CN113405877B

CN113405877B - Biological molecule extraction method

Info

Publication number: CN113405877B
Application number: CN202110868042.7A
Authority: CN
Inventors: 吴烨娴; 冯澄宇
Original assignee: Hicomp Microtech Suzhou Co ltd
Current assignee: Hicomp Microtech Suzhou Co ltd
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2024-04-02
Anticipated expiration: 2041-07-30
Also published as: CN113405877A

Abstract

The invention discloses a biomolecule extraction method, which comprises the following steps: s1, adding a sample and a diluent into a diluting component; adding buffer solution into the buffer component; s2, performing first centrifugation, and enabling a sample and a diluent to enter a first filter assembly for filtration; s3, stopping the first centrifugation; performing secondary centrifugation; s4, stopping the second centrifugation; performing third centrifugation, and allowing other substances of the mixed solution to enter a filtrate collecting assembly; s5, stopping the third centrifugation, and adding a first eluent and a second eluent into the first eluting component and the second eluting component; s6, performing fourth centrifugation; accelerating the fifth centrifugation; s7, stopping the fifth centrifugation, and performing the sixth centrifugation; s8, extracting exosomes from the eluent collecting assembly. According to the biomolecule extraction method, only the sample and the matched reagent are needed to be added, and the disc matched instrument automatically completes the dilution, mixing, filtering membrane, filtrate collection and eluent collection of the sample, so that no professional operator is needed.

Description

Biological molecule extraction method

Technical Field

The invention relates to the field of extraction methods of medical instruments for exosomes, in particular to a biomolecule extraction method.

Background

At present, a centrifugal microfluidic chip is often applied to the POCT field, and can conveniently and rapidly output a detection result without the operation of a professional technician. The method is characterized in that microfluidic structures such as a liquid storage tank, a detection tank, a valve and the like are integrated on a disc-shaped chip, and the flow of the microfluid is driven by centrifugal force, so that the detection and analysis of a sample are realized. The centrifugal microfluidic chip can finish operations such as pretreatment, uniform mixing, accurate volume quantification, detection and the like of a sample. In recent years, centrifugal microfluidic chips have been rapidly developed with the advantages of integration, multi-parallel detection, high throughput, low cost, automation, and the like, and have been widely used in the fields of biochemical detection, immunoassay, nucleic acid detection, biomolecule enrichment, food safety, and the like.

Extracellular vesicles are important mediators of protein, mRNA, miRNA and lipid transport to complete the communication pathway between cells and are classified into three classes according to their size and occurrence, including exosomes, microvesicles and apoptotic bodies. Wherein the exosomes are packaging vesicles having a diameter of about 40-100nm secreted by a variety of cells, containing specific proteins, lipids, cytokines or genetic material. Exosomes derived from different tissues not only have their specific protein molecules, but also contain key molecules for their function. In recent years, as exosome research continues to be in depth, the application of exosome has been related to the fields of tumor treatment, medical foundation and immunization, and parasite; clinical studies have been directed to the cardiovascular system, endocrine and metabolic systems, and the like.

In the prior art, a commercial exosome extraction kit needs a large-scale refrigerated centrifuge, has a plurality of operation steps, consumes a long time, needs professional experimenters to perform manual operation, is easy to make mistakes in the operation process, and causes the problems of insufficient exosome amount, insufficient exosome purity and the like.

Disclosure of Invention

In view of the above, the main object of the present invention is to provide a method for extracting biomolecules, which integrates a plurality of micro valves and filter membranes on a chip and solves the problems of complex operation, insufficient quantity of extracted exosomes, insufficient purity of extracted exosomes, etc. in human samples.

In order to achieve the above purpose, the technical scheme of the invention is realized as follows:

a method of biomolecule extraction comprising the steps of:

s1, adding a sample and a diluent into a diluting component; adding buffer solution into the buffer component;

s2, performing first centrifugation, and enabling a sample and a diluent to enter a first filter assembly for filtration; and then enters a buffer assembly to be mixed with the buffer solution to form mixed solution;

s3, stopping the first centrifugation; performing secondary centrifugation, and allowing the mixed solution to enter a second filtering component; the secretion of the mixed solution is enriched in the second filter assembly, and other substances enter the liquid separation assembly;

S4, stopping the second centrifugation; performing third centrifugation, and allowing other substances of the mixed solution to enter a filtrate collecting assembly;

s5, stopping the third centrifugation, adding the first eluent into the first eluting component, and adding the second eluent into the second eluting component;

s6, performing fourth centrifugation; the first eluent enters the second filter assembly, and the second eluent remains in the second filter assembly; the fifth centrifugation is accelerated, and the first eluent enters the liquid separation component through the second filtering component;

s7, stopping the fifth centrifugation, and performing the sixth centrifugation, wherein the second eluent enters the second filtering assembly and further enters the liquid separating assembly, and the first eluent and the second eluent both enter the eluent collecting assembly;

s8, extracting exosomes from the eluent collecting assembly.

The outlet end of the dilution assembly is connected with the first filter assembly, the outlet end of the first filter assembly is connected with the buffer assembly, the outlet end of the buffer assembly is connected with the liquid separation assembly, the liquid separation assembly is also connected with the outlet end of the second elution assembly, and the outlet end of the liquid separation assembly is respectively connected with the filtrate collection assembly and the eluent collection assembly;

the diluting component, the first filtering component, the second filtering component, the buffer component, the first eluting component, the second eluting component, the liquid separating component, the filtrate collecting component and the eluent collecting component are all ring groove structures.

In a preferred embodiment, the dilution assembly comprises: a first reservoir for adding and storing a diluent; and a sample tank for adding and storing the sample, wherein the first liquid storage tank and the outlet end of the sample tank are connected with a first filtering component.

In a preferred embodiment, the first reservoir and the sample reservoir are both of annular configuration and are disposed adjacent to each other; and an outlet is formed in the position, close to the first liquid storage tank and the sample tank, and is connected with the first filtering component.

In a preferred embodiment, the upper end of the sample tank is provided with a sample filling hole

In a preferred embodiment, the first filter assembly comprises: the device comprises a diluting component, a buffer component, a first filter membrane groove, a through hole and a impurity removing filter membrane, wherein one end of the first filter membrane groove is connected with the diluting component in a conducting mode, the other end of the first filter membrane groove is provided with the through hole and the buffer component are connected, and the end part, close to the first filter membrane groove, of the through hole is provided with the impurity removing filter membrane.

In a preferred embodiment, the side wall of the through hole close to the end part of the first filter membrane tank is obliquely arranged, and the included angle between the side wall and the bottom surface is 30-150 degrees.

In a preferred embodiment, the side wall of the through hole close to the end of the first filter membrane tank is obliquely arranged, and the included angle between the side wall and the bottom surface is 120 degrees.

In a preferred embodiment, the size of the ultrafiltration membrane is 0.22-0.03 microns.

In a preferred embodiment, the diameter of the through hole is 0.5-3mm.

In a preferred embodiment, the cushioning assembly comprises: a mixing tank, a second reservoir for storing buffer solution; the inlet end of the mixing tank is connected with the outlet end of the first filtering component in a conducting way, and the outlet end of the second liquid storage tank is connected with the mixing tank.

In a preferred embodiment, the depth of the mixing tank is 1-5mm.

In a preferred embodiment, the second filter assembly comprises: a second filter membrane tank; the inlet end of the second filter membrane tank is connected with the outlet end of the buffer assembly through a first siphon runner, and the outlet end of the second filter membrane tank is connected with the liquid separation assembly;

in a preferred embodiment, the outlet end of the filter membrane tank is provided with a biomolecular filter membrane.

In a preferred embodiment, the side wall of the outlet end of the second filter membrane tank is arranged obliquely, and the included angle between the side wall and the bottom surface is 30-150 degrees.

In a preferred embodiment, the side wall of the outlet end of the second filter membrane tank is inclined, and the included angle between the side wall and the bottom surface is 120 degrees.

In a preferred embodiment, the width of the first siphon flow passage is 0.1-0.5mm, and the depth of the first siphon flow passage is 0.1-0.5mm.

In a preferred embodiment, the first elution assembly comprises: and the outlet end of the third liquid storage tank is connected with the second filter membrane tank.

In a preferred embodiment, the second elution assembly comprises: a fourth reservoir, a transition tank; the fourth liquid storage tank is connected with the transition tank through a capillary valve in a conducting way, and the outlet end of the transition tank is connected with the second filter membrane tank through a fourth siphon runner in a conducting way.

In a preferred embodiment, the depth of the third reservoir is 3-5mm, the width of the outlet channel is 3-5mm, and the depth of the outlet channel is the same as the depth of the third reservoir;

in a preferred embodiment, the capillary valve has a width of 0.1-0.2mm, a depth of 0.1-0.2mm, and a length of 2-5mm.

In a preferred embodiment, the liquid separation assembly comprises: the inlet end of the liquid separating tank is connected with the outlet end of the second filtering component, and the two ends of the liquid separating tank are respectively connected with the filtrate collecting component and the eluent collecting component through the second siphon flow passage and the third siphon flow passage.

In a preferred embodiment, the second filter assembly outlet end has a pore size of 0.5-3mm in diameter.

In a preferred embodiment, the second siphon flow channel has a smaller hydrophilic index than the third siphon flow channel.

In a preferred embodiment, the filtrate collection assembly comprises: the inlet end of the filtrate collecting tank is connected with the second siphon runner, and the overflow port of the filtrate collecting tank is connected with the filtrate collecting tank.

In a preferred embodiment, the filtrate collection tank is a waste liquid quantitative structure having a quantitative size of waste liquid of 100 to 300. Mu.l.

In a preferred embodiment, the eluent collection assembly comprises: the extraction tank, extraction tank one end fourth siphon runner is connected, extraction tank one end has seted up and has got the liquid hole.

In a preferred embodiment, the dilution module, the first filter module, the second filter module, the buffer module, the first elution module, the second elution module, the liquid separation module, the filtrate collection module and the eluent collection module are respectively and centrally symmetrically arranged.

In a preferred embodiment, the step S1 is: adding human samples into the sample tank, adding diluent into the first liquid storage tank, and adding buffer into the second liquid storage tank;

The step S2 is as follows: performing first centrifugation on the chip, enabling the sample and the diluent to enter a first filter membrane tank together, enabling the buffer solution to enter a mixing tank, enabling the sample and the diluent to pass through a impurity removing filter membrane in the first filter membrane tank after centrifugation for a certain time, and mixing the sample and the diluent with the buffer solution in the mixing tank;

the step S3 is as follows: after stopping the first centrifugation, the mixed solution fills the first siphon flow passage; performing secondary centrifugation, enabling the mixed solution to enter a second filter membrane tank, and enabling the mixed solution to pass through a biological molecular filter membrane in the second filter membrane tank and enter a liquid separation tank after centrifugation for a certain time; the exosomes in the mixed solution are enriched on a biological molecular filtering membrane;

the step S4 is as follows: after stopping the second centrifugation, the mixed liquid is filled in the second siphon flow channel preferentially, when the third siphon flow channel is not filled, the third centrifugation is carried out, the mixed liquid enters a waste liquid quantitative structure in the filtrate collecting tank, and the redundant mixed liquid enters a waste liquid overflow tank;

the step S5 is as follows: adding a first eluent into a third liquid storage tank of the chip, and adding a second eluent into a fourth liquid storage tank;

the step S6 is as follows: performing fourth centrifugation, namely low-speed centrifugation, wherein the first eluent enters a second filter membrane tank, and the second eluent is thrown into a fourth liquid storage tank; carrying out fifth centrifugation, namely high-speed centrifugation, wherein the first eluent enters a liquid separating tank through a biological molecular filtering membrane, and the second eluent breaks through a capillary valve and enters a transition tank;

The step S7 is as follows: stopping centrifugation for the fifth time, and filling the second eluent entering the transition groove with the fourth siphon flow passage; performing a sixth centrifugation, allowing the second eluent to enter a second filter membrane tank and pass through a biological molecule filter membrane to enter a liquid separation tank, and allowing the first eluent and the second eluent to enter an extraction tank together;

the step S8 is as follows: the extract containing exosomes was removed from the chip through the extraction tank.

The biomolecule extraction method has the following beneficial effects:

according to the biomolecule extraction method, the outlet end of a dilution component is connected with a first filter component, the outlet end of the first filter component is connected with a buffer component, the outlet end of the buffer component is connected with a liquid separation component, the liquid separation component is also connected with the outlet end of a second elution component, and the outlet end of the liquid separation component is respectively connected with a filtrate collection component and an eluent collection component; the diluting component, the first filtering component, the second filtering component, the buffer component, the first eluting component, the second eluting component, the liquid separating component, the filtrate collecting component and the eluent collecting component are all ring groove structures.

The problems that a commercial exosome extraction kit in the prior art needs a large-scale refrigerated centrifuge, is multiple in operation steps, long in time consumption, needs professional experimenters to perform manual operation, is easy to make mistakes in the operation process, and causes insufficient exosome amount to be extracted, insufficient exosome purity and the like are solved.

According to the biomolecule extraction method, only a sample (such as whole blood, plasma, urine and the like) and a matched reagent (a reagent used for extracting exosome biomolecules) are added, and the dilution, mixing, filtering membrane and filtrate collection and eluent collection of the sample are automatically completed by a disc matched instrument, so that a professional operator is not required. For example: pretreatment of plasma, enrichment of exosome biomolecules and elution of exosome biomolecules without professional operators. The ultrafiltration membrane for sample filtration and the exosome biomolecule enrichment filter membrane are integrated on the disc, and the use amount of the filter membrane and the reagent is 1/10 of that of the traditional filter and the centrifugal column, so that the cost is low, and the extraction efficiency is high. The sample for extracting the exosome biomolecules is 1/5 of that of the traditional method, so that the blood sampling amount of patients can be reduced. Cell fragments and impurities in the sample are separated from the exosome biomolecule extracting solution, and the extracted exosome biomolecule has high purity. The extraction of exosome biomolecules from 2 samples can be performed simultaneously. The extraction of the whole exosome biomolecules can be completed within 15 minutes within the time period required for the extraction of the whole exosome biomolecules. At present, centrifugal microfluidic chip technology is not used for extracting exosome biomolecules of human blood samples. Wherein the biomolecules comprise all circulating tumor cells, exosomes, specific proteins, etc. enriched with the filter membrane.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a perspective view of a method of biomolecule extraction according to one embodiment of the present disclosure;

fig. 2 is a schematic structural view of a chip flow channel structure of a biomolecule extraction method according to one embodiment of the present disclosure;

FIG. 3 is a cross-sectional view of a filter membrane built in a chip of a method for extracting biomolecules according to an embodiment of the present disclosure;

FIG. 4 is a schematic diagram of chip waste and extract separation according to a biomolecular extraction method according to one embodiment of the present disclosure;

FIG. 5 is a schematic diagram of on-chip addition of a blood sample, diluent, and buffer according to a biomolecule extraction method in one embodiment of the present disclosure;

FIG. 6 is a schematic diagram of a first centrifugation of a chip, a dilution of a sample followed by filtration of the sample with a buffer in a chip mixing tank, according to one embodiment of the disclosure;

FIG. 7 is a schematic diagram of a second centrifugation of a chip with a biomolecular extraction method according to one embodiment of the present disclosure, after passing through a biomolecular filtration membrane, the mixed solution into a separation tank;

FIG. 8 is a schematic diagram of a quantitative structure of a third centrifugation mixed solution of a chip entering a filtrate collection tank and filling the filtrate collection tank, according to a method of extracting biomolecules according to an embodiment of the present disclosure;

FIG. 9 is a schematic diagram of a biomolecule extraction method according to one embodiment of the present disclosure, when a first eluent and a second eluent are added on a chip;

FIG. 10 is a schematic diagram of a second eluent unable to break through a capillary valve after entering a second filter tank for incubation with a biomolecule filter at low rotational speed according to a fourth centrifugation of a chip of a biomolecule extraction method in one embodiment of the present disclosure;

FIG. 11 is a schematic diagram of a fourth centrifugation of a chip of a biomolecular extraction method at high rotational speeds with a first eluent passing through a biomolecular filtration membrane into a separation tank and a second eluent breaking through a capillary valve into a transition tank, according to one embodiment of the present disclosure;

fig. 12 is a schematic diagram of a fifth centrifugation eluent from a chip passing through a biomolecule filter membrane into an extraction tank together with a first eluent according to an embodiment of the present disclosure.

[ Main reference numerals Specification ]

1. A dilution assembly; 11. a first reservoir; 12. a sample tank; 121. a sample liquid adding hole;

2. a first filter assembly; 21. a first filter membrane tank; 22. a through hole; 23. removing impurity filter membranes;

3. a buffer assembly; 31. a mixing tank; 32. a second reservoir;

4. a second filter assembly; 41. a second filter membrane tank; 42. a first siphon flow passage;

5. a liquid separation component; 51. a liquid dividing tank; 52. a second siphon flow passage; 53. a third siphon flow passage;

6. a first elution assembly; 61. a third reservoir;

7. a second elution assembly; 71. a fourth reservoir; 72. a transition groove; 73. a capillary valve; 74. a fourth siphon flow passage;

8. a filtrate collection assembly; 81. a filtrate collecting tank; 82. a filtrate collecting tank;

9. an eluent collection assembly; 91. an extraction tank; 92. and a liquid taking hole.

Detailed Description

The method for extracting biomolecules according to the present invention will be described in further detail with reference to the accompanying drawings.

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

It should be noted that the terms "first," "second," and the like in the description and claims of the present application and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that embodiments of the present application described herein may be capable of being practiced otherwise than as specifically illustrated and described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Spatially relative terms, such as "above … …," "above … …," "upper surface at … …," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial location relative to another device or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "above" or "over" other devices or structures would then be oriented "below" or "beneath" the other devices or structures. Thus, the exemplary term "above … …" may include both orientations of "above … …" and "below … …". The device may also be positioned in other different ways (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

As shown in fig. 1 to 12, the biomolecule extraction method comprises the following steps:

s1, adding a sample and a diluent into a diluting component 1; adding buffer solution into the buffer component 3;

S2, performing first centrifugation, and enabling a sample and a diluent to enter a first filter assembly 2 for filtration; and enters a buffer component 3 to be mixed with the buffer liquid to form mixed liquid;

s3, stopping the first centrifugation; performing secondary centrifugation, and allowing the mixed solution to enter a second filter assembly 4; the secretion of the mixed liquor is enriched in the second filter component 4, and other substances enter the liquid separation component 5;

s4, stopping the second centrifugation; performing third centrifugation, and allowing other substances of the mixed solution to enter a filtrate collecting assembly 8;

s5, stopping the third centrifugation, adding a first eluent into the first eluting component 6, and adding a second eluent into the second eluting component 7;

s6, performing fourth centrifugation; the first eluent enters the second filter assembly 4, and the second eluent remains in the second elution assembly 7; after the fifth centrifugation acceleration, the first eluent passes through the second filter assembly 4 and enters the liquid separation assembly 5;

s7, stopping the fifth centrifugation, and performing the sixth centrifugation, wherein the second eluent enters the second filtering assembly 4 and further enters the liquid separating assembly 5, and the first eluent and the second eluent enter the eluent collecting assembly 9;

s8, extracting exosomes from the eluent collection assembly 9.

The outlet end of the diluting component 1 is connected with the first filtering component 2, the outlet end of the first filtering component 2 is connected with the buffer component 3, the outlet end of the buffer component 3 is connected with the liquid separating component 5, the liquid separating component 5 is also connected with the outlet end of the second eluting component 7, and the outlet end of the liquid separating component 5 is respectively connected with the filtrate collecting component 8 and the eluent collecting component 9;

the diluting component 1, the first filtering component 2, the second filtering component 4, the buffer component 3, the first eluting component 6, the second eluting component 7, the liquid separating component 5, the filtrate collecting component 8 and the eluent collecting component 9 are all ring groove structures.

In one embodiment:

the diluting assembly 1 includes: a first reservoir 11 for adding and storing a diluent; a sample tank 12 for adding and storing a sample, and the outlet ends of the first reservoir 11 and the sample tank 12 are connected to the first filter assembly 2.

The first liquid storage tank 11 and the sample tank 12 are of annular structures and are adjacently arranged; an outlet is formed at a position where the first liquid storage tank 11 and the sample tank 12 are close to each other, and is connected with the first filtering component 2.

The upper end of the sample tank 12 is provided with a sample liquid adding hole 121

The first filter assembly 2 comprises: the first filter membrane tank 21, first filter membrane tank 21 one end with diluting subassembly 1 switches on and is connected, through-hole 22 has been seted up to first filter membrane tank 21 other end with buffer module 3 is connected the through-hole 22 is provided with the edulcoration filter membrane 23 near the tip in first filter membrane tank 21.

The side wall of the through hole 22 close to the end part of the first filter membrane tank 21 is obliquely arranged, and the included angle between the side wall and the bottom surface is 30-150 degrees.

The side wall of the through hole 22 close to the end part of the first filter membrane tank 21 is obliquely arranged, and the included angle between the side wall and the bottom surface is 120 degrees.

The aperture of the impurity removing filter membrane 23 is an ultrafiltration membrane with the aperture of 0.22-0.03 micrometers.

The diameter of the through hole 22 is 0.5-3mm.

In one embodiment:

the cushion assembly 3 includes: a mixing tank 31, a second reservoir 32 for storing buffer solution; the inlet end of the mixing tank 31 is connected with the outlet end of the first filter assembly 2 in a conducting way, and the outlet end of the second liquid storage tank 32 is connected with the mixing tank 31.

The depth of the mixing tank 31 is 1-5mm.

The second filter assembly 4 comprises: a second filter membrane tank 41; the inlet end of the second filter membrane tank 41 is connected with the outlet end of the buffer assembly 3 through a first siphon runner 42, and the outlet end of the second filter membrane tank 41 is connected with the liquid separation assembly 5;

the outlet end of the filter membrane tank is provided with a biological molecular filter membrane.

The side wall of the outlet end of the second filter membrane tank 41 is obliquely arranged, and the included angle between the side wall and the bottom surface is 30-150 degrees.

The side wall of the outlet end of the second filter membrane tank 41 is inclined, and the included angle between the side wall and the bottom surface is 120 degrees.

The width of the first siphon flow passage 42 is 0.1-0.5mm, and the depth of the first siphon flow passage 42 is 0.1-0.5mm.

The first eluting assembly 6 comprises: and a third liquid storage tank 61, wherein the outlet end of the third liquid storage tank 61 is connected with the second filter membrane tank 41.

The second eluting assembly 7 comprises: a fourth reservoir 71, a transition 72; the fourth liquid storage tank 71 is connected with the transition tank 72 in a conducting way through a capillary valve 73, and the outlet end of the transition tank 72 is connected with the second filter membrane tank 41 in a conducting way through a fourth siphon flow passage 74.

The depth of the third liquid storage groove 61 is 3-5mm, the width of the outlet flow channel is 3-5mm, and the depth of the outlet flow channel is the same as the depth of the third liquid storage groove 61;

the capillary valve 73 has a width of 0.1-0.2mm, a depth of 0.1-0.2mm, and a length of 2-5mm.

In one embodiment:

the liquid separation assembly 5 comprises: the inlet end of the liquid separating groove 51 is connected with the outlet end of the second filtering component 4, and two ends of the liquid separating groove 51 are respectively connected with the filtrate collecting component 8 and the eluent collecting component 9 through a second siphon runner 52 and a third siphon runner 53.

The aperture of the outlet end of the second filter component 4 is 0.5-3mm in diameter.

The second siphon flow passage 52 has a smaller hydropathic index than the third siphon flow passage 53.

The filtrate collection assembly 8 comprises: the inlet end of the filtrate collecting tank 81 is connected with the second siphon runner 52, and the overflow port of the filtrate collecting tank 81 is connected with the filtrate collecting tank 82.

The filtrate collecting tank 81 is a waste liquid quantitative structure, and the quantitative size of the waste liquid quantitative structure is 100-300 mu l of waste liquid.

The eluent collection assembly 9 comprises: the extraction tank 91, the fourth siphon runner 74 in extraction tank 91 one end connects, extraction tank 91 one end has seted up and has got liquid hole 92.

In one embodiment:

the dilution component 1, the first filtering component 2, the second filtering component 4, the buffer component 3, the first eluting component 6, the second eluting component 7, the liquid separating component 5, the filtrate collecting component 8 and the eluent collecting component 9 are respectively and centrally symmetrically arranged.

In a preferred embodiment, the step S1 is: the human sample is added into the sample tank 12, the diluent is added into the first liquid storage tank 11, and the buffer is added into the second liquid storage tank 32;

the step S2 is as follows: performing first centrifugation on the chip, enabling the sample and the diluent to enter the first filter membrane tank 21 together, enabling the buffer to enter the mixing tank 31, enabling the sample and the diluent to pass through the impurity removing filter membrane 23 in the first filter membrane tank 21 after centrifugation for a certain time, and mixing with the buffer in the mixing tank 31;

The step S3 is as follows: after stopping the first centrifugation, the mixed liquid fills the first siphon flow passage 42; performing secondary centrifugation, enabling the mixed solution to enter a second filter membrane tank 41, and enabling the mixed solution to pass through a biological molecular filter membrane in the second filter membrane tank 41 and enter a liquid separation tank 51 after centrifugation for a certain period of time; the exosomes in the mixed solution are enriched on a biological molecular filtering membrane;

the step S4 is as follows: after stopping the second centrifugation, the mixed solution fills the second siphon runner 52 preferentially, and when the third siphon runner 53 is not filled, the third centrifugation is performed, the mixed solution enters a waste liquid quantitative structure in the filtrate collecting tank 81, and the redundant mixed solution enters a waste liquid overflow tank;

the step S5 is as follows: adding the first eluent into the third liquid storage tank 61 of the chip, and adding the second eluent into the fourth liquid storage tank 71;

the step S6 is as follows: performing a fourth centrifugation, i.e., a low-speed centrifugation, wherein the first eluent enters the second filter membrane tank 41, and the second eluent is thrown into the fourth liquid storage tank 71; performing fifth centrifugation, namely high-speed centrifugation, wherein the first eluent enters a liquid separating tank through a biological molecular filtering membrane, and the second eluent breaks through a capillary valve 73 and enters a transition groove 72;

the step S7 is as follows: after stopping the fifth centrifugation, the second eluent entering the transition channel 72 fills the fourth siphon flow channel 74; a sixth centrifugation is performed, the second eluent enters the second filter membrane tank 41 and passes through the biological molecule filter membrane to enter the liquid separating tank 51, and the first eluent and the second eluent enter the extraction tank 91 together;

The step S8 is as follows: the extract containing exosomes was removed from the chip through extraction tank 91.

Fig. 1 is a schematic perspective view, the overall structure of the chip is divided into an upper layer and a lower layer, the upper layer of the chip is provided with various groove structures, liquid adding holes, liquid taking holes 92, various flow channels and air holes, and the lower layer of the chip is a polyester film. The upper layer of the chip is provided with a channel which faces downwards and is tightly bonded with the lower layer of the chip, the chip can accommodate a plurality of detection units, and can simultaneously extract exosomes of a plurality of samples, 2 detection units are exemplified in the figure, and 2 samples can be simultaneously extracted. Of course, designs of 3, 4, 5, etc. are also possible.

Fig. 2 is a plan view of a single unit of the upper layer of the chip. Comprises the following steps: sample tank 12, first reservoir 11, second reservoir 32, third reservoir 61, fourth reservoir 71, transition tank 72, first filter tank 21, second filter tank 41, mixing tank 31, liquid separation tank 51, filtrate collection tank 81, extraction tank 91, first siphon flow path 42, second siphon flow path 52, third siphon flow path 53, fourth siphon flow path 74, capillary valve 73, sample addition hole 121, and liquid taking hole 92. Wherein each siphon flow passage needs to be subjected to surface hydrophilic treatment, and the hydrophilic index of the second siphon flow passage 52 is smaller than that of the third siphon flow passage 53.

Fig. 3 is a cross-sectional view of a filter membrane built in a chip, taking the impurity removing filter membrane 23 as an example, the impurity removing filter membrane 23 is bonded to the side wall surface of the first filter membrane tank 21 by ultrasonic welding, laser welding or an adhesive method. The angle between the side wall surface and the bottom surface of the first filter membrane tank 21 is 90-150 degrees, and 120 degrees is preferable in the illustration. The first filter membrane tank 21 and the mixing tank 31 are connected by a through hole 22 penetrating the side wall surface, and the diameter of the through hole 22 is 0.5-3mm, preferably 2mm.

FIG. 4 is a schematic diagram of the separation of chip waste and extract. The chip divides liquid structure includes: the second filter membrane tank 41, the liquid separation tank 51, the second siphon flow passage 52, the third siphon flow passage 53, the filtrate collection tank 81, and the extraction tank 91. Wherein the filtrate collection tank 81 comprises: a waste liquid quantitative structure and a waste liquid overflow tank. When the mixed solution (sample+diluent+buffer solution) enters the liquid separation tank 51 through the biomolecule filter membrane in the second filter membrane tank 41 to become a waste solution, the waste solution can fill the second siphon runner 52 preferentially because the hydrophilicity index of the second siphon runner 52 is smaller than that of the third siphon runner 53, and the third siphon runner 53 cannot fill in a short time because the hydrophilicity is relatively poor. After re-centrifugation, the waste liquid enters the filtrate collecting tank 81 through the second siphon flow passage 52, fills the waste liquid quantitative structure in the filtrate collecting tank 81, and the surplus waste liquid enters the waste liquid overflow tank.

When the eluting solution is used for eluting the exosomes enriched on the biological molecular filtering membrane, the eluting solution enters the liquid dividing groove 51 through the biological molecular filtering membrane to become extracting solution, and the liquid is filled in the liquid waste quantitative structure in the filtrate collecting groove 81 connected with the outlet of the second siphon flow passage 52, so that the air column in the second siphon flow passage 52 cannot be discharged, and therefore, the second siphon flow passage 52 cannot be opened to become a normally closed valve. When the second siphon flow channel 52 is filled with the extracting solution, the extracting solution is centrifuged again and then enters the extracting tank 91 through the third siphon flow channel 53, so that the on-chip liquid separating structure separates the waste liquid and the extracting solution into two different tanks.

Specifically, the exosomes of the samples were extracted by the following steps:

as shown in FIG. 5, 100. Mu.l of sample was added to the sample well 12 of the chip, 100. Mu.l of diluent was added to the first reservoir 11, and 200. Mu.l of buffer was added to the second reservoir 32.

As shown in FIG. 6, the first centrifugation of the chip was performed at 7000rpm for 5 minutes, and the sample and the diluent were mixed in the first filter tank 21 and introduced into the mixing tank 31 through the impurity removing filter 23. After passing through the impurity removal filter 23, the diluted sample is filtered for impurities and impurity proteins, and mixed with a buffer solution in the mixing tank 31.

As shown in fig. 7, after the mixed solution (sample+diluent+buffer) fills the first siphon flow passage 42, it is centrifuged again at 3400rpm for 1min. The mixed solution passes through the biological molecular filtering membrane and enters the liquid separating tank 51 to become waste liquid, and exosomes in the mixed solution are enriched on the biological molecular filtering membrane.

As shown in fig. 8, due to the action of the liquid separation structure of the chip (the specific principle is described in detail in fig. 4), the chip is centrifuged again at 3400rpm for 1min, the waste liquid enters the filtrate collecting tank 81 and fills the waste liquid quantitative structure in the filtrate collecting tank 81, and the surplus waste liquid enters the waste liquid overflow tank.

As shown in fig. 9, the first eluent is added to the third liquid storage tank 61 on the chip, the second eluent is added to the fourth liquid storage tank 71, and the exosomes enriched on the biomolecule filter membrane are prepared for elution.

As shown in FIG. 10, the chip was subjected to low-speed centrifugation at 300rpm for 1min. Since the outlet of the third liquid storage tank 61 is not provided with a valve, the eluent enters the second filter membrane tank 41 at a low rotation speed, but cannot pass through the biological molecule filter membrane, so that the first eluent is fully contacted with the exosome on the biological molecule filter membrane for a period of time, and the incubation effect is achieved. And the capillary valve 73 is arranged at the outlet of the fourth liquid storage groove 71, the width of the capillary valve 73 is 0.1-0.3mm, the depth is 0.1-0.3mm, the width is preferably 0.2mm, the depth is 0.1mm, and the eluent 2 can not break through the capillary valve 73 at low rotation speed and still stays in the fourth liquid storage groove.

As shown in FIG. 11, after the chip was centrifuged at a low rotation speed for 1min, the rotation speed was immediately increased to 3400rpm and the chip was centrifuged for 1min. The first eluent is used for eluting most of the exosomes on the biological molecular filtering membrane, then passing through the biological molecular filtering membrane and entering the liquid separation tank to be an extracting solution, the second eluent breaks through the capillary valve 73 and enters the transition tank 72, after centrifugation is stopped, the first eluent is filled in the third siphon flow channel 53, and the second eluent is filled in the fourth siphon flow channel 74.

As shown in fig. 12, the chip is centrifuged at high speed at 8800rpm for 1min, the second eluent passes through the bio-molecular filtration membrane to elute the remaining exosomes on the bio-molecular filtration membrane, and the first eluent and the second eluent enter the extraction tank 91 together because the third siphon flow channel 53 is opened, and the extracting solution with exosomes can be sucked out of the disc through the liquid-extracting hole 92 at the upper end of the extraction tank 91 for subsequent analysis and detection.

When the centrifugal microfluidic chip is used, only an operator needs to add the corresponding sample and reagent, and the disc can automatically finish:

1. the pretreatment of the sample comprises the steps of diluting the sample, passing the diluted sample through an ultrafiltration membrane, removing cell fragments and impurities, and mixing the filtered sample with a buffer solution;

2. Exosome enrichment, which comprises the steps of enriching exosomes on a filter membrane, and enabling waste liquid in a sample to pass through the filter membrane;

3. exosome elution comprises the steps of incubating eluent and a filter membrane, fully eluting the exosome on the filter membrane and eluting for a plurality of times to ensure that the exosome on the filter membrane is completely eluted, and ensuring that the purity of the extracted exosome is high because the eluent is not contacted with waste liquid.

The whole exosome extraction process comprises the following steps: pretreatment of samples, enrichment of exosomes and elution of exosomes can be completed within 15Min, large-scale freezing and centrifuging equipment is not needed, professional experimenters do not need to perform manual operation, and the whole extraction process is highly automated and good in repeatability.

The commercial exosome extraction kit needs a large-scale refrigerated centrifuge, has a plurality of operation steps, consumes a long time, needs professional experimenters to perform manual operation, is easy to make mistakes in the operation process, and causes the problems of insufficient exosome amount, insufficient exosome purity and the like.

In order to solve the problems of complex extraction operation, insufficient exosome extraction quantity, insufficient purity of the extracted exosome and the like of human plasma, the invention provides an exosome extraction centrifugal microfluidic chip, which is integrated with various micro-valves and filter membranes, the whole extraction process can be completed within 15Min, large-scale freezing and centrifugation equipment is not needed, no professional experimenters do manual operation, and the whole extraction process is highly automatic and has good repeatability.

The above embodiments illustrate the present invention by taking the extraction of exosomes as an example, and the centrifugal microfluidic chip of the present invention can also be used for the extraction of other biomolecules, including all circulating tumor cells, exosomes, and specific proteins enriched with a filter membrane.

The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention.

Claims

1. A method of extracting a biomolecule, comprising the steps of:

s1, adding a sample and a diluent into a diluting component (1); adding buffer solution into the buffer component (3);

s2, performing first centrifugation, and enabling a sample and a diluent to enter a first filter assembly (2) for filtration; and enters a buffer component (3) to be mixed with the buffer liquid to form mixed liquid;

s3, stopping the first centrifugation; performing secondary centrifugation, and allowing the mixed solution to enter a second filter assembly (4); the secretion of the mixed liquor is enriched in the second filter component (4), and other substances enter the liquid separation component (5);

s4, stopping the second centrifugation; performing a third centrifugation, and introducing other substances of the mixed solution into a filtrate collecting assembly (8);

s5, stopping the third centrifugation, adding a first eluent into the first eluting component (6), and adding a second eluent into the second eluting component (7);

S6, performing fourth centrifugation; the first eluent enters the second filter assembly (4), and the second eluent remains in the second elution assembly (7); the first eluent enters the liquid separating component (5) through the second filtering component (4) after the fifth centrifugation is accelerated;

s7, stopping the fifth centrifugation, and performing the sixth centrifugation, wherein the second eluent enters the second filtering assembly (4) and further enters the liquid separating assembly (5), and the first eluent and the second eluent both enter the eluent collecting assembly (9);

s8, extracting exosomes from the eluent collection assembly (9).

2. The method for extracting a biological molecule according to claim 1, wherein,

the outlet end of the diluting component (1) is connected with the first filtering component (2), the outlet end of the first filtering component (2) is connected with the buffer component (3), the outlet end of the buffer component (3) is connected with the liquid separating component (5), the liquid separating component (5) is also connected with the outlet end of the second eluting component (7), and the outlet end of the liquid separating component (5) is respectively connected with the filtrate collecting component (8) and the eluent collecting component (9);

the diluting component (1), the first filtering component (2), the second filtering component (4), the buffer component (3), the first eluting component (6), the second eluting component (7), the liquid separating component (5), the filtrate collecting component (8) and the eluent collecting component (9) are all ring groove structures.

3. The method of biomolecule extraction according to claim 2, wherein the dilution assembly (1) comprises: a first reservoir (11) for adding and storing a dilution liquid; a sample tank (12) for adding and storing a sample, and a first filter assembly (2) connected to the first reservoir (11) and the outlet end of the sample tank (12).

4. A method of extracting a biological molecule according to claim 3, wherein the first reservoir (11) and the sample reservoir (12) are each of annular configuration and are disposed adjacently; an outlet is formed in the position, close to the first liquid storage groove (11) and the sample groove (12), and is connected with the first filtering component (2).

5. The method for extracting a biological molecule according to claim 4, wherein a sample filling hole (121) is provided at an upper end of the sample tank (12).

6. The method of biomolecule extraction according to claim 4, wherein the first filter assembly (2) comprises: the device comprises a diluting component (1), a first filter membrane groove (21), a through hole (22) and a buffer component (3), wherein one end of the first filter membrane groove (21) is connected with the diluting component (1) in a conducting mode, the other end of the first filter membrane groove (21) is provided with the through hole (22) and is connected with the buffer component (3), and a impurity removing filter membrane (23) is arranged at the end part, close to the first filter membrane groove (21), of the through hole (22).

7. The method according to claim 6, wherein the side wall of the through hole (22) near the end of the first filter membrane tank (21) is inclined at an angle of 30-150 ° to the bottom surface.

8. The method according to claim 7, wherein the side wall of the through hole (22) near the end of the first filter membrane tank (21) is inclined at an angle of 120 ° to the bottom surface.

9. The method according to claim 7, characterized in that the size of the ultrafiltration membrane (23) is 0.22-0.03 μm.

10. The method of extracting biomolecules according to claim 6, wherein the diameter of the through hole (22) is 0.5-3mm.

11. The method of biomolecule extraction according to claim 6, wherein the buffer assembly (3) comprises: a mixing tank (31), a second reservoir (32) for storing a buffer solution; the inlet end of the mixing tank (31) is connected with the outlet end of the first filtering component (2) in a conducting mode, and the outlet end of the second liquid storage tank (32) is connected with the mixing tank (31).

12. The method of biomolecule extraction according to claim 11, wherein the depth of the mixing tank (31) is 1-5mm.

13. The method of biomolecule extraction according to claim 11, wherein the second filter assembly (4) comprises: a second filter membrane tank (41); the inlet end of the second filter membrane groove (41) is connected with the outlet end of the buffer component (3) through a first siphon runner, and the outlet end of the second filter membrane groove (41) is connected with the liquid separating component (5);

14. The method of extracting biomolecules as claimed in claim 13, wherein the outlet side wall of the second filter membrane tank (41) is inclined at an angle of 30-150 ° to the bottom surface.

15. The method of extracting biomolecules as claimed in claim 14, wherein the outlet side wall of the second filter tank (41) is inclined at an angle of 120 ° to the bottom surface.

16. The method according to any one of claims 13 to 15, wherein the width of the first siphon flow passage is 0.1 to 0.5mm and the depth of the first siphon flow passage is 0.1 to 0.5mm.

17. The method of biomolecule extraction according to any one of claims 13-16, wherein the first elution assembly (6) comprises: and the outlet end of the third liquid storage groove (61) is connected with the second filter membrane groove (41).

18. The method of biomolecule extraction according to claim 17, wherein the second elution assembly (7) comprises: a fourth liquid storage tank (71) and a transition tank (72); the fourth liquid storage tank (71) is in conductive connection with the transition tank (72) through a capillary valve (73), and the outlet end of the transition tank (72) is in conductive connection with the second filter membrane tank (41) through a fourth siphon runner (74).

19. The method of extracting biomolecules according to claim 18, wherein the depth of the third liquid storage tank (61) is 3-5mm, the width of the outlet flow passage is 3-5mm, and the depth of the outlet flow passage is the same as the depth of the third liquid storage tank (61);

the capillary valve (73) has a width of 0.1-0.2mm, a depth of 0.1-0.2mm and a length of 2-5mm.

20. The method of biomolecule extraction according to claim 11, wherein the liquid separation module (5) comprises: the inlet end of the liquid separating groove (51) is connected with the outlet end of the second filtering component (4), and two ends of the liquid separating groove (51) are respectively connected with the filtrate collecting component (8) and the eluent collecting component (9) through the second siphon flow passage (52) and the third siphon flow passage (53).

21. The method of extracting biomolecules according to claim 20, wherein the pore diameter of the outlet end of the second filter assembly (4) is 0.5-3mm.

22. The method of biomolecule extraction according to claim 20, wherein the second siphon flow channel (52) has a smaller hydrophilicity index than the third siphon flow channel (53).

23. The method of biomolecule extraction according to any one of claims 20-22, wherein the filtrate collection assembly (8) comprises: the device comprises a filtrate collecting tank (81) and a filtrate collecting tank (82), wherein the inlet end of the filtrate collecting tank (81) is connected with a second siphon runner (52), and the overflow port of the filtrate collecting tank (81) is connected with the filtrate collecting tank (82).

24. The method for extracting biomolecules according to claim 23, wherein said filtrate collecting tank (81) is a waste liquid quantitative structure having a quantitative size of waste liquid of 100 to 300 μl.

25. The method of biomolecule extraction according to any one of claims 20-22, wherein the eluent collection assembly (9) comprises: the extraction tank (91), draw groove (91) one end fourth siphon runner (74) to connect, draw groove (91) one end to offer liquid hole (92).

26. The method for extracting biomolecules according to claim 20, wherein the dilution assembly (1), the first filter assembly (2), the second filter assembly (4), the buffer assembly (3), the first eluting assembly (6), the second eluting assembly (7), the liquid separation assembly (5), the filtrate collection assembly (8) and the eluent collection assembly (9) are respectively and centrally symmetrically arranged.

27. The method according to claim 26, wherein the step S1 is: a human sample is added into a sample tank (12), a diluent is added into a first liquid storage tank (11), and a buffer is added into a second liquid storage tank (32);

the step S2 is as follows: performing first centrifugation on the chip, enabling the sample and the diluent to enter a first filter membrane tank (21), enabling the buffer solution to enter a mixing tank (31), enabling the sample and the diluent to pass through a impurity removing filter membrane (23) in the first filter membrane tank (21) after centrifugation for a certain time, and mixing the sample and the diluent with the buffer solution in the mixing tank (31);

the step S3 is as follows: after stopping the first centrifugation, the mixed solution fills the first siphon flow passage; performing secondary centrifugation, enabling the mixed solution to enter a second filter membrane tank (41), enabling the mixed solution to pass through a biological molecular filter membrane in the second filter membrane tank (41) after being centrifuged for a certain time, and enabling the mixed solution to enter a liquid separation tank (51); the exosomes in the mixed solution are enriched on a biological molecular filtering membrane;

the step S4 is as follows: after stopping the second centrifugation, the mixed liquid is filled in the second siphon flow channel (52) preferentially, when the third siphon flow channel (53) is not filled, the third centrifugation is carried out, the mixed liquid enters a waste liquid quantitative structure in the filtrate collecting tank (81), and the redundant mixed liquid enters a waste liquid overflow tank;

The step S5 is as follows: adding a first eluent into a third liquid storage tank (61) of the chip, and adding a second eluent into a fourth liquid storage tank (71);

the step S6 is as follows: performing a fourth centrifugation, i.e. a low-speed centrifugation, the first eluent entering the second filter membrane tank (41), the second eluent being thrown away in the fourth reservoir (71); carrying out fifth centrifugation, namely high-speed centrifugation, wherein the first eluent enters a liquid separating tank through a biological molecular filtering membrane, and the second eluent breaks through a capillary valve (73) door and enters a transition tank (72);

the step S7 is as follows: after stopping the fifth centrifugation, the second eluent entering the transition channel (72) fills the fourth siphon flow channel (74); performing a sixth centrifugation, allowing the second eluent to enter a second filter membrane tank (41) and pass through a biological molecular filter membrane to enter a liquid separation tank (51), and allowing the first eluent and the second eluent to enter an extraction tank (91) together;

the step S8 is as follows: the extract containing exosomes is removed from the chip through an extraction tank (91).