CN109486667B

CN109486667B - Fluid control and processing cartridge

Info

Publication number: CN109486667B
Application number: CN201810431898.6A
Authority: CN
Inventors: 梁骞; 于浩
Original assignee: Delta Electronics International Singapore Pte Ltd
Current assignee: Delta Electronics International Singapore Pte Ltd
Priority date: 2017-09-11
Filing date: 2018-05-08
Publication date: 2022-06-28
Anticipated expiration: 2038-05-08
Also published as: CN109486667A

Abstract

A fluid control and processing cartridge for a nucleic acid analysis device includes a cartridge body and a reaction chip. The cassette body comprises a plurality of grooves and a plurality of flow channels, the grooves are suitable for storing at least one sample, a plurality of biochemical reagents and buffer solution, and the flow channels are connected with the grooves. The reaction chip is combined with the cassette body and comprises a plurality of detection grooves, at least one main fluid channel and at least one gas release flow channel, wherein the main fluid channel is connected with the detection grooves and is suitable for distributing a sample into the detection grooves, and the gas release flow channel is connected with the detection grooves and is suitable for releasing gas from the detection grooves.

Description

Fluid control and processing cartridge

Technical Field

The present invention relates to a fluid control and processing cartridge, and more particularly, to a fluid control and processing cartridge for a nucleic acid analysis device.

Background

In Vitro Diagnostics (IVD) is becoming increasingly important In modern medical practice. In recent years, due to the demand for rapid diagnosis and decentration of medical institutions, a Point-of-care testing (POCT) technique capable of performing testing on site with a minimum of trained technicians and human errors is widely used for many applications. Generally, POCT refers to a simple medical examination that can be performed at the bedside, i.e., at the time and place of care for the patient, by means of specially designed devices and disposable test strips or cassettes. A variety of techniques for realizing POCT have been developed, including biochemical, immunological, and molecular biological techniques, among which molecular diagnosis is considered as the most promising technique to lead the future market.

Traditional molecular diagnostics are performed by trained technicians at a central laboratory using sophisticated equipment and following a first-column default procedure. Furthermore, most central laboratory tests collect large numbers of samples for high throughput testing only when overall operating time and cost effectiveness are required. Alternatively, POCT platforms may be provided that integrate these devices in a desktop or handheld size device, and emphasize portability and flexibility. Most POCT devices based on molecular manipulation are used with disposable cartridges for diagnostic purposes, and in fact some of the functionality originally present in the corresponding instrument is removed from the platform and integrated into the fluidic circuit of the disposable cartridge.

Therefore, the development of disposable cassettes is important in the development of POCT products, and it is necessary to provide a cassette design for all-in-one nucleic acid analysis device to realize and improve POCT.

Disclosure of Invention

It is an object of an embodiment of the present invention to provide a fluid control and processing cartridge for a nucleic acid analysis device to precisely control fluid flow direction and dynamic fluid behavior in the cartridge, thereby facilitating nucleic acid amplification and detection.

To achieve the above objects, one embodiment of the present invention provides a fluid control and processing cartridge for a nucleic acid analysis device, which includes a cartridge body and a reaction chip. The cassette body comprises a plurality of grooves and a plurality of runners, the plurality of grooves are suitable for storing at least one sample, a plurality of biochemical reagents and buffer solutions, and the plurality of runners are connected with the plurality of grooves; the reaction chip is combined with the cassette body and comprises a plurality of detection grooves, at least one main fluid channel and at least one gas release flow channel, wherein the main fluid channel is connected with the detection grooves and is suitable for distributing the samples to the detection grooves, and the gas release flow channel is connected with the detection grooves and is suitable for releasing gas from the detection grooves.

In one embodiment, the gas release channels are substantially narrower than the main flow channels.

In one embodiment, the primary fluid passageway includes a plurality of wide runner sections, a plurality of narrow runner sections, and a plurality of slot entrance runners.

In one embodiment, each wide runner section is aligned with one of the plurality of detection grooves and connected to the corresponding detection groove through the corresponding groove entrance runner, and each narrow runner section is connected between two adjacent wide runner sections.

In one embodiment, the flow resistance of the narrow flow path portion is higher than the total flow resistance of the wide flow path portion and the groove entrance flow path.

In one embodiment, the flow resistance of the narrow flow path portion is 2 to 20 times higher than the total flow resistance of the wide flow path portion and the groove entrance flow path.

In one embodiment, the flow resistance of the gas discharge flow channel is 2 to 500 times higher than the flow resistance of the narrow flow channel portion.

In one embodiment, the cross-sectional area of the channel entrance runner is significantly smaller than the wide runner section.

In one embodiment, the reaction chip is disposed on one side of the cassette body.

In one embodiment, each of the detection slots has at least one flat surface.

In one embodiment, the reaction chip is substantially in the shape of a regular polygon.

In one embodiment, the reaction chip further comprises at least one sample loading hole for loading a sample into the cassette.

In one embodiment, the reaction chip further comprises a plurality of sample loading holes for loading different samples into the cartridge.

In one embodiment, the cassette is installed in a housing of the nucleic acid analyzer, and the reaction chip includes at least one alignment slot, which is capable of aligning with at least one positioning element on the housing.

In one embodiment, the reaction chip includes at least one sample inlet, and the cassette body includes at least one conduit connected to the sample inlet for delivering the sample to the reaction chip.

In one embodiment, the cassette body further comprises a plurality of openings disposed on a bottom surface thereof, and the openings are communicated with the grooves through the flow channels.

In one embodiment, at least one of the bottom or top of the detection chamber includes a thin, light-transmissive wall or membrane through which light can pass.

In one embodiment, the detection slot has a light-transmissive front wall through which light can pass.

The fluid control and processing cartridge for the nucleic acid analysis device has a well-designed flow channel geometry, and can precisely control the flow direction and dynamic fluid behavior of the fluid in the reaction chip, so that the sample can be sequentially and smoothly distributed to each detection groove, thereby promoting subsequent nucleic acid amplification and detection. Furthermore, by arranging the multiple detection tanks and the multiple optical units, the multiple nucleic acid analysis and multiple color detection of multiple tanks can be achieved.

Drawings

FIG. 1 shows a schematic view of a nucleic acid analysis apparatus according to an embodiment of the present invention.

FIG. 2 shows the nucleic acid analysis apparatus with the tank of FIG. 1 opened.

FIG. 3 shows the locking and releasing mechanism between the adapter cartridge and the bottom slot.

Fig. 4 and 5 show the cassette at different angles.

FIG. 6 shows a top view of the reaction chip.

Fig. 7 shows a schematic view of a fluid circuit as it flows through the wide runner section and into the narrow runner section and the slot entrance runner.

Fig. 8A and 8B are schematic views showing the fluid circuit when the first detection tank is full.

Fig. 9 shows the liquid dispensing and oil sealing sequence for the adapter cartridge.

FIG. 10 shows a cross-sectional view of a test well.

FIG. 11 shows the internal structure of a cell body of a nucleic acid analysis apparatus.

Fig. 12 shows the structure of the rotary drive unit, the cartridge, and the optical unit.

FIG. 13 shows a flowchart of the operation of the nucleic acid analysis apparatus.

FIG. 14 shows a many-to-one cassette that can detect multiple samples.

Figure 15 shows the liquid dispensing and oil sealing procedure for three-to-one cartridge.

The reference numbers are as follows:

1: trough body

11: top trough body

12: bottom trough body

121: chamber

13: hinge assembly

14: fastening piece

141: hook part

15: release ring

151: convex part structure

16: release actuator

17: locating piece

2: fluid transfer unit

3: temperature control unit

31: heating device

32: heat radiator

33: fan blade

4: rotary drive unit

41: cartridge holder

411: magnetic member

5: optical unit

51: light source

52: photodetector

6: cartridge

61: buckle slot

62: reaction chip

621: detection tank

622: principal fluidic passage

623: alignment groove

624: sample loading hole

625: sample inlet

626: sample outlet

627: thin walls or films

628: film(s)

629: front wall

63: cartridge body

631: trough

632: flow passage

633: opening of the container

634: pipeline

64: magnetic material

71. 71': wide runner section

72: narrow runner section

73: channel inlet flow passage

74: gas release flow channel

740: end segment

Detailed Description

Some embodiments which embody features and advantages of the invention will be described in detail in the description which follows. As will be realized, the invention is capable of modifications in various obvious respects, all without departing from the scope of the present invention, and the description and drawings are to be regarded as illustrative in nature, and not as restrictive.

The embodiment of the invention provides a full-function integrated (all-in-one) nucleic acid analysis device, which adopts an isothermal amplification mode, and integrates a fluid conveying unit, a temperature control unit, a rotary driving unit, an optical unit and the like on a single device, so that the processes of sample purification, nucleic acid extraction, nucleic acid amplification, optical detection and the like can be carried out on the full-function integrated device, and real-time nucleic acid analysis is realized. In particular, embodiments of the present invention provide a fluid control and processing cartridge for a nucleic acid analysis device that precisely controls fluid flow direction and dynamic fluid behavior in the cartridge to facilitate subsequent nucleic acid amplification and detection.

FIG. 1 is a schematic view showing a nucleic acid analysis apparatus according to an embodiment of the present invention, and FIG. 2 is a view showing the nucleic acid analysis apparatus of FIG. 1 in which the nucleic acid analysis apparatus is in an open state and a cassette is removed from the nucleic acid analysis apparatus. As shown in FIG. 1 and FIG. 2, the nucleic acid analysis apparatus 100 includes a tank 1, a fluid transport unit 2, a temperature control unit 3, a rotation driving unit 4, and at least one optical unit 5. The housing 1 may be opened to install a fluid control and processing cassette 6 therein. In one embodiment, the fluid control and processing cartridge 6 may be a disposable cartridge. The fluid delivery unit 2 is connected with the interior of the tank 1 and is adapted to deliver reagents in the cassette 6 for sample purification and/or nucleic acid extraction. The temperature control unit 3 is disposed in the tank 1 and adapted to provide a default temperature for nucleic acid amplification. The rotation driving unit 4 is connected to the housing 1 and adapted to rotate the cartridge 6 within the housing 1 by a default procedure. In one embodiment, the rotational drive unit 4 may grip the cassette 6 as needed. At least one optical unit 5 is disposed on the tank 1 and includes a plurality of optical components for detection, such as nucleic acid detection or sample reaction detection.

In one embodiment, the tank 1 comprises a top tank 11 and a bottom tank 12. The top tank 11 and the bottom tank 12 are connected by a hinge (hinge)13, but not limited thereto. The bottom channel 12 has a chamber 121 specifically designed to receive the cassette 6 therein. The top housing 11 may be opened to allow the cassette 6 to be placed in the cavity 121 of the bottom housing 12, and when the top housing 11 is closed, an enclosed space is formed in the housing 1.

In one embodiment, the shape of the tank 1 may be, but not limited to, cylindrical, spherical, cubic, conical or olive shape, and the tank 1 may be made of, but not limited to, metal, ceramic, polymer, wood, glass or other materials that provide good thermal insulation.

The bottom housing 12 is connected to the fluid delivery unit 2 by a tube or channel, and when the cassette 6 is installed in the bottom housing 12, the cassette 6 is locked and in close contact with the fluid delivery unit 2 to prevent leakage. For example, the cassette 6 may be locked to the bottom slot 12 by at least one locking element, wherein the locking element may include, but is not limited to, a clip (clip).

FIG. 3 shows the locking and releasing mechanism between the adapter cartridge and the bottom slot. As shown in fig. 2 and 3, the cylindrical body of the cartridge 6 includes at least one locking groove 61, and the bottom groove 12 includes at least one locking member 14, a release ring 15 and a release actuator 16. The bottom of the fastener 14 is fixed, and the top has a hook 141. The fastener 14 may be made of a polymer or a metal sheet having elasticity. When the cartridge 6 is placed in the cavity 121 of the bottom slot 12, a user may press the cartridge 6 to engage and lock the hook 141 of the fastener 14 with the locking groove 61 of the cartridge 6, so that the cartridge 6 is tightly contacted with the fluid delivery unit 2. The release ring 15 surrounds the cylindrical body of the cartridge 6 and abuts against the bottom surface of the hook 141. The release ring 15 is slidable over a distance and is connected to a release actuator 16, for example a solenoid actuator. When the cassette 6 is to be released, the releasing actuator 16 is triggered to pull the releasing ring 15, so that the protrusion 151 of the releasing ring 15 pushes the fastening member 14 to separate the hook 141 from the locking groove 61, thereby releasing the cassette 6.

In one embodiment, the fastener 14 may be manually operated by a user or may be automated by the device upon command. Of course, the locking and releasing mechanism is not limited to the above-mentioned fastener 14, and other locking components capable of locking and releasing the cartridge 6 may be used.

Fig. 4 and 5 show the cassette at different angles. As shown in FIGS. 4 and 5, the cassette 6 includes a planar reaction chip 62 for nucleic acid amplification and detection, and a cassette body 63 including a plurality of grooves 631 and a plurality of channels 632, the plurality of channels 632 are connected to the plurality of grooves 631 for fluid delivery of sample processing, purification, and nucleic acid extraction (only a portion of the grooves 631 and the channels 632 are shown in FIG. 4 to avoid confusion due to too many lines). In one embodiment, the biochemical reagents and buffers are pre-loaded into the cassette body 63 and stored in the grooves 631, and the sample is loaded into the cassette 6 through a sample loading hole 624 on the top surface of the reaction chip 62 and stored in one of the grooves 631. In one embodiment, the cassette body 63 may be, but is not limited to, a cylindrical body. The cassette body 63 further includes a plurality of openings 633 formed on the bottom surface of the cassette body 63, and the openings 633 are communicated with the grooves 631 through the flow channels 632. The shape of the opening 633 may be, but is not limited to, circular, rectangular, or other regular or irregular shapes.

In one embodiment, the reaction chip 62 comprises a planar fluid chip and is disposed on one side of the cartridge body 63, such as the top of the cartridge body 63. The reaction chip 62 is combined with the cassette body 63 and includes a plurality of detection grooves 621, at least one main fluid channel 622 and at least one gas release flow channel 74 (shown in FIG. 6). The detection tank 621 is used for nucleic acid amplification and detection, the main fluid channel 622 is connected to the detection tank 621 and adapted to distribute the sample into the detection tank 621, and the gas release flow channel 74 is connected to the detection tank 621 and adapted to release gas from the detection tank 621. In one embodiment, the detection chamber 621 contains reagents for nucleic acid amplification and/or detection. For example, the detection wells 621 may be coated with reagents for nucleic acid amplification and/or detection, such as reagents comprising different fluorescent dyes.

In some embodiments, the reaction chip 62 and the cassette body 63 can be made of, but not limited to, metal, thermoplastic, glass, rubber, and silicone. The cartridge body 63 may be rigid or deformable depending on the fluid driving method.

In some embodiments, the manufacturing method of the reaction chip 62 and the cassette body 63 may be, but not limited to, Computer Numerical Control (CNC) machining, 3D printing (or additive manufacturing), injection molding (injection molding), layer-to-layer stacking (layer-to-layer stacking), hot press molding (hot forming), laser ablation (laser ablation), thermoplastic molding (thermoforming), photolithography (photolithography), soft lithography (soft lithography), electron beam lithography (e-beam lithography), or a combination of any of the foregoing methods.

In some embodiments, the reaction chip 62 and the cassette body 63 are bonded together in advance, and the bonding method can be, but not limited to, thermal bonding (thermal bonding), solvent bonding (solvent bonding), adhesive bonding (adhesive bonding), ultrasonic bonding (ultrasonic bonding), laser welding (laser welding) or a combination of any of the foregoing methods to form a permanent bonding structure. In some embodiments, the reaction chip 62 and the cassette body 63 are separated and then coupled together by a user using a structure designed for coupling with each other, such as a snap fit or a screw.

The number of detection wells 621 is not limited, and the device of the present invention can perform multiplexed (multiplexing) nucleic acid analysis. In one embodiment, the reaction chip 62 is substantially regular polygon in shape, such that the reaction chip 62 has a plurality of planar side surfaces, which can be aligned with the optical unit 5 to facilitate light focusing. The number of planar sides depends on the number of detection slots 621. Of course, the shape of the reaction chip 62 is not limited to a regular polygon, and it may be a circle or other shapes because light can be focused on the sample in the detection well 621 by the arrangement of the optical elements of the optical unit 5.

In one embodiment, the reaction chip 62 further includes at least one alignment slot 623, and the bottom slot 12 further includes at least one positioning element 17 (as shown in fig. 2), and the positioning element 17 includes a positioning pin, for example. When the cassette 6 is placed in the cavity 121 of the bottom slot 12, the alignment slot 623 of the cassette 6 is aligned with the positioning element 17 of the bottom slot 12, which facilitates the loading of the cassette 6, and by this alignment, the cassette 6 can be automatically aligned with the fluid delivery unit 2 through the flow channel or pipe of the bottom slot 12, and each optical unit 5 is aligned with one of the detection slots 621. In one embodiment, each of the detecting slots 621 has at least one flat surface. For example, the detection slot 621 may be rectangular, and during the nucleic acid detection process, the detection slot 621 has one plane aligned with the light source 51 of the optical unit 5 and another plane aligned with the light detector 52 of the optical unit 5.

In operation, once a sample is loaded, the cartridge 6 is placed into the nucleic acid analysis device 100 and subjected to fluid processing by the fluid delivery unit 2. The fluid delivery unit 2 and the cassette 6 operate simultaneously to perform sample purification, nucleic acid extraction and fluid delivery, thereby realizing a fully automatic apparatus. Fluid transport may be accomplished, but is not limited to, via pneumatic (pneumatic), vacuum (vacuum), piston (plunger), chamber deformation (chamber deformation), thermal-induced expansion (thermal-induced expansion), acoustic forces (acoustics), centrifugal forces (centrifugal force), or other methods that may accomplish sample processing within the cartridge body 63.

In one embodiment, the fluid is pneumatically driven through the microchannels and the apertures. For example, the fluid delivery unit 2 is similar to the fluid integration module described in taiwan patent application No. 105123156 (claiming priority of singapore patent application No. 10201605723Y, filed 2016, 7, 13), filed 2016 by the applicant of the present invention on 22/7, and the entire contents of the aforementioned application are hereby incorporated by reference and will not be described herein again. Briefly, the fluid delivery unit 2 of the present embodiment includes the fluid manifold portion, the rotary valve stator, the rotary valve rotor, the rotary valve housing, and the fluid source described in taiwan patent application No. 105123156. The fluid manifold portion has a plurality of microchannels, which are connected to the grooves 631 of the cassette 6 through the bottom opening 633 of the cassette 6. Because the through holes and/or grooves on the rotary valve stator and the rotary valve rotor have corresponding alignment relationships when the rotary valve rotor rotates, when the rotary valve rotor rotates to different positions, the switching of multiple fluid paths can be realized, and the fluid operation of the cassette 6 can be further regulated and controlled. Therefore, the reagent stored in the cassette 6 can be transported to the position to be transported by the pneumatic force provided by the pump of the fluid transport unit 2, thereby automatically performing the sample purification and nucleic acid extraction processes. Of course, the fluid delivery unit is not limited to the above-mentioned design, and any other type of fluid delivery unit can be used as long as it can achieve multiple fluid delivery and path switching functions in the cassette 6 without departing from the scope of the present disclosure.

After sample purification and nucleic acid extraction are completed, the sample with the extracted nucleic acids is dispensed into the detection slot 621 of the cartridge 6 for subsequent nucleic acid amplification and detection. FIG. 6 shows a top view of the reaction chip 62. Reaction chip 62 includes at least one sample inlet 625 and at least one sample outlet 626. The cassette body 63 includes at least one conduit 634 (shown in FIG. 4) connected to the sample inlet 625 of the reaction chip 62. Once sample processing is complete, the sample with extracted nucleic acids is delivered to the reaction chip 62 via the conduit 634 and sample inlet 625 for nucleic acid amplification and detection.

The primary fluid channel 622 is specifically designed to distribute the sample evenly into each detection slot 621 and to substantially fill the detection slot 621 without air bubbles. As shown in fig. 4 and 6, the main fluid passage 622 includes a plurality of wide flow path portions 71, 71 '(71' indicates the next wide flow path portion), a plurality of narrow flow path portions 72, and a plurality of short groove inlet flow paths 73. Each wide runner portion 71, 71 'is aligned with a detection slot 621 and connected to the detection slot 621 through the corresponding slot entrance runner 73, and each narrow runner portion 72 is connected between two adjacent wide runner portions 71, 71'. Once the liquid sample is fed from the sample inlet 625 by the pressure difference driving, the liquid first fills the wide flow path portion 71 corresponding to the first detection groove 621, and then, the liquid further flows along the main flow path 622 and is delayed due to the high flow resistance (flow resistance) caused by the abruptly contracted flow path sectional area. At this time, the liquid enters the detection groove 621 through the groove inlet flow passage 73, and the gas remaining in the detection groove 621 is pushed out by the inflowing liquid through the gas release flow passage 74 and flows to the adjacent detection groove 621. Since the surfaces of the flow channels are hydrophobic or treated to be hydrophobic, the surface tension in the fine flow channels substantially repels the inflow of liquid. Since the gas releasing flow channel 74 is significantly narrower than all

other flow channels

71, 71', 72, 73, so that the liquid is difficult to flow into the gas releasing flow channel 74, the residual gas in the gas releasing flow channel 74 also isolates each detection slot 621, and prevents the sample from being contaminated between adjacent detection slots 621. When the detecting groove 621 is filled with liquid, the fluid further overcomes the flow resistance of the narrow flow path portion 72 and then moves to the next wide flow path portion 71' corresponding to the next detecting groove 621, so as to fill the next detecting groove 621, and these operations are repeated until all the detecting grooves 621 are sequentially filled. Finally, the remaining liquid is drawn out of the main fluid channel 622, and the fluid that is not compatible with the sample, such as oil or liquid wax, is then injected into the main fluid channel 622, and the well inlet channel 73 serves as a capillary valve to prevent the sample from flowing out of the test well 621. Therefore, the detection chambers 621 filled with the purified sample are isolated and sealed by the immiscible fluid, thereby preventing contamination therebetween and reducing sample evaporation during the nucleic acid amplification process.

In one embodiment, the gas release channel 74 is directly connected to each detection slot 621 without any branches, and is substantially circular. In addition, the end segment 740 of the gas release flow path 74 is connected to the last detection well 621 and the flow path to the sample outlet 626 for gas release from the last detection well 621.

The pressure-driven flow in a rectangular flow channel under low Reynolds number conditions according to the classical Hagen-Poiseuille (classical Hagen-Poiseuille) method is represented by:

ΔP＝RQ＝αμQL/WH³ (1)

where Δ P is the driving pressure gradient, R is the flow resistance, Q is the volumetric flow rate, L, W and H represent the flow channel length, width and height, respectively, μ is the fluid viscosity, α is a dimensionless parameter dependent on the aspect ratio,

α＝12[1-(192H/π⁵W)tanh(πW/2H)]-1 (2)

the following flow resistance can be obtained from the formula (1)

R＝ΔP/Q＝12μL/WH³(1-0.63H/W)＝μa (3)

Wherein

a＝12L/WH³(1-0.63H/W) (4)

It is clear from equations (3) and (4) that the flow resistance depends directly on two factors, namely the fluid viscosity and the channel geometry. For the selected fluid, the flow resistance of the designed flow channel can be estimated by calculating the parameter a, and the time required for the fluid to pass through the flow channel can be estimated by using the flow resistance. In a fluid circuit, the overall flow resistance follows ohm's law. For example, when the liquid flows through the wide flow path portion 71 and enters the narrow flow path portion 72, the high flow resistance of the narrow flow path portion 72 significantly retards most of the flow velocity, and thus switches the flow to the low flow resistance path of the slot inlet flow path 73. The flow resistance at the narrow flow path portion 72 is higher than the total flow resistance of the wide flow path portion 71 and the groove inlet flow path 73, and generally the former is higher than the latter by a factor of 2 to 20. Fig. 7 shows a schematic view of the fluid circuit as the fluid flows through the wide runner section 71 and into the narrow runner section 72 and the slot entrance runner 73. Subscripts L and G denote liquid and gas fluids, respectively, W denotes a

detection cell

621, and 71 to 74 denote different flow channel portions shown in fig. 6. Since the viscosity of a gas is typically thousands of times lower than the viscosity of a liquid, the flow resistance associated with the gas is negligible compared to the flow resistance of the same flow channel filled with the liquid. The above calculations are based on the assumption of high Weber number conditions, where the inertial force of the fluid is much stronger than the capillary force due to surface tension. When fluid is drawn out at the completion of the step of dispensing the fluid into the detection well 621, the flow rate can be controlled so that the capillary force acts to stop the flow of the fluid at the well inlet channel 73.

Once the dispensed sample occupies the detection well 621, the gas originally in the well is pushed out through the gas release flow channel 74 and flows to the adjacent detection well 621. To minimize liquid flow into the gas discharge channels 74, the cross-sectional area of the gas discharge channels 74 is significantly smaller than all of the other channels. That is, the gas discharge channels 74 are designed to discharge gas and have an extremely high flow resistance to the flow of liquid, so that the gas discharge channels 74 selectively pass the gas while excluding the inflow of liquid. Fig. 8A and 8B are schematic diagrams illustrating the fluid circuit when the first detection groove 621 is filled. As shown in fig. 8A, the flow resistance of the gas discharge flow channel 74 is much greater than that of all other flow channels, and thus it can be assumed that the liquid flow is interrupted here, as shown in fig. 8B. The flow resistance of the gas release flow path 74 is usually 2 to 500 times higher than that of the narrow flow path portion 72, in which case when an external driving pressure is applied, the fluid slowly passes through the narrow flow path portion 72 and reaches the inlet of the next detection groove 621. Since the path through the gas release flow passage 74 is blocked, the only direction to fill the next detection cell 621 is through the cell inlet flow passage 73 of the next detection cell 621.

By the above method, the flow channel geometry can be carefully designed to precisely control the fluid direction and dynamic fluid behavior in the reaction chip 62. With respect to the withdrawal of residual sample, the flow rate can also be carefully controlled to utilize the cell inlet channel 73 as a capillary valve. In most instruments, the pump pressure is limited to a certain range due to hardware characteristics. By using the method of the present invention, the flow channel geometry is designed by calculating the dynamic fluid circuit at each stage, so that the pumping pressure and the distribution pressure can be the same and can be performed by the same pump. In other words, with a well-designed flow channel geometry, the dispensing and withdrawing of fluids can be driven by the same drive source. In some embodiments, the flow channel geometry may not be the same in each detection slot 621 depending on the pressure distribution in the system.

Fig. 9 shows the liquid dispensing and oil sealing sequence for the adapter cartridge. The cartridge 6 utilizes a piezoelectric micropump to deliver the liquid sample from the cartridge body 63 to the reaction chip 62 through the sample inlet 625. As shown in the sub-diagrams a, b, and c of FIG. 9, the liquid sample can be sequentially and smoothly distributed to each detection well 621 via the wide flow path portions 71 and 71', the narrow flow path portion 72, and the well inlet flow path 73. After each of the test wells 621 is filled, the remaining liquid sample is pumped out through the sample outlet 626 to remove the liquid sample in the wide flow-path portions 71, 71' and the narrow flow-path portion 72, but keep the liquid sample in the test well 621, and then separate each test well 621 with immiscible oil or liquid wax, as shown in the sub-diagram d of FIG. 9. Since the oil is light permeable and has a reflectivity close to that of the light transmissive material (thermoplastic material) of the reaction chip 62, the primary fluid channel 622 image in the sub-image d of fig. 9 is hidden, although not readily visible, indicating that the oil sealing procedure was successful. It is clear that the flow of fluid over the reaction chip 62 can be precisely controlled by the flow channel geometry designed according to the present invention.

In each of the detection vessels 621, the dry reagent may be pre-loaded so that each of the detection vessels 621 can serve as an independent reaction unit. In some embodiments, each reaction chip 62 includes 2 to 100 detection wells 621, so that multiplex detection (multiplexing detection) can be realized. Once the sample distribution is complete, the dry reagent is ready to mix with and dissolve in the sample in the test well 621. By controlling the dispensing fluid rate and the dissolution rate, contamination between adjacent test wells 621 can be avoided. In addition, the dry reagents in each detection well 621 may also be covered with a nucleic acid-friendly chemical, such as paraffin (paraffin). Once the sample distribution is complete, the chemicals coated dissolve at a temperature reached by the heating process, allowing the reagents coated underneath to be released and further mixed.

In some embodiments, each detection well 621 has a volume of 1 μ L to 200 μ L. The design of the detection slot 621 also facilitates optical detection. FIG. 10 shows a cross-sectional view of the detection well 621. The sample is distributed from the wide flow path portions 71, 71' and filled into the detection groove 621 via the groove inlet flow path 73. The cross-sectional area of the channel inlet channel 73 is significantly smaller than the wide channel portions 71, 71', and thus can act as a capillary valve for passive flow control. In some embodiments, the detection slot 621 has a thin wall 627 on the bottom surface during the manufacturing process, and the top surface of the reaction chip 62 is sealed by a film 628 to form a closed slot. In some embodiments, the reaction chip 62 has a through detection well 621, and the detection well 621 is sealed by a top film 628 and a bottom film 627. The thin bottom wall or membrane 627 includes an optical wall or film that ensures that excitation light efficiently reaches the detection well 621 through the thin bottom wall or membrane 627. Meanwhile, the detection well 621 has an optical front wall 629, so that a fluorescent signal emitted from a sample can pass through the front wall 629 of the detection well 621 with a low loss and maintain a high signal-to-noise ratio (S/N ratio). In one embodiment, the bottom thin wall or membrane 627 and the front wall 629 are light transmissive for light to pass through. In some embodiments, at least one of the bottom or top of the detection well 621 includes a thin, light-transmissive wall or film through which light can pass. In some embodiments, optical components having optical power (refractive index or refractive index) are not used in the system, in other words, the system does not have any lenses or mirrors in addition to the optically thin walls or films.

FIG. 11 shows the tank inside structure of the nucleic acid analyzing apparatus, with the fluid transport unit 2 removed and the outline of the tank 1 and the rotation driving unit 4 indicated by dotted lines, to more clearly show the tank 1 inside structure. As shown in fig. 2 and 11, the temperature control unit 3 includes a heater 31, a heat sink 32, and a plurality of blades 33. The heat sink 32 includes a plurality of heat radiating fins which are disposed around the heater 31 and mounted on the heater 31 so that heat generated from the heater 31 can be rapidly dissipated. The fan blades 33 are mounted on the rotary driving unit 4 and driven by the rotary driving unit 4, and the fan blades 33 rotate to generate an air flow toward the heat sink 32 to accelerate the heat mixing in the enclosed tank 1.

In some embodiments, the nucleic acid analysis apparatus 100 can be designed for isothermal nucleic acid amplification, so that only a fixed temperature is required, and thermal cycling control of three different temperature intervals is not required, and thus, the temperature control unit 3 can be significantly simplified. In addition, the tank 1 of the nucleic acid analysis apparatus 100 is designed to have good thermal insulation, so that the internal temperature can be easily maintained. Once the well 1 is in a uniform temperature environment, heat loss from the detection well 621 and the sample flowing to the environment can be minimized, and the temperature of the well 1 and the sample in each detection well 621, which are integrally closed, is substantially the same during the nucleic acid amplification and/or detection process, regardless of whether the cartridge 6 is in a rotating or stationary state.

The temperature control unit 3 provides a desired temperature inside the tank body 1 during operation, wherein the temperature control is not affected by the number and shape of the detection tanks 621. In one embodiment, the temperature control unit 3 further comprises at least one temperature sensor for controlling the accuracy of the temperature.

In one embodiment, the temperature control unit 3 heats the sample in a non-contact manner, such as, but not limited to, hot air convection, heat dissipation, infrared heating, microwave heating, or laser heating.

Alternatively, the temperature control unit 3 may heat the sample by contact heating. In one embodiment, the temperature control unit 3 is disposed in the bottom tank 12, and the detection tank 621 and the sample therein are directly heated by the temperature control unit 3 in a heat conduction manner.

In one embodiment, the temperature control unit 3 includes a detachable heater that can contact the detection groove 621 during amplification for the purpose of excellent heat conduction, and the heater can be removed from the cartridge 6 if necessary so that the cartridge 6 can be rotated.

The rotation driving unit 4 is mounted on the top tank body 11. The rotation driving unit 4 can be, but not limited to, a motor, and it can also be an electromagnetic device, a manual operation, a spring, a clockwork or other components, and can hold and rotate the cassette 6 at a predetermined angle, and sequentially pass each detection slot 621 through and align each optical unit 5. In one embodiment, the rotational drive unit 4 comprises a stepper motor that can drive the rotation of the fan blades 33 and the cassette 6 in different modes.

Fig. 12 shows the structures of the rotary drive unit 4, the cassette 6, and the optical unit 5. As shown in fig. 2 and 12, the rotary drive unit 4 further includes a cassette holder 41 for holding and rotating the cassette 6. Once the cassette 6 is held, it is driven by the rotation driving unit 4 to rotate in the slot 1. There are various mechanisms by which the cartridge 6 may be retained and released. In one embodiment, the cassette holder 41 includes a magnetic member 411, the magnetic member 411 includes a magnet, and accordingly, the cassette 6 may embed a magnetic member 64 in the reaction chip 62 during the manufacturing process, and the magnetic member 64 includes an iron sheet. When the cassette 6 is locked by the fastener 14, there is a slight gap of about 0.5mm to 3mm between the top surface of the cassette 6 and the cassette holder 41, and in this case, the rotary drive unit 4 drives only the rotation of the fan blade 33. Once the cassette 6 and the fastener 14 are released, the magnetic force between the magnetic member 411 and the magnetic member 64 causes the cassette 6 to move toward the cassette holder 41, so that the cassette 6 is held and fixed by the cassette holder 41 of the rotary drive unit 4, and thus the cassette 6 can freely rotate in the housing 1 during nucleic acid amplification and/or detection, and a slight gap exists between the bottom surface of the cassette 6 and the fluid delivery unit 2.

In other embodiments, the cassette holder 41 may be an electromagnetic device, a screw, a nut, a press-fit part (press-fit part), a friction part (friction part), a grip (grip), a pincer (pincer), an epoxy (epoxy), a chemical bonding (chemical bonding) or other forms, as long as it can hold the cassette 6 according to the use requirement.

In one embodiment of the present invention, the nucleic acid analysis device 100 includes a plurality of optical units 5. The optical unit 5 includes optical components such as a light source, lenses, filters, and a photodetector to enable optical detection so that the sample can be detected in real time during nucleic acid amplification. As shown in fig. 11 and 12, the optical unit 5 includes at least one light source 51 and at least one light detector 52. The light sources 51, such as Light Emitting Diodes (LEDs), are embedded in the slot body 1, and during operation, each light source 51 is aligned with one of the detection slots 621 of the cassette 6 to provide an effective light source for detection. Once the cartridge 6 is held, a photodetector 52, such as a photodiode (photodiode), is aligned with one of the detection slots 621 of the cartridge 6 for detection and analysis. The rotation of the cassette 6 allows each detection slot 621 to sequentially pass through different optical units 5. in one embodiment of the present invention, each optical unit 5 can provide light with a unique wavelength, thereby providing light with different colors for fluorescence detection, so that the nucleic acid analysis device 100 can simultaneously detect multiple targets and realize multiplex detection.

In one embodiment, the nucleic acid analysis apparatus 100 includes a controller for controlling the operations of the fluid delivery unit 2, the temperature control unit 3, the rotation driving unit 4, and the optical unit 5. In one embodiment, the controller may also control the release of the fasteners 14.

Since the isothermal amplification method is adopted, the entire system can be significantly simplified, and the nucleic acid analysis apparatus 100 can be designed to be compact, even smaller than a general cup. In one embodiment, the nucleic acid analysis device 100 has a height of 100mm to 120mm and a width of 80mm to 100 mm. Since the nucleic acid analysis device 100 is a cup size, it is portable and suitable for POC diagnosis.

In one embodiment, the nucleic acid analysis apparatus 100 is designed for isothermal amplification, and thus can be used for performing all isothermal amplification methods, such as nucleic acid sequence-based amplification (NASBA), Strand Displacement Amplification (SDA), helicase-dependent amplification (HAD), loop-mediated isothermal amplification (loop-mediated amplification), Recombinase Polymerase Amplification (RPA), and Nicking Enzyme Amplification (NEAR).

FIG. 13 is a flow chart showing the operation of the nucleic acid analysis apparatus, in which bold arrows indicate the flow of operation, white boxes indicate a number of major actions, white diamond boxes indicate major steps in completing the operation, gray boxes indicate core hardware components of the apparatus, communication from the controller to the core hardware components is indicated by dashed arrows, and the reaction by the core hardware components to generate default functions is indicated by thin line arrows. The operation flow of the nucleic acid analysis device 100 will be described below with reference to FIGS. 1 to 13.

The first step is to perform a manual operation. The top tank 11 of the nucleic acid analyzing apparatus 100 is opened. The sample is introduced into the cartridge 6 through the sample loading hole 624 of the reaction chip 62, wherein the reagents for sample purification and nucleic acid extraction have been previously introduced into the groove 631 of the cartridge body 63. After the sample is loaded into the cassette 6, the cassette 6 is loaded into the bottom slot 12, and once the cassette 6 is placed in the cavity 121 of the bottom slot 12, the positioning member 17 on the bottom slot 12 can assist the cassette 6 to align with the fluid delivery unit 2. In addition, by pressing down the cassette 6, the fastener 14 locks the cassette 6 and brings the cassette 6 into close contact with the fluid delivery unit 2. The top tank 11 is then closed for sample processing.

The second step performs sample purification and nucleic acid extraction. In this step, the sample processing procedure is performed in the cassette 6, and reagents such as biochemical buffers are transported to the position to be transported by the fluid transport unit 2. After sample purification and nucleic acid extraction are completed, the sample with the extracted nucleic acids is dispensed to the detection slot 621 of the cartridge 6 for subsequent nucleic acid amplification and/or detection.

In the third step, the tank body 1 is heated by the temperature control unit 3. In this step, the heater 31 is activated to perform heating. The rotation driving unit 4 drives the rotation of the fan blades 33 to harmonize the temperature inside the tank body 1 and generate an air flow toward the heat sink 32 to accelerate the thermal mixing inside the closed tank body 1. In addition, a temperature sensor may monitor the sample temperature.

The fourth step performs nucleic acid amplification and nucleic acid detection. When the sample temperature reaches the predetermined value, the rotation of the fan blade 33 is stopped and the fastener 14 is unlocked to release the cartridge 6, while the heater 31 is continuously operated to maintain the temperature. Then, the cassette holder 41 holds the cassette 6, and isothermal amplification is started. Once the cartridge 6 is held, the cartridge 6 is driven by the rotation driving unit 4 to rotate within the slot 1. The cassette 6 may be rotated a certain angle so that the detection slot 621 is aligned with the optical unit 5 and stationary for a short period of time (e.g., 200 milliseconds) for detection. Thus, each detection slot 621 may pass through a series of light sources having different colors, and the emitted light may be detected by a light detector 52 (e.g., a photodiode).

After the detection is completed, the detection result is transmitted to a cloud or digital device, such as a personal computer, a tablet or a smart phone, via a USB, bluetooth or a network, and the slot 1 is opened to discard the cartridge 6.

In the above embodiment, the cassettes 6 are exemplified as one-to-one cassettes, i.e., one sample at a time is tested. However, in some embodiments, the cassette 6 may have more than one sample loading aperture 624, or the cassette 6 may be a many-to-one cassette, i.e., multiple (e.g., X) samples at a time. FIG. 14 shows a many-to-one cassette, such as an eight-to-one cassette, that can detect multiple samples. The reaction chip 62 includes eight sample loading wells 624 for loading eight different samples into the cassette 6. In the cassette body 63 and the reaction chip 62, the entire internal space is divided into eight sub-sections, each of which is responsible for processing and detecting one sample. In some embodiments, the subcomponents may share some common slots, such as a waste fluid slot. Therefore, when a single cassette is installed in a nucleic acid analysis device, many pairs of cassettes can have flexible throughput, and a user can add a plurality of different samples into the single cassette for detection without changing the device, so that the cassette has flexible throughput (1-X) and does not increase hardware cost, and therefore, the cassette and the device provided by the embodiment of the invention can be used as a simple, convenient and economic technical scheme for achieving medium and high throughput. Figure 15 shows the liquid dispensing and oil sealing procedure for three-to-one cartridge. The cassette space is divided into three sub-sections in which the liquid distribution and oil sealing processes are sequentially performed in a clockwise direction, the colored liquids in the three sub-sections representing three different sample extracts to be added to the cassette 6.

In summary, the present invention provides a full-function integrated (all-in-one) nucleic acid analysis apparatus, which can be applied to an isothermal amplification method, and integrates a fluid delivery unit, a temperature control unit, a rotation driving unit and an optical unit into a single apparatus, so that processes such as sample purification, nucleic acid extraction, nucleic acid amplification and/or nucleic acid analysis can be performed on the full-function integrated apparatus to achieve real-time nucleic acid analysis. In particular, in some embodiments, the fluid control and processing cassettes for nucleic acid analysis devices have well-designed flow channel geometries that precisely control the flow direction and dynamic fluid behavior of the fluid in the reaction chip, allowing for sequential and smooth distribution of the sample to each detection well, thereby facilitating subsequent nucleic acid amplification and detection. Furthermore, by arranging multiple detection tanks and multiple optical units, multiple nucleic acid analyses and multiple color detection can be achieved. In addition, since the entire system is significantly simplified, the nucleic acid analysis device can be designed compactly, and thus, is portable and suitable for POC diagnosis, and also significantly reduces the cost of nucleic acid analysis. In addition, the nucleic acid analysis device has good sensitivity and specificity, and flexible detection flux.

While the present invention has been described in detail in connection with the above embodiments, it will be apparent to those skilled in the art that various modifications may be made thereto without departing from the scope of the invention as set forth in the appended claims.

Claims

1. A fluid control and processing cartridge for a nucleic acid analysis device, comprising:

the cartridge body comprises a plurality of grooves and a plurality of flow channels, the grooves are suitable for storing at least one sample, a plurality of biochemical reagents and buffer solutions, the flow channels are connected with the grooves and connected with a pump so as to drive fluid to be conveyed by the pneumatic force provided by the pump; and

a reaction chip combined with the cassette body and including a sample inlet, multiple detection grooves, at least one main fluid channel and at least one gas release channel, wherein the main fluid channel is connected with the detection grooves and is suitable for distributing the sample to the detection grooves, the gas release channel is directly connected with each of the detection grooves and is suitable for releasing gas from the detection grooves, the sample inlet is connected with the main fluid channel, the main fluid channel includes multiple wide channel parts, multiple narrow channel parts and multiple detection groove inlet channels, each wide channel part is aligned with one of the detection grooves and is connected with the corresponding detection groove through the corresponding detection groove inlet channel, and each narrow channel part is connected between two adjacent wide channel parts, wherein the gas release channel is connected with the wide channel part of the main fluid channel, The narrow flow channel part and the detection groove inlet flow channel are both obviously narrower, when a liquid is driven by the pneumatic force to feed in from the sample inlet, the liquid is firstly filled in the wide flow channel part of a first detection groove corresponding to the plurality of detection grooves and is delayed by an adjacent narrow flow channel part, and enters the first detection groove through the detection groove inlet flow channel corresponding to the first detection groove, and the gas remained in the first detection groove can be pushed out by the inflowing liquid through the gas release flow channel and flows to the adjacent detection groove, when the first detection groove is filled with the liquid, the liquid further overcomes the flow resistance of the adjacent narrow flow channel by the pneumatic force, enters the wide flow channel part corresponding to the next detection groove of the first detection groove, and is delayed by the other adjacent narrow flow channel part, and enters the next detection groove through the detection groove inlet flow channel corresponding to the next detection groove, thereby filling the next test slot.

2. The fluid control and processing cassette of claim 1, wherein the narrow flow channel portion has a higher flow resistance than a total flow resistance of the wide flow channel portion and the detection slot inlet channel.

3. The fluid control and processing cassette of claim 2, wherein the narrow flow path portion has a flow resistance 2 to 20 times higher than the total flow resistance of the wide flow path portion and the detection slot inlet flow path.

4. The fluid control and processing cassette of claim 1, wherein the gas release flow channel has a flow resistance 2 to 500 times higher than the flow resistance of the narrow flow channel portion.

5. The fluid control and processing cassette of claim 1, wherein the cross-sectional area of the test slot inlet flow channel is substantially smaller than the wide flow channel portion.

6. The fluid control and processing cassette of claim 1, wherein the reaction chip is disposed on one side of the cassette body.

7. The fluid control and processing cassette of claim 1, wherein each of the detection wells has at least one flat surface.

8. The fluid control and processing cassette of claim 1, wherein the reaction chip is substantially in the shape of a regular polygon.

9. The fluid control and processing cassette of claim 1, wherein the reaction chip further comprises at least one sample loading well for adding the sample to the cassette.

10. The fluid control and processing cassette of claim 1, wherein the reaction chip further comprises a plurality of sample loading wells for adding different samples to the cassette.

11. The fluid control and processing cassette of claim 1, wherein the cassette is mounted in a housing of the nucleic acid analysis device, and the reaction chip comprises at least one alignment slot that is alignable with at least one positioning element on the housing.

12. The fluid control and processing cassette of claim 1, wherein the reaction chip comprises at least one sample inlet port, and the cassette body comprises at least one conduit connected to the sample inlet port for delivering the sample to the reaction chip.

13. The fluid control and processing cassette of claim 1, wherein the cassette body further comprises a plurality of openings in a bottom surface thereof, the openings communicating with the channels through the flow channels.

14. The fluid control and processing cassette of claim 1, wherein at least one of the bottom or top of the detection well comprises a thin, light-transmissive wall or membrane through which light can pass.

15. The fluid control and processing cassette of claim 1, wherein the detection slot has a light-transmissive front wall through which light can pass.