Fully integrated centrifugal microfluidic chip for nucleic acid detection, and nucleic acid detection method therefor

Abstract

Provided in the present application are a fully integrated centrifugal microfluidic chip for nucleic acid detection, and a nucleic acid detection method therefor. The microfluidic chip of the present application is specifically provided with a plurality of washing-solution distribution reservoirs. Compared with conventional techniques in which a washing solution is instantaneously transferred from a nucleic acid capture reservoir during centrifugation, the microfluidic chip of the present application cleverly uses centrifugal forces and is designed for graded cleaning in a small volume manner multiple times, thereby enhancing the cleaning efficiency and avoiding the inhibitory effect of lysis buffer and sample matrix on amplification. Moreover, the microfluidic chip of the present application integrates the functions of sample lysis, nucleic acid purification and amplification detection, and only a sample needs to be added to the chip; and the entire analysis process is completed within the fully sealed chip. The microfluidic chip of the present application features a simple structure and process, enables parallel multiple-sample multiple-indicator testing, and is especially suitable for the field of in-vitro diagnostic POCT.

Description

Centrifugal full-integrated nucleic acid detection microfluidic chip and nucleic acid detection method thereof TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro diagnosis, and particularly relates to a centrifugal full-integrated nucleic acid detection microfluidic chip and a nucleic acid detection method thereof. BACKGROUND

[0002] Molecular diagnosis is the only in vitro diagnostic method that can diagnose, prevent and customize treatment plans for diseases in the early stage. Nucleic acid detection based on polymerase chain reaction (PCR) as the gold standard of molecular diagnosis has important value in clinical practice. Traditional PCR reaction needs to be completed in a professional partition laboratory to avoid aerosol pollution, and is strongly dependent on professional equipment and personnel, with extremely high cost and maintenance cost.

[0003] Microfluidic technology has the ability to integrate all functions such as sample pretreatment, reaction and detection on a chip with a size of centimeter. In recent years, molecular POCT (Point-of-Care Testing) chips based on microfluidic technology can effectively solve the above-mentioned problems of PCR.

[0004] However, the nucleic acid detection process involves complex liquid manipulation steps such as sample lysis, nucleic acid extraction and purification, amplification and detection, and needs to be truly completed in a fully sealed chip environment to avoid aerosol pollution. Most current technical solutions use pressure-driven cartridge microfluidic chips to achieve this, integrating multiple micropumps and microvalves to sequentially control the fluid. The chip has a complex structure, high cost of supporting peripheral equipment and poor stability.

[0005] Centrifugal-driven microfluidic chips are simpler than pressure-driven ones. By centrifugal direction and speed, fluid can be manipulated in different areas of the chip, and has the advantage of simple assembly structure. However, because it is not convenient to synchronize mechanical valve control during the centrifugation process, the selection of valves for fluid control on the centrifugal chip is limited, which directly increases the difficulty of applying such chips to nucleic acid detection and becomes a technical difficulty.

[0006] Patent CN118185746A discloses a full-integrated microfluidic nucleic acid analysis chip and a nucleic acid analysis method, which integrates nucleic acid detection steps on a centrifugal microfluidic chip. However, the mixing of samples and lysis solution needs to be manually mixed outside the chip and then added. The liquid reagent is in a hollow liquid pool formed in the chip. This kind of reagent liquid pool is interconnected and cannot play a storage role. It still needs to be manually added before use. It does not truly meet the on-site analysis needs of "sample in-result out".

[0007] Patent CN118048231A discloses a centrifugal nucleic acid detection microfluidic chip, the internal design structure of the whole centrifugal microfluidic chip is complex, but the front and rear liquid pools of the detection process are all controlled by siphon valves, the siphon valves are affected by the rotation speed, the properties of the liquid and the properties of the chip surface, and the patent only relies on the precise control of all processes by the four-stage siphon valve, which is difficult to achieve in essence. In addition, the nucleic acid extraction and purification process of the sample has a great influence on the result accuracy, but the chip structure in the patent has not integrated this part of the function even if it is complex.

[0008] Patent CN118146930A discloses a single-disk full-extraction chip based on centrifugal microfluidics, which pre-embeds liquid reagents required in the nucleic acid detection process in the form of a liquid capsule, and integrates the nucleic acid extraction and purification process, but after the reaction reagents of each step are released from the liquid capsule, they directly pass through the buffer cavity and the silica gel membrane area into the waste liquid pool under the action of centrifugation. On the one hand, the reagents and the sample do not have a sufficient mixing time, and on the other hand, the transfer of the liquid is completed instantaneously during the centrifugation process, and it is difficult to ensure that the washing liquid or the eluent fully washes the nucleic acid on the silica membrane in this short period of time.

[0009] In summary, it is necessary to develop a new type of centrifugal full-integrated nucleic acid detection microfluidic chip and a nucleic acid detection method thereof, which focuses on adding multiple washing liquid distribution pools to realize staged washing and further reduce the influence of reagent or impurity residues in the extraction process on the detection results. SUMMARY

[0010] The present application provides a centrifugal full-integrated nucleic acid detection microfluidic chip and a nucleic acid detection method thereof. The microfluidic chip described in the present application focuses on adding multiple washing liquid distribution pools. Compared with the conventional technology in which the washing liquid is instantaneously transferred from the nucleic acid capture pool during centrifugation, the microfluidic chip described in the present application ingeniously utilizes the centrifugal force, is designed for staged washing in small amounts and multiple times, enhances the washing efficiency, and avoids the inhibitory effect of the lysis solution and the sample matrix on amplification. Moreover, the microfluidic chip described in the present application integrates the functions of sample lysis, nucleic acid purification, and amplification detection, and only needs to add the sample to the chip to complete the above-mentioned analysis full process in the fully sealed chip. The microfluidic chip structure and process described in the present application are simple, can detect multiple samples and multiple indicators in parallel, and are particularly suitable for the field of point-of-care testing (POCT).

[0011] In a first aspect, the present application provides a centrifugal full-integrated nucleic acid detection microfluidic chip, which comprises a reagent layer located on the upper layer and a structure layer located on the lower layer, and the upper and lower layers are arranged around the same centrifugal shaft and are tightly combined.

[0012] The reagent layer comprises at least one set of liquid capsule reagent assembly, the liquid capsule reagent assembly comprises a lysis solution liquid capsule, a washing liquid liquid capsule and an elution liquid liquid capsule, and the reagent layer is also provided with a sample adding tube, a gas hole is provided beside the sample adding tube to ensure that the fluid flows smoothly during the sample adding process.

[0013] The structure layer comprises at least one set of sample lysis functional modules, nucleic acid extraction and purification functional modules and amplification functional modules which are distributed along the radial direction in sequence; the sample lysis functional module comprises a sample pool, a lysis liquid pool and a mixing pool, and the sample pool and the lysis liquid pool are independently communicated with the mixing pool; the nucleic acid extraction and purification functional module comprises a nucleic acid capture pool, an eluent pool, a total cleaning liquid pool, a sorting pool, a buffer pool and an extraction waste liquid pool, the mixing pool is communicated with the nucleic acid capture pool, and the eluent pool is communicated with the nucleic acid capture pool; the total cleaning liquid pool is communicated with at least two cleaning liquid distribution pools respectively, the at least two cleaning liquid distribution pools are distributed along the radial direction in sequence and are communicated with each other, and the cleaning liquid distribution pool which is distributed farthest along the radial direction is communicated with the nucleic acid capture pool; the nucleic acid capture pool is communicated with the sorting pool, and the left and right sides of the sorting pool are respectively communicated with the extraction waste liquid pool and the buffer pool; the amplification functional module is communicated with the buffer pool, and is used for performing quantitative and amplification reactions.

[0014] The sample adding tube, the lysis liquid capsule, the cleaning liquid capsule and the eluent capsule located in the upper reagent layer are respectively corresponded with the sample pool, the lysis liquid pool, the total cleaning liquid pool and the eluent pool located in the lower structure layer, so that the sample liquid in the sample adding tube, the lysis liquid in the lysis liquid capsule, the cleaning liquid in the cleaning liquid capsule and the eluent in the eluent capsule can enter the corresponding liquid pools respectively.

[0015] The microfluidic chip described in the application highlights the addition of a plurality of cleaning liquid distribution pools. Compared with the conventional technology in which the cleaning liquid is instantaneously transferred from the nucleic acid capture pool during centrifugation, the microfluidic chip described in the application ingeniously utilizes the centrifugal force to design a small number of multiple-stage cleaning, thereby enhancing the cleaning efficiency and avoiding the inhibitory effect of the lysis liquid and the sample matrix on amplification. Moreover, the microfluidic chip described in the application integrates the functions of sample lysis, nucleic acid purification and amplification detection, and only needs to add a sample to the chip to complete the above-mentioned analysis process in a fully sealed chip. The microfluidic chip described in the application has simple structure and process, can perform parallel multi-sample and multi-index detection, and is particularly suitable for the field of point-of-care testing (POCT).

[0016] It should be noted that, in order to realize parallel multi-sample and multi-index detection, two or more sets of liquid capsule reagent assemblies can be distributed symmetrically along the radial direction on the reagent layer, and correspondingly, two or more sets of sample lysis functional modules, nucleic acid extraction and purification functional modules and amplification functional modules which are distributed along the radial direction in sequence can be provided on the structure layer.

[0017] It should be noted that in order to realize that the upper layer and the lower layer are arranged around the same centrifugal shaft and are closely combined, the reagent layer and the structure layer are respectively provided with a central rotating ring and a positioning clamping groove; the sample adding tube is often designed adjacent to the liquid capsule reagent assembly to reduce the reagent flow path; if not otherwise specified in the present application, the structure layer is connected in communication through micro-channels.

[0018] As a preferred technical solution of the present application, the amplification function module comprises a quantitative pool, an amplification pool and an amplification waste pool; at least one quantitative pool is arranged in the circumferential direction and is in communication with each other; the quantitative pool farthest from the buffer pool is in communication with the amplification waste pool; the quantitative pool and the amplification pool correspond one by one and are distributed in the radial direction in sequence;

[0019] The mixing pool and the nucleic acid capture pool are in communication through a first siphon valve; the quantitative pool closest to the buffer pool is in communication with the buffer pool through a second siphon valve; the quantitative pool and the amplification pool are in communication through a flow resistance valve, the flow resistance valve comprises an inlet channel, a breakthrough valve port and an overflow channel, the inlet channel is located at the bottom of the quantitative pool and makes the bottom of the quantitative pool in communication with the breakthrough valve port, the overflow channel is located at the top of the amplification pool and makes the top of the amplification pool in communication with the breakthrough valve port, and there is gas in the breakthrough valve port.

[0020] The flow resistance valve structure commonly used in the prior art centrifugal chip is a flow channel with a small cross section, but on the outside of the large radius circumference, it is difficult to block the liquid from being released too early even under low speed conditions. A gas chamber, i.e. a breakthrough valve port, is designed in the middle of the flow resistance valve in the present application, which adopts a top-in and bottom-out form. Only under the condition of breaking through the air resistance of the gas chamber at high speed, the fluid can pass through the inlet channel, the breakthrough valve port and the overflow channel of the flow resistance valve in sequence and enter the corresponding amplification pool. That is, the flow resistance valve of the present application can effectively block.

[0021] As a preferred technical solution of the present application, the structure layer further comprises at least one gas buffer pool, the gas buffer pool is in communication with the extraction waste liquid pool, at least two cleaning liquid distribution pools and the amplification waste pool through a gas channel, and the mixing pool is in communication with the extraction waste liquid pool through a gas channel, so that gas-liquid circulation is realized in the whole chip.

[0022] It should be noted that the structure layer of the present application further comprises at least one gas buffer pool, which is arranged closest to the central region. Through the arrangement of the gas channel, all liquid pools are in communication with the gas buffer pool, so that the gas pressure balance can be realized during the transfer of liquid between different liquid pools, the influence of positive pressure or negative pressure on flow control is prevented, and precise control of fluid is realized in a sealed internal environment.

[0023] As a preferred technical solution of the present application, a switching valve is arranged at the communication position of the nucleic acid capture pool and the sorting pool, and is arranged inside the sorting pool. The switching valve can switch between the following two states:

[0024] State 1: The nucleic acid capture pool is communicated with the extraction waste liquid pool through the sorting pool.

[0025] State 2: The nucleic acid capture pool is communicated with the buffer pool through the sorting pool.

[0026] Optionally, the nucleic acid capture pool is a silicon membrane pool provided with a silica gel membrane.

[0027] It should be noted that if no switching valve is arranged inside the sorting pool, the sorting pool can be designed to have a structure with left and right branch chambers, and the left and right branch chambers are respectively communicated with the extraction waste liquid pool and the buffer pool. By adjusting the forward / reverse centrifugal rotation, the state switching of the nucleic acid capture pool being communicated with the extraction waste liquid pool and the buffer pool through the sorting pool can also be achieved.

[0028] As a preferred technical solution of the present application, the lysing solution pool, the total washing liquid pool and the eluent pool are respectively provided with a liquid capsule puncture needle, so that the sealed aluminum foil of the upper layer liquid capsule can be punctured and the internal reagent can be released under the pressing action.

[0029] As a preferred technical solution of the present application, the extraction waste liquid pool is provided with a water-absorbing material, such as water-absorbing cotton, etc., for increasing the liquid containing volume and preventing the backflow of waste liquid.

[0030] In the second aspect, the present application provides a nucleic acid detection method based on the centrifugal full-integrated nucleic acid detection microfluidic chip of the first aspect, the lysing solution capsule is encapsulated with a lysing solution, the washing liquid capsule is encapsulated with a washing liquid, the eluent capsule is encapsulated with an eluent, and the buffer pool is pre-embedded with a freeze-dried enzyme ball.

[0031] The nucleic acid detection method comprises the following steps:

[0032] Step S1: Injecting a sample liquid into the sample pool of the microfluidic chip through a sample adding tube, and sealing the air hole beside the sample adding tube, so that the microfluidic chip and the sample adding tube form a fully closed internal environment.

[0033] Step S2: Lysis:

[0034] The microfluidic chip is centrifuged to make the residual sample liquid in the sample tube completely transferred to the sample pool; the lysis liquid bag is pressed to make the lysis liquid released into the lysis liquid pool, the microfluidic chip is centrifuged, the sample liquid and the lysis liquid are transferred to the mixing pool for sufficient mixing and lysis, the liquid after lysis is introduced into the nucleic acid capture pool to realize nucleic acid capture; the microfluidic chip is centrifuged, and the liquid after nucleic acid capture is introduced from the nucleic acid capture pool into the extraction waste liquid pool through one side of the sorting pool;

[0035] Step S3: fractional cleaning:

[0036] The cleaning liquid bag is pressed to make the cleaning liquid released into the total cleaning liquid pool, and under the extrusion pressure of the liquid bag, the cleaning liquid is introduced into at least two cleaning liquid distribution pools respectively, the microfluidic chip is centrifuged, and the cleaning liquid in the at least two cleaning liquid distribution pools is introduced into the nucleic acid capture pool in a sequence from far to near according to the distance from the center, the fractional cleaning is completed, and the waste liquid after cleaning is introduced from the nucleic acid capture pool into the extraction waste liquid pool through one side of the sorting pool;

[0037] Step S4: elution:

[0038] The elution liquid bag is pressed to make the elution liquid released into the elution liquid pool; the microfluidic chip is centrifuged in a reverse direction opposite to the fractional cleaning, the elution liquid elutes the captured nucleic acid from the nucleic acid capture pool, and the eluted nucleic acid is introduced from the nucleic acid capture pool into the buffer pool through the other side of the sorting pool and mixed with the freeze-dried enzyme balls in the buffer pool;

[0039] Step S5: amplification:

[0040] The microfluidic chip is centrifuged in a direction opposite to the fractional cleaning, so that the liquid after mixing and reconstitution is introduced into the amplification functional module to perform quantitative and amplification reactions.

[0041] The nucleic acid detection method described in the application adopts the aforementioned centrifugal full-integrated nucleic acid detection microfluidic chip to perform the nucleic acid detection method, which belongs to non-medical diagnosis purposes, the chip environment is completely sealed after adding the sample to be detected, there is no zero aerosol pollution problem, one chip can detect multiple samples at the same time, all reagents for nucleic acid extraction and purification are integrated, and the entire analysis process is completed by driving only the centrifugal force; in particular, based on the structural design of the multiple cleaning liquid distribution pools, fractional cleaning of the cleaning liquid can be realized in a small amount and multiple times, the cleaning efficiency is enhanced, and the inhibition effect of the lysis liquid and the sample matrix on amplification is avoided.

[0042] As a preferred technical solution of the application, in step S2, after lysis is completed, the centrifugal rotation of the microfluidic chip is stopped, and under the capillary action, the liquid after lysis breaks through the first siphon valve and enters the nucleic acid capture pool from the mixing pool;

[0043] In step S5, after the re-dissolving mixing is completed, the centrifugal rotation of the microfluidic chip is stopped, and under the capillary action, the liquid after the re-dissolving mixing breaks through the second siphon valve from the buffer pool to the quantitative pool of the amplification functional module;

[0044] In the opposite direction of the hierarchical washing, the microfluidic chip is centrifugally rotated, and the eluted sample is distributed into at least one quantitative pool, and the excess eluted sample enters the amplification waste pool; the high-speed centrifugal breaks through the gas resistance of the break valve port of the flow resistance valve, so that the eluted sample in each quantitative pool sequentially passes through the inlet channel, the break valve port and the overflow channel of the flow resistance valve, and enters the corresponding amplification pool to complete the quantitative and amplification reaction.

[0045] As a preferred technical solution of the present application, in step S2, the sample liquid and the lysis liquid are transferred and mixed in the mixing pool by the ultrasonic module and / or forward and reverse reciprocating centrifugation of the instrument.

[0046] As a preferred technical solution of the present application, in step S4, the eluted nucleic acid is re-dissolved and mixed with the freeze-dried enzyme ball in the buffer pool by forward and reverse reciprocating centrifugation.

[0047] Compared with the prior art, the present application has at least the following beneficial effects:

[0048] (1) The microfluidic chip described in the present application integrates sample lysis, nucleic acid purification and amplification detection functions, only needs to add a sample to the chip, and completes the above-mentioned analysis whole process in a fully sealed chip; the microfluidic chip structure and process described in the present application are simple, can detect multiple samples and multiple indexes in parallel, and is particularly suitable for the field of point-of-care testing (POCT);

[0049] (2) The washing liquid is released step by step: the microfluidic chip described in the present application highlights the addition of multiple washing liquid distribution pools, and compared with the conventional technology in which the washing liquid is transferred from the nucleic acid capture pool instantaneously in the centrifugal process, the microfluidic chip described in the present application ingeniously utilizes the centrifugal force, is designed to be a small amount of multiple hierarchical washing, enhances the washing efficiency, and avoids the inhibitory effect of the lysis liquid and the sample matrix on amplification;

[0050] (3) Internal gas-liquid circulation design of the chip: the microfluidic chip described in the present application is designed with at least one gas buffer pool in the structure layer, and is arranged closest to the center region; through the arrangement of the gas channel, all liquid pools are connected with the gas buffer pool, so that the gas pressure balance can be realized during the transfer of the liquid between different liquid pools, the influence of positive pressure or negative pressure on the flow control is prevented, and the precise control of the fluid is realized in the sealed internal environment;

[0051] (4) High-efficiency flow resistance valve design: the flow resistance valve structure commonly used in the prior art centrifugal chip is a flow channel with a small cross section, but on the outside of the large radius circumference, it is difficult to block the liquid from releasing too early, even under low speed conditions. The application designs a gas chamber in the middle of the flow resistance valve, i.e. a breakthrough valve port, adopts a form of going in from the bottom and out from the top, and only under the condition of breaking through the air resistance of the gas-filled chamber at high speed, the fluid can sequentially pass through the inlet channel, the breakthrough valve port and the overflow channel of the flow resistance valve, and enter the corresponding amplification pool, i.e. the flow resistance valve described in the application can effectively block. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a structural schematic diagram of the centrifugal full-integrated nucleic acid detection microfluidic chip according to Embodiment 1 of the application;

[0053] Figure 2 is an exploded view of the structure of Figure 1;

[0054] Figure 3 is a schematic diagram of the reagent layer in Figure 1;

[0055] Figure 4 is a schematic diagram of the structure layer in Figure 1;

[0056] Figure 5 is a structural schematic diagram of the flow resistance valve according to Embodiment 1 of the application;

[0057] Figure 6 is a structural schematic diagram of the centrifugal full-integrated nucleic acid detection microfluidic chip with a switching valve according to Embodiment 2 of the application;

[0058] Figure 7 is a schematic diagram of the structure layer in the centrifugal full-integrated nucleic acid detection microfluidic chip according to Embodiment 3 of the application;

[0059] Figure 8 is a comparison diagram of the point pigment dry point characterization of the cleaning effect on the silicon membrane in the nucleic acid capture pool according to the application;

[0060] Figure 9 is a comparison diagram of the liquid interception effect on the quantitative pool according to the application;

[0061] In the figure: 1-reagent layer; 2-structure layer; 11-rotation ring; 12-positioning clamping groove; 13-sample loading tube; 14-air hole; 15-lysis liquid capsule; 16-elution liquid capsule; 17-cleaning liquid capsule; 21-gas buffer pool; 22-sample pool; 23-lysis liquid pool; 24-total cleaning liquid pool; 25-liquid capsule puncture needle; 26-elution liquid pool; 27-mixing pool; 28-extraction waste liquid pool; 29-cleaning liquid distribution pool; 30-first siphon valve; 31-second siphon valve; 32-nucleic acid capture pool; 33-sorting pool; 34-buffer pool; 35-quantitative pool; 36-flow resistance valve; 37-amplification pool; 38-switching valve; 39-amplification waste liquid pool; 291-first distribution pool flow channel; 292-second distribution pool flow channel; 293-third distribution pool flow channel; 361-inlet channel; 362-breakthrough valve port; 363-overflow channel. DETAILED DESCRIPTION

[0062] The technical solutions of the present application are further illustrated below in conjunction with the accompanying drawings and through specific embodiments.

[0063] To better illustrate the present application and facilitate the understanding of the technical solutions of the present application, the typical but non-limiting embodiments of the present application are as follows:

[0064] Embodiment 1

[0065] The present embodiment provides a centrifugal full-integrated nucleic acid detection microfluidic chip, as shown in FIGS. 1-4, which comprises a reagent layer 1 located at the upper layer and a structure layer 2 located at the lower layer, and the two layers are arranged around the same centrifugal shaft and are tightly combined; the sample loading layer 1 is composed of a circular plastic disc and an assembly thereon, and a rotating ring 11 is distributed at the center, two positioning clamping grooves 12 are symmetrically distributed on both sides of the rotating ring 11, and the rotating ring 11 is matched and fixed with the centrifugal shaft of the peripheral equipment; the structure layer 2 comprises all fluid functional structures in the nucleic acid detection process, and a rotating ring 11 of the same size as the rotating ring 11 in the reagent layer 1 is distributed at the center, two positioning clamping grooves 12 are symmetrically distributed on both sides of the rotating ring 11, and the rotating ring 11 is matched and fixed with the centrifugal shaft of the peripheral equipment;

[0066] The reagent layer 1 comprises two groups of symmetrically distributed liquid capsule reagent assemblies, the liquid capsule reagent assemblies comprise a lysis liquid capsule 15, a washing liquid capsule 17 and an elution liquid capsule 16; a sample loading tube 13 is also provided near the center of the reagent layer 1, and a gas hole 14 is provided beside the sample loading tube 13 to ensure that the fluid flows smoothly during the sample loading process;

[0067] The structure layer 2 comprises two groups of sample lysis functional modules, nucleic acid extraction and purification functional modules and amplification functional modules distributed along the radial direction in sequence; the sample lysis functional module comprises a sample pool 22, a lysis liquid pool 23 and a mixing pool 27, the sample pool 22 and the lysis liquid pool 23 are independently communicated with the mixing pool 27; the nucleic acid extraction and purification functional module comprises a nucleic acid capture pool 32, an eluent pool 26, a total cleaning liquid pool 24, a sorting pool 33, a buffer pool 34 and an extraction waste liquid pool 28, the mixing pool 27 is communicated with the nucleic acid capture pool 32, and the eluent pool 26 is communicated with the nucleic acid capture pool 32; the total cleaning liquid pool 24 is communicated with three cleaning liquid distribution pools 29 respectively, the three cleaning liquid distribution pools 29 are distributed along the radial direction in sequence and are communicated with each other, and the cleaning liquid distribution pool 29 farthest from the radial direction is communicated with the nucleic acid capture pool 32; the nucleic acid capture pool 32 is communicated with the sorting pool 33, the sorting pool 33 is designed to have a structure with two branch cavities on the left and right, and the two branch cavities are communicated with the extraction waste liquid pool 28 and the buffer pool 34 respectively; the amplification functional module is located at the outermost periphery of the chip and comprises eight quantitative pools 35, eight amplification pools 37 and an amplification waste liquid pool 39; the eight quantitative pools 35 are arranged along the circumferential direction and are communicated with each other; the quantitative pool 35 farthest from the buffer pool 34 is communicated with the amplification waste liquid pool 39; the quantitative pool 35 is corresponding to the amplification pool 37 and is distributed along the radial direction in sequence; the amplification pool 37 is pre-embedded with probe primers of different indexes, and combined with the multi-color fluorescence channel of the peripheral instrument, the multi-index parallel detection can be realized; and according to the detection requirement, the quantitative pool 35 and the amplification pool 37 can be designed in different numbers.

[0068] The mixing pool 27 is communicated with the nucleic acid capture pool 32 through a first siphon valve 30; the quantitative pool 35 closest to the buffer pool 34 is communicated with the buffer pool 34 through a second siphon valve 31; the quantitative pool 35 is communicated with the amplification pool 37 through a flow resistance valve 36, as shown in FIG. 5, the flow resistance valve 36 comprises an inlet channel 361, a breakthrough valve port 362 and an overflow channel 363, the inlet channel 361 is located at the bottom of the quantitative pool 35 and makes the bottom of the quantitative pool 35 communicated with the breakthrough valve port 362, the overflow channel 363 is located at the top of the amplification pool 37 and makes the top of the amplification pool 37 communicated with the breakthrough valve port 362, and there is gas in the breakthrough valve port 362;

[0069] The structure layer 2 is provided with two gas buffer pools 21 close to the center of the circle, the gas buffer pools 21 are communicated with the extraction waste liquid pool 28, the three cleaning liquid distribution pools 29 and the amplification waste liquid pool 39 through gas channels respectively, and the mixing pool 27 is communicated with the extraction waste liquid pool 28 through a gas channel, so that the gas-liquid circulation is realized in the whole chip and the internal gas-liquid balance is formed.

[0070] The sample tube 13, the lysis liquid bag 15, the washing liquid bag 17 and the elution liquid bag 16 located in the upper reagent layer 1 correspond to the sample pool 22, the lysis liquid pool 23, the total washing liquid pool 24 and the elution liquid pool 26 located in the lower structural layer 2 respectively. Three liquid bag puncture needles 25 are arranged in the lysis liquid pool 23, the total washing liquid pool 24 and the elution liquid pool 26 respectively, so that the sealed aluminum foil of the upper liquid bag can be punctured and the internal reagent can be released under the pressing action. The extraction waste liquid pool 28 is provided with water-absorbing cotton for increasing the liquid containing volume and preventing backflow of waste liquid. The nucleic acid capture pool 32 is a silicon membrane pool provided with a silicon gel membrane.

[0071] Application Example 1

[0072] The application example provides a nucleic acid detection method based on the centrifugal full-integrated nucleic acid detection microfluidic chip of the application example 1. The lysis liquid bag 15 is packaged with lysis liquid, the washing liquid bag 17 is packaged with washing liquid, the elution liquid bag 16 is packaged with elution liquid, and the buffer pool 34 is pre-embedded with freeze-dried enzyme balls.

[0073] The nucleic acid detection method comprises the following steps:

[0074] The sample tube 13 containing the sample liquid is inserted into the microfluidic chip, the liquid sample in the sample tube 13 is squeezed into the sample pool 22, and the air hole 14 beside the sample tube 13 is sealed, so that the microfluidic chip and the sample tube 13 form a fully closed internal environment. Then the whole is put into a matching instrument, the parameters are set, and one-key operation is started. The following actions are automatically completed on the instrument:

[0075] Lysis: The microfluidic chip is centrifuged, and under the action of counterclockwise low-speed centrifugation, the residual sample liquid in the sample tube 13 is completely transferred to the sample pool 22. The instrument pressure rod presses the lysis liquid bag 15, so that the lysis liquid is released into the lysis liquid pool 23. The microfluidic chip is centrifuged, and under the action of counterclockwise medium-speed centrifugation, the sample liquid and the lysis liquid are transferred to the mixing pool 27 for sufficient mixing and lysis. Through the ultrasonic module or forward and reverse reciprocating centrifugation of the instrument, the sample reagent is fully mixed and lysed. After lysis is completed, the centrifugal rotation of the microfluidic chip is stopped, and under the action of capillary force, the lysed liquid breaks through the first siphon valve 30 from the mixing pool 27 to the nucleic acid capture pool 32, realizing nucleic acid capture. The microfluidic chip is centrifuged, and under the action of counterclockwise high-speed centrifugation, the liquid for nucleic acid capture is transferred from the nucleic acid capture pool 32 to the extraction waste liquid pool 28 through the left side of the sorting pool 33.

[0076] Fractionation washing: press the washing liquid bag 17, so that the washing liquid is released into the total washing liquid pool 24, while under the action of the liquid bag extrusion pressure, the washing liquid enters three radial series of washing liquid distribution pools 29 respectively, then the microfluidic chip is centrifuged, under the action of counterclockwise medium-speed centrifugation, because the centrifugal force is related to the radius of the liquid pool distance from the center and the centrifugal speed, according to the centrifugal speed from small to large, the liquid of the three washing liquid distribution pools 29 from far to near the center is transferred to the nucleic acid capture pool 32 in turn, completing a small number of multiple fractionation washing, enhancing the washing efficiency, and the waste liquid after washing enters the extraction waste liquid pool 28 from the left of the sorting pool 33 of the nucleic acid capture pool 32;

[0077] Elution: press the elution liquid bag 16, so that the elution liquid is released into the elution liquid pool 26, and the microfluidic chip is centrifuged, under the action of clockwise high-speed centrifugation, the elution liquid elutes the captured nucleic acid from the nucleic acid capture pool 32, and enters the buffer pool 34 from the right of the sorting pool 33; when all the elution liquid is transferred, the elution liquid is mixed with the freeze-dried enzyme ball in the buffer pool 34 by reciprocating forward and reverse low-speed rotation; after the completion of the reconstitution and mixing, the centrifugal rotation of the microfluidic chip is stopped, and the liquid after reconstitution and mixing breaks through the second syringe valve 31 under the action of capillary force, and enters the eight quantitative pools 35 of the amplification functional module from the buffer pool 34, and the excess eluted sample enters the amplification waste liquid pool 39;

[0078] Amplification: the microfluidic chip is centrifuged, under the action of clockwise high-speed centrifugation, the high-speed centrifugation breaks through the gas resistance of the breakthrough valve port 362 of the flow resistance valve 36, so that the eluted sample in each quantitative pool 35 enters the corresponding amplification pool 37 in turn through the inlet channel 361, the breakthrough valve port 362 and the overflow channel 363 of the flow resistance valve 36, and is mixed with the probe primer dry powder reagent in the amplification pool 37, and then the amplification is completed under the cyclic temperature control condition provided by the instrument.

[0079] In the nucleic acid detection method for non-medical diagnostic purposes in this application example, based on the centrifugal full-integrated nucleic acid detection microfluidic chip described in embodiment 1, on the one hand, the volume of the total washing liquid is 300 μL, and based on the depth of the residual pigment color, it can be judged that the fractionation washing efficiency is excellent, on the other hand, during the amplification process, the chip is synchronously rotated at a speed of 2000 rpm for 60 s, and the flow resistance valve effectively retains the liquid in the quantitative pool from entering the amplification pool in advance.

[0080] Embodiment 2

[0081] The embodiment provides a centrifugal full-integrated nucleic acid detection micro-fluidic chip, and the difference from the embodiment 1 is that a switching valve 38 is arranged at the inside of the sorting pool 33 and at the communication position of the nucleic acid capturing pool 32 and the sorting pool 33, the switching valve 38 can realize switching of the following two states: state 1: the nucleic acid capturing pool 32 is communicated with the extraction waste liquid pool 28 through the sorting pool 33; and state 2: the nucleic acid capturing pool 32 is communicated with the buffer pool 34 through the sorting pool 33; as shown in Figure 6, one end of the switching valve 38 is fixed away from the nucleic acid capturing pool 32, and the other end can swing.

[0082] Application example 2

[0083] The application example provides a nucleic acid detection method for non-medical diagnosis based on the centrifugal full-integrated nucleic acid detection micro-fluidic chip in the embodiment 2, and the difference from the application example 1 is that:

[0084] When the chip is started to rotate counterclockwise at high acceleration, the switching valve 38 is attached to the buffer pool 34 and closes the inlet of the buffer pool 34 under the action of inertia, and the waste liquid is transferred from the left open port to the extraction waste liquid pool 28; when the chip is started to rotate clockwise at high acceleration, the switching valve 38 is attached to the inlet of the extraction waste liquid pool 28 due to inertia, and the eluent is transferred to the buffer pool 34; thereby realizing directional transfer of the liquid.

[0085] In the nucleic acid detection method for non-medical diagnosis in the application example, the centrifugal full-integrated nucleic acid detection micro-fluidic chip in the embodiment 2 is adopted, on the one hand, the volume of the total cleaning liquid is 300 mu L, and the efficiency of the fractional cleaning is excellent according to the depth of the residual pigment color, and on the other hand, in the amplification process, the chip is synchronously rotated at a speed of 2000 rpm for 60 s, the flow resistance valve effectively retains the liquid in the quantitative pool from entering the amplification pool in advance, and the switching valve 38 effectively ensures directional transfer of the liquid, and loss of the liquid in the directional transfer is avoided.

[0086] Embodiment 3

[0087] The embodiment provides a centrifugal full-integrated nucleic acid detection microfluidic chip, which is different from the embodiment 1 only in that three washing liquid distribution pools 29 located at the same radius are replaced by three washing liquid distribution pools 29 different in the radius; as shown in Figure 7, the three washing liquid distribution pools 29 are distributed on different radii of the microfluidic chip, and the washing liquid distribution pool 29 farthest in the radial direction is in communication with the nucleic acid capture pool 32, and the washing liquid distribution pool 29 closest in the radial direction is in communication with the gas buffer pool 21; the three washing liquid distribution pools 29 are connected through flow channels of different resistance sizes, and the resistance size is controlled through the size of the flow channel, that is, the resistance of the third distribution pool flow channel 293 between the washing liquid distribution pool 29 closest in the radial direction and the middle washing liquid distribution pool 29 is greater than the resistance of the second distribution pool flow channel 292 between the middle washing liquid distribution pool 29 and the washing liquid distribution pool 29 farthest in the radial direction, and the resistance of the second distribution pool flow channel 292 between the middle washing liquid distribution pool 29 and the washing liquid distribution pool 29 farthest in the radial direction is greater than the resistance of the first distribution pool flow channel 291 between the washing liquid distribution pool 29 farthest in the radial direction and the nucleic acid capture pool 32; the design of the flow channel can further pull apart the speed difference between the hierarchical washing, and adapt to more kinds of liquid property washing liquid.

[0088] Application Example 3

[0089] The application example provides a nucleic acid detection method for non-medical diagnosis purposes based on the centrifugal full-integrated nucleic acid detection microfluidic chip in the embodiment 3, which is different from the application example 1 only in that:

[0090] When the liquid in the total washing liquid pool 24 is distributed into the three washing liquid distribution pools 29, the chip is first rotated at a low speed, the washing liquid in the washing liquid distribution pool 29 farthest in the radial direction is transferred into the nucleic acid capture pool 32 through the first distribution pool flow channel 291 to complete the first washing; then the chip is rotated at a medium speed, the washing liquid in the middle washing liquid distribution pool 29 is sequentially transferred into the nucleic acid capture pool 32 through the second distribution pool flow channel 292 and the first distribution pool flow channel 291 to complete the second washing; finally, the chip is rotated at a high speed, the washing liquid in the washing liquid distribution pool 29 closest in the radial direction is sequentially transferred into the nucleic acid capture pool 32 through the third distribution pool flow channel 293, the second distribution pool flow channel 292 and the first distribution pool flow channel 291 to complete the third washing, thereby realizing multi-step effective washing.

[0091] In the nucleic acid detection method for non-medical diagnosis purposes in the application example, the volume of the total washing liquid is 300 μL, and Figure 8(a) shows that the point pigment dry point on the silicon film in the nucleic acid capture pool characterizes the washing effect, that is, the place indicated by the arrow is lighter in color, indicating that the efficiency of hierarchical washing is higher.

[0092] Comparative Example 1

[0093] The present comparative example provides a centrifugal fully integrated nucleic acid detection microfluidic chip, which is different from the example 1 only in that three washing liquid dispensing pools 29 are completely omitted, i.e., the total washing liquid pool 24 is directly connected with the nucleic acid capture pool 32.

[0094] Comparative application example 1

[0095] The present comparative application example provides a nucleic acid detection method for non-medical diagnostic purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in the comparative example 1, which is different from the application example 1 only in that, in the washing process, the washing liquid in the total washing liquid pool 24 is directly and once transferred to the nucleic acid capture pool 32 to complete single washing, and the waste liquid after washing enters the extraction waste liquid pool 28 from the left of the sorting pool 33.

[0096] In the nucleic acid detection method for non-medical diagnostic purposes carried out by the present comparative application example using the centrifugal fully integrated nucleic acid detection microfluidic chip described in the comparative example 1, the volume of the total washing liquid is 300 μL, and FIG. 8(b) shows that the point pigment dry point on the silicon film in the nucleic acid capture pool characterizes the washing effect, i.e., the place indicated by the arrow, the color is darker, which indicates that the efficiency of single washing is lower.

[0097] As can be seen from the comparison of (a) and (b) in FIG. 8, it can be seen that the residual pigment color can be obviously compared under the same volume of washing liquid, and the efficiency of fractional washing is higher than that of single washing.

[0098] Comparative example 2

[0099] The present comparative example provides a centrifugal fully integrated nucleic acid detection microfluidic chip, which is different from the example 1 only in that the flow resistance valve 36 is completely omitted, and the quantitative pool 35 and the amplification pool 37 are connected through a microchannel with a smaller cross section.

[0100] Comparative application example 2

[0101] The present comparative application example provides a nucleic acid detection method for non-medical diagnostic purposes based on the centrifugal fully integrated nucleic acid detection microfluidic chip described in the comparative example 2, which is different from the application example 1 only in that, in the amplification process, the eluted sample in each quantitative pool 35 directly enters the corresponding amplification pool 37 through a microchannel with a smaller cross section.

[0102] Fig. 9(a) and (b) respectively show the flow blocking effect of the quantitative pool liquid in application example 1 and comparative application example 2, the distribution of the quantitative pool and the amplification pool is at the same radial distance from the center, Fig. 9(a) corresponds to the flow resistance valve, and Fig. 9(b) corresponds to the ordinary microchannel with small cross section, rotating at 2000 rpm for 60 s, it can be seen that the flow resistance valve can effectively block the quantitative pool liquid and avoid its early entering the amplification pool, but in the same speed, the quantitative pool liquid in comparative application example 2 is basically transferred to the amplification pool in advance.

[0103] The application is described above by the above-mentioned embodiments to illustrate the detailed structural features of the application, but the application is not limited to the above-mentioned detailed structural features, that is, it does not mean that the application must rely on the above-mentioned detailed structural features to be implemented. Those skilled in the art should understand that any improvement of the application, equivalent replacement of the components selected by the application and addition of auxiliary components, selection of specific modes, etc. fall within the protection scope and disclosure scope of the application.

[0104] The preferred embodiments of the application are described in detail above, but the application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the application, the technical solutions of the application can be subjected to various simple modifications, and these simple modifications all belong to the protection scope of the application.

[0105] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable means without contradiction, in order to avoid unnecessary repetition, the application will not further describe various possible combination modes.

[0106] In addition, various different embodiments of the application can also be combined arbitrarily, as long as it does not deviate from the idea of the application, it should also be considered as the disclosed content of the application.

Claims

1. A centrifugal full-integrated nucleic acid detection microfluidic chip, comprising a reagent layer (1) on the upper layer and a structure layer (2) on the lower layer, which are arranged around the same centrifugal axis and are tightly combined. wherein The reagent layer (1) comprises at least one set of liquid capsule reagent components, which comprises a lysis liquid capsule (15), a washing liquid capsule (17) and an elution liquid capsule (16); the reagent layer (1) is also provided with a sample loading tube (13), and a gas hole (14) is arranged beside the sample loading tube (13) to ensure that the fluid flows smoothly during the sample loading process; The structure layer (2) comprises at least one set of sample lysis functional modules, nucleic acid extraction and purification functional modules and amplification functional modules which are distributed along the radial direction; the sample lysis functional module comprises a sample pool (22), a lysis liquid pool (23) and a mixing pool (27), and the sample pool (22) and the lysis liquid pool (23) are independently communicated with the mixing pool (27), respectively; the nucleic acid extraction and purification functional module comprises a nucleic acid capture pool (32), an elution liquid pool (26), a total washing liquid pool (24), a sorting pool (33), a buffer pool (34) and an extraction waste liquid pool (28), the mixing pool (27) is communicated with the nucleic acid capture pool (32), and the elution liquid pool (26) is communicated with the nucleic acid capture pool (32); the total washing liquid pool (24) is communicated with at least two washing liquid distribution pools (29), respectively, and at least two washing liquid distribution pools (29) are sequentially distributed along the radial direction and are communicated, and the washing liquid distribution pool (29) farthest from the radial direction is communicated with the nucleic acid capture pool (32); the nucleic acid capture pool (32) is communicated with the sorting pool (33), and the left and right sides of the sorting pool (33) are respectively communicated with the extraction waste liquid pool (28) and the buffer pool (34); the amplification functional module is communicated with the buffer pool (34) for quantitative and amplification reaction; the amplification functional module comprises a quantitative pool (35), an amplification pool (37) and an amplification waste liquid pool (39); at least one quantitative pool (35) is arranged along the circumferential direction, and the quantitative pools (35) are communicated with each other; the quantitative pool (35) farthest from the buffer pool (34) is communicated with the amplification waste liquid pool (39); the quantitative pool (35) corresponds to the amplification pool (37) one by one and is sequentially distributed along the radial direction; the structure layer (2) further comprises at least one gas buffer pool (21), which is respectively communicated with the extraction waste liquid pool (28), at least two washing liquid distribution pools (29) and the amplification waste liquid pool (39) through a gas channel, and the mixing pool (27) is communicated with the extraction waste liquid pool (28) through a gas channel, so that the gas-liquid circulation is realized in the whole chip; The sample loading tube (13), the lysis liquid capsule (15), the washing liquid capsule (17) and the elution liquid capsule (16) on the upper reagent layer (1) are respectively corresponded to the sample pool (22), the lysis liquid pool (23), the total washing liquid pool (24) and the elution liquid pool (26) on the lower structure layer (2).

2. The centrifugal-type fully integrated nucleic acid detection microfluidic chip according to claim 1, wherein, The mixing pool (27) is communicated with the nucleic acid capture pool (32) through a first siphon valve (30); the quantitative pool (35) closest to the buffer pool (34) is communicated with the buffer pool (34) through a second siphon valve (31); the quantitative pool (35) is communicated with the amplification pool (37) through a flow resistance valve (36), the flow resistance valve (36) comprises an inlet channel (361), a breakthrough valve port (362) and an overflow channel (363), the inlet channel (361) is located at the bottom of the quantitative pool (35) and makes the bottom of the quantitative pool (35) communicated with the breakthrough valve port (362), the overflow channel (363) is located at the top of the amplification pool (37) and makes the top of the amplification pool (37) communicated with the breakthrough valve port (362), and gas exists in the breakthrough valve port (362).

3. The centrifugal-type fully integrated nucleic acid testing microfluidic chip according to claim 1, wherein, A switching valve (38) is arranged in the sorting pool (33) at the communication between the nucleic acid capture pool (32) and the sorting pool (33), and the switching valve (38) can realize switching between the following two states: State 1: the nucleic acid capture pool (32) is communicated with the extraction waste liquid pool (28) through the sorting pool (33); State 2: the nucleic acid capture pool (32) is communicated with the buffer pool (34) through the sorting pool (33).

4. The centrifugal-type fully integrated nucleic acid testing microfluidic chip according to claim 1, wherein, The lysis liquid pool (23), the total cleaning liquid pool (24) and the elution liquid pool (26) are respectively provided with liquid capsule puncture needles (25), so that the sealed aluminum foil of the upper liquid capsule can be punctured and the internal reagent can be released under the pressing action.

5. The centrifugal-type fully integrated nucleic acid testing microfluidic chip according to claim 1, wherein, The extraction waste liquid pool (28) is provided with water absorption material, which is used for increasing the liquid containing volume and preventing the backflow of waste liquid.

6. A method for nucleic acid detection for non-medical diagnostic purposes based on the centrifugal, fully integrated nucleic acid detection microfluidic chip according to any one of claims 1 to 5, wherein, The lysis liquid capsule (15) encapsulates lysis liquid, the cleaning liquid capsule (17) encapsulates cleaning liquid, the elution liquid capsule (16) encapsulates elution liquid, and the buffer pool (34) is pre-embedded with freeze-dried enzyme balls; The nucleic acid detection method comprises the following steps: Step S1: injecting sample liquid into the sample pool (22) of the microfluidic chip through the sample adding tube (13), and sealing the air hole (14) beside the sample adding tube (13) to form a fully closed internal environment of the microfluidic chip and the sample adding tube (13); Step S2 lysis: Centrifugal rotation of the microfluidic chip makes the residual sample liquid in the sample adding tube (13) completely transferred to the sample pool (22); the lysis liquid capsule (15) is pressed to release the lysis liquid into the lysis liquid pool (23), and the microfluidic chip is centrifugally rotated, so that the sample liquid and the lysis liquid are transferred to the mixing pool (27) for complete mixing and lysis, the liquid after lysis enters the nucleic acid capture pool (32) to realize nucleic acid capture, and the liquid after nucleic acid capture enters the extraction waste liquid pool (28) through one side of the sorting pool (33); Step S3 fractional cleaning: Press the cleaning liquid bag (17) to release the cleaning liquid into the total cleaning liquid pool (24), and under the extrusion pressure of the liquid bag, the cleaning liquid enters at least two cleaning liquid distribution pools (29) respectively. The microfluidic chip is rotated by centrifugation, and the cleaning liquid in the at least two cleaning liquid distribution pools (29) is sequentially introduced into the nucleic acid capture pool (32) from far to near according to the distance from the center, so as to complete the fractional cleaning. The waste liquid after cleaning enters the extraction waste liquid pool (28) from one side of the sorting pool (33) through the nucleic acid capture pool (32); Step S4 elution: Press the elution liquid bag (16) to release the elution liquid into the elution liquid pool (26); reverse the fractional cleaning to rotate the microfluidic chip by centrifugation. The elution liquid elutes the captured nucleic acid from the nucleic acid capture pool (32) and enters the buffer pool (34) through the other side of the sorting pool (33). The eluted nucleic acid is mixed with the freeze-dried enzyme ball in the buffer pool (34); Step S5 amplification: Reverse the fractional cleaning to rotate the microfluidic chip by centrifugation. The eluted sample is distributed to at least one quantitative pool (35). The excess eluted sample enters the amplification waste liquid pool (39). The liquid after reconstitution and mixing enters the amplification functional module to complete the quantitative and amplification reaction.

7. The nucleic acid detection method according to claim 6, wherein, In step S2, after the lysis is completed, the centrifugal rotation of the microfluidic chip is stopped. Under the capillary action, the liquid after lysis breaks through the first siphon valve (30) and enters the nucleic acid capture pool (32) from the mixing pool (27); In step S5, after the reconstitution and mixing are completed, the centrifugal rotation of the microfluidic chip is stopped. Under the capillary action, the liquid after reconstitution and mixing breaks through the second siphon valve (31) and enters the quantitative pool (35) of the amplification functional module from the buffer pool (34); Reverse the fractional cleaning to rotate the microfluidic chip by centrifugation. The eluted sample is distributed to at least one quantitative pool (35). The excess eluted sample enters the amplification waste liquid pool (39). High-speed centrifugation breaks through the gas resistance of the breakthrough valve port (362) of the flow resistance valve (36), so that the eluted sample in each quantitative pool (35) sequentially passes through the inlet channel (361), the breakthrough valve port (362) and the overflow channel (363) of the flow resistance valve (36), and enters the corresponding amplification pool (37) to complete the quantitative and amplification reaction.

8. The nucleic acid detection method according to claim 6, wherein, In step S2, the sample liquid and the lysis liquid are transferred to the mixing pool and mixed by ultrasonic module and / or forward and reverse reciprocating centrifugation.

9. The nucleic acid detection method according to claim 6, wherein, In step S4, the eluted nucleic acid is reconstituted and mixed with the freeze-dried enzyme ball in the buffer pool by forward and reverse reciprocating centrifugation.

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