tube 1 after passing through the filter component;

Wherein, the filter assembly includes a first screen plate 6, a water blocking layer 4, a filler layer 3 and a second screen plate 2 arranged in sequence from bottom to top.

The purification separation column provided in this application is mainly used for pre-processing the samples to be tested for clinical testing. Conventional clinical testing uses solid-phase extraction for pre-processing, which requires multi-step operations such as activation, adsorption, washing and elution, which involves The solution reagents obtained are also different. In order to simplify the pre-treatment steps, this application provides a purification separation column, which realizes one-step pre-treatment of the sample to be tested, greatly simplifying the pre-treatment operation process and reducing the amount of reagents. , improve sample processing efficiency; and no need to be careful during the process, lowering the operating threshold. When in use, the sample to be tested passes through the filter assembly from the bottom of the purification separation column and then enters the main chamber of the purification separation column. In the process of passing through the filter assembly, the impurities in the sample to be tested are initially separated through the first sieve plate 6. The polymer compound in the water-blocking layer 4 allows the organic phase 10 to pass smoothly, and expands rapidly when encountering the water phase 11. While blocking water, the organic phase 10 and the water phase 11 are separated, and the water phase 11 and the water phase 11 in the liquid to be filtered are separated. The protein precipitation is blocked under the water-blocking layer 4, and then the organic phase 10 continues to pass through the packing layer 3. The packing layer 3 is filled with adsorbent materials with specific adsorption functions. The selective adsorption of the adsorbent materials will Specific substances are adsorbed to purify the extraction solution. The second sieve plate 2 can filter some layered impurities and granular impurities missed in the previous layers to avoid clogging the chromatographic column.

Further, the filter assembly is located at the bottom of the inner cavity of the column tube 1, and the filtrate passes through the filter assembly from the bottom of the column tube 1 and then enters the inner cavity of the column tube 1.

Further, the filler layer 3 is filled with purification materials, and the purification materials include specific adsorption materials for adsorbing polypeptides and phospholipid compounds.

Further, the purification materials include silica gel, octadecyl bonded silica gel and its derivatives, octadecyl bonded silica gel and its derivatives, diatomaceous earth, activated carbon, N-propylethylenediamine, quartz sand, Any one or a combination of at least two of molecular sieves, hydrophilic lipophilic polymeric fillers, strong cation exchange resins, weak cation exchange resins, strong anion exchange resins or weak anion exchange resins.

It should be noted that considering that blood samples mainly contain proteins, peptides, fats (mainly phospholipid compounds), inorganic salts, etc., they need to be separated from the target substances to be measured so as not to affect subsequent detection, and try to avoid large matrix effects. Therefore, during the extraction process using acetonitrile (or a mixture with methanol), the protein in the sample precipitates first, and the inorganic salts in the sample still remain in the aqueous phase due to their strong water solubility. 11 Therefore, the purification filler used in this application is preferred. Specific adsorption materials that can adsorb polypeptides and phospholipid compounds to further remove impurities from the sample.

Further, the particle size of the purification material is 5 to 2000 μm.

Further, the filling mass of the purification material is 10 to 100 mg.

Furthermore, the aperture diameter of the first sieve plate 6 is the same as or different from the aperture diameter of the second sieve plate 2 .

Further, the pore diameter of the first sieve plate 6 is 5-50 μm, and the pore diameter of the second sieve plate 2 is 5-50 μm.

Further, the water-blocking layer 4 is a water-blocking film, and the pore size of the water-blocking film is 5 to 50 μm.

It should be noted that the water-blocking layer 4 defined in this application can adopt a sieve plate structure with a certain pore diameter, and the inner surface of the pores is coated with a polymer compound. The organic phase 10 can pass through the water-blocking layer 4, and the polymer compound encounters water. Then it absorbs water and expands, thus achieving the effect of hindering the passage of the water phase 11, and finally realizing the separation of the water phase 11 and the organic phase 10.

Furthermore, a filter layer 5 is provided between the first screen plate 6 and the water-blocking layer 4 .

During the pretreatment process of the sample to be tested using the purification separation column provided by this application, it is necessary to add an extraction agent, such as acetonitrile, etc. For blood samples, it mainly contains proteins and peptides. After adding the extraction agent, the protein in the blood sample Precipitates will form. By setting a filter layer 5 between the first sieve plate 6 and the water-blocking layer 4, the protein precipitate can be filtered and intercepted to avoid clogging the water-blocking layer 4 at the rear end.

Further, the filter layer 5 includes at least one filter membrane.

Further, the filter layer 5 includes at least two layers of filter membranes, and the pore sizes of the filter membranes are the same or different.

Further, the filter layer 5 includes at least two layers of filter membranes with different pore sizes, and the pore sizes of the filter membranes decrease sequentially from bottom to top.

Further, the filter layer 5 includes a first filter membrane 51 , a second filter membrane 52 and a third filter membrane 53 that are stacked sequentially from bottom to top. The pore size of the first filter membrane 51 ≥ that of the second filter membrane 52 The pore size ≥ the pore size of the third filter membrane 53 .

Specifically, the pore size of the first filter membrane 51 is 0.5-2.5 μm, the pore size of the second filter membrane 52 is 0.2-0.8 μm, and the pore size of the third filter membrane 53 is 0.1-0.5 μm.

In this application, when the pore sizes of the first filter membrane 51, the second filter membrane 52 and the third filter membrane 53 are different, they decrease in sequence from bottom to top, so that the purification separation column can adapt to the filtration of different liquids to be filtered, thereby expanding the purification efficiency. The applicable range of the separation column; at the same time, the pore sizes of each layer of filter membrane are different, which makes the separation process of the liquid to be filtered progressive layer by layer and enhances the separation effect of the liquid to be filtered.

In another specific embodiment, the present application provides a pre-treatment device for clinical detection, as shown in Figure 2. The pre-treatment device includes a liquid storage tube 9 and the above-mentioned purification separation column for pre-treatment of clinical detection. .

The liquid storage tube 9 is used in conjunction with the purification separation column. The extraction reagent is pre-dispensed in the liquid storage tube 9. The sample to be tested is added to the liquid storage tube 9. The sample to be tested is mixed with the extraction reagent and then allowed to stand for separation. layer, the purification and separation column is pressed into the liquid storage tube 9, and the upper organic phase 10 passes through the filter assembly from the bottom of the purification and separation column and enters the inner cavity of the column tube 1 of the purification and separation column.

Further, the inner cavity of the column tube 1 of the purification separation column is divided into a first liquid storage chamber and a second liquid storage chamber from bottom to top.

Further, the diameter of the outer wall of the column tube 1 corresponding to the first liquid storage chamber is smaller than the diameter of the outer wall of the column tube 1 corresponding to the second liquid storage chamber, so that the outer wall of the column tube 1 between the first liquid storage chamber and the second liquid storage chamber An annular step surface is formed at the purification separation column; after the purification separation column is pressed into the liquid storage tube 9, the open end surface of the liquid storage tube 9 is in contact with the annular step surface.

Furthermore, a sealing ring 7 is provided along the circumferential direction on the outer wall of the column tube 1 at the corresponding position of the filter assembly.

In this application, a sealing ring 7 is provided on the outer wall of the column tube 1 along the circumferential direction. After the purification separation column is pushed into the liquid storage pipe 9, the sealing ring 7 can close the gap between the purification separation column and the liquid storage pipe 9 to prevent The sample to be measured flows out from the gap between the purification separation column and the liquid storage tube 9, ensuring that the sample to be measured stored in the liquid storage tube 9 can only enter through the bottom opening of the purification separation column.

Furthermore, a cutout 8 is provided at the top opening of the column tube 1 of the purification and separation column. When the purification and separation column is pressed down toward the liquid storage pipe 9, the air in the liquid storage pipe 9 is discharged from the cutout 8.

In another specific embodiment, the present application provides a pre-processing method for clinical detection, which uses the above-mentioned pre-processing device for clinical detection to pre-process the sample to be tested. The pre-processing method includes:

Add the sample to be tested into the liquid storage tube 9. The sample to be tested is mixed with the pre-placed extraction reagent and left to stand. After the aqueous phase 11 and the organic phase 10 are initially separated, press the purification separation column into the liquid storage tube 9. The organic phase 10 passes through the filter assembly from the bottom of the purification separation column and enters the inner cavity of the column tube 1 of the purification separation column. The aqueous phase 11 is isolated by the filter assembly. The organic phase 10 entering the inner cavity is the sample to be tested after pretreatment. The processed sample to be tested is tested on the machine or the sample is reconstituted after nitrogen blowing and tested on the machine.

(1) The conventional operation process of the traditional dispersive solid phase extraction method includes: adding extraction reagents to the sample to be tested to form a mixed solution, adding a separation material package to the mixed solution, followed by vortex mixing, and then performing the first centrifugation. After the aqueous phase 11 and the organic phase 10 are separated, transfer the upper organic phase 10 to another container, then add the purifying agent and vortex for a second time to mix, let it stand or centrifuge, and then use a filter membrane to filter the obtained organic phase. 10 is transferred to another container, and finally the organic phase 10 is taken and tested on the machine or re-dissolved by nitrogen blowing and tested on the machine. It can be seen from this that the dispersive solid-phase extraction method requires multiple transfers of the organic phase 10. In addition, it also involves complex operations such as two centrifugations and two vortex mixings. In addition, the purifier used in the dispersive solid phase extraction method only relies on its adsorption for purification. In this application, the purifier packing is designed as a bed structure and filled into the column tube 1. When the extract liquid passes through the packing layer 3, it is purified with the help of purification. The adsorption effect of the agent can not only purify, but also intercept some impurities, thereby flowing out the purified supernatant.

Further, the extraction reagents include salts and extraction agents.

Further, the salts include any one or a combination of at least two of magnesium sulfate, sodium sulfate, sodium chloride or potassium chloride.

Preferably, the extraction agent includes acetonitrile, a mixture of acetonitrile and methanol, acidified acetonitrile or a mixture of acidified acetonitrile and methanol;

Further, the extraction reagent also includes a purification agent.

Further, the purification agent includes any one or a combination of at least two of N-propylethylenediamine, activated carbon or C18.

In another specific embodiment, the present application provides an application of the above-mentioned pre-processing device, which is used to pre-process the sample to be tested before clinical testing.

The applicant declares that the above are only specific implementation modes of the present application, but the protection scope of the present application is not limited thereto. Persons skilled in the technical field should understand that any person skilled in the technical field will not disclose any information disclosed in this application. Within the technical scope, changes or substitutions that can be easily imagined fall within the protection scope and disclosure scope of this application.

Claims

A purification separation column used for pre-treatment of clinical testing, which includes a column tube with at least one end open. A filter component is provided in the column tube. The liquid to be filtered enters from the outside of the column tube through the opening, and enters after passing through the filter component. column tube lumen;

Wherein, the filter assembly includes a first screen plate, a water blocking layer, a filler layer and a second screen plate arranged in sequence from bottom to top.
The purification separation column for pre-treatment of clinical detection according to claim 1, wherein the filter component is located at the bottom of the inner cavity of the column tube, and the filtered liquid passes through the filter component from the bottom of the column tube and then enters the column tube. cavity; cavity

Preferably, the filler layer is filled with purification materials, and the purification materials include specific adsorption materials for adsorbing polypeptides and phospholipid compounds;

Preferably, the purification materials include silica gel, octadecyl bonded silica gel and its derivatives, octadecyl bonded silica gel and its derivatives, diatomaceous earth, activated carbon, N-propylethylenediamine, quartz sand, Any one or a combination of at least two of molecular sieves, hydrophilic lipophilic polymeric fillers, strong cation exchange resins, weak cation exchange resins, strong anion exchange resins or weak anion exchange resins;

Preferably, the particle size of the purification material is 5 to 2000 μm;

Preferably, the filling mass of the purification material is 10 to 100 mg.
The purification separation column for clinical detection pre-treatment according to claim 1 or 2, wherein the pore diameter of the first sieve plate is the same as or different from the pore diameter of the second sieve plate;

Preferably, the pore diameter of the first sieve plate is 5-50 μm;

Preferably, the pore diameter of the second sieve plate is 5-50 μm;

Preferably, the water-blocking layer is a water-blocking film, and the pore size of the water-blocking film is 5 to 50 μm.
The purification separation column for clinical detection pretreatment according to any one of claims 1 to 3, wherein a filter layer is further provided between the first sieve plate and the water-blocking layer;

Preferably, the filter layer includes at least one filter membrane;

Preferably, the filter layer includes at least two layers of filter membranes, and the pore sizes of the filter membranes are the same or different;

Preferably, the filter layer includes at least two layers of filter membranes with different pore sizes, and the pore sizes of the filter membranes decrease sequentially from bottom to top;

Preferably, the filtration layer includes a first filter membrane, a second filter membrane and a third filter membrane stacked sequentially from bottom to top, and the pore size of the first filter membrane ≥ the pore size of the second filter membrane ≥ the third filter membrane aperture;

Preferably, the pore size of the first filter membrane is 0.5-2.5 μm;

Preferably, the pore size of the second filter membrane is 0.2-0.8 μm;

Preferably, the pore size of the third filter membrane is 0.1-0.5 μm.
A pretreatment device for clinical detection, which includes a liquid storage tube and a purification separation column for pretreatment of clinical detection according to any one of claims 1-4;

Wherein, the liquid storage tube is used in conjunction with the purification separation column, and the extraction reagent is pre-distributed in the liquid storage tube. The sample to be tested is added to the liquid storage tube, and the sample to be tested is mixed with the extraction reagent and left to stratify. The purification and separation column is pressed into the liquid storage tube, and the upper organic phase passes through the filter assembly from the bottom of the purification and separation column and enters the inner cavity of the column tube of the purification and separation column.
The pretreatment device for clinical testing according to claim 5, wherein the inner cavity of the column tube of the purification separation column is divided into a first liquid storage chamber and a second liquid storage chamber from bottom to top;

Preferably, the diameter of the outer wall of the column tube corresponding to the first liquid storage chamber is smaller than the diameter of the outer wall of the column tube corresponding to the second liquid storage chamber, so that an annular shape is formed at the outer wall of the column tube between the first liquid storage chamber and the second liquid storage chamber. Step surface; after the purification separation column is pressed into the liquid storage tube, the open end surface of the liquid storage tube contacts the annular step surface.
The pretreatment device for clinical testing according to claim 5 or 6, wherein a sealing ring is provided along the circumferential direction at the outer wall of the column tube at the corresponding position of the filter assembly;

Preferably, a cutout is provided at the top opening of the column tube of the purification separation column. When the purification separation column is pressed down toward the liquid storage pipe, the air in the liquid storage pipe is discharged through the cutout.
A pre-processing method for clinical detection, which uses the pre-processing device for clinical detection according to any one of claims 5-7 to pre-process the sample to be tested, the pre-processing method includes:

Add the sample to be tested into the liquid storage tube, mix the sample to be tested with the pre-placed extraction reagent and let it stand. After the aqueous phase and organic phase are initially separated, press the purification separation column into the liquid storage tube, and the organic phase will pass through the purification separation column. The bottom passes through the filter component and enters the inner cavity of the column tube of the purification separation column. The aqueous phase is isolated by the filter component. The organic phase entering the inner cavity is the sample to be tested after pretreatment. The sample to be tested after pretreatment is put on the machine for detection or nitrogen blowing. Then reconstitute and test on the machine.
The pretreatment method according to claim 8, wherein the extraction reagent includes salts and extraction agents;

Preferably, the salts include any one or a combination of at least two of magnesium sulfate, sodium sulfate, sodium chloride or potassium chloride;

Preferably, the extraction agent includes acetonitrile, a mixture of acetonitrile and methanol, acidified acetonitrile or a mixture of acidified acetonitrile and methanol;

Preferably, the extraction reagent further includes a purifying agent;

Preferably, the purification agent includes any one or a combination of at least two of N-propylethylenediamine, activated carbon or C18.
An application of the pre-processing device according to any one of claims 5 to 7, wherein the pre-processing device is used to pre-process the sample to be tested before clinical testing.