1. A biomolecule extraction method is characterized by comprising the following steps:

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

s2, centrifuging for the first time, and enabling the sample and the diluent to enter a first filtering component (2) for filtering; and enters a buffer component (3) to be mixed with the buffer solution to form a mixed solution;

s3, stopping the first centrifugation; carrying out second centrifugation, and enabling the mixed solution to enter a second filtering component (4); the secretion of the mixed liquid is enriched in the second filtering component (4), and other substances enter the liquid separating component (5);

s4, stopping the second centrifugation; performing third centrifugation, and allowing other substances in the mixed solution to enter a filtrate collection assembly (8);

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

s6, carrying out fourth centrifugation; the first eluent enters the second filtering component (4), and the second eluent still remains in the second eluting component (7); after the fifth centrifugation, the first eluent enters a liquid separation component (5) through a second filtering component (4);

s7, stopping the fifth centrifugation, performing sixth centrifugation, enabling the second eluent to enter a second filtering assembly (4) and further enter a liquid separating assembly (5), and enabling the first eluent and the second eluent to enter an eluent collecting assembly (9);

and S8, extracting exosomes from the eluent collection assembly (9).

2. The method of extracting biomolecules according to claim 1,

the outlet end of the dilution component (1) is connected with a first filtering component (2), the outlet end of the first filtering component (2) is connected with a buffering component (3), the outlet end of the buffering component (3) is connected with a liquid separating component (5), the liquid separating component (5) is also connected with the outlet end of a second elution component (7), and the outlet ends of the liquid separating component (5) are respectively connected with a filtrate collecting component (8) and an eluent collecting component (9);

dilute subassembly (1), first filtering component (2), second filtering component (4), buffer component (3), first elution subassembly (6), second elution subassembly (7), divide liquid subassembly (5) and filtrating and collect subassembly (8) and eluent and collect subassembly (9) and be the annular structure.

3. The biomolecule extraction method according to claim 2, characterized in that the dilution unit (1) comprises: a first reservoir (11) for adding and storing a diluent; a sample tank (12) for adding and storing the sample, wherein the first liquid storage tank (11) and the outlet end of the sample tank (12) are connected with the first filter component (2).

4. The biomolecule extraction method according to claim 3, wherein the first reservoir (11) and the sample tank (12) are both annular structures and are adjacently disposed; an outlet is arranged at the 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).

5. The biomolecule extraction method according to claim 4, wherein a sample well (121) is provided at an upper end of the sample tank (12).

6. The biomolecule extraction method according to claim 4, characterized in that the first filter assembly (2) comprises: first filter membrane groove (21), first filter membrane groove (21) one end with dilute subassembly (1) turn-on connection, through-hole (22) have been seted up to first filter membrane groove (21) other end with buffering subassembly (3) are connected the tip that through-hole (22) are close to first filter membrane groove (21) is provided with miscellaneous filter membrane (23).

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

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

9. The biomolecule extraction method of claim 7, wherein the membrane (23) is an ultrafiltration membrane having a pore size of 0.22-0.03 μm.

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

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

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

13. The biomolecule extraction method according to claim 11, characterized in that 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 flow channel, and the outlet end of the second filter membrane groove (41) is connected with the liquid separation component (5);

and a biomolecule filtering membrane is arranged at the outlet end of the filtering membrane tank.

14. The method for extracting biomolecules according to claim 13, wherein the side wall of the outlet end of the second membrane groove (41) is inclined at an angle of 30-150 ° with respect to the bottom surface.

15. The method for extracting biomolecules according to claim 14, wherein the side wall of the outlet end of the second membrane groove (41) is inclined at an angle of 120 ° with respect to the bottom surface.

16. The biomolecule extraction method of any one of claims 13 to 15, wherein the width of the first siphon channel is 0.1 to 0.5mm and the depth of the first siphon channel is 0.1 to 0.5 mm.

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

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

19. The biomolecule extraction method of claim 18, wherein the third reservoir (61) has a depth of 3-5mm, the outlet channel has a width of 3-5mm, and the outlet channel has a depth equal to the depth of the third reservoir (61);

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

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

21. The biomolecule extraction method of claim 20, wherein the outlet end of the second filter unit (4) has a pore size of 0.5-3mm in diameter.

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

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

24. The method as claimed in claim 23, wherein the filtrate collection tank (81) is a quantitative waste liquid structure, and the quantitative size of the quantitative waste liquid structure is 100-300 μ l waste liquid.

25. The biomolecule extraction method according to any one of claims 20 to 22, wherein the eluent collection assembly (9) comprises: the liquid extracting device comprises an extracting groove (91), wherein a fourth siphon flow channel (74) is connected with one end of the extracting groove (91), and a liquid extracting hole (92) is formed in one end of the extracting groove (91).

26. The biomolecule extraction method according to claim 20, wherein two dilution units (1), two first filter units (2), two second filter units (4), two buffer units (3), two first elution units (6), two second elution units (7), two liquid separation units (5), two filtrate collection units (8), and two eluent collection units (9) are provided in a central symmetry manner.

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

the step S2 is: carrying out first centrifugation on the chip, enabling the sample and the diluent to enter a first filter membrane groove (21) together, enabling the buffer solution to enter a mixing groove (31), and after centrifuging for a certain time, enabling the sample and the diluent to pass through an impurity removal filter membrane (23) in the first filter membrane groove (21) and to be mixed with the buffer solution in the mixing groove (31);

the step S3 is: stopping the first centrifugation, and filling the first siphon flow channel with the mixed solution; carrying out second centrifugation, enabling the mixed solution to enter a second membrane filtering tank (41), and after the mixed solution is centrifuged for a certain time, enabling the mixed solution to pass through a biomolecule filtering membrane in the second membrane filtering tank (41) and enter a liquid separating tank (51); the exosome in the mixed solution is enriched on the biomolecule filtering membrane;

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

the step S5 is: 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: performing a fourth centrifugation, namely low-speed centrifugation, wherein the first eluent enters the second filter membrane tank (41), and the second eluent is thrown and stays in the fourth liquid storage tank (71); performing fifth centrifugation, namely high-speed centrifugation, wherein the first eluent enters the liquid separation pool through the biomolecular filtering membrane, and the second eluent breaks through a capillary valve (73) and enters a transition groove (72);

the step S7 is: after the fifth centrifugation is stopped, the second eluent entering the transition groove (72) fills the fourth siphon flow channel (74); carrying out sixth centrifugation, enabling the second eluent to enter a second filtering film groove (41) and penetrate through the biomolecular filtering film to enter a liquid separating groove (51), and enabling the first eluent and the second eluent to enter an extraction groove (91) together;

the step S8 is: the extract containing exosomes is taken out from the chip through an extraction tank (91).

Background

At present, centrifugal microfluidic chips are often applied to the field of POCT, and detection results can be conveniently and quickly output without the operation of professional technicians. The device 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 microfluid is driven by centrifugal force, so that the detection and analysis of a sample are realized. The centrifugal microfluidic chip can complete the operations of 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 applied to 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 intercellular communication pathways, and are classified into three categories, including exosomes, microvesicles, and apoptotic bodies, according to their size and occurrence. Wherein the exosome is a packaging vesicle with a diameter of about 40-100nm, is secreted by various cells, and contains specific proteins, lipids, cytokines or genetic materials. Exosomes derived from different tissues not only have their specific protein molecules, but also contain key molecules for their function. In recent years, with the continuous and deep research of exosome, the application of the exosome relates to the fields of tumor treatment, medical foundation and immunity, and parasite; clinical studies have been directed to the cardiovascular system, endocrine-metabolic system, and the like.

In the prior art, a commercial exosome extraction kit needs a large-scale refrigerated centrifuge, has multiple operation steps and long time consumption, needs manual operation of professional experimenters, and easily makes mistakes in the operation process, so that the problems of insufficient amount of extracted exosomes, insufficient exosome purity and the like are caused.

Disclosure of Invention

In view of the above, the main objective of the present invention is to provide a biomolecule extraction method with integrated chip with multiple micro valves and filter membranes, which solves the problems of complicated biomolecule extraction operation, insufficient amount of extracted exosomes, insufficient purity of extracted exosomes, etc. in human samples.

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

a biomolecule extraction method comprises the following steps:

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

s2, centrifuging for the first time, and enabling the sample and the diluent to enter a first filtering assembly for filtering; and enters a buffer component to be mixed with the buffer solution to form a mixed solution;

s3, stopping the first centrifugation; carrying out second centrifugation, and enabling the mixed solution to enter a second filtering component; the secretion of the mixed liquid is enriched in the second filtering component, and other substances enter the liquid separating component;

s4, stopping the second centrifugation; centrifuging for the third time, and allowing other substances in the mixed solution to enter a filtrate collecting assembly;

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

s6, carrying out fourth centrifugation; the first eluent enters the second filtering component, and the second eluent still remains in the second eluting component; accelerating the fifth centrifugation, and allowing the first eluent to enter a liquid separation component through a second filtering component;

s7, stopping the fifth centrifugation, performing sixth centrifugation, enabling the second eluent to enter a second filtering assembly and further enter a liquid separation assembly, and enabling the first eluent and the second eluent to enter an eluent collection assembly;

and S8, extracting exosomes from the eluent collection assembly.

The outlet end of the diluting component is connected with a first filtering component, the outlet end of the first filtering component is connected with a buffering component, the outlet end of the buffering component is connected with a liquid separating component, the liquid separating component is also connected with the outlet end of the second eluting component, and the outlet end of the liquid separating component is respectively connected with a filtrate collecting component and an eluent collecting component;

the diluting component, the first filtering component, the second filtering component, the buffering component, the first eluting component, the second eluting component, the liquid separating component, the filtrate collecting component and the eluent collecting component are all of annular groove structures.

In a preferred embodiment, the dilution assembly comprises: a first reservoir for adding and storing a diluent; 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 liquid storage tank and the sample tank are both annular structures and are adjacently arranged; and an outlet is formed at 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 groove is provided with a sample liquid adding hole

In a preferred embodiment, the first filter assembly comprises: the device comprises a first filter membrane groove, one end of the first filter membrane groove is in conduction connection with the diluting component, the other end of the first filter membrane groove is provided with a through hole which is connected with the buffering component, and an impurity removing filter membrane is arranged at the end part, close to the first filter membrane groove, of the through hole.

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

In a preferred embodiment, the membrane has a pore size of 0.22-0.03 μm.

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

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

In a preferred embodiment, the mixing tank has a depth of 1 to 5 mm.

In a preferred embodiment, the second filter assembly comprises: a second membrane filtering groove; the inlet end of the second filter membrane groove is connected with the outlet end of the buffer assembly through a first siphon flow channel, and the outlet end of the second filter membrane groove 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 groove is obliquely arranged and forms an included angle of 30-150 degrees with the bottom surface.

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

In a preferred embodiment, 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 0.5 mm.

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 liquid storage tank and a transition tank; the fourth liquid storage tank is communicated and connected with the transition tank through a capillary valve, and the outlet end of the transition tank is communicated and connected with the second filter membrane tank through a fourth siphon flow channel.

In a preferred embodiment, the third reservoir has a depth of 3-5mm, the outlet channel has a width of 3-5mm, and the outlet channel has a depth equal to the depth of the third reservoir;

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

In a preferred embodiment, the liquid separation assembly comprises: and the inlet end of the liquid separating tank is connected with the outlet end of the second filtering component, and 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 channel and the third siphon flow channel.

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

In a preferred embodiment, the second siphon flow channel has a lower hydrophilicity index than the third siphon flow channel.

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

In a preferred embodiment, the filtrate collection tank is a waste liquid quantitative structure, and the quantitative size of the waste liquid quantitative structure is 100-.

In a preferred embodiment, the eluent collection assembly includes: the extraction tank is connected with a fourth siphon flow channel at one end of the extraction tank, and a liquid taking hole is formed in one end of the extraction tank.

In a preferred embodiment, the dilution assembly, the first filtering assembly, the second filtering assembly, the buffer assembly, the first elution assembly, the second elution assembly, the liquid separation assembly, the filtrate collection assembly and the eluent collection assembly are respectively arranged in a central symmetry manner, and two components are arranged in the central symmetry manner.

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

the step S2 is: centrifuging the chip for the first time, enabling the sample and the diluent to enter a first membrane filtering groove together, enabling the buffer solution to enter a mixing groove, and after centrifuging for a certain time, enabling the sample and the diluent to pass through an impurity removal filter membrane in the first membrane filtering groove and be mixed with the buffer solution in the mixing groove;

the step S3 is: stopping the first centrifugation, and filling the first siphon flow channel with the mixed solution; carrying out second centrifugation, allowing the mixed solution to enter a second membrane filtering tank, and allowing the mixed solution to pass through a biomolecular filtration membrane in the second membrane filtering tank and enter a liquid separating tank after the mixed solution is centrifuged for a certain time; the exosome in the mixed solution is enriched on the biomolecule filtering membrane;

the step S4 is: after stopping the second centrifugation, preferentially filling the second siphon flow channel with the mixed liquid, carrying out third centrifugation when the third siphon flow channel is not filled, allowing the mixed liquid to enter a waste liquid quantitative structure in the filtrate collecting tank, and allowing the redundant mixed liquid to enter a waste liquid overflow tank;

the step S5 is: 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: performing a fourth centrifugation, namely low-speed centrifugation, wherein the first eluent enters the second filter membrane tank, and the second eluent is thrown into and stays in the fourth liquid storage tank; performing fifth centrifugation, namely high-speed centrifugation, wherein the first eluent enters the liquid separation pool through the biomolecular filter membrane, and the second eluent breaks through the capillary valve and enters the transition groove;

the step S7 is: stopping the fifth centrifugation, and filling the fourth siphon flow channel with the second eluent entering the transition groove; carrying out sixth centrifugation, enabling the second eluent to enter a second filtering film groove and penetrate through a biomolecular filtering film to enter a liquid separating groove, and enabling the first eluent and the second eluent to enter an extraction groove together;

the step S8 is: the extract containing exosomes was taken out 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 the dilution component is connected with the first filtering component, the outlet end of the first filtering component is connected with the buffering component, the outlet end of the buffering component is connected with the liquid separating component, the liquid separating component is also connected with the outlet end of the second elution component, and the outlet ends of the liquid separating component are respectively connected with the filtrate collecting component and the eluent collecting component; the diluting component, the first filtering component, the second filtering component, the buffering component, the first eluting component, the second eluting component, the liquid separating component, the filtrate collecting component and the eluent collecting component are all of annular groove structures.

The problems that a commercial exosome extraction kit in the prior art needs a large-scale refrigerated centrifuge, the operation steps are multiple, the time consumption is long, manual operation is needed by professional experimenters, errors are easy to occur in the operation process, the amount of extracted exosomes is insufficient, the exosome purity is insufficient 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 for exosome biomolecule extraction) disc matched instrument are required to be added to automatically complete sample dilution, mixing, filtration membrane, filtrate collection and eluent collection, and professional operators are not required. For example: pretreatment of plasma, enrichment of exosome biomolecules and elution of exosome biomolecules, and no professional operator is needed. The ultrafiltration membrane for filtering the sample and the exosome biomolecule enrichment filter membrane are integrated on the disc, and the usage amount of the filter membrane and the reagent is 1/10 of the traditional filter and a centrifugal column, so that the cost is low and the extraction efficiency is high. The sample for extracting the exosome biomolecule is 1/5 of the traditional method, and the blood sampling amount of a patient can be reduced. Cell debris and impurities in the sample are separated from the exosome biomolecule extracting solution, and the extracted exosome biomolecule has high purity. Exosome biomolecule extraction of 2 samples can be performed simultaneously. The extraction of the whole exosome biomolecule can be completed within 15 minutes within a time period required by the extraction of the whole exosome biomolecule. At present, a centrifugal microfluidic chip technology is not used for exosome biomolecule extraction of human blood samples. Wherein the biological molecules comprise all circulating tumor cells, exosomes, special proteins and the like which are enriched by the filter membrane.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

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

FIG. 2 is a schematic structural diagram of a chip flow channel structure of a biomolecule extraction method according to an embodiment of the present disclosure;

FIG. 3 is a sectional view of a filter built in a chip according to a biomolecule extraction method according to an embodiment of the present disclosure;

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

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

FIG. 6 is a schematic diagram of a first centrifugation of a chip, sample dilution, and removal of a hybridization filter and mixing with a buffer in a chip mixing tank according to an embodiment of the present disclosure;

FIG. 7 is a schematic diagram of a second centrifugation of the chip and a second separation of the mixed solution into a separation tank after passing through a biomolecule filtering membrane according to an embodiment of the present disclosure;

FIG. 8 is a diagram illustrating a quantitative structure of a third centrifugation step of a chip for mixing a liquid with a filtrate collection tank and filling the filtrate collection tank with the mixture, according to an embodiment of the present disclosure;

FIG. 9 is a schematic diagram of a first eluent and a second eluent being added to a chip of a biomolecule extraction method according to an embodiment of the present disclosure;

FIG. 10 is a schematic diagram of a fourth centrifugation of the chip with a low rotation rate of the first eluent entering the second membrane tank to incubate with the biomolecule filter membrane, the second eluent failing to break through the capillary valve, according to an embodiment of the present disclosure;

FIG. 11 is a schematic diagram of a fourth centrifugation of the chip with a first eluent passing through a biomolecular filter membrane into a liquid separation tank and a second eluent breaking through a capillary valve into a transition tank at a high rotation speed according to an embodiment of the present disclosure;

fig. 12 is a schematic view of a chip for a biomolecule extraction method according to an embodiment of the present disclosure, wherein an eluate is fifth centrifuged through a biomolecule filtering membrane and enters an extraction tank together with a first eluate.

[ description of main reference symbols ]

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 membrane filtration tank; 22. a through hole; 23. removing impurities and filtering the solution;

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

4. a second filter assembly; 41. a second membrane filtering groove; 42. a first siphon flow path;

5. a liquid separating component; 51. a liquid separating tank; 52. a second siphon flow channel; 53. a third siphon flow channel;

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 channel;

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

9. an eluent collection assembly; 91. an extraction tank; 92. and (6) liquid taking holes.

Detailed Description

The biomolecule extraction method of the present invention will be described in further detail with reference to the accompanying drawings and examples of the present invention.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

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 according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the application described herein are, for example, capable of operation in sequences other than those illustrated or otherwise 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 … …," "above … …," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial relationship 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 of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is turned over, devices described as "above" or "on" other devices or configurations would then be oriented "below" or "under" the other devices or configurations. Thus, the exemplary term "above … …" can include both an orientation of "above … …" and "below … …". The device may be otherwise variously oriented (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 includes the following steps:

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

s2, centrifuging for the first time, and enabling the sample and the diluent to enter the first filtering component 2 for filtering; and enters the buffer component 3 to be mixed with the buffer solution to form a mixed solution;

s3, stopping the first centrifugation; carrying out second centrifugation, and enabling the mixed solution to enter a second filtering component 4; the secretion of the mixed liquid is enriched in the second filtering component 4, and other substances enter the liquid separating component 5;

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

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

s6, carrying out fourth centrifugation; the first eluent enters the second filtering component 4, and the second eluent still remains in the second eluting component 7; after the fifth centrifugation, the first eluent enters the liquid separation component 5 through the second filtering component 4;

s7, stopping the fifth centrifugation, performing sixth centrifugation, enabling the second eluent to enter a second filtering component 4 and further enter a liquid separating component 5, and enabling the first eluent and the second eluent to enter an eluent collecting component 9;

and S8, extracting exosomes from the eluent collection assembly 9.

The outlet end of the dilution component 1 is connected with a first filtering component 2, the outlet end of the first filtering component 2 is connected with a buffering component 3, the outlet end of the buffering component 3 is connected with a liquid separating component 5, the liquid separating component 5 is also connected with the outlet end of a second elution component 7, and the outlet ends of the liquid separating component 5 are respectively connected with a filtrate collecting component 8 and an eluent collecting component 9;

dilute subassembly 1, first filter assembly 2, second filter assembly 4, buffering subassembly 3, first elution subassembly 6, second elution subassembly 7, divide liquid subassembly 5 and filtrating collection subassembly 8 and eluent collection subassembly 9 and be the annular structure.

In one embodiment:

the dilution assembly 1 comprises: a first reservoir 11 for adding and storing a diluent; a sample tank 12 for adding and storing a sample, and the first reservoir 11 and an outlet end of the sample tank 12 are connected to the first filter module 2.

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

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

The first filter assembly 2 comprises: first filter membrane groove 21, first filter membrane groove 21 one end with dilute 1 turn-on connection of subassembly, the first filter membrane groove 21 other end seted up through-hole 22 with buffer module 3 is connected the tip that through-hole 22 is close to first filter membrane groove 21 is provided with miscellaneous filter membrane 23.

The side wall of the through hole 22 close to the end part of the first filter membrane groove 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 of the first filter membrane groove 21 is inclined, 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 micron.

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

In one embodiment:

the cushion assembly 3 includes: a mixing tank 31, a second reservoir tank 32 for storing a buffer solution; the inlet end of the mixing tank 31 is in conduction connection with the outlet end of the first filtering component 2, and the outlet end of the second reservoir tank 32 is connected with the mixing tank 31.

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

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 flow channel 42, and the outlet end of the second filter membrane groove 41 is connected with the liquid separation component 5;

and a biomolecule filtering membrane is arranged at the outlet end of the filtering membrane tank.

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

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

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

The second elution assembly 7 comprises: a fourth reservoir 71, a transition tank 72; the fourth reservoir 71 and the transition tank 72 are in communication connection through a capillary valve 73, and an outlet end of the transition tank 72 is in communication connection with the second filter tank 41 through a fourth siphon flow passage 74.

The depth of the third liquid storage tank 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 tank 61;

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

In one embodiment:

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

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

The second siphon flow path 52 has a lower hydrophilic index than the third siphon flow path 53.

The filtrate collection assembly 8 comprises: the inlet end of the filtrate collecting tank 81 is connected with the second siphon flow channel 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-.

The eluent collection assembly 9 includes: and one end of the extraction groove 91 is connected with the fourth siphon flow channel 74, and one end of the extraction groove 91 is provided with a liquid taking hole 92.

In one embodiment:

the two dilution assemblies 1, the first filtering assemblies 2, the second filtering assemblies 4, the buffer assemblies 3, the first eluting assemblies 6, the second eluting assemblies 7, the liquid separating assemblies 5, the filtrate collecting assemblies 8 and the eluent collecting assemblies 9 are respectively arranged in a central symmetry mode.

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

the step S2 is: carrying out first centrifugation on the chip, enabling the sample and the diluent to enter a first membrane filtering groove 21 together, enabling the buffer solution to enter a mixing groove 31, and after centrifuging for a certain time, enabling the sample and the diluent to pass through an impurity removal filter membrane 23 in the first membrane filtering groove 21 and to be mixed with the buffer solution in the mixing groove 31;

the step S3 is: after the first centrifugation is stopped, the first siphon flow passage 42 is filled with the mixed liquid; performing second centrifugation, allowing the mixed solution to enter the second membrane filtering tank 41, and allowing the mixed solution to pass through a biomolecular filtration membrane in the second membrane filtering tank 41 and enter the liquid separating tank 51 after the mixed solution is centrifuged for a certain time; the exosome in the mixed solution is enriched on the biomolecule filtering membrane;

the step S4 is: after the second centrifugation is stopped, the mixed liquid is preferentially filled in the second siphon flow channel 52, 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: 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: performing a fourth centrifugation, i.e. low speed centrifugation, wherein the first eluent enters the second membrane filtration tank 41, and the second eluent is thrown and stays in the fourth liquid storage tank 71; performing fifth centrifugation, namely high-speed centrifugation, wherein the first eluent enters the liquid separation pool through the biomolecular filter membrane, and the second eluent breaks through the capillary valve 73 and enters the transition tank 72;

the step S7 is: after the fifth centrifugation is stopped, the second eluent entering the transition tank 72 fills the fourth siphon flow channel 74; performing sixth centrifugation, allowing the second eluent to enter the second membrane filtering tank 41 and penetrate through the biomolecular filtering membrane to enter the liquid separating tank 51, and allowing the first eluent and the second eluent to enter the extraction tank 91;

the step S8 is: the extract containing exosomes was taken out from the chip through the extraction tank 91.

Fig. 1 is a schematic diagram of a three-dimensional structure, 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 chip upper strata takes runner face down and the inseparable bonding of chip lower floor, and the chip can hold a plurality of detecting element, can carry out the exosome of a plurality of samples simultaneously and draw, has exemplified 2 detecting element in the picture, can carry out the exosome of 2 samples simultaneously and draw. Of course, the number of the grooves can be 3, 4, 5, etc.

Fig. 2 is a plan view of a single unit on a chip. Comprises the following steps: the device comprises a sample tank 12, a first reservoir tank 11, a second reservoir tank 32, a third reservoir tank 61, a fourth reservoir tank 71, a transition tank 72, a first filter membrane tank 21, a second filter membrane tank 41, a mixing tank 31, a liquid dividing tank 51, a filtrate collecting tank 81, an extraction tank 91, a first siphon flow channel 42, a second siphon flow channel 52, a third siphon flow channel 53, a fourth siphon flow channel 74, a capillary valve 73, a sample liquid adding hole 121 and a liquid taking hole 92. Wherein each siphon flow passage needs to be surface-hydrophilized, and particularly the second siphon flow passage 52 has a hydrophilicity index smaller than that of the third siphon flow passage 53.

FIG. 3 is a cross-sectional view of the filter membrane built in the chip, and the impurity removing filter membrane 23 is bonded to the side wall surface of the first filter membrane groove 21 by ultrasonic welding, laser welding or gluing, for example. The included angle between the side wall surface and the bottom surface of the first filter film groove 21 is 90-150 degrees, and the included angle is preferably 120 degrees in the figure. The first filter film groove 21 and the mixing groove 31 are connected through a through hole 22 penetrating through the side wall surface, and the diameter of the through hole 22 is 0.5-3mm, preferably 2 mm.

FIG. 4 is a schematic diagram showing the separation of chip waste liquid and extract liquid. Chip divides liquid structure includes: a second filter membrane tank 41, a liquid separating tank 51, a second siphon flow channel 52, a third siphon flow channel 53, a filtrate collecting tank 81 and an extraction tank 91. Wherein the filtrate collection tank 81 contains: waste liquid ration structure and waste liquid overflow tank. When the mixed solution (sample + diluent + buffer solution) enters the liquid separation tank 51 through the biomolecular filtration membrane in the second filtration membrane tank 41 to become waste liquid, the second siphon flow channel 52 is preferentially filled with the waste liquid because the hydrophilicity index of the second siphon flow channel 52 is smaller than that of the third siphon flow channel 53, and the third siphon flow channel 53 cannot be filled in a short time because of relatively poor hydrophilicity. After the centrifugation, the waste liquid enters the filtrate collecting tank 81 through the second siphon flow channel 52, and fills the waste liquid quantitative structure in the filtrate collecting tank 81, and the excess waste liquid enters the waste liquid overflow tank.

When the exosome enriched on the biomolecule filtering membrane is eluted by using the eluent, the eluent enters the liquid separation tank 51 through the biomolecule filtering membrane to become the extracting solution, and the air column in the second siphon flow channel 52 cannot be discharged because the liquid is filled in the waste liquid quantitative structure in the filtrate collecting tank 81 connected at the outlet of the second siphon flow channel 52, so that the second siphon flow channel 52 cannot be opened and becomes a normally closed valve. When the second siphon flow channel 52 is filled with the extracting solution, the extracting solution centrifuged again enters the extracting groove 91 through the third siphon flow channel 53, and the on-chip liquid separating structure completes the separation of the waste liquid and the extracting solution into two different grooves.

Specifically, the exosomes of the sample are extracted by the following steps:

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

As shown in FIG. 6, the chip was centrifuged for the first time at 7000rpm for 5min, and the sample and the diluent were mixed in the first membrane tank 21 and introduced into the mixing tank 31 through the impurity removing filter 23. After the diluted sample passes through the impurity removing filter 23, impurities and impurity proteins in the sample are filtered and mixed with a buffer solution in the mixing tank 31.

As shown in FIG. 7, after the first siphon channel 42 was filled with the mixture (sample + diluent + buffer), the mixture was centrifuged again at 3400rpm for 1 min. The mixed liquid passes through the biomolecule filtering membrane and enters the liquid separating tank 51 to become waste liquid, and the exosome in the mixed liquid is enriched on the biomolecule filtering membrane.

As shown in FIG. 8, due to the function of the chip liquid-separating structure (the specific principle is described in detail in FIG. 4), centrifugation is performed 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 excess waste liquid enters the waste liquid overflow tank.

As shown in FIG. 9, the first eluent is added into the third reservoir 61 and the second eluent is added into the fourth reservoir 71 on the chip to prepare for eluting the exosomes enriched on the biomolecule filtering membrane.

As shown in FIG. 10, the chip was centrifuged at 300rpm for 1min at a low rotation speed. Because no valve is arranged at the outlet of the third liquid storage tank 61, the eluent enters the second filtering membrane tank 41 at a low rotating speed but cannot pass through the biomolecular filtering membrane, so that the first eluent is in full contact with exosomes on the biomolecular filtering membrane for a period of time to play a role in incubation. And a capillary valve 73 is arranged at the outlet of the fourth liquid storage tank 71, the width of the capillary valve 73 is 0.1-0.3mm, the depth of the capillary valve 73 is 0.1-0.3mm, the preferred width is 0.2mm, and the depth of the capillary valve is 0.1mm, so that the eluent 2 can not break through the capillary valve 73 at a low rotating speed and still stays in the fourth liquid storage tank.

As shown in FIG. 11, after the chip was centrifuged at a low speed for 1min, the speed was immediately increased to 3400rpm, and the chip was centrifuged for 1 min. Most of the exosomes on the biomolecule filtering membrane are eluted by the first eluent, then the first eluent passes through the biomolecule filtering membrane and enters the liquid separation pool to form extracting solution, the second eluent breaks through the capillary valve 73 and enters the transition groove 72, after the centrifugation is stopped, the third siphon flow channel 53 is filled with the first eluent, and the fourth siphon flow channel 74 is filled with the second eluent.

As shown in fig. 12, the chip is centrifuged at 8800rpm for 1min, the second eluent passes through the biomolecule filtering membrane to elute the remaining exosomes on the biomolecule filtering membrane, the first eluent and the second eluent enter the extracting 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-taking hole 92 at the upper end of the extracting tank 91 for subsequent analysis and detection.

When the centrifugal microfluidic chip disclosed by the invention is used, an operator only needs to add corresponding samples and reagents, and the disk automatically finishes the following steps:

1. pre-treating a sample, namely diluting the sample, diluting the diluted sample by an ultrafiltration membrane, removing cell debris and impurities, and mixing the filtered sample with a buffer solution;

2. exosome enrichment, comprising enriching exosomes on a filter membrane through which waste fluid in a sample passes;

3. and (3) eluting the exosomes, wherein the elution comprises incubation of eluent and a filter membrane, the exosomes on the filter membrane are fully eluted, and the exosomes on the filter membrane are completely eluted by multiple times of elution, and the eluent is not contacted with waste liquid, so that the purity of the extracted exosomes is high.

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

The commercialized exosome extraction kit needs a large-scale refrigerated centrifuge, has multiple operation steps and long time consumption, needs manual operation of professional experimenters, and easily makes mistakes in the operation process, so that the problems of insufficient amount of extracted exosomes, insufficient exosome purity and the like are caused.

In order to solve the problems of complex operation, insufficient amount of extracted exosomes, insufficient purity of extracted exosomes and the like in human plasma, the invention provides an exosome extraction centrifugal microfluidic chip which integrates various micro valves and filter membranes, the whole extraction process can be finished within 15Min, large-scale freezing and centrifuging equipment is not needed, professional experimenters are not needed for manual operation, the whole extraction process is highly automated, and the repeatability is good.

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

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention.