Three-dimensional robot sequencing device

Abstract

A nucleotide sequencing device comprising a plurality of microfluidic chips configured for nucleic acid sequencing and a reagent distribution manifold having at least one distribution port wherein the manifold is operable to move in at least two dimensions relative to the plurality of microfluidic chips to allow reagent to be distributed from the manifold to the microfluidic chips, there is no need to use a common line between the dispensing port and the microfluidic chip when certain reagents are requested.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 381,070, filed Oct. 26, 2022, the subject matter of which is incorporated herein by reference in its entirety. Background Art

[0003] Microfluidic devices / chips are widely used in bio / medical applications. Especially in next-generation sequencing (NGS) systems, such devices are used to immobilize template nucleic acid molecules derived from biological samples and then introduce a repetitive flow of synthesis sequencing reagents to attach labeled nucleotides to specific positions in the template sequence. A series of labeled signals are detected and decoded to reveal the nucleotide sequence of the template molecule, such as the immobilized and / or amplified nucleic acid template molecules attached to the inner surface of the microchannels of the device.

[0004] Typical devices use integrated pumps and valves to control the flow / delivery of different reagents. The common pipelines between the valves and the microfluidic chip are inevitable and additional flushing volumes are required during reagent exchange. A large amount of reagent is wasted to flush the common pipelines, especially when the microfluidic channels are very small. Summary of the Invention

[0005] This application describes a new design of a nucleotide sequencing device that saves reagents during fluid exchange. The nucleotide sequencing device includes: a plurality of microfluidic chips configured for nucleotide sequencing; and a reagent distribution manifold that is discontinuously connected to the microfluidic chips to distribute or deliver nucleotide sequencing reagents to each microfluidic chip. Each microfluidic chip includes a microchannel having an inlet configured to receive a reagent and an outlet in fluid communication with a waste collection unit. The reagent distribution manifold includes at least one reagent distribution port. When a chip requests certain reagents, the reagent distribution manifold is operable to move relative to the plurality of microfluidic chips in at least two or three dimensions to allow at least one distribution port to form a leak-proof fluid connection with each inlet of the microchannel after the movement. At least one distribution port is configured to disconnect from the inlet of the microchannel after delivering the reagent and / or when the reagent distribution manifold moves. Advantageously, compared with conventional nucleotide sequencing devices, the common pipeline between the valve / pump and the microfluidic channel is not shared. Not sharing the common pipeline for delivering reagents to the microchannel can significantly improve the use of expensive reagents and reduce the operating cost of the nucleotide sequencing device.

[0006] In some embodiments, the device includes a platform that generally extends in the x-y plane of an x-y-z coordinate system. A plurality of microfluidic chips are disposed on the surface of the platform, and a reagent dispensing manifold is positioned above the platform in the z direction. The device further includes a robotic arm that can move the reagent dispensing manifold in the x direction, y direction, and / or z direction to position a reagent dispensing port above an inlet of a corresponding microfluidic chip.

[0007] In some embodiments, the device includes at least one reagent reservoir that is in fluid communication with the reagent dispensing manifold and at least one dispensing port, and at least one fluid pump configured to pump a reagent from the reagent reservoir to the manifold and through the manifold to the at least one dispensing port. The fluid pump can include a selector valve that is operable to control a selected reagent from the reagent reservoir to be pumped through the manifold to the at least one dispensing port. The fluid pump can include a vacuum actuator or a pressure or syringe pump to facilitate transfer of the reagent from the reagent reservoir for dispensing by the dispensing port.

[0008] In some embodiments, the device further includes a heating unit configured to heat the plurality of microfluidic chips and an imaging module for imaging the microfluidic channels of the corresponding microfluidic chips. The imaging module can be disposed in an imaging area on the platform that is separate from a sequencing area where the plurality of microfluidic chips are disposed for receiving reagents and sequencing. The robotic arm can transfer a microfluidic chip from the sequencing area to the imaging area. The robotic arm can include a vacuum aspiration device to hold the microfluidic chip for transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Illustrates the layout of a robotic sequencing device having an aggregation / sequencing station, a chip storage area, and an optical imaging / scanning station.

[0010] Figure 2 Illustrates a microfluidic chip having an inlet in a top glass and an outlet in a bottom glass.

[0011] Figure 3 Illustrates a schematic diagram of a reagent delivery unit.

[0012] Figure 4 Illustrates a prototype instrument with 3-axis movement, a reagent delivery unit, a heating stage, and a waste collection unit.

[0013] Figure 5 Illustrates a reagent / fluid manifold and dispensing port with Z-axis movement.

[0014] Figure 6 Illustrates a vacuum-driven reagent delivery setup.

[0015] Figure 7Illustrated is a pressure-driven reagent delivery arrangement. Detailed Description

[0016] The embodiments described herein relate to devices and systems for delivering reagents to microfluidic channels and, in particular, to nucleotide sequencing devices and systems that can achieve more efficient use and minimal waste of reagents during fluid exchange to reduce the time and cost of nucleotide sequencing. The devices and systems can be used, for example, for comparative genomics sequencing, tracking gene expression, microRNA sequence analysis, epigenomics, aptamer and phage display library profiling, and other sequencing applications. The devices and systems herein include different combinations of optical, mechanical, fluidic, thermal, electrical, and computational devices / aspects. Advantages conferred by the devices and systems described herein include, but are not limited to: (i) reduced device and system manufacturing complexity, operation, and cost, (ii) significantly reduced consumption costs (e.g., compared to those of currently available nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexible flow control when combined with microfluidic components (e.g., syringe pumps and diaphragm valves, etc.), and (v) flexible system throughput.

[0017] Figure 1 Illustrated is a schematic diagram of a robotic nucleotide sequencing device 10 in accordance with embodiments described herein. The nucleotide sequencing device 10 includes a plurality of microfluidic chips 12 configured for nucleotide sequencing and a reagent delivery unit 14. The microfluidic chips 12 can be arranged on a surface 30 of a platform 32 generally extending in the x-y plane of an x-y-z coordinate system.

[0018] Each microfluidic chip 12 ( Figure 2 ) includes a microchannel 20 that extends generally the length of the microfluidic chip 12 from an inlet 22 on an upper surface 24 of the microfluidic chip 12 to an outlet 26 on an opposite lower surface 28 of the microfluidic chip 12. The inlet 22 can be configured to receive reagents from a reagent distribution manifold 34 of the reagent delivery unit 14, and the outlet 26 can be in fluid communication with a waste collection unit (not shown).

[0019] The platform 30 includes a sequencing area or stage 40 that is configured to be loaded with more than one microfluidic chip 12 and to run different recipes in different channels 20 and chips 12. This design can enable a very flexible sequencing instrument with a flexible throughput of from 10M to 5000M reads on the same instrument, and sequencing libraries can be loaded into channels 20 at any time in any available channel 20.

[0020] The inlet 22 of the microfluidic chip 12 can face upward toward the reagent dispensing manifold 14, and the outlet 26 can face downward to connect to the waste port of the waste collection unit. This design also enables stacking of the microfluidic chips 12 during aggregation or other chemical steps, as the outlet 26 of the first microfluidic chip 12 can be stacked on the inlet 22 of the second microfluidic chip 12, which can simplify the manufacturing process of the microfluidic chip 12 and provide further cost reduction compared to traditional designs.

[0021] The microchannels 20 can have a height and width on the order of 1 nm to 1000 μm. For example, in some embodiments, the microchannels can have a depth of 1 - 50 μm, 1 - 100 μm, 1 - 150 μm, 1 - 200 μm, 1 - 250 μm, 1 - 300 μm, 50 - 100 μm, 50 - 200 μm, or 50 - 300 μm or greater than 300 μm or a depth within a range defined by any two of these values. In some embodiments, the microchannels can have a length less than 0.1 mm, between 0.1 mm and 0.5 mm, between 0.1 mm and 1 mm, between 0.1 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 25 mm, between 0.1 mm and 50 mm, between 0.1 mm and 100 mm, between 0.1 mm and 150 mm, between 0.1 mm and 200 mm, between 0.1 mm and 250 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 1 mm and 50 mm, between 1 mm and 100 mm, between 1 mm and 150 mm, between 1 mm and 200 mm, between 1 mm and 250 mm, between 5 mm and 10 mm, between 5 mm and 25 mm, between 5 mm and 50 mm, between 5 mm and 100 mm, between 5 mm and 150 mm, between 5 mm and 200 mm, between 1 mm and 250 mm or greater than 250 mm or a length within a range defined by any two of these values. In some embodiments, the microchannel length can be in the micrometer range.

[0022] The material for the microfluidic chip 12 used to fabricate the nucleotide sequencing device 10 described herein is typically optically transparent to facilitate use with spectroscopy - or imaging - based detection techniques. In some cases, the entire microfluidic chip 12 will be optically transparent. In some cases, only a portion of the microfluidic chip 12 (e.g., an optically transparent "window") will be optically transparent.

[0023] The microfluidic chip 12 can be fabricated by a combination of microfabrication processes. The method of fabricating the microfluidic chip 12 can include: providing a surface; and forming at least one channel on the surface. The fabrication method can further include: providing a first substrate having at least a first flat surface, wherein the first surface has a plurality of channels; providing a second substrate having at least a second flat surface; and bonding the first flat surface of the first substrate to the second flat surface of the second substrate. In some examples, the channels on the first surface have an open top side and a closed bottom side, and the second surface is bonded to the first surface through the bottom side of the channel and thus leaves the open top side of the channel unaffected. In some cases, the methods described herein further include providing a third substrate having a third flat surface and bonding the third surface to the first surface through the open top side of the channel. The bonding conditions can include, for example, heating the substrates or applying an adhesive to one of the flat surfaces of the first or second substrate.

[0024] Typically, because microfluidic chips are microfabricated, the substrate materials are selected based on their compatibility with known microfabrication techniques (e.g., lithography, wet chemical etching, laser ablation, laser irradiation, air abrasion techniques, injection molding, embossing, and other techniques). The substrate materials are also typically selected based on their compatibility with the full range of conditions to which the microfluidic chip may be exposed, the full range of conditions including extreme pH, temperature, salt concentration, and the application of irradiation or electric fields. Thus, in some preferred aspects, the substrate materials can include silica - based substrates such as borosilicate glass, quartz, and other substrate materials.

[0025] In some embodiments, the fabrication of the microfluidic chip 12 includes laminating or bonding two or more substrate layers in order to produce the chip 12. For example, in a microfluidic chip, the microfluidic elements of the chip are typically produced by laser irradiation, etching, or otherwise fabricating features into the surface of a first substrate. Then a second substrate is laminated or bonded to the surface of the first substrate to seal these features and provide the fluid elements of the chip, such as fluid channels.

[0026] See again Figure 1, the reagent dispensing manifold 34 is positioned above the platform 32 and the microfluidic chip 12 in the z-direction and is configured to move relative to the microfluidic chip 12 in at least two dimensions or three dimensions (e.g., the x, y, and / or z directions) by the robotic arm 50. When the chip 12 requests certain reagents, the reagent dispensing manifold 14 can be configured to dispense reagents from a reagent reservoir (not shown) to the inlets of the microchip 12 disposed on the platform 30 through at least one reagent dispensing port 52. At least one dispensing port 52 can form a leak-proof fluid connection with each inlet of the microchannel after the movement of the reagent dispensing manifold 34. At least one dispensing port 52 is configured to disconnect from the inlet of the microchannel after delivering the reagent and / or when the reagent dispensing manifold 34 moves. The reagent delivery unit 14 and the reagent dispensing manifold 34 are capable of sucking a large amount of liquid into at least one dispensing port 52 and depositing those amounts of liquid from at least one dispensing port 52. The movement and operation of the reagent delivery unit 14 and the reagent dispensing manifold 34 are generally controlled by a processor (not shown) such that the reagent dispensing operation can be automated.

[0027] Advantageously, the reagent delivery unit 14 can be configured such that any pumps, sensors, sample identification verifiers, and other items move with it, and thus minimize the number of control lines that cross the device 10 or the platform 30 during use and reduce the likelihood that such control lines will become entangled during the movement of the reagent delivery unit 14. In some embodiments, the reagent delivery unit 14 and the reagent dispensing manifold 34 are the only items that undergo movement and remain in communication with other components fixed at different points within the device 10. The reagent delivery unit 14 can also be configured to align at least one dispensing port 52 with the microfluidic chip inlet 22 using an electrified alignment plate.

[0028] Figure 3 The components of the reagent delivery unit 100, as further described herein, are schematically illustrated. Figure 3The layout of the components in [the device] is for convenience only, and those skilled in the art will understand that other arrangements are possible depending on the circumstances and other factors. The reagent delivery unit 100 includes a reagent delivery manifold 102 that has two dispensing ports 104, 106 mounted to the reagent delivery manifold 102. Other numbers of dispensing ports (such as 1, 3, 4, 5, 6, 7, 8, 9, and 10) are consistent therewith. The dispensing ports 104, 106 are fluidly connected via separate connection lines 114, 116 to corresponding reagent reservoirs 110, 112 such that separate or discrete reagents are transferred from the reagent reservoirs 110, 112 through the corresponding connection lines 114, 116 and the reagent dispensing manifold 102 and directly to the corresponding dispensing ports 104, 106 without traveling through a common line. The reagent dispensing manifold 102 is movably attached to the mounting portion 122 of the reagent delivery unit 100 via a connection member 120. The relative position of the reagent dispensing manifold 102 and the mounting portion 122 in the illustrated z-direction can be controlled by a Z motor 130 that is electrically coupled via an electrical connection to the connection member 120 and the mounting portion 122. In some embodiments, the Z motor 130 may receive instructions from a processor (not shown) via an electrical connection. In other embodiments, the Z motor 130 may control the relative position of the reagent dispensing manifold 102 and the mounting portion 122 by moving the reagent dispensing manifold 102. In other embodiments, the Z motor 130 is coupled to the mounting portion 122 and effects a similar relative movement of the mounting portion 122 and the support member 102. Such relative movement can be achieved by any suitable mechanical moving device (such as a gear drive or a rack and pinion assembly or a lead screw), the details of which are not shown in Figure 3 the [device].

[0029] Optionally, the reagent delivery unit 100 may include a sensor (not shown) that is configured to sense when the vertical movement of the reagent dispensing manifold 102 or the mounting portion 122 is impeded and provide a suitable signal directly to a processor (not shown) via an electrical connection (not shown), or indirectly (not shown) via a printed circuit board (not shown). Thus, depending on the matter of design choice, the sensor may be mounted on the reagent dispensing manifold 102 or on the mounting portion 122.

[0030] Valves 140, 142 are associated with each connection line 114, 116 and dispensing port 104, 106 and are used to control the operation of each dispensing port 104, 106, for example by controlling when to reduce pressure, thereby causing a suction operation, or increasing pressure, thereby causing a dispensing operation. Each valve 140, 142 is connected to (including being in fluid communication with) the reagent dispensing manifold 102.

[0031] The operation of the reagent delivery unit 100 is typically controlled by a printed circuit board (PCB) (not shown) to which the reagent delivery unit is connected via electrical connectors. Thus, the aspiration and dispensing operations can be precisely controlled by signals from the PCB, enabling precise volume control. In some embodiments, the reagent delivery unit 100 needs to be calibrated so that the amount of time for forcing or aspirating air required to dispense or aspirate a desired volume of reagent is known. Thus, for example, the time between valve opening and valve closing, which is controlled by a signal, is known and can be incorporated into the control software.

[0032] Figure 4 is an image illustrating an example of a nucleotide sequencing device 200 that includes a reagent delivery unit 202 as described herein. Those of ordinary skill in the art will understand that such components, their relative configurations, numbers, and orientations are exemplary, and the degrees of freedom of movement and the accuracy of positioning and dispensing consistent with the description herein can be achieved by other such configurations. For example, in cases where one or more mounting portions are shown, other embodiments may have different numbers of mounting portions.

[0033] The nucleotide sequencing device 200 includes a gantry 204 that provides movement of the attached reagent delivery unit 202 relative to a platform 206, and a plurality of microchips 208 are arranged on the platform 206. The gantry 204 includes a horizontal track 210 to allow the reagent delivery unit 202 to move in the x-direction, and the horizontal track 210 is controlled by a controller assembly. Orthogonally arranged tracks 212, 214 allow the attached reagent delivery unit 202 to move in the y-direction of the tracks 212, 214.

[0034] Control bands 220, 222 are provided to be orthogonal to each other and provide movement of the reagent delivery 202 in two orthogonal directions, the two orthogonal directions generally being in the x-y plane of the x-y-z coordinate system along the horizontal track 210 and the orthogonally arranged tracks 212, 214. The control bands 220, 222 may also hold cables and are arranged to allow movement in the horizontal plane. The control bands 220, 222 allow easy movement of the reagent delivery 202 without tangling various cables. The cables may supply control signals to a control assembly that houses circuitry to control the operation of the reagent delivery unit 202 and the pump / valve 230 of the reagent delivery unit 202. Thereby, the reagent delivery unit can move in two horizontal directions (x-y axes).

[0035] The vertically movable extension shaft 232 of the reagent delivery unit 202 connected to the mounting portion 234 provides movement of the reagent distribution manifold 236 and the reagent distribution port 238 of the reagent delivery unit 202 in the Z direction. Cables can supply control signals to the reagent delivery unit 202 connected to a motor to effect vertical movement and thereby allow control of such movement.

[0036] The gantry 204 and the shaft 232 thus generally allow three translational degrees of freedom of the reagent distribution manifold 236 and the distribution port. Other embodiments not described herein may include gantries having less than three translational degrees of freedom. Thus, the gantry provides two axes of belt-driven sliders driven by an encoded stepper motor. The gantry sliders can be mounted on a frame of structural angle aluminum or other equivalent material, particularly metal or metal alloy. The sliders aligned in the x and y directions respectively facilitate movement of the dispenser across the microfluidic chip array and in a direction along a given holder. The Z-axis of the gantry can be associated with a variable force sensor that can be configured to control the degree of vertical movement of the shaft, the mounting portion, and the dispenser port during a reagent dispensing operation.

[0037] The translational movement of the reagent delivery unit 202, the reagent distribution manifold 236, and the distribution port 236 in three dimensions can be controlled by a microprocessor (not shown). Each distribution port 238 includes a separate connecting line 240 to a corresponding reagent reservoir 250. This design enables simplification of the assembly of the nucleotide sequencing device 200, minimizes reagent contamination and sample cross-contamination between different examples of the operation of the device 200, increases pumping efficiency (minimum dead volume), and enables easy maintenance and repair of the device 200. This arrangement also enables easy upgrading of features in the reagent delivery unit 202 and the reagent distribution manifold 236, such as distribution ports and connecting lines to different reagent reservoirs and individual and independent pump control for each reagent distribution port.

[0038] It will be understood that the reagent delivery unit 202 can be configured to perform fluid transfer operations simultaneously on two or more distribution ports, such as when operating under instructions received from one or more electrical controllers.

[0039] Figure 5 is the image of the reagent distribution manifold 236 of the reagent delivery unit 202 having Z-axis movement Figure 4 The reagent distribution manifold 236 is in fluid communication with a connecting line 240 that is in fluid communication with a pump and / or valve 230 and a reagent reservoir 250. The distribution ports 238 are mounted to the reagent distribution manifold 236, which is attached to the shaft 232 of the reagent delivery unit and allows the reagent distribution ports 236 to move vertically up and down. The mounting can be via mechanical fasteners, such as one or more screws.

[0040] The reagent dispensing port 238 includes an O-ring 260 that can form a leak-proof fluid connection with the inlet of a microchannel (not shown) after moving the reagent dispensing manifold 236 and / or the dispensing port 238. Any expensive reagents (such as incorporation mixtures, lysis mixtures, or enzyme mixtures) will have their own dispensing ports, and their own dispensing ports eliminate the need to flush a shared common volume.

[0041] In most conventional nucleotide sequencing device designs that include a common pipeline from different reagent reservoirs to a single reagent dispensing port, the common pipeline can have a length ranging from about 30 mm to about 300 mm, where the inner diameter ranges from about 0.5 mm to about 2 mm. The volume of the common pipeline can range from 6 μL to 1000 μL. The common volume consumes most of the reagents for the sequencing protocol, especially for small flow chip specifications. A microfluidic channel of 31×3.2×0.08 mm only requires about 8 μL of reagent to fill the entire channel, which is less than the common pipeline volume of about 20 μL (if the inner diameter is 0.5 mm and the length is 100 mm). The common pipeline volume limits the minimum reagent consumption of small-sized sequencing instruments and can prevent further price reduction of low-throughput sequencing instruments.

[0042] In contrast, for the nucleotide sequencing devices described herein, by dispensing all expensive reagents to dedicated reagent dispensing ports, the minimum reagent consumption is proportional to the size of the microfluidic channel rather than the common pipeline. In addition, the reagent injected into the microfluidic channel can be withdrawn to the reagent reservoir by sucking or aspirating the reagent back from the channel to the dispensing port, because there is no common cross-contamination between the reagents, which is common in traditional designs that use common pipelines.

[0043] In some embodiments, the reagent dispensing port 238 can be configured such that a force upward against the port 238, such as when the O-ring 260 of the dispensing port 238 encounters the inlet of the microfluidic chip, can be sensed by relative movement between the reagent dispensing port 238 and a force sensor (not shown). The force sensor can communicate with a processor or controller on a PC board, and the processor or controller at least controls the vertical movement of the dispensing port 238 such that the processor or controller can send an instruction to stop the vertical movement of the dispenser port 238 when receiving an appropriate signal from the force sensor.

[0044] The reagent delivery unit 202 can be configured to dispense reagents into the microfluidic chip. Generally, the reagent delivery unit 202 is configured to receive or dispense an amount of reagent of about 10 μl or less (such as an amount of fluid in the range of about 0.1 μl to about 10 μl) in a single operation.

[0045] The nucleotide sequencing device described herein can provide fluid flow control capabilities for delivering a sample or reagent to one or more microchannels of a microfluidic chip connected to a dispensing port. Reagents and buffers can be stored in bottles, kits, buffer cartridges, or other suitable containers connected to a reagent dispensing manifold through tubing and valve manifolds. The device can also include a processed sample reservoir and a waste reservoir in the form of a bottle, cartridge, or other suitable container for collecting the fluid downstream of these microfluidic chips. In some embodiments, the fluid flow control module can provide programmable switching of the flow between different sources, e.g., sample or reagent reservoirs or bottles located within the device and different dispensing ports of the microchannels of the microfluidic chip. In some embodiments, the fluid flow control module can provide programmable switching of the flow between sample reservoirs, waste reservoirs, etc., connected to the dispensing ports of the system. In some cases, the sample, reagent, and / or buffer can be stored within reservoirs integrated into the reagent dispensing manifold itself.

[0046] The control of the fluid flow through the reagent dispensing manifold, reagent dispensing ports, and the microchannels of the microfluidic chip will typically be carried out by using pumps (or other fluid actuation mechanisms) and valves (e.g., programmable pumps and valves). Examples of pumps include, but are not limited to, syringe pumps, programmable syringe pumps, peristaltic pumps, diaphragm pumps, etc. Examples of valves include, but are not limited to, check valves, electromechanical two-way or three-way valves, pneumatic two-way and three-way valves, etc. In some embodiments, the fluid flow through the reagent dispensing manifold, reagent dispensing ports, and the microchannels of the microfluidic chip can be controlled by applying positive pneumatic pressure to one or more inlets of the reagent and buffer containers, or to inlets incorporated into the reagent dispensing manifold, or by creating a vacuum at one or more outlets of the waste reservoir, or at one or more outlets incorporated into the microchannels of the microfluidic chip. For example, as Figure 6 shown, in the pressure-driven mode, the selected reagent is aspirated from the reagent pool via a selector valve integrated with the pump and injected into the microfluidic channel via the selector valve of the pump. In this case, the outlet of the microfluidic channel can lead to the atmosphere or be connected to a vacuum source. As Figure 7 shown, in the vacuum-driven mode, the outlet of the microfluidic channel is connected to a vacuum source, such as a syringe pump or a vacuum generator. The upstream of the microfluidic channel is controlled by a valve that will be opened during reagent delivery.

[0047] In some cases, different modes of fluid flow control are utilized at different points during the assay or analysis process, e.g., forward flow (relative to the inlet and outlet of a given microchannel of the microfluidic chip), reverse flow, oscillatory or pulsatile flow, or a combination thereof. In some applications, for example, during the assay wash / rinse steps, oscillatory or pulsatile flow can be applied to facilitate complete and efficient exchange of the fluid within one or more microchannels of the microfluidic chip.

[0048] In some cases, different fluid flow rates can be utilized at different points in the assay or analysis process workflow. For example, in some cases, the volumetric flow rate can vary from -100 μl / sec to +100 μl / sec. In some embodiments, the absolute value of the volumetric flow rate can be at least 0.001 μl / sec, at least 0.01 μl / sec, at least 0.1 μl / sec, at least 1 μl / sec, at least 10 μl / sec, or at least 100 μl / sec. In some embodiments, the absolute value of the volumetric flow rate can be at most 100 μl / sec, at most 10 μl / sec, at most 1 μl / sec, at most 0.1 μl / sec, at most 0.01 μl / sec, or at most 0.001 μl / sec. The volumetric flow rate at a given time point can have any value within this range, for example, a forward flow rate of 2.5 μl / sec, a reverse flow rate of -0.05 μl / sec, or a value of 0 ml / sec (i.e., flow stopped).

[0049] Referring again to Figure 4 , the nucleotide sequencing device 200 can include temperature control functionality for the purpose of facilitating the accuracy and reproducibility of assay or analysis results. Examples of temperature control components that can be incorporated into the device 200 for controlling the temperature of the individual or corresponding microfluidic chip 12 include resistive heating elements, infrared light sources, Peltier heating or cooling devices, heat sinks, thermistors, thermocouples, etc. In some embodiments, the temperature control module or heating stage 300 (or "temperature controller") can provide a programmable temperature change at a specified, adjustable time prior to performing a particular assay or analysis step. In some cases, the temperature controller can provide a programmable temperature change over a specified time interval. In some embodiments, the temperature controller can further provide a temperature cycle between two or more set temperatures with a specified frequency and ramp rate such that thermal cycling for an amplification reaction can be performed.

[0050] In some embodiments, the nucleotide sequencing device 10 can further include imaging capabilities 400 for imaging the microchannels of the microfluidic chip, such as optical imaging or other spectroscopic measurement capabilities. As Figure 1 shown, the imaging capabilities can be separated on the device 10 from the region 40 where nucleic acid sequencing (e.g., heating / sequencing chemistry stage) occurs. The microfluidic chip 12 selected for imaging can be moved and repositioned by a robotic arm via a vacuum chuck (not shown) to the designed imaging stage 400 of the platform 30. Once removed from the heat source at the heating / sequencing chemistry stage and with the help of a heat sink at the imaging stage 400, the microfluidic chip 12 can naturally cool to room temperature.

[0051] The imaging capabilities can include any one of a variety of imaging modalities known to those skilled in the art (including bright field, dark field, fluorescence, luminescence, or phosphorescence imaging). In some embodiments, the microfluidic chip includes a window that allows at least a portion of the microchannel to be illuminated and imaged.

[0052] In some embodiments, single-wavelength excitation and emission fluorescence imaging can be performed. In other embodiments, dual-wavelength excitation and emission (or multi-wavelength excitation or emission) fluorescence imaging can be performed. In some cases, the imaging module is configured to acquire video images. The choice of imaging modality can affect the design of the microfluidic chip, since all or a portion of the microfluidic chip will necessarily need to be optically transparent in the spectral range of interest. In some embodiments, a series of images can be "tiled" to produce a single high-resolution image of the microchannels within the microfluidic chip.

[0053] The spectroscopy or imaging module can include, for example, a microscope equipped with a CCD camera with CMOS. In some cases, the spectroscopy or imaging module can include, for example, a custom instrument configured to perform a particular spectroscopy or imaging technique of interest. Generally, the hardware associated with the imaging module can include a light source, a detector, and other optical components, as well as a processor or computer.

[0054] Any of a variety of light sources can be used to provide imaging or excitation light, including but not limited to tungsten filament lamps, tungsten-halogen lamps, arc lamps, lasers, light-emitting diodes (LEDs), or laser diodes. In some cases, a combination of one or more light sources and additional optical components, such as lenses, filters, apertures, diaphragms, mirrors, etc., can be configured as an illumination system (or subsystem).

[0055] Any of a variety of image sensors can be used for imaging purposes, including but not limited to photodiode arrays, charge-coupled device (CCD) cameras, or complementary metal-oxide semiconductor (CMOS) image sensors. As used herein, an "imaging sensor" can be a one-dimensional (linear) or two-dimensional array sensor. In many cases, a combination of one or more image sensors and additional optical components (e.g., lenses, filters, apertures, diaphragms, mirrors, etc.) can be configured as an imaging system (or subsystem). In some cases, for example, in the case where spectral measurements are performed by the system rather than imaging, suitable detectors can include but are not limited to photodiodes, avalanche photodiodes, and photomultiplier tubes.

[0056] The hardware components of the spectral measurement or imaging module may further include various optical components for guiding, shaping, filtering, or focusing the light beam passing through the system. Examples of suitable optical components include, but are not limited to, lenses, mirrors, prisms, apertures, diffraction gratings, stained glass filters, long-pass filters, short-pass filters, band-pass filters, narrow-band interference filters, wide-band interference filters, dichroic reflectors, optical fibers, optical waveguides, etc. In some cases, the spectral measurement or imaging module may further include one or more translation stages or other motion control mechanisms for moving the capillary flow cell device and the cartridge relative to the illumination and / or detection / imaging subsystem, or vice versa.

[0057] In some embodiments, the nucleotide sequencing device may further include a computer (or processor) and a computer-readable medium that includes code for providing image processing and analysis capabilities. Examples of image processing and analysis capabilities that may be provided by software include, but are not limited to, manual, semi-automatic, or fully automatic image exposure adjustment (e.g., white balance, contrast adjustment, signal averaging, and other noise reduction capabilities, etc.), automated edge detection and object identification (e.g., for identifying clusters of clonal amplification of fluorescently labeled oligonucleotides on the surface of a microchannel), automated statistical analysis (e.g., for determining the number of clusters of clonal amplification of oligonucleotides identified per unit area on the surface of a microchannel, or for automated nucleotide base calling in nucleic acid sequencing applications), and manual measurement capabilities (e.g., for measuring the distance between clusters or other objects, etc.). Optionally, the instrument control and image processing / analysis software may be written as separate software modules. In some embodiments, the instrument control and image processing / analysis software may be incorporated into an integrated package.

[0058] In some examples, the device may include a computer (or processor) and a computer-readable medium that includes code for providing a user interface and manual, semi-automated, or fully automated control of all system functions (e.g., control of fluid modules, temperature control modules, and / or spectroscopic or imaging modules) and other data analysis and display options. The system computer or processor may be an integrated component of the device (e.g., a microprocessor or motherboard embedded within the device) or may be a stand-alone module, such as a mainframe computer, personal computer, or laptop computer that is part of a system that includes the device. Examples of fluid control functions provided by the system control software include, but are not limited to, volumetric fluid flow rate, fluid flow rate, sample and reagent addition, buffer addition, and the timing and duration of rinse steps. Examples of temperature control functions provided by the system control software include, but are not limited to, specifying temperature set points and control of the timing, duration, and ramp rate for temperature changes. Examples of spectroscopic measurement or imaging control functions provided by the system control software include, but are not limited to, autofocus capabilities, control of illumination or excitation light exposure time and intensity, control of image acquisition rate, exposure time, and data storage options.

[0059] In some embodiments, the disclosed system may include one or more processors or computers. The processor may be a hardware processor such as a central processing unit (CPU), a graphics processing unit (GPU), a multi-purpose processing unit, or a computing platform. The processor may be composed of any of a variety of suitable integrated circuits, microprocessors, logic devices, field programmable gate arrays (FPGAs), etc. In some cases, the processor may be a single-core or multi-core processor, or multiple processors may be configured for parallel processing. Although the present disclosure is described with reference to processors, other types of integrated circuits and logic devices are also applicable. The processor may have any suitable data operation capabilities. For example, the processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations.

[0060] The processor or CPU may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location. The instructions may be directed to the CPU, which may then program or otherwise configure the CPU to implement, for example, the system control methods of the present disclosure. Examples of operations performed by the CPU may include fetch, decode, execute, and write-back.

[0061] Some processors are processing units of a computer system. The computer system may implement cloud-based data storage and / or computing. In some cases, the computer system may be operably coupled to a computer network ("network") by means of a communication interface. The network may be the Internet, an intranet and / or an extranet, an intranet and / or an extranet communicating with the Internet, or a local area network (LAN). In some cases, the network is a telecommunications and / or data network. The network may include one or more computer servers, which may implement distributed computing, such as cloud-based computing.

[0062] The computer system may also include a computer memory or memory location (e.g., random access memory, read-only memory, flash memory), an electronic storage unit (e.g., a hard disk), a communication interface for communicating with one or more other systems (e.g., a network adapter), and peripheral devices such as a cache, other storage units, data storage units, and / or an electronic display adapter. In some cases, the communication interface may allow the computer to communicate with one or more additional devices. The computer may be capable of receiving input data from the connected devices for analysis. The memory unit, storage unit, communication interface, and peripheral devices may communicate with the processor or CPU via a communication bus (solid line), such as may be incorporated into a motherboard. The memory or storage unit may be a data storage unit (or data repository) for storing data. The memory or storage unit may store files, such as drivers, libraries, and saved programs. The memory or storage unit may store user data, such as user preferences and user programs.

[0063] The system control, image processing, and / or data analysis methods described herein may be implemented by machine-executable code stored in an electronic storage location (e.g., memory or electronic storage unit) of the computer system. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by the processor. In some cases, the code may be retrieved from the storage unit and stored in the memory for ready access by the processor. In some cases, the electronic storage unit may be excluded, and the machine-executable instructions are stored in the memory.

[0064] In some cases, the code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code. In some cases, the code may be compiled during runtime. The code may be provided in a programming language selected such that the code can be executed in a pre-compiled or interpreted manner.

[0065] Some aspects of the apparatus, systems, and methods provided herein may be embodied in software. Aspects of the technology may be considered a "product" or "article of manufacture" that typically takes the form of machine (or processor) executable code and / or associated data carried on or embodied in a type of machine-readable medium. The machine executable code may be stored on an electronic storage unit such as a memory (e.g., read only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memories or associated modules of a computer, processor, etc., such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory storage for a software program at any time. All or part of the software may sometimes be communicated via the Internet or various other remote communication networks. Such communication can, for example, enable the loading of software from one computer or processor into another, such as from a management server or host computer into the computer platform of an application server. Thus, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as used across physical interfaces between local devices via wired and optical landline networks and via various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media that carry software. As used herein, unless restricted to non-transitory, tangible "storage" media, the term such as computer or machine "readable medium" refers to any medium that participates in providing instructions to a processor for execution.

[0066] In some cases, the system control, image processing, and / or data analysis methods of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented in software when executed by a central processing unit.

[0067] In some embodiments, the devices and systems described herein can be used for nucleic acid sequencing of samples. Nucleic acid sequencing provides a non-limiting example of the application of the disclosed devices. Many "second-generation" and "third-generation" sequencing technologies utilize large-scale parallel, cyclic array methods for sequencing by synthesis (SBS), where the accurate decoding of single-stranded template oligonucleotide sequences tethered to a solid support depends on successfully classifying the signals generated by the stepwise addition of A nucleotides, G nucleotides, C nucleotides, and T nucleotides to a complementary oligonucleotide strand by a polymerase. These methods typically require the oligonucleotide templates to be modified with known adapter sequences of a fixed length and attached to a solid support (e.g., the microchannel surface of the disclosed microfluidic chip) in a random or patterned array by hybridization to surface-fixed probes with known sequences complementary to the adapter sequences, and then probed by a series of cyclic single-base addition primer extension reactions to identify the base sequence in the template oligonucleotide using, for example, fluorescently labeled nucleotides. Thus, these methods require the use of miniaturized fluidic systems that provide precise, reproducible control of the timing of the flow cells into which the reagents are introduced for the sequencing reaction, as well as small volumes to minimize the consumption of expensive reagents.

[0068] The devices and systems described herein can also be used in a variety of applications, such as sequencing assays, to improve the efficient use of expensive reagents. For example, a method for sequencing a nucleic acid sample and a second nucleic acid sample can include: delivering a plurality of oligonucleotides to the microchannels of at least a partially transparent microfluidic chip; delivering a first nucleic acid sample to the microchannels; delivering a plurality of non-specific reagents to the microchannels through a first reagent dispensing port; delivering a specific reagent to the microchannels through a second dispensing port different from the first dispensing port; and visualizing the sequencing reaction in the microchannels. In some aspects, the described method can include selecting a plurality of oligonucleotides to sequence a eukaryotic genome. In some aspects, the described method can include selecting a plurality of oligonucleotides to sequence a prokaryotic genome. In some aspects, the described method can include selecting a plurality of oligonucleotides to sequence a transcriptome.

[0069] The devices and systems described can also be used in a method for reducing the reagents used in a sequencing reaction, the method including providing a first reagent in a first reservoir; providing a second reagent in a second reservoir, wherein each of the first reagent in the first reservoir and the second reagent in the second reservoir is sequentially dispensed or introduced into the microchannels of the microfluidic chip through separate connecting pipelines and reagent dispensing ports, and the separate connecting pipelines and reagent dispensing ports are discontinuously fluidly connected to the microchannels of the microfluidic chip.

[0070] From the foregoing description of the present invention, those skilled in the art will perceive improvements, changes, and modifications. The improvements, changes, and modifications of those skilled in the art are intended to be covered by the appended claims. All references, publications, and patents cited in this application are hereby incorporated by reference in their entirety.

Claims

1. A nucleotide sequencing device, comprising: a plurality of microfluidic chips configured for nucleic acid sequencing, wherein each microfluidic chip includes a microchannel having an inlet configured to receive a reagent and an outlet in fluid communication with a waste collection unit; and a reagent distribution manifold including at least one reagent distribution port, the reagent distribution manifold being operable to move relative to the plurality of microfluidic chips in at least two dimensions to allow the at least one distribution port to form a leak-proof fluid connection with each inlet of the microchannel after movement, wherein the at least one distribution port is configured to disconnect from the inlet of the microchannel after delivering the reagent and / or when the reagent distribution manifold moves.

2. The apparatus according to claim 1, further comprising a platform extending generally in the x-y plane of the x-y-z coordinate system, wherein, The plurality of microfluidic chips are arranged on the surface of the platform, and the reagent distribution manifold is positioned above the platform in the z-direction.

3. The device according to claim 2, further comprising a robotic arm that moves the reagent distribution manifold in the x-direction, y-direction, and / or z-direction to position the distribution port above the inlet of the corresponding microfluidic chip.

4. The device according to claim 1, further comprising at least one reagent reservoir in fluid communication with the reagent distribution manifold and the at least one distribution port.

5. The device according to claim 4, further comprising at least one fluid pump configured to pump the reagent from the reagent reservoir through the manifold to the at least one distribution port.

6. The device according to claim 5, wherein, The fluid pump includes a selector valve that is operable to control the reagent selected from the reagent reservoir for pumping through the manifold to the at least one distribution port.

7. The device according to claim 5, wherein The fluid pump includes a vacuum driver.

8. The device according to claim 5, wherein, The fluid pump includes a pressure or syringe pump.

9. The device according to claim 1, further comprising a heating unit configured to heat the plurality of microfluidic chips.

10. The device according to claim 2, further comprising an imaging module for imaging the microfluidic channels of the corresponding microfluidic chips.

11. The device according to claim 1, wherein, The imaging module is arranged in an imaging area on the platform separate from the sequencing area, where the plurality of microfluidic chips are arranged for receiving the reagent.

12. The device according to claim 11, including a robotic arm for transferring the microfluidic chip from the sequencing area to the imaging area.

13. The apparatus according to claim 12, wherein, The robotic arm includes a vacuum suction device for fixing the microfluidic chip to be transferred.

14. The device according to claim 1, having no common pipeline between the distribution port and the inlet of the microfluidic chip.

15. The device according to claim 1, wherein, The reagent and the microchannel are configured for nucleotide sequencing.

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