Column purification device and column purification method thereof

Abstract

The invention relates to a column purification device and a column purification method. The column purification apparatus includes: a base configured to rotate about a rotation axis, and the base includes: at least one collection portion, one of the at least one collection portion including a receiving portion, a first cavity and a second cavity located outside the receiving portion in a radial direction with respect to the rotation axis; and at least one reaction container, one of the at least one reaction container is arranged in the accommodating part, and one reaction container is configured to move in the accommodating part under the condition that the base rotates along different directions so as to communicate with the first cavity or the second cavity through the filter column. In this way, by rotating the base in different directions, the reaction liquid in the reaction container can enter different cavities to complete different experimental operations, so that the column method nucleic acid automatic purification is allowed to be realized, and the required consumables and cost are reduced.

Description

Technical Field

[0001] The present disclosure relates to the field of molecular biology, and more particularly, to a column purification device and a column purification method. Background Art

[0002] The separation and purification of biomolecules at the submicroscopic level to extract biomolecules of the required purity has attracted widespread attention in the academic community. Common methods for nucleic acid extraction include the magnetic bead method, column method, phenol-chloroform method, etc. The phenol-chloroform method requires multiple centrifugation and phase separation, and contains toxic volatile solvents, so it is difficult to automate. The magnetic bead method is the easiest method to automate, and its development is relatively mature. There are already many commercial products and representative instruments. The magnetic field of these instruments controls the adsorption, release and transfer of magnetic beads in reagents and samples, thereby realizing the automated extraction process of nucleic acids.

[0003] Column purification is the use of filter columns (also known as adsorption columns) to purify substances. In this method, special adsorption materials are usually used to fill the filter column. For deoxyribonucleic acid (DNA) purification, a common adsorption material can be a silica gel membrane. Under specific conditions, such as appropriate salt concentration and pH environment, the target molecule (such as DNA) will specifically bind to the adsorption material, while other impurities will not be adsorbed or the binding force is very weak. After the target molecule is adsorbed on the column, the target molecule can be eluted from the adsorption material by changing the conditions of the solution, thereby achieving separation from the impurities. Summary of the invention

[0004] In a first aspect of the present disclosure, a column purification device is provided. The column purification device comprises: a base, which is configured to rotate around a rotation axis, and the base comprises: at least one collecting part, one of the at least one collecting part comprises a receiving part, a first cavity and a second cavity located outside the receiving part in a radial direction relative to the rotation axis; at least one reaction container, a corresponding one of the at least one reaction container is arranged in the receiving part, and the one reaction container is configured to move in the receiving part when the base rotates in different directions, so as to communicate with the first cavity or the second cavity via a filter column for adsorbing a target substance.

[0005] The second aspect of the present disclosure provides a column purification method. The column purification method comprises: adding a first reaction liquid to at least one reaction container of a column purification device according to any one of claims 1 to 3; driving an active component to rotate the base of the column purification device in a first direction, so that the first reaction liquid enters the first cavity of the column purification device; in response to the base stopping rotation, adding a second reaction liquid to at least one reaction container; driving an active component to rotate the base of the column purification device in a second direction, so that the second reaction liquid enters the second cavity of the column purification device.

[0006] Additional features and advantages of the present disclosure will be set forth in the following detailed description and will be apparent to those skilled in the art from that description or may be recognized by practicing the embodiments as described in the following description and in conjunction with the claims and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to better understand the above and other purposes, features, advantages and functions of the present disclosure, reference may be made to the preferred embodiments shown in the accompanying drawings. The same reference numerals in the accompanying drawings refer to the same components. It should be understood by those skilled in the art that the accompanying drawings are intended to schematically illustrate the preferred embodiments of the present disclosure and have no limiting effect on the scope of the present disclosure, and the components in the drawings are not drawn to scale.

[0008] Figure 1A and 1B shows a cross-sectional view of an exemplary column purification apparatus according to an embodiment of the present disclosure;

[0009] Figure 2A-2C Different schematic diagrams of exemplary column purification devices according to embodiments of the present disclosure are shown;

[0010] Figure 3 A flow chart showing an exemplary column purification method according to an embodiment of the present disclosure is shown;

[0011] Figures 4A-4G A schematic diagram showing an exemplary column purification process according to an embodiment of the present disclosure is shown;

[0012] Figure 5 A fluorescence analysis comparison diagram of the distribution of nucleic acid fragments extracted according to an embodiment of the present disclosure and the distribution of nucleic acid fragments extracted from a control group is shown. DETAILED DESCRIPTION

[0013] Now, with reference to the accompanying drawings, the specific embodiments of the present disclosure are described in detail. What is described here is only the preferred embodiment of the present disclosure. Those skilled in the art can think of other ways to implement the present disclosure based on the preferred embodiment, and the other ways also fall within the scope of the present disclosure.

[0014] Unless expressly stated otherwise, it is not intended to require a specific orientation for any device. First of all, it should be noted that the directional terms and positional terms in this disclosure should be understood as relative directions and positions, rather than absolute directions and positions. The directional terms and positional terms in this disclosure can be interpreted with reference to the exemplary structures shown in the accompanying drawings. Therefore, in any device claim that does not actually describe the order or direction of individual components, or in the case where there is no additional specific description in the claims or description that the steps must be limited to a specific order or the specific order or direction of the components of the device is not described, it is not intended to infer their order or direction in any way.

[0015] As discussed above, column purification has certain advantages over the other two purification schemes. For example, through high-speed centrifugation, there are almost no impurities such as washing liquid remaining in the eluent; elution can be performed with a trace volume (~10 microliters); the elution volume is precisely controllable and the loss is small; with the assistance of the expansion column, samples in a larger volume range can be extracted. These advantages make column extraction still one of the mainstream directions of nucleic acid extraction. However, since the test tubes need to be repeatedly replaced during the column nucleic acid extraction process, it is difficult to achieve semi-automation. In addition, a variety of consumables such as tubes need to be used during use, and the preparation of consumables is relatively cumbersome; during operation, the consumables need to be moved with a gripper, making it difficult to miniaturize the instrument.

[0016] In view of this, the embodiment of the present disclosure provides a device that can control the direction of liquid flow and realize nucleic acid extraction and purification by mechanical drive. The column purification device includes a base that can rotate in two different directions, positive and negative. A reaction container for accommodating reaction reagents is accommodated in the collection part of the base. When the base rotates in one direction, the reaction container abuts against one end of the accommodating part and communicates with the first cavity. Under the push of the accommodating part, the reaction container rotates together, and since the first cavity is located radially outside, the reaction reagent in the reaction container flows toward the first cavity under the action of centrifugal force to achieve the first reaction operation. Relatively, when the base rotates in the opposite direction, the reaction container abuts against the other end of the accommodating part and communicates with the second cavity. Under the push of the accommodating part, the reaction container rotates together, and since the second cavity is also located radially outside, the reaction reagent in the reaction container flows toward the second cavity under the action of centrifugal force to achieve the second reaction operation.

[0017] According to the embodiments of the present disclosure, the cavity to which the reaction container is docked is controlled by rotating in different directions to perform different reactions. In this way, there is no need for a robotic arm or gripper, nor is there a need to manually place consumables or replace test tubes, and column-based automated nucleic acid purification can be achieved, thereby reducing the required consumables and costs.

[0018] The following will refer to Figures 1A to 5To describe the structure and operation principle of the column purification device and the use method thereof according to the present disclosure. Figure 1A FIG. 2 shows a cross-sectional view of an exemplary column purification device 10 according to an embodiment of the present disclosure when rotating in a first direction D1. Figure 1A As shown, the column purification apparatus 10 includes a housing 11. The housing 11 contains a column purification device 100, a driving device 12 for driving the column purification device 100, and a controller 13 for controlling the operation of components in the column purification apparatus 10.

[0019] The column purification device 100 includes a base 110. The base 110 can rotate along a first direction D1 around a rotation axis R under the drive of a driving device 12. The base 110 includes one or more collecting parts 111. Here, each collecting part in the one or more collecting parts 111 is identical in structure and function. At least one collecting part 111 includes at least one accommodating part 112, at least one first cavity 113 and at least one second cavity 114. In the radial direction relative to the rotation axis R (i.e., perpendicular to the rotation axis R and pointing away from the rotation axis R), at least one first cavity 113 and at least one second cavity 114 (in the radial direction relative to the rotation axis R) are arranged. Figure 1B ) is located outside at least one accommodating portion 112.

[0020] The column purification device 100 also includes a reaction assembly 120. The reaction assembly 120 includes at least one reaction vessel 121 and a vessel holder 122 for connecting at least one reaction vessel 121. At least one reaction vessel 121 is movably disposed in a corresponding accommodation portion 112 in at least one collecting portion 111. One side of at least one accommodation portion of at least one collecting portion 111 facing the rotation axis R is open, so that the vessel holder 122 can extend in the cavity in the middle of the base 110 and connect at least one reaction vessel 121. At least one reaction vessel 121 also includes a liquid outlet pipe 123. The liquid outlet pipe 123 extends from the side wall of the reaction vessel toward the first cavity 113 in a radial direction. In the liquid outlet pipe 123, a filter column 124 is arranged.

[0021] It should be understood that the receiving portion may also have other structures. For example, the receiving portion may be a slide groove, and the reaction container is independently arranged in the corresponding slide groove and can slide in the slide groove. In such an embodiment, the reaction component 120 does not include a reaction bracket.

[0022] The column purification device 100 further includes a cover 130. The cover 130 is covered on the base 110 to cover the opening at the top of the first cavity 113. The cover 130 includes a cover body 131 and a cover wall 132 formed at the edge of the cover body 131 and extending in a direction away from the cover body 131. The cover wall 132 includes a groove 133 extending in the circumferential direction. Correspondingly, a protrusion 125 extending outward from the outer wall is provided at a position adjacent to the upper end on the outer wall of the base 110 away from the rotation axis R. The protrusion 125 is slidably provided in the groove 133.

[0023] exist Figure 1A In the illustrated embodiment, the base 110 is rotating in the first direction D1. At this time, at least one reaction container 121 abuts against the end wall of one side of the receiving portion 112 and rotates together with the base 110. Since the first cavity 113 is located radially outward, the reaction reagent in the reaction container 121 flows toward the first cavity through the filter column 112 under the action of centrifugal force, thereby completing a type of reaction operation.

[0024] Figure 1B A cross-sectional view of an exemplary column purification device 10 according to an embodiment of the present disclosure when rotating in the second direction D2 is shown. After the reaction operation is completed, the base 110 can be driven by the driving device 12 to change direction to rotate along the second direction D2. At the beginning of the change of direction, due to the lack of abutment, at least one reaction vessel 121 will not rotate with the base 110, thereby generating a relative movement of the base 110 relative to at least one reaction vessel 121 in the second direction D2. Until the base 110 rotates a certain angle, at least one reaction vessel 121 contacts the end wall on the other side of the corresponding receiving portion, and starts to rotate with the base 110 under the push of the end wall.

[0025] Figure 1B As shown in Figure 1A The column purification device 10 is rotated in a second direction D2 opposite to the first direction D1. Figure 1B As shown, the base 110 is rotating in the second direction D2. At this time, at least one reaction container 121 abuts against the end wall on the other side of the receiving portion 112 and rotates with the base 110. Since the second cavity 114 is located radially outward, the reaction reagent in the reaction container 121 flows toward the second cavity 113 through the filter column 112 under the action of centrifugal force, thereby completing another type of reaction operation.

[0026] It should be understood that although Figure 1A and Figure 1BIn the illustrated embodiment, the driving device 12 directly drives the base 110 . The driving device 12 may also directly drive the reaction assembly 120 or the cover 130 to indirectly rotate the base 110 in a predetermined direction.

[0027] The following will refer to FIG. 2A to FIG. 2C The structural details of the column purification device 100 will be described. Figure 2A Schematic diagram showing the appearance of an exemplary column purification device 100 in an assembled state according to an embodiment of the present disclosure. Figure 2A As shown, the column purification device 100 includes a base 110 and a cover 130. A protrusion 125 extending outward from the outer wall is provided at a position adjacent to the upper end of the outer wall of the base 110 that is away from the rotation axis R. The cover 130 is covered on the base 110 to cover the opening at the top of the first cavity 113. The cover 130 includes a cover body 131 and a cover wall 132 formed at the edge of the cover body 131 and extending in a direction away from the cover body 131. The cover wall 132 includes a groove 133 extending in the circumferential direction. Correspondingly, the protrusion 125 can be slidably arranged in the groove 133.

[0028] Figure 2B FIG. 1 shows a schematic exploded view of an exemplary column purification device 100 according to an embodiment of the present disclosure. Figure 2B As shown, the column purification device 100 includes three parts as a whole, including a base 110, a reaction assembly 120 and a cover 130. The base 110 can rotate along a first direction D1 around the rotation axis R under the drive of the driving device 12. The base 110 includes a base body 201. The base body 201 extends circumferentially around the rotation axis R and forms a ring. The base 110 also includes an end side of the base body 201 in a direction parallel to the rotation axis R, that is, in Figure 2B The base body 201 in the illustrated embodiment has a first surface 215 at the top end side. In contrast, the base 110 further includes a second surface 216 located at the other end side, i.e., the bottom end side, of the base body 201. The base body 201 further includes an inner wall 213 facing the rotation axis R and an outer wall 214 facing away from the rotation axis R. A plurality of protrusions 125 are provided on the outer wall 214 adjacent to the first surface 215.

[0029] At least one collecting portion 111 is provided in the base body 201. Here, the base 110 includes eight collecting portions. The at least one collecting portion 111 is uniformly distributed in the circumferential direction around the rotation axis R, that is, the angles between two adjacent collecting portions relative to the rotation axis R are the same. Each collecting portion in the at least one collecting portion 111 is identical in structure and function. Therefore, for the purpose of brevity, only one collecting portion 202 in the at least one collecting portion 111 is described in detail below. The collecting portion 202 includes a receiving portion 112 for receiving a reaction container, a first cavity 113 for receiving a reaction reagent from the reaction container, and a second cavity 114 for receiving a reaction reagent from the reaction container. The first cavity 113, the second cavity 114, and the receiving portion 112 extend from the first surface 215 to the second surface 216 in a direction toward the second surface 216, respectively. Thus, a first opening 206 of the first cavity 113, a second opening 207 of the second cavity 114, and the receiving portion 112 are formed on the first surface 215. In the radial direction relative to the rotation axis R, the first cavity 113 and the second cavity 114 are located outside the accommodating portion 112 , that is, the distance between the first cavity 113 and the second cavity 114 and the rotation axis R is greater than the distance between the accommodating portion 112 and the rotation axis R.

[0030] The receiving portion 112 extends from the first stopper 203 to the second stopper 204 circumferentially around the rotation axis R and opens at the inner wall 213 of the base body 201. The receiving portion 112 includes a slide groove 205 circumferentially opened on the side wall of the receiving portion 112 around the rotation axis R.

[0031] The column purification device 100 also includes a reaction assembly 120. The reaction assembly 120 includes at least one reaction vessel 121 and a container support 122 for connecting at least one reaction vessel 121. The reaction assembly 120 can be placed in the middle of the base 110 as a whole so that at least one reaction vessel 121 is properly placed in the corresponding accommodation portion. At the same time, the size of the container support 122 is set so that at least one reaction vessel 121 is stably against the side wall of the corresponding accommodation portion. Thus, the movement of at least one reaction vessel maintains the consistency of movement under the limitation of the reaction support. In addition, due to the integrated design of at least one reaction vessel and the reaction support, the overall mass of the reaction assembly is increased, the inertia is improved, and the reaction operation of the embodiment of the present disclosure is more easily realized.

[0032] The at least one reaction container 121 is also evenly distributed around the rotation axis R in the circumferential direction corresponding to the at least one collection portion 111. Each reaction container in the at least one reaction container 121 is identical in structure and function. Therefore, for the purpose of brevity, only one reaction container 221 in the at least one reaction container is described in detail below. Figure 2B In the illustrated embodiment, the reaction vessel 221 includes a container body 222 having a shape relative to a body of revolution. The axis of the container body 222 is parallel to the rotation axis R and perpendicular to the reaction support 122. The container body 222 includes an inverted conical bottom, a first truncated cone portion extending upward from the bottom, and a second truncated cone portion extending upward from the first truncated cone portion. The diameter of the first truncated cone portion increases in the upward direction, while the diameter of the second truncated cone portion decreases, thereby forming a body of revolution having the largest cross-sectional area perpendicular to the axis in the middle. Here, the "upward" direction refers to the direction from the second surface 216 of the base 110 to the first surface 215 in the assembled state.

[0033] A liquid outlet pipe 223 is provided at the position where the side wall 224 of the container body 222 is located at the largest cross-sectional area. The liquid outlet pipe 223 extends from the side wall 224 in the radial direction away from the rotation axis R. A liquid outlet 225 is provided at the end of the liquid outlet pipe 223. The shape of being wide in the middle and narrow at the upper and lower ends forms a suitable slope to ensure that under the action of centrifugal force, the reagent and the eluent are better gathered at the liquid outlet pipe 223 and flow out from the liquid outlet 225, without causing the reagent and the eluent to gather at the top or bottom of the reaction container 2221. In addition, the liquid outlet pipe 223 is arranged at the position of the side wall 224 at the largest distance from the rotation axis R, further ensuring the smoothness of the outflow of the reagent and the eluent.

[0034] The cover 130 includes a circular cover body 131 and a cover wall 132 extending in a downward direction formed at the edge of the cover body 131. The cover wall 132 includes a plurality of grooves 133 extending in a circumferential direction. Figure 2B In the illustrated embodiment, the number of the grooves 133 is 8 corresponding to the protrusions 125, and the width of the grooves 133 is greater than the width of the protrusions 125. The grooves 133 have a first end 224 and a second end 225 at the sidewall. In the assembled state, the protrusions 125 of the base 110 can be slidably arranged in the grooves 133, that is, they can be opened from the first end 224 and the second end 225. The cover 131 of the lid 130 is also provided with openings for the first cavity, the second cavity and the reaction vessel. For example, when the protrusion 125 is located in the middle of the groove 133, the opening 242 for the first cavity, the opening 243 for the second cavity and the opening 241 for the reaction vessel are aligned with the top openings of the first cavity, the second cavity and the reaction vessel, respectively, so that reagents can be added to the reaction vessel, or the reacted liquid can be taken out from the first cavity or the second cavity. When the protrusion 125 is located at other positions of the groove 133, the openings for the first cavity, the second cavity and the reaction container are staggered with the top openings of the first cavity, the second cavity and the reaction container respectively, so as to achieve the closure of the corresponding top openings.

[0035] In some embodiments, when the base 110 is driven to rotate along the first direction D1, the protrusion 125 moves relative to the groove 133 along the first direction D1 from the middle of the groove 133 toward the first end 224. After reaching the first end 224, the protrusion 125 abuts against the first end 224 and pushes the cover 130 to rotate together. At this time, the opening on the cover 130 is staggered with the top opening of the cavity and the reaction container to achieve closure. Correspondingly, when the base 110 is driven to rotate along the second direction D2, the protrusion 125 moves relative to the groove 133 along the first direction D1 toward the second end 225. After reaching the second end 225, the protrusion 125 abuts against the second end 225 and pushes the cover 130 to rotate together.

[0036] Figure 2C A perspective view of an example base 110 is shown according to an embodiment of the present disclosure. Figure 2C The details of the first cavity 113, the second cavity 114 and the slideway 205 are shown. Figure 2C As shown, one end of the slideway 205 includes a first channel 208 extending from the side wall 210 of the receiving portion to the first inlet 211 of the first cavity 113 in a radial direction. When the reaction container 221 abuts against the first stopper 203, the liquid outlet pipe 223 abuts against the side wall of the first channel 208, so that the liquid outlet pipe 223 is relatively stationary in the first channel 208. At this time, the liquid outlet 225 of the liquid outlet pipe 223 is aligned with the first inlet 211 of the first cavity 113. For example, the connecting line of the liquid outlet pipe 223, the liquid outlet 225 and the first inlet 211 is in a radial direction D3 relative to the rotation axis R. The other end of the slideway 205 includes a second channel 209 extending from the side wall 210 of the receiving portion to the second inlet 212 of the second cavity 114 in a radial direction. When the reaction container 221 abuts against the second stopper 204, the liquid outlet pipe 223 abuts against the side wall of the second channel 209, so that the liquid outlet pipe 223 is relatively stationary in the second channel 209. At this time, the liquid outlet 225 of the liquid outlet pipe 223 is aligned with the second inlet 212 of the second cavity 114. For example, the line connecting the liquid outlet pipe 223, the liquid outlet 225 and the second inlet 212 is in a radial direction relative to the rotation axis R.

[0037] exist Figure 2C In the illustrated embodiment, the first cavity 113 is in the shape of a truncated cone having an axis parallel to the rotation axis R. The cross-sectional area of ​​the first cavity 113 gradually increases from the first surface 215 toward the second surface 216. The second cavity 114 is in the shape of a cylinder having an inverted conical bottom. The volume of the second cavity 114 is smaller than the volume of the first cavity 113.

[0038] Figure 3 FIG. 3 shows a flow chart of an exemplary column purification method 300 according to an embodiment of the present disclosure. The column purification method 300 may be, for example, Figure 1A and Figure 1B The column purification device 10 shown in the figure is used for execution, and the column purification device in the method 300 corresponds to Figures 2A to 2C The column purification device 100 in the embodiment shown. For the purpose of discussion, the following will be combined with Figures 1A to 2C To describe the column purification method 300. Figure 3 As shown, at 302, method 300 includes adding a first reaction solution to at least one reaction vessel of a column purification device. Figures 1A to 2C In the illustrated embodiment, the lid 130 is rotated to an open state so that the opening on the lid 130 for at least one reaction vessel 121 is aligned with the top opening of at least one reaction vessel 121. After alignment, the reaction reagent is added to the at least one reaction vessel 121 for reaction. It should be understood that since the reaction vessel can move in the accommodating portion, when the opening on the lid 130 for at least one reaction vessel 121 is aligned with the top opening of at least one reaction vessel 121, the opening on the lid 130 for the first cavity and the second cavity is not necessarily aligned with the top opening of the first cavity and the second cavity.

[0039] At 304, method 300 includes driving an active component to rotate the base of the column purification device in a first direction, so that the first reaction liquid enters the first cavity of the column purification device. In some embodiments, the base of the column purification device is directly driven by the driving device 12, and the active component is the base. In this embodiment, the driving device 12 directly drives the base 110 to rotate in the first direction D1. When the base 110 starts to rotate, due to inertia, the reaction component 120 and the cover 130 are approximately stationary, thereby forming a relative rotation of the base 110 relative to the reaction component 120 and the cover 130 along the first direction D1. During the relative rotation, the reaction container 221 in the reaction component 120 moves toward the first stopper 203 in the receiving portion 112. When the reaction container 221 reaches the first stopper 203, the reaction container 221 abuts against the first stopper 203 and communicates with the first cavity 113. At the same time, the protrusion 125 on the outer wall of the base 110 moves in the groove 133 of the cover wall 132 of the cover 130 toward the first end 244 of the groove 133. When the protrusion 125 reaches the first end 244, the protrusion 125 abuts against the first end 244. At this time, due to the relative displacement between the cover and the reaction vessel and the base, the opening on the cover 130 is misaligned with the top opening of the cavity and the reaction vessel, thereby achieving the closure of the cavity and the reaction vessel. Afterwards, the first stopper 203 and the protrusion 125 push the reaction vessel 221 and the cover 130 respectively, so that they rotate with the base 110 in the first direction D1. During the rotation of the reaction vessel 221 along the first direction D1, the first reaction liquid in the reaction vessel 221 flows toward the first cavity 113 via the liquid outlet pipe 223 under the action of centrifugal force. A part of the first reaction liquid is adsorbed in the filter column of the liquid outlet pipe 233, and the remaining part of the first reaction liquid enters the first cavity 113.

[0040] In some alternative embodiments, the reaction support of the column purification device is directly driven by a driving device, and the active component is the reaction support. In this embodiment, the driving device directly drives the reaction support to rotate in a first direction. When the reaction support starts to rotate, due to inertia, the base and the cover are approximately stationary, thereby forming a relative rotation of the reaction support and the connected reaction vessel relative to the base and the cover along the first direction. During the relative rotation, the reaction vessel moves toward the first stop portion in the accommodating portion. In this embodiment, the first stop portion is located downstream of the second stop portion in a first direction. When the reaction vessel reaches the first stop portion, the reaction vessel abuts against the first stop portion and is communicated with the first cavity.

[0041] Afterwards, the reaction vessel pushes the base so that it rotates along the first direction with the reaction assembly. When the base starts to rotate, the lid is still approximately stationary, thereby forming the base relative to the lid in the first direction. The protrusion on the outer wall of the base moves toward the first end of the groove in the groove of the cover wall of the lid. When the protrusion reaches the first end, the protrusion abuts against the first end. At this time, due to relative displacement between the lid, the reaction vessel and the base, the opening on the lid is misaligned with the top opening of the cavity and the reaction vessel, thereby achieving the closure of the cavity and the reaction vessel. Afterwards, the reaction vessel pushes the first stopper so that the protrusion pushes the lid, thereby causing the lid and the base to rotate together with the reaction assembly in the first direction. During the rotation of the reaction vessel in the first direction, the first reaction liquid in the reaction vessel flows toward the first cavity via the liquid outlet pipe under the action of centrifugal force. A portion of the first reaction liquid is adsorbed in the filter column of the liquid outlet pipe, and the remaining portion of the first reaction liquid enters the first cavity.

[0042] In some other embodiments, the lid of the column purification device is directly driven by a driving device, and the active component is a lid. In this embodiment, the driving device directly drives the lid to rotate along a first direction. When the lid starts to rotate, due to inertia, the base and the reaction component are approximately stationary, thereby forming a relative rotation of the lid relative to the reaction component and the base along a first direction. During relative rotation, the first end of the groove of the cover wall of the lid (here, the second end in the first direction downstream of the first end) moves toward the projection on the outer wall of the base. When the first end arrives at the projection, the first end is against the projection. At this point, due to relative displacement between the lid and the reaction vessel and the base, the opening on the lid is misaligned with the top opening of the cavity and the reaction vessel, thereby achieving the closure of the cavity and the reaction vessel.

[0043] Afterwards, the lid pushes the base so that it rotates along the first direction with the lid. When the base starts to rotate, the reaction assembly is still approximately stationary, thereby forming a rotation of the lid and the base relative to the reaction assembly in the first direction. The first stopper of the base moves toward the reaction vessel of the reaction assembly. When the first stopper reaches the reaction vessel, the first stopper abuts against the reaction vessel. Afterwards, the first stopper pushes the reaction vessel so that the reaction assembly and the base rotate together with the lid in the first direction. At this time, the reaction vessel is connected to the first cavity. During the rotation of the reaction vessel in the first direction, the first reaction liquid in the reaction vessel flows toward the first cavity via the liquid outlet pipe under the action of centrifugal force. A portion of the first reaction liquid is adsorbed in the filter column of the liquid outlet pipe, and the remaining portion of the first reaction liquid enters the first cavity.

[0044] At 306, method 300 includes adding a second reaction solution to at least one reaction vessel after the base stops rotating. When the base stops rotating, the reaction vessel is located at the first stopper, and the opening of the lid is misaligned with the top opening of the reaction vessel. The lid is rotated to an open state so that the opening on the lid for the reaction vessel is aligned with the top opening of the reaction vessel again. After alignment, the second reaction solution is added to the reaction vessel.

[0045] At 308, the method 300 includes driving the active component to rotate the base of the column purification device 100 in the second direction, so that the second reaction liquid enters the second cavity of the column purification device. In some embodiments, the base 110 of the column purification device 100 is directly driven by the driving device 12, and the active component is the base 110. In this embodiment, the driving device 12 directly drives the base 110 to rotate in the second direction D2. When the base 110 starts to rotate, due to inertia, the reaction component 120 and the cover 130 are approximately stationary, thereby forming a relative rotation of the base 110 relative to the reaction component 120 and the cover 130 in the second direction D2. During the relative rotation, the reaction container 221 in the reaction component 120 moves toward the second stopper 204 in the receiving portion 112. When the reaction container 221 reaches the second stopper 204, the reaction container 221 abuts against the second stopper 204 and communicates with the second cavity 114. At the same time, the protrusion 125 on the outer wall of the base 110 moves in the groove 133 of the cover wall 132 of the cover 130 toward the second end 245 of the groove 133. When the protrusion 125 reaches the second end 245, the protrusion 125 abuts against the second end 245. At this time, due to the relative displacement between the cover and the reaction container and the base, the opening on the cover 130 is misaligned with the top opening of the cavity and the reaction container, thereby achieving the closure of the cavity and the reaction container. Afterwards, the second stopper 204 and the protrusion 125 push the reaction container 221 and the cover 130 respectively, so that they rotate with the base 110 in the second direction D2. During the rotation of the reaction container 221 along the second direction D2, the second reaction liquid in the reaction container 221 flows toward the second cavity 114 through the liquid outlet pipe 223 under the action of centrifugal force. The second reaction liquid rinses the filter column of the liquid outlet pipe 233 to extract part of the reaction liquid adsorbed in the filter column, and flows into the second cavity 114 together.

[0046] In some alternative embodiments, the reaction support of the column purification device is directly driven by the driving device, and the active component is the reaction support. In this embodiment, the driving device directly drives the reaction support to rotate along the second direction. When the reaction support starts to rotate, due to inertia, the base and the cover are approximately stationary, thereby forming a relative rotation of the reaction support and the connected reaction container relative to the base and the cover along the second direction. During the relative rotation, the reaction container moves toward the second stop portion in the accommodating portion. In this embodiment, the first stop portion is located downstream of the second stop portion in the first direction. When the reaction container reaches the second stop portion, the reaction container abuts against the second stop portion and is communicated with the second cavity.

[0047] Afterwards, the reaction container pushes the base so that it rotates along the second direction with the reaction component. When the base starts to rotate, the cover is still approximately stationary, thereby forming the base relative to the cover in the second direction. The protrusion on the outer wall of the base moves in the groove of the cover wall of the cover toward the second end of the groove. When the protrusion reaches the second end, the protrusion abuts on the second end. At this time, due to the relative displacement between the cover and the reaction container and the base, the opening on the cover is misaligned with the top opening of the cavity and the reaction container, thereby achieving the closure of the cavity and the reaction container. Afterwards, the reaction container pushes the second stopper so that the protrusion pushes the cover, so that the cover and the base rotate together with the reaction component in the second direction. During the rotation of the reaction container in the second direction, the second reaction liquid in the reaction container flows toward the second cavity via the liquid outlet pipe under the action of centrifugal force. The second reaction liquid rinses the filter column of the liquid outlet pipe to extract part of the reaction liquid adsorbed in the filter column, and flows into the second cavity together.

[0048] In some other embodiments, the lid of the column purification device is directly driven by a driving device, and the active component is a lid. In this embodiment, the driving device directly drives the lid to rotate along the second direction. When the lid starts to rotate, due to inertia, the base and the reaction component are approximately stationary, thereby forming a relative rotation of the lid relative to the reaction component and the base along the second direction. During the relative rotation, the second end of the groove of the cover wall of the lid (here, the second end in the first direction downstream of the first end) moves towards the projection on the outer wall of the base. When the second end arrives at the projection, the second end is against the projection. At this point, due to relative displacement between the lid and the reaction vessel and the base, the opening on the lid is misaligned with the top opening of the cavity and the reaction vessel, thereby achieving the closure of the cavity and the reaction vessel.

[0049] Afterwards, the lid pushes the base so that it rotates along the second direction with the lid. When the base starts to rotate, the reaction assembly is still approximately stationary, thereby forming a rotation of the lid and the base relative to the reaction assembly in the second direction. The second stopper of the base moves toward the reaction container of the reaction assembly. When the second stopper reaches the reaction container, the second stopper abuts against the reaction container. Afterwards, the second stopper pushes the reaction container so that the reaction assembly and the base rotate together with the lid in the second direction. At this time, the reaction container is connected to the second cavity. During the rotation of the reaction container in the second direction, the second reaction liquid in the reaction container flows toward the second cavity via the liquid outlet pipe under the action of centrifugal force. The second reaction liquid rinses the filter column of the liquid outlet pipe to extract part of the reaction liquid adsorbed in the filter column, and flows into the second cavity together.

[0050] After the rotation is completed, the substance adsorbed in the filter column when the base rotates in the first direction is eluted into the second cavity during the rotation of the base in the second direction, thereby obtaining the purified target substance. The cover 130 can be rotated to align the opening on the cover 130 with the top opening of the second cavity, so as to remove the reaction mixture containing the target substance in the second cavity.

[0051] The following will refer to Figures 4A to 4G To describe the process of conducting an experiment using the column purification device of an embodiment of the present disclosure. Figures 4A to 4G A schematic diagram of different steps of the experimental process is shown. In this experiment, the experimental conditions of the control group are: the sample type is cell-free plasma, the target product is cell-free polyoxyribonucleic acid (cfDNA), the extraction reagent is QIAamp Circulating Nucleic Acid Kit, and the extraction is performed according to the standard operating procedures of QIAamp Circulating Nucleic Acid Kit. The experimental conditions of the experimental group are: the sample type is the same cell-free plasma, the target product is cfDNA, and the column purification method of the embodiment of the present disclosure is performed using the column purification device of the embodiment of the present disclosure.

[0052] The first step: adding a first reaction liquid into at least one reaction container of a column purification device. Figure 4A A schematic diagram of the first step in the experimental process is shown. Figure 4A As shown, the cover 130 is directly driven by the driving device as an active component to rotate. The cover 130 is rotated to an open state so that the opening 401 on the cover 130 for the reaction container 420 is aligned with the top opening 421 of the reaction container 420. Figure 4AIn the illustrated embodiment, the opening 402 for the first cavity and the opening 403 for the second cavity of the cover 130 are also aligned with the top opening 412 of the first cavity and the top opening 413 of the third cavity, respectively. At the same time, the protrusion 414 of the base 110 is located in the middle of the groove 404 of the cover wall 132 of the cover 130. After alignment, the first reaction liquid 440 is added to the reaction container 420 for reaction. Here, the first reaction liquid 440 is a mixture of a sample, a lysate, a proteinase K, and a binding solution. Afterwards, the first reaction liquid 440 is incubated in the reaction container 420 for 30 minutes.

[0053] Step 2: driving the active component to rotate the base of the column purification device in a first direction, so that the first reaction liquid enters the first cavity of the column purification device. Figure 4B and Figure 4C Figure 2 shows a schematic diagram of the second step in the experimental process. Figure 4B As shown, the lid 130 of the column purification device 100 is directly driven by the driving device as an active component. The driving device directly drives the lid 130 to rotate along the first direction D1. When the lid 130 starts to rotate, due to inertia, the base 110 and the reaction assembly 120 are approximately stationary, thereby forming the relative rotation of the lid 130 relative to the reaction assembly 120 and the base 110 along the first direction D1. During the relative rotation, the first end 405 (here, the second end 406 is downstream of the first end 404 in the first direction) of the groove 404 of the cover wall 132 of the lid 130 moves toward the protrusion 414 on the base 110. When the first end 405 arrives at the protrusion 414, the first end 405 abuts against the protrusion 414. At this time, due to the relative displacement between the cover 130, the reaction vessel 420 and the base 110, the openings 401, 402 and 403 on the cover 130 are staggered with the top openings 421, 412 and 413, thereby achieving the closure of the reaction vessel 420 and the cavity.

[0054] Afterwards, the cover 130 pushes the base 110 so that it rotates along the first direction D1 with the cover 130. When the base 110 starts to rotate, the reaction assembly 120 is still approximately stationary, thereby forming a rotation of the cover 130 and the base 110 relative to the reaction assembly 120 in the first direction D1. The first stopper 416 of the accommodating portion 415 in the base 110 moves toward the reaction vessel 420. When the first stopper 416 reaches the reaction vessel 420, the first stopper 416 abuts against the reaction vessel 420. Afterwards, the first stopper 416 pushes the reaction vessel 420 so that the reaction assembly 120 and the base 110 rotate together with the cover 130 in the first direction D1. At this time, the reaction vessel 420 is connected to the first cavity 421. Thus, the startup process is completed.

[0055] like Figure 4C As shown, during the process in which the reaction assembly 120 and the base 110 rotate together with the cover 130 in the first direction D1, the first reaction liquid 440 in the reaction container 420 flows toward the first cavity 410 through the liquid outlet pipe 422 located in the first channel 419 under the action of centrifugal force. A portion of the first reaction liquid 440 is adsorbed in the filter column 430 of the liquid outlet pipe 422, and the remaining portion of the first reaction liquid 440 enters the first cavity 410. In some embodiments, a washing liquid can also be used as a reaction liquid and the first and second steps are repeated.

[0056] Step 3: Add the second reaction liquid into at least one reaction container. Figure 4D Figure 2 shows a schematic diagram of the third step in the experimental process. Figure 4D As shown, after the lid stops rotating, the reaction vessel 4210 is located at the first stopper 416, and the openings 401, 402, and 403 of the lid 130 are staggered with the top opening 421. At this time, the lid 130 is rotated to the open state so that the opening 401 on the lid 130 is aligned with the top opening 421 again. After alignment, the second reaction liquid 450 is added to the reaction vessel 210. Here, the second reaction liquid 450 is an eluent.

[0057] Step 4: driving the active component to rotate the base of the column purification device 100 in the second direction, so that the second reaction liquid enters the second cavity of the column purification device. Figure 4E and 4F Figure 2 shows a schematic diagram of the fourth step in the experimental process. Figure 4E As shown, the driving device directly drives the lid 130 to rotate along the second direction D2. When the lid 130 starts to rotate, due to inertia, the base 110 and the reaction assembly 120 are approximately stationary, thereby forming a relative rotation of the lid 130 relative to the reaction assembly 120 and the base 110 along the second direction D2. During the relative rotation, the second end 406 of the groove 404 of the lid 130 moves toward the protrusion 414 on the outer wall of the base 110. When the second end 406 reaches the protrusion 414, the second end 406 abuts against the protrusion 414. At this time, due to the relative displacement between the lid 130 and the reaction vessel 420 and the base 110, the opening on the lid 130 is misaligned with the top opening of the cavity and the reaction vessel, thereby achieving the closure of the cavity and the reaction vessel.

[0058] Afterwards, the cover 130 pushes the base 110 to rotate along the second direction D2 with the cover 130. When the base 110 starts to rotate, the reaction assembly 120 is still approximately stationary, thereby forming a rotation of the cover 130 and the base 110 relative to the reaction assembly 120 in the second direction D2. The second stopper 417 of the accommodating portion 415 of the base 110 moves toward the reaction container 420. When the second stopper 417 reaches the reaction container 420, the second stopper 417 abuts against the reaction container 420. Afterwards, the second stopper 417 pushes the reaction container 420 to rotate the reaction assembly 120 and the base 110 along the cover 130 in the second direction D2. At this time, the reaction container 420 is connected to the second cavity 411.

[0059] like Figure 4F As shown, during the rotation of the reaction assembly 120 and the base 110 along with the cover 130 in the second direction D2, the second reaction liquid 450 in the reaction container 420 flows toward the second cavity 411 through the liquid outlet pipe 422 in the second channel 419 under the action of centrifugal force. The second reaction liquid 450 rinses the filter column 430 in the liquid outlet pipe 422 to extract part of the reaction substances adsorbed in the filter column 430, and flows into the second cavity 411 together.

[0060] Step 5: Take out the eluate containing the biomolecules from the second chamber. Figure 4G Figure 2 shows a schematic diagram of the fifth step in the experimental process. Figure 4G As shown, after the rotation is completed, the substances adsorbed in the filter column 430 when the base 110 rotates along the first direction D1 are eluted into the second cavity 411 during the rotation of the base 110 along the second direction D2, thereby obtaining the purified target biomolecules. The cover 130 is rotated to align the opening 403 on the cover 130 with the top opening 413 of the second cavity 411, so as to remove the target biomolecules in the second cavity 411.

[0061] The following will refer to Figure 5 To describe FIG. 4A to FIG. 4G Experimental results in the examples shown. Figure 5 The fluorescence analysis comparison of the distribution of nucleic acid fragments extracted according to the embodiment of the present disclosure and the distribution of nucleic acid fragments extracted from the control group is shown. The same volume of nucleic acid extracted from the present disclosure and the control group was analyzed by labchip. The results are shown in Figure 5 As shown, the fragment distribution of cfDNA extracted by the present disclosure is consistent with that of the control group, with the main peak at around 170bp, which is consistent with the theoretical main peak position of cfDNA, and the main peak intensity is consistent with that of the control group. There are obvious two peaks, and the position of the two peaks is around 350bp. The results show that the present disclosure successfully extracted cfDNA, and the content and fragment distribution of cfDNA are consistent with the gold standard (control group).

[0062] The above description of various embodiments of the present disclosure is provided to a person of ordinary skill in the relevant art for the purpose of description. It is not intended that the present disclosure be exclusive or limited to a single disclosed embodiment. As above, a person of ordinary skill in the field of the above teachings will understand the various substitutions and variations of the present disclosure. Therefore, although some alternative embodiments are specifically described, a person of ordinary skill in the art will understand or relatively easily develop other embodiments. The present disclosure is intended to include all substitutions, modifications and variations described herein, as well as other embodiments that fall within the spirit and scope of the present disclosure described above.

Claims

1. A column purification device (100), comprising: A base (110) is configured to rotate around a rotation axis (R), and the base (110) comprises: at least one collecting portion (111), wherein one collecting portion (202) of the at least one collecting portion (111) comprises a receiving portion (112), a first cavity (113) and a second cavity (114) located outside the receiving portion (112) in a radial direction relative to the rotation axis (R); At least one reaction container (121), one reaction container (221) of the at least one reaction container (121) is arranged in the accommodating portion (112), and the one reaction container (221) is configured to move in the accommodating portion (112) when the base (110) is rotated in different directions, so as to communicate with the first cavity (113) or the second cavity (114) via a filter column (124) for adsorbing a target substance.

2. The column purification device (100) according to claim 1, wherein the receiving portion (112) extends from the first stop portion (203) to the second stop portion (204), and The reaction container (221) is configured to abut against the first stopper (203) and communicate with the first cavity (113) when the base (110) rotates along a first direction (D1) among the different directions, and to abut against the second stopper (204) and communicate with the second cavity (114) when the base (110) rotates along a second direction (D2) opposite to the first direction.

3. The column purification device (100) according to claim 2, wherein the one reaction container (221) is further configured as: When the base (110) rotates along the first direction (D1) relative to the one reaction container (221), the base (110) moves toward the first stopper (203); and When the base (110) rotates along the second direction (D1) relative to the one reaction container (221), it moves toward the first stopper (203).

4. The column purification device (100) according to claim 2, wherein the one reaction container (221) comprises a liquid outlet (225) provided on a side of the side wall (224) facing away from the rotation axis (R), the liquid outlet (225) is open in the radial direction, and The first cavity (113) has a first inlet (211) in the radial direction toward the rotation axis (R), and the second cavity (114) has a second inlet (212) in the radial direction toward the rotation axis (R), The liquid outlet (225) is configured to be aligned with the first inlet (211) when the one reaction container (221) abuts against the first stopper (203), and to be aligned with the second inlet (212) when the one reaction container (221) abuts against the second stopper (204).

5. The column purification device (100) according to claim 4, wherein the one reaction container (221) further comprises: a liquid outlet pipe (223) extending from the side wall (224) in the radial direction away from the rotation axis (R), The liquid outlet (225) is located at the end of the liquid outlet pipe (223).

6. The column purification device (100) according to claim 5, wherein the housing (112) comprises: A accommodating portion side wall (210) extending circumferentially around the rotation axis (R); A first channel (208) extending from the accommodating portion side wall (210) along the radial direction to the first inlet (211); A second channel (209) extending from the accommodating portion side wall (210) along the radial direction to the second inlet (212); as well as a slide groove (205) extending circumferentially from the first channel (208) to the second channel (209) around the rotation axis (R), The liquid outlet pipe (223) is arranged in the chute (205) so as to be movable from the first channel (208) to the second channel (209).

7. The column purification device (100) according to claim 5, wherein the filter column (124) is located in the liquid outlet pipe (223).

8. The column purification device (100) according to claim 1, wherein the base (110) comprises: A base body (201) extending circumferentially around the rotation axis (R); a first surface (215) located at one end side of the base body (201) in a direction parallel to the rotation axis (R); and The second surface (216) is located at the other end side of the base body (201), The first cavity (113), the second cavity (114) and the accommodating portion (112) extend from the first surface (215) in a direction toward the second surface (216) to a position adjacent to the second surface (216).

9. The column purification device (100) according to claim 8, wherein the receiving portion (112) is open at the inner wall (213) of the base body (201) facing the rotation axis (R), and The column purification device (100) further comprises the container holder (122), wherein the container holder (122) extends between the side walls of at least two reaction containers among the at least one reaction container (121) facing the rotation axis (R), so that the at least two reaction containers abut against the inner walls of the corresponding receiving portions.

10. The column purification device (100) according to claim 9, wherein the at least one reaction vessel (121) is evenly distributed at the edge of the vessel holder (122) relative to the rotation axis (R).

11. The column purification device (100) according to claim 8, further comprising a cover (130), wherein the cover (130) is movably disposed on the first surface (215) relative to the base (110), and the cover (130) comprises a plurality of through holes corresponding to the first cavity (113), the second cavity (114) and the receiving portion (112), The plurality of through holes are configured to be staggered with the first cavity (113), the second cavity (114) and the accommodating portion (112) when the base (110) rotates.

12. A column purification device (100) according to claim 11, wherein a protrusion (125) is provided on the outer wall (214) of the base body (201) which is away from the rotation axis (R), and the cover (130) is formed with a cover wall (132) at the edge extending in a direction toward the second surface (216) to the protrusion (125), the cover wall (132) includes a groove (132) extending circumferentially from the first end (224) to the second end (225), and the protrusion (125) can be slidably arranged in the groove (132) between the first end (224) and the second end (225).

13. The column purification device (100) according to claim 12, wherein the protrusion (125) is configured to abut against the first end (224) or the second end (225) when the base (110) rotates in the different directions.

14. The column purification device (100) according to claim 1, wherein the size of the middle portion of the one reaction container (221) in a direction parallel to the rotation axis (R) is larger than the sizes at both ends.

15. A column purification method comprising: Adding a first reaction liquid containing a target substance into at least one reaction container (121) of the column purification device (100) according to any one of claims 1 to 14; driving an active component to rotate the base (110) of the column purification device (100) along a first direction (D1), thereby allowing the first reaction liquid to enter a first cavity (113) of the column purification device (100); In response to the base (110) stopping rotating, adding a second reaction liquid into the at least one reaction container (121); and The active component is driven to rotate the base (110) of the column purification device (100) along a second direction (D2), thereby allowing the second reaction liquid to enter the second cavity (114) of the column purification device (100) to obtain the target substance.

16. The method of claim 15, wherein the active component comprises one of: The base (110); a vessel holder (122) connected to the at least one reaction vessel (121); or The cover (130) of the column purification device (100). The method according to claim 15 , wherein the first reaction liquid is a reagent including a biomolecule, and the second reaction liquid is an eluent.

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