Reagent nozzle pipette mixing system and method

Abstract

The present disclosure relates to reagent nozzle pipette mixing systems and methods. An analytical instrument is provided that can perform analytical operations on an analyte combined with a plurality of reagents prior to being introduced into a flow cell. The instrument can include a nozzle pipette that aspirates reagents from a container along with the analyte. The reagents can be directed to a volume and repeatedly moved into and out of the volume by cycling of a pump. The reagents can be ejected by the nozzle pipette into a destination container, facilitating vortexing in the container to enhance mixing. The reagents and template are effectively mixed in an automated or semi-automated manner via the repeated aspiration and ejection of the nozzle pipette.

Description

[0001] This application is a divisional application of the application filed on December 21, 2017, with application number 201780037939.7, entitled "Reagent Nozzle Pipette Mixing System and Method".

[0002] Cross-reference of related applications

[0003] This application claims priority to U.S. Patent Application No. 15 / 841,098, filed December 13, 2017, which claims priority to U.S. Patent Application No. 62 / 442,765, filed January 5, 2017, and also claims the benefit of UK (GB) Patent Application No. 1704760.6, filed March 24, 2017, which also claims priority to U.S. Patent Application No. 62 / 442,765; all of these prior applications are incorporated herein by reference in their entirety. background

[0004] Instruments for sequencing relevant molecules (specifically, DNA, RNA, and other biological samples) have been developed and will continue to be developed. Prior to sequencing, samples of relevant molecules are prepared to form libraries or templates. These libraries or templates are mixed with reagents and ultimately introduced into a flow cell, where individual molecules attach to sites and are amplified to enhance detectability. The sequencing operation then involves a cycle of repeating the following steps: binding molecules at sites, labeling the bound components, imaging the components at sites, and processing the resulting image data.

[0005] In such sequencing systems, a fluid system (or subsystem) provides the flow of substances (e.g., reagents) under the control of a control system, such as a programmed computer and appropriate interfaces. Overview

[0006] Details of one or more embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages will become apparent from the description, drawings, and claims.

[0007] In some embodiments, a system may be provided comprising: a flow path fluidly connected to a flow cell to support an analyte in an analytical system; a fluid system that aspirates a reagent from a reagent container, mixes the reagent, sprays the mixed reagent into a destination container, and delivers the mixed reagent from the destination container to the flow path; and a nozzle pipette fluidly in communication with the fluid system, the nozzle pipette comprising an elongated body having a central cavity extending between its ends and a nozzle insert disposed at a distal end of the central cavity, wherein the nozzle pipette is used to aspirate the mixed reagent from the container via the nozzle insert and spray the mixed reagent back into the destination container.

[0008] In some embodiments of the system, the size of the nozzle and the inner cavity can be configured to promote vortex mixing in the destination container as the reagent is discharged from the nozzle pipette via the nozzle insert and enters the destination container.

[0009] In some embodiments of the system, the cavity may have a nominal inner diameter of about 0.5 mm, and the nozzle insert may be a tubular insert with a nominal inner diameter of about 0.25 mm.

[0010] In some embodiments of the system, the distal end of the nozzle suction tube may have a wedge shape having facets that meet at a vertex offset relative to the central axis of the nozzle suction tube.

[0011] In some embodiments of the system, the nozzle insert may have a distal end whose shape corresponds to the wedge shape of the distal end of the nozzle suction tube.

[0012] In some implementations of the system, the wedge shape may include four facets that meet at the apex.

[0013] In some implementations of the system, the nozzle can extend to a nominal distance of 2 mm from the bottom surface of the destination container.

[0014] In some embodiments of the system, the system may include multiple additional pipettes for aspirating individual reagents; these additional pipettes may not have a nozzle insert.

[0015] In some embodiments of the system, the suction nozzle can be used to accelerate the mixed container material to a flow velocity of at least about 1600 mm / s at a flow rate of at least about 5,000 μL / min.

[0016] In some embodiments, a system may be provided comprising: a flow cell supporting relevant analytes in an analytical system; a fluid system for aspirating reagents, mixing the reagents, spraying the mixed reagents into a destination container, and delivering the mixed reagents from the destination container to the flow cell; a nozzle pipette in fluid communication with the fluid system, the nozzle pipette comprising an elongated body having a central cavity extending between its ends and a nozzle located at a distal end of the elongated body, wherein the nozzle reduces the nominal inner diameter of the central cavity; and control circuitry operatively coupled to the fluid system for controlling the fluid system such that the fluid system: aspirates a group of reagents one by one, sprays the reagents from the group of reagents into the destination container via the nozzle, aspirates the group of reagents from the destination container via the nozzle for mixing, and sprays the group of mixed reagents back into the destination container via the nozzle.

[0017] In some embodiments of the system, the nozzle may include an insert that is inserted into the central cavity at the distal end of the nozzle suction tube.

[0018] In some implementations of this system, the destination container may contain the analyte to be sequenced.

[0019] In some implementations of the system, the central cavity may have a nominal inner diameter of 0.5 mm, and the nozzle may have a nominal inner diameter of 0.25 mm.

[0020] In some embodiments of the system, the distal end of the nozzle suction tube may have a wedge shape having facets that meet at a vertex offset relative to the central axis of the nozzle suction tube.

[0021] In some such embodiments of the system, the nozzle may have a distal end whose shape corresponds to the wedge shape of the distal end of the nozzle suction tube.

[0022] In some embodiments, a method may be provided comprising: a) actuating a pump to draw multiple reagents sequentially from corresponding plurality of reagent containers; b) actuating the pump to spray the reagents into a destination container via a nozzle pipette in fluid communication with the pump, the nozzle pipette comprising an elongated body having a central cavity extending between its ends and a nozzle located at a distal end of the elongated body, wherein the nozzle reduces the nominal inner diameter of the central cavity; c) actuating the pump to draw the reagents from the destination container and via the nozzle pipette to further mix the reagents; and d) actuating the pump to spray the reagents from the nozzle pipette and spray the reagents back into the destination container.

[0023] In some embodiments of the method, the size of the nozzle and the inner cavity may be configured to promote vortex mixing in the destination container as the reagent is discharged from the nozzle suction tube via the nozzle and enters the destination container.

[0024] In some embodiments of the method, the central cavity may have a nominal inner diameter of 0.5 mm, and the nozzle may include an insert having a nominal inner diameter of 0.25 mm inserted into the central cavity.

[0025] In some embodiments of the method, the distal end of the nozzle suction tube may have a wedge shape having facets that meet at a vertex offset relative to the central axis of the nozzle suction tube.

[0026] In some embodiments of the method, the reagent may include at least three reagents with different specific gravities.

[0027] In some implementations of the method, the method may further include performing (b) and (c) one or more times before performing (d).

[0028] Details of one or more embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages will become apparent from the description, drawings, and claims. It should be noted that the relative dimensions in the following figures may not be drawn to scale. Brief description of the attached diagram

[0029] These and other features, aspects, and advantages of the invention will become better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters denote like parts throughout the drawings, wherein:

[0030] Figure 1 A graphical overview of an example sequencing system in which the revealed technology can be used;

[0031] Figure 2 for Figure 1 A graphical overview of a sequencing system and a fluid system;

[0032] Figure 3 for Figure 1 A graphical overview of the processing and control system of an example sequencing system;

[0033] Figure 4 A perspective view of an example of a reagent manifold with a selector valve;

[0034] Figure 5 for Figure 4 A top view of an example manifold and valve configuration;

[0035] Figure 6A A graphical view configured for an example of aspirating and mixing reagents and sample templates, and Figure 6B Demonstrates how to add striate to reagents and sample templates before mixing;

[0036] Figure 7 A graphical view of an example of how reagents to be mixed can be individually aspirated into a mixing volume;

[0037] Figure 8 The graphic portion of the container is intended to illustrate an example of a destination container for a mixed reagent and sample template, using a nozzle pipette to spray the mixed reagent into the container.

[0038] Figures 9A to 9D Examples of nozzle pipettes that can be used to mix reagents;

[0039] Figure 10 A graphical representation of an example cycle of aspirating and mixing reagents and sample template; and

[0040] Figure 11 A flowchart illustrating the example logic used for aspirating and mixing reagents and sample templates. Detailed description

[0041] Figure 1 This describes an embodiment of a sequencing system 10 configured to process molecular samples, which can be sequenced to determine their components, component ordering, and bulk sample structure. The system includes an instrument 12 for receiving and processing biological samples. A sample source 14 provides a sample 16 that, in many cases, will include tissue samples. Sample sources may include, for example, individuals or entities such as humans, animals, microorganisms, plants, or other donors (including environmental samples), or any other individual containing relevant organic molecules whose sequences are to be determined. The system can be used with samples other than those obtained from organisms, including synthetic molecules. In many cases, the molecules will include DNA, RNA, or other molecules with base pairs whose sequences define genes and variants with specific functions of ultimate interest.

[0042] Sample 16 is introduced into sample / library preparation system 18. This system can isolate, split, and otherwise prepare samples for analysis. The resulting library contains relevant molecules of a length conducive to sequencing operations. The resulting library is then provided to instrument 12, where sequencing is performed. In practice, a library, sometimes referred to as a template, is combined with reagents in an automated or semi-automated process and then introduced into a flow cell prior to sequencing.

[0043] exist Figure 1 In the embodiments described herein, the instrument includes a flow cell or array 20 that houses a sample library. The flow cell includes one or more fluid channels that allow sequencing chemistry reactions to occur, including the attachment of molecules from the library and amplification at locations or sites detectable during sequencing operations. For example, the flow cell / array 20 may include sequencing templates fixed at locations or sites on one or more surfaces. A “flow cell” may include patterned arrays, such as microarrays, nanoarrays, etc. In practice, locations or sites may be arranged in regular repeating patterns, complex non-repetitive patterns, or randomly configured on one or more surfaces of a support. To enable sequencing chemistry reactions, the flow cell also allows the introduction of substances for reactions, rinsing, etc., including various reagents, buffers, and other reaction media. Substances flow through the flow cell and may contact relevant molecules at individual sites.

[0044] In the instrument, flow cell 20 is mounted on a movable stage 22, which in this embodiment is movable in one or more directions as indicated by reference numeral 24. For example, flow cell 20 may be provided in the form of a removable and replaceable cartridge that can connect to a port on the movable stage 22 or other components of the system to allow delivery of reagents and other fluids to or from flow cell 20. The stage is associated with an optical detection system 26 that directs radiation or light 28 to the flow cell during sequencing. The optical detection system may use various methods, such as fluorescence microscopy, to detect analytes placed at sites within the flow cell. By way of a non-limiting example, optical detection system 26 may use confocal scanning to generate progressively pixelated image data, which can be analyzed to locate individual sites within the flow cell and determine the type of nucleotides most recently attached or bound to each site. Other suitable imaging techniques may also be used, such as techniques that scan one or more radiation points along the sample, or techniques that use a "step and shoot" imaging method. The optical detection system 26 and the stage 22 can cooperate to maintain a static relationship between the flow cell and the detection system while acquiring a regional image, or, as mentioned, the flow cell can be scanned in any suitable mode (e.g., point scan, line scan, "step and shoot" scan).

[0045] While many different techniques can be used to image or, more broadly, to detect molecules at a site, the currently covered implementation utilizes confocal optical imaging at the wavelength that leads to the excitation of fluorescent labeling. The label, excited by absorption spectroscopy, transmits a fluorescent signal by means of its emission spectrum. Optical detection system 26 is configured to capture such signals to allow resolution processing of pixelated image data for analysis of the signal emission site, and to process and store the resulting image data (or data derived therefrom).

[0046] In sequencing operations, the cycling process or procedure is performed automatically or semi-automatically. In automatic or semi-automatic processes, reactions are facilitated, such as by single nucleotides or oligonucleotides, followed by rinsing, imaging, and unpacking in preparation for subsequent cycles. The sample library, prepared for sequencing and immobilized on the flow cell, can undergo several such cycles before all useful information is extracted from the library. An optical detection system generates image data by scanning the flow cell (and its sites) during each cycle of the sequencing operation using electronic detection circuitry (e.g., a camera or imaging electronics or chip). The resulting image data can then be analyzed to locate individual sites within the image data and to analyze and characterize the molecules present at those sites, such as by referring to the specific color or wavelength of light detected at a particular location (a characteristic emission spectrum of a specific fluorescent label), as indicated by groups or clusters of pixels in the image data at that location. For example, in DNA or RNA sequencing applications, four common nucleotides can be represented by distinguishable fluorescence emission spectra (wavelengths or wavelength ranges of light). Values ​​corresponding to each nucleotide can then be assigned to each emission spectrum. Based on this analysis, and by tracking the cycle values ​​determined for each region, individual nucleotides and their order can be determined for each region. These sequences can then be further processed to assemble longer segments, including genes, chromosomes, etc. As used in this disclosure, the terms "automatic" and "semi-automatic" mean that once the operation begins, or once the procedure including the operation begins, the operation is performed by a system program or configuration with little or no human interaction.

[0047] In the illustrated embodiment, reagent 30 is drawn or aspirated into the flow cell via valve 32. The valve may be located via, for example, a pipette or tube. Figure 1 (Not shown) Reagents are drawn from a container or vessel containing the reagents. Valve 32 allows selection of reagents based on a specified sequence of operations performed. The valve may further receive a command to direct the reagents into the flow cell 20 via flow path 34. The used reagents are withdrawn or discharged from the flow cell via flow path 36. In the illustrated embodiment, pump 38 is used to move reagents through the system. The pump may also provide other useful functions, such as measuring reagents or other fluids passing through the system, drawing air or other fluids, etc. An additional valve 40 downstream of pump 38 allows the used reagents to be properly directed to disposal vessels or containers 42.

[0048] The instrument further includes a series of circuits that help command the operation of various system components, monitor the operation of the components via feedback from sensors, collect image data, and at least partially process the image data. Figure 1In the implementation described herein, the control / monitoring system 44 includes a control system 46 and a data acquisition and analysis system 48. Both systems will include one or more processors (e.g., digital processing circuitry, such as microprocessors, multi-core processors, FPGAs, or any other suitable processing circuitry) and associated memory circuitry 50 (e.g., solid-state memory devices, dynamic memory devices, onboard and / or offboard memory devices, etc.) capable of storing machine-executable instructions for controlling, for example, one or more computers, processors, or other similar logic devices to provide certain functionality. A dedicated or general-purpose computer may at least partially constitute the control system and the data acquisition and analysis system. The control system may include circuitry configured (e.g., programmed) to process commands for any other useful functions related to fluid dynamics, optics, stage control, and instrumentation. The data acquisition and analysis system 48 interfaces with the optical inspection system to command the movement of the optical inspection system or the stage, or both, the emission of light for cyclic detection, the reception or processing of returned signals, etc. The instrument may also include various interfaces as indicated by reference numeral 52, such as operator interfaces that allow control and monitoring of the instrument, transfer of samples, initiation of automated or semi-automated sequencing operations, generation of reports, etc. Finally, Figure 1 In one implementation, an external network or system 54 may be coupled to and cooperate with the instrument, for example, for analysis, control, monitoring, servoing and other operations.

[0049] It should be noted that, although in Figure 1 The document describes a single flow cell and fluidic pathway, as well as a single optical detection system; however, some instruments may accommodate more than one flow cell and fluidic pathway. For example, in the currently covered implementation, two such configurations are provided to enhance sequencing and yield. In practice, any number of flow cells and pathways can be provided. These flow cells and pathways utilize the same or different reagent receiving containers, disposal receiving containers, control systems, image analysis systems, etc. When multiple fluidic systems are provided, they may be controlled individually or in a coordinated manner. It should be understood that the phrase “fluidly connected” may be used herein to describe a connection between two or more components that fluidly communicates with each other; similarly, “electrically connected” may be used to describe an electrical connection between two or more components. The phrase “fluid insertion” may be used, for example, to describe a specific ordering of components. For example, if component B is fluidly inserted between components A and C, the fluid flowing from component A to component C will flow through component B before reaching component C.

[0050] Figure 2 illustrate Figure 1An example fluid system for a sequencing system. In the illustrated embodiment, flow cell 20 includes a series of paths or pathways 56A and 56B, which can be grouped in pairs for containing fluid substances (e.g., reagents, buffers, reaction media) during sequencing operations. Pathway 56A is coupled to a common line 58 (first common line), while path 56B is coupled to a second common line 60. A bypass line 62 is also provided to allow fluid to bypass the flow cell without entering it. As mentioned above, a series of dishes or containers 64 allow for the storage of reagents and other fluids that can be used during sequencing operations. A reagent selector valve 66 is mechanically coupled to a motor or actuator (not shown) to allow selection of one or more reagents to be introduced into the flow cell. The selected reagent then proceeds to a common line selector valve 68, which similarly includes a motor (not shown). The common line selector valve can be commanded to select one or more of common lines 58 and 60, or both common lines, so that reagent 64 flows in a controlled manner to passages 56A and / or 56B, or to select bypass line 62 so that one or more reagents flow through the bypass line. It should be noted that other useful operations can be achieved via the bypass line, such as the ability to fill all reagents (and liquids) into the reagent selector valve (and the common line selector valve) without evacuating air via the flow cell, the ability to perform cleaning of reagent channels and pipettes independently of the flow cell (e.g., automatic or semi-automatic cleaning), and the ability to perform diagnostic functions on the system (e.g., pressure and volume delivery tests).

[0051] The used reagent exits the flow cell via a line coupled between the flow cell and pump 38. In the illustrated embodiment, the pump includes a syringe pump with a pair of syringes 70, which are controlled and moved by actuator 72 to aspirate and eject reagents and other fluids during different operations of the testing, validation, and sequencing cycles. The pump assembly may include various other parts and components, including valves, gauges, actuators, etc. (not shown). In the illustrated embodiment, pressure sensors 74A and 74B sense the pressure on the pump inlet line, while pressure sensor 74C senses the pressure output from the syringe pump.

[0052] Fluid supplied by the system is pumped into the used reagent selector valve 76. This valve allows selection of one of several flow paths for used reagents and other fluids. In the illustrated embodiment, a first flow path leads to a first used reagent receiving container 78, while a second flow path leads to a second used reagent receiving container 82 via a flow meter 80. Depending on the reagents used, it may be advantageous to collect the reagents, or certain reagents, in separate containers for disposal, and the used reagent selector valve 76 allows for this control.

[0053] It should be noted that the valves within the pump assembly allow various fluids, including reagents, solvents, cleaning agents, air, etc., to be drawn by the pump and injected or circulated via one or more of a common line, bypass line, and flow tank. Furthermore, as mentioned above, in the currently covered embodiments, under common control... Figure 2 The paper presents two parallel implementations of a fluid dynamics system. Each component of the fluid dynamics system can be part of a single sequencing instrument and can perform sequencing operations in parallel on different flow cells and sample libraries.

[0054] The fluid dynamics system operates under commands from a control system 46 that implements specified protocols for testing, validation, sequencing, etc. These specified protocols are pre-established and include a series of events or operations for the activity, such as aspirating reagents, aspirating air, aspirating other fluids, spraying such reagents, air, and fluids, etc. The protocols will allow coordination of such fluid operations with other instrument operations, such as reactions in a flow cell, imaging of the flow cell and its components, etc. In the illustrated embodiment, the control system 46 uses one or more valve interfaces 84 and a pump interface 86, the valve interfaces 84 being configured to provide command signals to valves, and the pump interface 86 being configured to command the operation of pump actuators. Various input / output circuits 88 may also be provided for receiving and processing feedback, such as from pressure sensors 74A to 74C and flow meters 80.

[0055] Figure 3 This describes certain functional components of the control / monitoring system 44. As described, the memory circuitry 50 stores specified routines executed during testing, debugging, fault diagnosis, servo operation, and sequencing. Many such protocols and routines can be implemented and stored in the memory circuitry, and these protocols and routines can be updated or changed from time to time. Figure 3 As described herein, these protocols and routines may include fluid control protocol 90, which controls various valves, pumps, and any other fluid actuators, and receives and processes feedback from fluid sensors and flow and pressure sensors, such as valves. Stage control protocol 92 allows, for example, the movement of the flow cell as needed during imaging. Optical component control protocol 94 allows commands to be issued to the imaging assembly to illuminate portions of the flow cell and to receive returned signals for processing. Image acquisition and processing protocol 96 allows at least partial processing of image data to extract useful data for sequencing. Other protocols and routines may be provided in the same or different memory circuitry as indicated by reference numeral 98. In practice, the memory circuitry may be provided as one or more memory devices, such as both volatile and non-volatile memory. This memory may be internal to the instrument, and some memory may be off-board.

[0056] One or more processors 100 access the stored protocol and implement the protocol on the instrument. As mentioned above, the processing circuitry can be a dedicated computer, a general-purpose computer, or part of any suitable hardware, firmware, and software platform. The processors and the operation of the instrument can be commanded by a human operator via operator interface 101. The operator interface allows for testing, debugging, troubleshooting, and servicing, as well as reporting any problems that may occur in the instrument. The operator interface also allows for initiating and monitoring sequencing operations.

[0057] Figure 4 This describes a valve assembly for drawing reagents and other fluids from a container and delivering them to a flow cell. Valve assembly 102 includes a manifold structure 104, in which channels are formed to define flow paths for the reagents and other fluids. Figure 4 As can be seen, valves 66 and 68 are driven and controlled by motors 106 and 108. One or more motor interfaces or connectors 110 provide power and (if necessary) signals to and from the motors. As mentioned above, the motors (and thus the valves) are controlled by control circuitry during testing, commissioning, and servoing, as well as during sequencing operations.

[0058] The reagent and fluid pathways within the manifold are coupled to pipette 112, which, during operation, draws reagents and other fluids from separate containers (not shown). Typically... Figure 4 The flow paths of the reagents and fluids specified by reference numeral 114 may be formed by molding, etching, or any other suitable process to allow the reagents and fluids to move from the pipette to the valve when the pump described above is commanded to aspirate the reagents and fluids. At least one of the pipettes is configured as a nozzle pipette 116 to aid in mixing reagents during sequencing operations (e.g., prior to reaction and imaging). Figure 4 The document also describes a mixing volume configured as channel 118, in which reagents and fluids can be drawn and moved for mixing. In some embodiments, the mixing volume may be part or all of bypass line 62. For example, reagents may be drawn into bypass line 62 in the desired sequence, but the reagents do not traverse the entire length of the bypass line (this allows the reagents to be delivered for disposal). Once the bypass line (or the portion of it that serves as the mixing volume) is loaded with the desired sequence of reagents, a valve can be used to switch the end of the bypass line through which the reagents are introduced to connect fluidly to a flow path leading to, for example, a destination container, so that the entire set of reagents loaded into the bypass line can then be drained back from the bypass line and into the destination container. In other embodiments, the mixing volume may be, for example, a destination container to which the premixed fluid will be delivered, or may be, for example, a separate destination container, for example, a destination container that is completely emptied before delivery of the selected reagents.

[0059] Figure 5This is a top view of valve assembly 102. Also here, valves 66 and 68 are visible in the manifold and coupled to the flow paths of reagents and fluids. Reagent selector valve 66 receives reagents via a self-priming tube and directs aspirated fluid to common line selector valve 68. Mixing channel 118 is coupled to the common line selector valve to allow mixing of reagents, as described below. Figure 5 The diagram also shows a port 120 disposed in the manifold to allow coupling of the manifold to a pipette. One of the ports 120 (indicated by reference numeral 122) is coupled to a nozzle pipette to allow injection of reagents into a destination container and to allow extraction of reagents from the destination container for mixing. For example, the destination container may be a container, tube, or other vessel designed to contain reagents. For example, the destination container may serve as a temporary working volume to which reagents and / or other materials can be transferred, for example by mixing, to prepare reagents and / or other materials for delivery to a flow cell. Thus, once reagents and other fluids have been prepared in the destination container, the reagents and other fluids can be transferred from the destination container to the flow cell.

[0060] Figure 6A The present embodiment describes the mixing channel 118 used for mixing and the reagent flow path. As mentioned above, the mixing channel 118 is coupled to a common line selector valve 68, which in turn is coupled to the outlet of a reagent selector valve 66. The mixing channel 118 is also coupled to a pump 38 to allow for the suction and ejection of reagents and fluids, as described below. Figure 6A In the embodiments described herein, reagent containers or vessels 124, 126, and 128 store reagents 130, 132, and 134, respectively. In this example, another or destination container 136 stores a pre-prepared sample template or library 138. For mixing operations, reagents 130, 132, and 134 are pre-mixed and then combined with template 138. To allow such pre-mixing, via... Figure 6A The reagents are drawn into the mixing channel 118 one by one through separate flow paths indicated by reference numeral 140. Another flow path 142 allows the reagents, along with the template, to be deposited in the destination container 136. In the illustrated embodiment, the mixing channel 118 forms a serpentine internal volume with a loop 144, which allows the desired volume of reagents to be drawn and mixed in a relatively compact region of the manifold.

[0061] In the currently covered embodiments, reagents 130, 132, and 134 have different fluid properties that pose a challenge to mixing. For example, the reagents have different densities, and there can be substantial differences in viscosity and oil interfacial tension. For example, in the currently covered embodiments, the viscosity varies between approximately 1.5 cP and 50 cP, such as 2.4 cP at 25°C, while the oil interfacial tension varies between approximately 5.0 dynes / cm and approximately 19.2 dynes / cm. By comparison, the template can still have different densities and lower viscosity (e.g., approximately 1 cP at 25°C) and different oil interfacial tensions (e.g., approximately approximately 9.8 dynes / cm). Figure 6B The diagram illustrates the stripes of the reagents and template in destination container 136 when unmixed. In the illustrated embodiment, the template comprises approximately 30% of the total volume, while reagent 130 comprises approximately 22%, reagent 132 comprises approximately 42%, and reagent 134 comprises approximately 6%. In this context, the term "approximately" means that the indicated value is not precise, and the actual value may vary within the range of those values ​​indicated in a manner that does not substantially alter the operation.

[0062] To enable automated mixing of reagents and templates, the fluid dynamics system and its controls allow for the selective aspiration of reagents one by one into the mixing channel, injection into the destination container, and cyclical withdrawal and re-injection for mixing. Figure 7 This describes the currently covered technique for reagent aspiration. As shown, reagents 130, 132, and 134 are aspirated one by one via the control of valve 66. Reagents are directed to the mixing channel via a common line selector valve, aspirating several volume groups of each reagent, as indicated by reference numerals 146, 148, 150, 152, and 154. To reduce pressure spikes during mixing, the pump can also draw in a large volume of air before aspirating the reagents. The air volume provides a buffer to limit both positive and negative pressure spikes during mixing. Figure 7 In the implementation scheme described herein, the aspirated air will be positioned at the upper left of the reagent assembly. Additionally, a liquid buffer can be aspirated to aid in the filling, rinsing, and displacing of the reagents. Once... Figure 7 As described above, if aspiration is performed, the valve can then be controlled to allow the pump to inject the reagent into the template 138, which will be preloaded into the template container 136 described above.

[0063] In another technique, where three or more reagents can be selected for mixing in a destination container, at least two of the selected reagents for mixing are introduced sequentially and repeatedly into a mixing channel, while at least one other selected reagent is held in reserve until the reagents introduced sequentially into the mixing channel have been completely delivered to the mixing channel. The reserved reagent can then be added to the mixing channel all at once. For example, with ABCABCABCABCABC (which can be derived, for example, from similar to...) Figure 7 In contrast to the techniques described, if reagents A and B are repeatedly introduced into a mixing channel one after another, followed by the introduction of the retained reagent C, the reagents in the mixing channel will typically separate into ABABABABABC. This technique is considered advantageous in preventing or reducing the occurrence of undesirable reaction byproducts of some reagents. For example, the retained reagent may react with one of other reagents isolated in one particular manner, but with two or more of other reagents combined in another manner. The latter may be the desired reaction that may occur after the reagents have been thoroughly mixed, while the former may occur during premixing when the reagents are still relatively separated and can only be mixed with directly adjacent reagents. In another instance, the retained reagent may react with the material forming the structure of the mixing channel and produce undesirable byproducts. Since introducing reagents one by one into the mixing channel can take several minutes—for example, 5 minutes, 10 minutes, 15 minutes, or longer, depending on the number and quantity of each reagent required—retaining the introduction of potentially problematic reagents until other reagents have been delivered into the mixing channel significantly reduces the amount of time spent on contact between the retained reagents and the structure of the mixing channel, thereby reducing the likelihood of generating unwanted reaction byproducts. Of course, in such embodiments, the retained reagents may not benefit from the premixing that other reagents benefit from, but reducing the likelihood of unwanted reaction byproducts may be more important than the lack of premixing of the retained reagents. Specifically, if the retained reagent is a low-viscosity liquid, the lack of premixing of the retained reagents may ultimately have little effect.

[0064] Using channel-like mixing volumes, for example, volumes much longer than their width (e.g., at least 10X, 100X, 150X to 170X, 160X, 200X, or 500X longer than their width), allows sequentially delivered reagents to maintain a relatively stratified configuration relative to each other within the channel by reducing the interfacial contact interface area between each layer of reagent (the reagent is a liquid and therefore likely to diffuse to some extent across this boundary into each other over time; therefore, the boundary / contact interface area mentioned herein should be understood theoretically; however, reducing these theoretical areas slows down the rate of diffusion). Additionally, for reagents that may be slightly immiscible with each other, mixing volumes, such as those spherically shaped or having a large width-to-length ratio, allow various reagent doses delivered to the mixing volume to float within the mixing volume and potentially recombine with earlier doses of the same reagent, thus losing the stratification achievable with channel-like mixing volumes. For example, a mixing channel with a diameter or width of approximately 2.25 mm and a length of approximately 360 mm can provide favorable stratification of the delivered reagents during the premixing process. Once the mixing volume has been loaded with the required amounts of multiple reagents, the contents of the mixing volume can be delivered to the destination container (a portion of the fluid in the mixing volume may be lost to the dead volume of the fluid system; the total volume of reagents delivered to the mixing volume can be calibrated to account for this loss). After delivery to the destination container, the delivered premixed reagents can be repeatedly aspirated and ejected back into the destination container to facilitate further mixing. In some embodiments, the premixed (or pre-mixed) reagents can be aspirated from the destination container and pulled back into the mixing volume before being ejected back into the destination container. Thus, in such embodiments, the premixed reagents can be repeatedly moved into and out of the mixing volume during the aspirate / ejection mixing operation.

[0065] It has been found that using mixing channels with nozzle pipettes promotes vortexing in the destination container and provides excellent mixing of reagents and templates, even when the fluid properties of the reagents differ substantially. Furthermore, these structures and technologies enable automated mixing with little or no human interaction. Figure 8 and Figures 9A to 9C This section describes an example nozzle suction tube used in these techniques. For example... Figure 8 As shown, the nozzle suction tube has an elongated body and a tip 156 at its distal end, the elongated body having a central inner cavity (cavity) extending along its length. The nozzle is positioned at the tip to reduce the inner diameter of the suction tube at this location, thereby increasing the velocity of fluid drawn in and ejected through the suction tube. In the illustrated embodiment, the nozzle is formed as an insert 158 ​​received in the distal end or tip of the suction tube. Other structures, such as caps, machined, formed, forged regions, etc., can form the nozzle.

[0066] In the illustrated embodiment, the nominal outer diameter 160 of the pipette is approximately 0.125 inches (3.175 mm), and the nominal inner diameter 162 is 0.020 inches ± 0.001 inches (0.508 mm). On the other hand, the nominal inner diameter 164 of the nozzle is 0.010 inches ± 0.001 inches (0.254 mm, although some embodiments may feature a nozzle inner diameter ranging from at most 0.20 mm to 0.28 mm). Of course, other sizes and dimensions can be used to provide the desired mixing. Furthermore, in the illustrated embodiment, the nozzle pipette 116 is positioned at a height 166 approximately 2 mm above the bottom of the container 138. When the reagent is injected into the container, then, as indicated by reference numeral 168, the mixing in the container is enhanced by increasing the speed at which the reagent moves through the nozzle, thereby increasing the vortex within the container. Figure 8 Arrow 170 indicates that the mixed reagents are allowed to rise in the container, as indicated by reference number 172.

[0067] Figure 9A The distal end of the nozzle straw is described in slightly more detail. As can be seen in the figure, the nominal inner diameter 162 of the straw is reduced by the nozzle insert 158, in this case to approximately half the inner diameter of the straw (in this example, the nozzle insert is tubular in shape). Figure 9B , Figure 9C and Figure 9D The presently illustrated distal end best illustrates the current covered form. As shown here, the nozzle straw has a faceted lower end comprising four facets 174, giving the nozzle straw tip a wedge-shaped appearance. The straw has a centerline 176, and the facets meet at vertices 178 offset or eccentric relative to the centerline 176. This geometry of the distal end reduces or avoids dragging or scraping of the container when the straw is lowered into the container or when the container is raised around the straw. However, it should be noted that in the illustrated embodiment, the insert has a lower profile that matches the tip profile (e.g., one or more of the angled facets). In other words, the shape of the insert can conform to the faceted or wedge shape of the distal end of the nozzle straw. Furthermore, it should be noted that in the currently covered embodiment, the straw and nozzle are made of engineering plastics such as polyetheretherketone (PEEK). Such materials provide chemical resistance to reagents and any solvents used in the process.

[0068] Figure 10 This is a graphical representation of an instance cycle involving aspiration, mixing, and spraying of reagents and sample templates. Figure 11 This is a flowchart illustrating an example of the logic used for aspirating and mixing reagents and sample templates. Figure 10In this process, reference numeral 180 specifies the suction, mixing, and ejection cycles, wherein the pressure applied by the pump is indicated by shaft 182, and the cycle time is indicated by shaft 184. Negative pressure indicates the suction of one or more reagents, while positive pressure indicates ejection. The process can be considered to include a “transfer” sequence 186, followed by a “mixing” sequence 196, as discussed below.

[0069] follow Figure 11 The flowchart shows that control logic 204 can begin by evacuating air at 206 to remove existing liquid from the flow path, where previous mixtures of reagents may have been delivered. For example, any residual liquid remaining in the flow path 142 connecting reagent selector valve 66 and destination container 136 can be evacuated by air (i.e., replacing the liquid with air) so that any new mixture of reagents subsequently delivered to the destination container via flow path 142 does not mix with the residual liquid. The transfer sequence can then proceed in... Figure 11 The infusion sequence begins with the infusion sequence indicated by reference numeral 208. This infusion sequence is initiated by... Figure 10 The series of negative pressure or aspiration events, collectively indicated by reference number 188, generally allow reagents to be drawn into the system first. Return to Figure 11 Slightly more specifically, an aspirable buffer, as indicated at 210, may be used. This buffer may comprise a liquid selected to be non-reactive or relatively inert relative to the reagent and may serve as an incompressible working fluid extending at least partially between the pump and the reagent to allow for more precise metering of the reagent entering the mixing volume in the following steps, if necessary. The first reagent may then be aspirated during the filling event, as in Figure 11 As indicated at position 212, any number of other reagents are then aspirated via aspirating the final reagent at position 214. For example, in the currently covered embodiment, three such reagents are aspirated during the perfusion sequence.

[0070] exist Figure 11In the logic described above, the reagents to be mixed are then aspirated using transfer sequence 218. The transfer sequence continues by aspirating the first reagent as indicated at 220, and then each of the additional reagents is aspirated one by one until the final reagent is aspirated as indicated at 222. As previously stated, in the currently covered embodiment, three reagents are aspirated in this sequence. As mentioned above, in the currently covered embodiment, a certain number of reagent groups are aspirated in relatively small amounts to generate a series of reagents and thereby promote premixing. Therefore, at 224, the logic can determine whether all groups of reagents have been aspirated, and if not, it returns to 220 to continue aspirating additional groups. It should also be noted that in the currently covered embodiment, all groups contain all the reagents selected for mixing, but this is not necessary. Furthermore, different volumes or quantities of reagents can be aspirated in various groups. Once all reagents have been aspirated, control can proceed beyond the transfer sequence. The transfer sequence is generated by... Figure 10 The negative pressure event is indicated by reference number 190.

[0071] like Figure 11 As shown in the document, and if it is from Figure 10 Each individual negative (and positive) pressure event is clearly defined, and each successive aspiration (or jet) of a reagent or premixed reagent involves controlling one or more of the valves described above, as well as the pump. That is, to aspirate an individual reagent, the reagent selector valve will shift to direct negative pressure to the pipette of the corresponding container of the selected reagent. The pump will similarly be commanded to aspirate the reagent (or air, buffer, or template) and deliver the aspirated fluid according to a specified protocol. This mixing protocol will be predetermined and stored in the memory circuitry described above, and will be performed automatically or semi-automatically based on sequencing operations also defined in the memory circuitry. These protocols are executed by processing and control circuitry, which commands the operation of the valves and pump via appropriate interface circuitry.

[0072] Once all reagents have been aspirated, the aspirated fluid can be sprayed into the destination container, as shown in... Figure 11 As indicated at location 226. As mentioned above, in the currently covered embodiment, this is accomplished via a nozzle pipette, wherein mixing is initiated by increasing the velocity of the reagent through the nozzle and creating a vortex in the destination container. This spray into the destination container is... Figure 10 Positive pressure event 192 is indicated. In some implementations, it can be as follows: Figure 11 Reference numeral 228 indicates the need for further aspiration. The aspirated reagent can then be sprayed into the destination container. This sequence can be used as shown in... Figure 11 The suction air is indicated by reference number 230 and Figure 10Before the negative pressure event 194 (e.g., to remove as much liquid as possible from the bypass line, mixing channel, template channel, and pipette). It should also be noted that in some embodiments, during aspiration and spraying, the nozzle pipette or container, or both, may be moved relative to the other (e.g., vertically) to further aid in mixing the streaked sample and reagents.

[0073] After aspiration and partial premixing in the mixing volume or channel by the operations described above, mixing is performed by repeatedly moving the reagent through a nozzle pipette in the channel and between the channel and the destination container. For this purpose, a series of mixing cycles are performed with mixing sequence 234. In this sequence, the combined reagent and template are aspirated at 236 and sprayed back into the destination container at 238. At 240, logic repeatably determines whether all these desired mixing cycles have been performed and continues until all such cycles are complete. Figure 10 In the graphical illustration, cycles are collectively indicated by reference numeral 198. As can be seen, each cycle involves a relatively short negative pressure event followed by a relatively short positive pressure event. These events efficiently aspirate the combined reagents and template into the mixing volume or channel via a nozzle pipette, and gradually transfer the mixed reagents and template back to the destination container via the nozzle. While any desired volume can be displaced during this process, in the currently covered embodiment, approximately 2,000 μL is aspirated from and sprayed into the destination container in each mixing cycle, but other embodiments may dispense approximately 500 μL or 1,500 μL, depending on the size of the flow cell used. At the end of the mixing process, the mixed reagents and template can be transferred back to the destination container to continue sequencing operations.

[0074] It should be noted that in the current embodiment, the nozzle effectively increases the velocity of the reagent (and the mixed reagent) during suction and jetting. This increase in velocity increases the kinetic energy used to aid mixing. For example, in the currently covered embodiment, the nozzle accelerates the mixture to a minimum of about 1600 mm / s at a flow rate of at least about 5,000 μL / min.

[0075] Unless a specific order or sequence is explicitly indicated, the use of ordinal indicators such as (a), (b), (c)... or similar in this disclosure and claims shall be understood not to convey any particular order or sequence. For example, if there are three steps labeled (i), (ii), and (iii), it shall be understood that, unless otherwise indicated, these steps may be performed in any order (or even simultaneously if not otherwise prohibited). For example, if step (ii) involves disposing of an element produced in step (i), then step (ii) may be considered to occur at a point after step (i). Similarly, if step (i) involves disposing of an element produced in step (ii), the reverse shall be understood.

[0076] It should also be understood that the use of “for” (e.g., “a valve for switching between two flow paths”) can be replaced by language such as “configured to” (e.g., “a valve configured to switch between two flow paths”) or similar terms.

[0077] When used for reference quantities or similar quantifiable attributes, unless otherwise indicated, terms such as “about,” “approximately,” “substantially,” “nominal,” or similar terms should be understood to include values ​​within ±10% of the specified value.

[0078] In addition to the embodiments listed in this disclosure, the following additional embodiments should also be understood to be within the scope of this disclosure:

[0079] Implementation Scheme 1: A system comprising: a flow cell supporting an analyte in an analytical system; a fluid system for aspirating a reagent, mixing the reagent, and spraying the mixed reagent into a destination container; and a nozzle pipette in fluid communication with the fluid system, the nozzle pipette comprising an elongated body having a central cavity extending between its ends and a nozzle insert disposed at a distal end, the nozzle pipette aspirating a reagent from the container via the nozzle insert and spraying the mixed reagent back into the destination container.

[0080] Implementation Scheme 2: The system of Implementation Scheme 1, wherein the size of the nozzle and the inner cavity is configured to promote vortex mixing in the inner cavity when the reagent is aspirated into the pipette via the nozzle insert.

[0081] Implementation Scheme 3: The system of Implementation Scheme 1, wherein the cavity has a nominal inner diameter of about 0.5 mm and the nozzle insert has a nominal inner diameter of about 0.25 mm.

[0082] Implementation Scheme 4: The system of Implementation Scheme 1, wherein the distal end of the nozzle suction tube has a wedge shape, the wedge shape having a vertex offset relative to the central axis of the nozzle suction tube.

[0083] Implementation Scheme 5: The system of Implementation Scheme 4, wherein the nozzle insert has a distal end whose shape corresponds to the wedge shape of the distal end of the nozzle suction tube.

[0084] Implementation Scheme 6: The system of Implementation Scheme 4, wherein the wedge shape includes four facets that meet at the apex.

[0085] Implementation Scheme 7: The system of Implementation Scheme 1, wherein the length of the nozzle suction tube extends to a nominal distance of 2 mm from the bottom surface of the container.

[0086] Implementation Scheme 8: The system of Implementation Scheme 1 includes a plurality of additional pipettes for aspirating separate reagents, wherein the additional pipettes do not include a nozzle insert.

[0087] Implementation Scheme 9: The system of Implementation Scheme 1, wherein the pipette nozzle accelerates the mixed container material to at least about 1600 mm / s at a flow rate of at least about 5,000 μL / min.

[0088] Implementation Scheme 10: A system comprising: a flow cell supporting relevant analytes in an analytical system; multiple reagents disposed in separate containers; a fluid system that aspirates the reagents, mixes the reagents, and sprays the mixed reagents into a destination container; a nozzle pipette in fluid communication with the fluid system, the nozzle pipette including an elongated body having a central cavity extending between its ends and a nozzle at a distal end; and control circuitry operatively coupled to the fluid system to command the fluid system to aspirate the multiple reagents one by one, spray the multiple reagents into the destination container via the nozzle, aspirate the multiple reagents from the destination container via the nozzle for mixing, and spray the mixed reagents back into the container via the nozzle.

[0089] Implementation Scheme 11: The system of Implementation Scheme 10, wherein the nozzle includes an insert in the distal end of the nozzle suction tube.

[0090] Implementation Scheme 12: The system of Implementation Scheme 10, wherein the destination container includes the analyte to be sequenced.

[0091] Implementation Scheme 13: The system of Implementation Scheme 10, wherein the cavity has a nominal inner diameter of 0.5 mm and the nozzle has a nominal inner diameter of 0.25 mm.

[0092] Implementation Scheme 14: The system of Implementation Scheme 10, wherein the distal end of the nozzle suction tube has a wedge shape, the wedge shape having an apex offset relative to the central axis of the nozzle suction tube.

[0093] Implementation Scheme 15: The system of Implementation Scheme 14, wherein the nozzle has a distal end whose shape corresponds to the wedge shape of the distal end of the nozzle suction tube.

[0094] Implementation Scheme 16: A method comprising: actuating a pump to draw multiple reagents from a destination container containing an analyte to be analyzed; drawing the multiple reagents to mix the multiple reagents via a nozzle pipette in fluid communication with the pump, the nozzle pipette including an elongated body having a central cavity extending between its ends and a nozzle at a distal end; and actuating the pump to spray the mixed reagents into the destination container.

[0095] Implementation Scheme 17: The method of Implementation Scheme 16, wherein the size of the nozzle and the inner cavity is configured to promote vortex mixing in the inner cavity when the reagent is drawn into the pipette through the nozzle.

[0096] Implementation Scheme 18: The method of Implementation Scheme 16, wherein the cavity has a nominal inner diameter of 0.5 mm and the nozzle includes an insert having a nominal inner diameter of 0.25 mm.

[0097] Implementation Scheme 19: The method of Implementation Scheme 16, wherein the distal end of the nozzle suction tube has a wedge shape having a vertex offset relative to the central axis of the nozzle suction tube.

[0098] Implementation Scheme 20: The method of Implementation Scheme 16, wherein the reagent comprises at least three reagents with different specific gravities.

[0099] It should be understood that all combinations of the foregoing concepts (assuming such concepts are not inconsistent with each other) are intended to constitute part of the inventive subject matter disclosed herein. Specifically, all combinations of the claimed subject matter appearing at the end of this disclosure are intended to constitute part of the inventive subject matter disclosed herein. It should also be understood that terms expressly used herein that may also appear in any disclosure incorporated by reference should have a meaning substantially consistent with the specific concepts disclosed herein.

Claims

1. A reagent nozzle straw mixing system comprising: a flow cell that supports an analyte of interest in an analysis system; a fluidic system that aspirates reagents, mixes the reagents, ejects the mixed reagents into a destination vessel, and delivers the mixed reagents from the destination vessel to the flow cell, wherein the fluidic system includes a plurality of reagent flow paths, another flow path, a mixing channel, and a selector valve; a nozzle straw in fluid communication with the fluid system, the nozzle straw including an elongate body and a nozzle at a distal end of the elongate body, the elongate body having a central lumen extending between a proximal end thereof and the distal end, wherein the nozzle reduces an inner diameter of the central lumen, and wherein, the plurality of reagent flow paths pass through the central lumen; and a control circuit operably coupled to the fluidic system, the control circuit to control the fluidic system to cause the fluidic system to: (a) actuate a pump to aspirate a plurality of reagents from a plurality of reagent vessels, each reagent vessel of the plurality of reagent vessels storing a reagent of the plurality of reagents, one at a time, to the mixing channel via the plurality of reagent flow paths and the selector valve, wherein each reagent flow path of the plurality of reagent flow paths is in fluid communication to a respective reagent vessel of the plurality of reagent vessels, the mixing channel, and the selector valve; (b) actuate the pump to eject the plurality of reagents from the mixing channel into the destination vessel via the other flow path different from the plurality of reagent flow paths, the selector valve, and the nozzle straw, the nozzle straw in fluid communication to the other flow path, the mixing channel, the selector valve, and the pump; (c) actuate the pump to aspirate the plurality of reagents from the destination vessel into the other flow path via the nozzle straw; and (d) actuate the pump to eject the plurality of reagents from the other flow path via the nozzle straw and eject the plurality of reagents back into the destination vessel. an inner diameter of the nozzle ranges between 0.20 mm and 0.28 mm.

2. The system of claim 1, wherein the nozzle comprises an insert inserted in the central lumen at the distal end of the nozzle suction tube, and wherein, 3. The system of claim 1, wherein the destination vessel contains an analyte to be sequenced.

4. The system of claim 1, wherein the central lumen has a nominal inner diameter of 0.5 mm and the nozzle has a nominal inner diameter of 0.25 mm.

5. The system of claim 1, wherein a distal end of the nozzle straw has a wedge shape with facets that meet at an apex that is offset relative to a central axis of the nozzle straw.

6. The system of claim 5, wherein the nozzle has a distal end that is shaped to conform to the wedge shape of the distal end of the nozzle straw.

7. A reagent nozzle straw mixing method comprising: (a) actuate a pump to aspirate a plurality of reagents from a plurality of reagent vessels, each reagent vessel of the plurality of reagent vessels storing a reagent of the plurality of reagents, one at a time, to the mixing channel via the plurality of reagent flow paths and the selector valve, wherein each reagent flow path of the plurality of reagent flow paths is in fluid communication to a respective reagent vessel of the plurality of reagent vessels, the mixing channel, and the selector valve; (a) actuating the pump to draw a plurality of reagents from a plurality of reagent containers, each of the plurality of reagent containers storing a reagent of the plurality of reagents, one at a time, through a plurality of reagent flow paths and a selector valve to the mixing channel, wherein, (b) actuate the pump to eject the plurality of reagents from the mixing channel into the destination vessel via the other flow path different from the plurality of reagent flow paths, the selector valve, and the nozzle straw, the nozzle straw in fluid communication to the other flow path, the mixing channel, the selector valve, and the pump; (c) actuate the pump to aspirate the plurality of reagents from the destination vessel into the other flow path via the nozzle straw; and (d) actuate the pump to eject the plurality of reagents from the other flow path via the nozzle straw and eject the plurality of reagents back into the destination vessel. an inner diameter of the nozzle ranges between 0.20 mm and 0.28 mm. (b) actuating the pump to draw the plurality of reagents from the destination vessel, through the nozzle straw, and into the another flow path; and (c) actuating the pump to eject the plurality of reagents from the another flow path, through the nozzle straw, and back into the destination vessel.

8. The method of claim 7, wherein the nozzle and the central lumen are sized to promote vortex mixing in the destination vessel as the reagents are expelled from the nozzle straw through the nozzle and into the destination vessel.

9. The method of claim 7, wherein the central lumen has a nominal inner diameter of 0.5 mm, and the nozzle includes an insert inserted into the central lumen and having a nominal inner diameter of 0.25 mm.

10. The method of claim 7, wherein a distal end of the nozzle straw has a wedge shape with facets meeting at an apex that is offset from a central axis of the nozzle straw.

11. The method of claim 7, wherein the reagents include at least three reagents having different specific gravities.

12. The method of claim 7, further comprising performing one or more repetitions of (b) and (c) prior to performing (d). ​

Images