Devices for liquid handling

Abstract

A liquid dispensing device is provided. Aspects of the device include an actuator with a movable shaft, a central channel, an elongate body, such as a cylinder, including a proximal end and a distal end, the distal end including an orifice, and a plunger including a proximal end and a distal end, the proximal end being mechanically coupled to the movable shaft, and at least a portion of the distal end of the plunger being movably positioned within the central channel. Systems incorporating the liquid handling devices are also provided, which may be provided as arrays of liquid handling devices as well as methods of using the devices and systems, for example, in liquid dispensing applications. [Selection diagram 1]

Description

[Background technology]

[0001] Liquid dispensing devices are known in the art. Problems with prior art for dispensing small amounts (e.g., 10 nL to 300 nL) are not related to the actual dispensing, but rather to how the dispensed liquid is handled in the device, including, for example, delivery of the liquid to the point where dispensing occurs. Most frequently, systems for dispensing small amounts of liquid must have a large reservoir of fluid present to dispense, even though they only dispense small nanoliter amounts.

[0002] Unfortunately, fluids of interest, e.g., experimental samples, are often only available in microliter (μL) or nanoliter (nL) quantities, which represents a significant problem for liquid handling devices.

[0003] To use small amounts of fluid in current dispensing systems, it is necessary to have large amounts of a "working" fluid plus the target liquid. These fluids must also be separated from one another during the aspirating and dispensing of the target fluid. This is complex and inefficient, requiring a complicated arrangement of tubing, valves, reservoirs, and pumps. The target fluid must also be kept from mixing with the "working" fluid. This may involve the use of an "air gap" to separate the two fluids, or the placement of a third, more viscous fluid between the two fluids. All of this increases the complexity and difficulty of controlling the aspirating and dispensing processes of a liquid handling device.

[0004] As mentioned above, a second consideration that represents a limitation of the state of the art is that small amounts of dispensed liquid in current dispensing systems often require large amounts of liquid to be supplied to the dispensing mechanism. This creates a "dead volume" of liquid supplying the dispensing mechanism, where the amount of liquid present in the supply reservoir and in the supply lines feeding the dispensing mechanism is wasted and never actually dispensed into, for example, a multiwell plate or chip. One configuration of a prior art liquid dispensing instrument is shown in FIG. 15, which depicts a microsolenoid nanofluidic device (MSND). Summary of the Invention

[0005] The inventors have recognized that there is a need in the art for improved and simplified systems for liquid handling that reduce the complexity of devices for dispensing very small amounts of sample or reagent, e.g., nanoliter (nL) amounts of liquid, there is a need in the art for liquid handling devices that do not produce large dead volumes of reagent solution or sample liquid, and there is a need in the art for liquid handling systems that are capable of accommodating reagent solutions and samples having very small volumes, e.g., 10 nL to 300 nL.

[0006] The present disclosure relates to devices and systems for liquid processing (e.g., aspirating and dispensing) small volumes of liquid samples, e.g., about 10 nL to about 300 nL. The present disclosure provides solutions and advantages over the prior art. In some aspects, the devices and systems described herein comprise an actuator, e.g., a linear actuator, operatively coupled to a plunger that is movably positioned within a central channel of an elongate body, e.g., a cylinder, such that movement of the actuator causes corresponding movement of the plunger, thereby either aspirating liquid into the elongate body or dispensing liquid out of the elongate body.

[0007] In some aspects, the present disclosure provides devices for liquid processing, the devices comprising: (a) an actuator, e.g., a linear actuator, having a movable shaft; (b) an elongate body, e.g., a cylinder, having a central channel, a proximal end, and a distal end, the distal end comprising an orifice; and (c) a plunger having a proximal end and a distal end, (i) the proximal end is mechanically coupled to the movable shaft, and (ii) at least a portion of the distal end of the plunger is movably positioned within the central channel of the cylinder.

[0008] In another aspect, the present disclosure provides a system, i.e., an apparatus, for simultaneously processing multiple liquid samples, the system comprising: (i) a nanosyringe array comprising a plurality of nanosyringes, each nanosyringe comprising: (a) an actuator, e.g., a linear actuator, having a movable shaft; (b) an elongate body, e.g., a cylinder, having a central channel, a proximal end, and a distal end, the distal end comprising an orifice; (c) a plunger having a proximal end and a distal end, the proximal end mechanically coupled to the movable shaft and at least a portion of the distal end of the plunger movably positioned within the central channel of the elongate body; The nanosyringes are configured to dispense liquid, and in some cases also to aspirate liquid.

[0009] In some cases, the system further comprises (ii) a computer control system and optionally an operably coupled user interface for controlling the actuator, and thereby controlling the nanosyringe liquid dispensing, and in some cases, the liquid aspiration. The system may be configured to accommodate a multi-well device comprising a plurality of fluidically isolated wells, e.g., the system is capable of positioning the multi-well device in a configuration to receive liquid dispensed from the nanosyringe.

[0010] In yet another aspect, the present disclosure provides a method for dispensing liquid into wells of a multi-well device. An embodiment of the method includes:

[0011] (a) Providing: (i) A nanosyringe, (A) an actuator, such as a linear actuator, having a movable shaft; (B) an elongate body, e.g., a cylinder, having a central channel, a proximal end, and a distal end, the distal end comprising an orifice; (C) a plunger having a proximal end and a distal end, the proximal end being mechanically coupled to the movable shaft, and at least a portion of the distal end of the plunger being movably positioned within the central channel of the elongate body; A nanosyringe comprising: (ii) providing a multi-well device comprising a plurality of fluidly separated wells; (b) positioning the multi-well device in a configuration to receive liquid dispensed from the nanosyringe, the method further comprising: (c) positioning the distal end of the cylinder in a source reservoir, e.g., a source reservoir plate, containing the liquid to be dispensed; (d) retracting the actuator shaft, thereby retracting the plunger in the central channel, thereby drawing liquid into the central channel; (e) repositioning the distal end of the elongate body over a well of the multi-well device; (f) extending the linear actuator shaft, thereby extending a plunger within the central channel, thereby dispensing liquid from the central channel into the wells of the multi-well device.

[0012] A complete and enabling description of the subject technology, and the advantages of this technology over the prior art, is set forth in the specification, examples, and accompanying drawings provided herein. It is to be understood that this disclosure is not intended to limit the scope of the invention to the particular embodiments described herein, but that those skilled in the art will recognize that it provides sufficient guidance to make and use embodiments not expressly described or shown herein. [Brief explanation of the drawings]

[0013] Various features of exemplary embodiments of the present invention are described below with reference to the drawings, which include the following figures:

[0014] [Figure 1] 1 provides a schematic diagram of a nanosyringe of the present disclosure showing the coaxial arrangement of the linear actuator shaft and the nanosyringe plunger. [Figure 2] 2 provides a schematic diagram showing a detailed portion of the nanosyringe shown in FIG. 1. [Figure 3] 1 provides a schematic diagram of a nanosyringe of the present disclosure showing the non-coaxial arrangement of the linear actuator shaft and the nanosyringe plunger. [Figure 4] 1 provides a schematic diagram of one embodiment of a nanosyringe of the present disclosure incorporating a plunger ceral at the distal end of the plunger. [Figure 5A] Alternatively, a view of a nanosyringe of the present disclosure is provided having a nanosyringe plunger extending into the nanosyringe barrel. [Figure 5B] Alternatively, a view of a nanosyringe of the present disclosure is provided with the nanosyringe plunger withdrawn from the nanosyringe barrel. [Figure 6] 1 provides an illustration of a nanosyringe herein containing a liquid during a dispensing stage. [Figure 7A]For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 7B] For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 7C] For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 7D] For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 7E] For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 7F] For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 7G] For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 7H] For example, embodiments of the distal end of a nanosyringe cylinder are shown, including a cylinder with an integral orifice, as well as a cylinder with an orifice plate. [Figure 8] 1 shows a schematic diagram of a liquid treatment device of the present disclosure. [Figure 9A] 1 shows a schematic diagram of a liquid treatment device according to one embodiment of the present disclosure. [Figure 9B] 1 shows a schematic diagram of a liquid treatment device according to another embodiment of the present disclosure. [Figure 10] 1 shows a schematic diagram of a nanosyringe array that can be used in the liquid treatment devices of the present disclosure. [Figure 11] 1 shows a schematic diagram of a nanosyringe array that can be used in the liquid treatment devices of the present disclosure. [Figure 12] 1 shows a schematic diagram of a nanosyringe array that can be used in the liquid treatment devices of the present disclosure. [Figure 13] 1 shows a schematic diagram of a movable stage containing a multiwell chip that can be used in the liquid handling device of the present disclosure. [Figure 14] FIG. 1 shows a schematic diagram of a single nanosyringe that can be used in the liquid treatment devices of the present disclosure. [Figure 15] 1 shows a general schematic diagram of a liquid handling device known in the art, namely a microsolenoid nanofluidic device (MSND). The MSND system uses a combination of a microsolenoid valve, a gas pressure source (helium), and a syringe pump that dispenses liquid either via pressure derived from the pressure source from a fluid reservoir, or via aspiration via the action of the syringe pump. [Figure 16] 1 shows the results of a test to examine the dispensing accuracy of the nanosyringe of the present disclosure, tested in the range of 30 nL to 300 nL using water as the dispensing liquid. [Figure 17A] 1 shows the results of an experiment testing the reproducibility of a 35 nL dispense volume study. [Figure 17B] 1 shows the results of an experiment testing the reproducibility of a 100 nL dispense volume study. [Figure 18] Results of experiments testing the accuracy of liquid dispensing events over a volume range of 20 nL to 50 nL are shown. [Figure 19] Shown is an image of an ICELL8 chip containing 5,184 nanowells after a test dispense pattern. [Figure 20A] 1 provides a schematic diagram of one embodiment of a liquid handling device described herein, namely a nanosyringe device further comprising a side port on the nanosyringe cylinder. [Figure 20B] 1 provides a schematic diagram of one embodiment of a liquid handling device described herein, namely a nanosyringe device further comprising a side port on the nanosyringe cylinder. [Figure 21A]1 provides a schematic diagram of one embodiment of a liquid handling device described herein, namely a nanosyringe device that uses a coupling between a linear actuator shaft and a plunger that is not a permanent mechanical coupling. [Figure 21B] 1 provides a schematic diagram of one embodiment of a liquid handling device described herein, namely a nanosyringe device that uses a coupling between a linear actuator shaft and a plunger that is not a permanent mechanical coupling. [Figure 22] 1 provides graphs illustrating one embodiment of the velocity and acceleration characteristics of the linear actuator, and therefore the plunger, of a nanosyringe device. [Figure 23] 1 provides a workflow schematic illustrating the use of the SMART-Seq® Pro application protocol by Takara Bio USA, Inc. for transcriptome analysis of single cells, as used with the nanosyringe system described herein. [Figure 24A] 5 shows images obtained after a PCR reagent dispensing test into an ICELL8 (registered trademark) 184-well chip. [Figure 24B] 5 shows images obtained after a PCR reagent dispensing test into an ICELL8 (registered trademark) 184-well chip. [Figure 25] 1 shows the qPCR linearity of two liquid dispensing systems of the present disclosure compared to prior art devices. DETAILED DESCRIPTION OF THE INVENTION

[0015] It is understood that various configurations of the subject technology will be readily apparent to those skilled in the art from this disclosure, and various configurations of the subject technology have been shown and described by way of example. As will be understood, the subject technology is capable of other different configurations, and its several details are capable of modification in various other respects, all without departing from the scope of the technology. Accordingly, the summary, drawings, and detailed description are to be regarded as illustrative in nature and not restrictive.

[0016] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The accompanying drawings are incorporated herein and constitute a part of the detailed description. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. In some instances, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject technology.

[0017] Unless otherwise defined, scientific, mechanical, engineering, and technical terms used in connection with the teachings provided herein have the meanings commonly understood by those skilled in the art. The terminology used herein in describing the disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0018] definition As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted as the alternative ("or"). For example, the term "A and / or B" includes A, B, and (A and B). Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

[0019] When a range of values ​​is presented, each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure. For example, if a range of 1 μM to 8 μM is recited, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, and 7 μM are also expressly intended to be disclosed. The range also includes all non-integer values, such as 2.2 μM, 6.33333 μM, etc.

[0020] The term "about" refers to a range of ±10% of the value, unless the context of the disclosure dictates otherwise or contradicts such an interpretation; for example, "about 5" means 4.5 to 5.5, "about 100" means 90 to 100, etc. For example, in a list of numerical values ​​such as "about 49, about 50, about 55," "about 50" means less than half of one or more intervals between the preceding and succeeding values, e.g., a range extending from greater than 49.5 to less than 52.5. Furthermore, phrases referring to a value "less than about" or a value "greater than about" should be understood in light of the definition of the term "about" provided herein.

[0021] As used herein, "substantially" means sufficient to function for its intended purpose. Thus, the term "substantially" allows for slight but insignificant variations from absolute or perfect conditions, dimensions, measurements, results, etc., that would be expected by one of ordinary skill in the art but that do not significantly affect overall performance. When used in reference to a numerical value, or a parameter or characteristic that can be expressed as a numerical value, "substantially" means within 10%, or within 5% or less, e.g., within 2% or within 1%, of the target value.

[0022] As used herein, the term "plurality" refers to any integer greater than 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 1,000, 10,000, 100,000, 1×10 6 It could be more than that.

[0023] As used herein, the unit length "millimeter" refers to a unit of 0.001 meters, commonly written as mm. As used herein, the unit length "micron" refers to and is synonymous with the term micrometer, which is 0.000001 meters and is also written as μm. One millimeter is equal to 1,000 microns.

[0024] As used herein, the term "mil", which is synonymous with the term "thou", refers to a unit of length that is one thousandth of an inch. That is, 1 mil is synonymous with 0.001 inch. 1 mil is equivalent to 0.0254 mm, which is equivalent to 25.4 microns.

[0025] As used herein, the unit of "microliter" is 0.000001 (1 x 10 -6 ) liters, commonly written as μL. As used herein, the unit volume of a "nanoliter" is 0.000000001 (1 x 10 -9 ) refers to the unit of liter, commonly written as nL. 1 microliter is equivalent to 1,000 nanoliters.

[0026] As used herein, the terms "operably coupled," "operably coupled," or similar expressions refer to the interaction of two or more components that are physically joined or otherwise arranged to achieve some desired result. The juxtaposition, or coupling, of the components may be a physical joint or a non-physical joint, such as a functional joint. The terms "operably coupled" or "operably coupled" are intended to have a relative meaning. In some embodiments, two components that are operably coupled are in a relationship that achieves some functional result. For example, a light switch is operably coupled to a light bulb in a lighting fixture when the light switch can be used to operate the light bulb. In this example, the light switch and the light bulb are operably coupled and also electrically coupled.

[0027] As used herein, the term "fluidically coupled" refers to two components capable of fluid communication, whereby fluid has the ability to move between the components. For example, fluidly coupled components can include liquid communication via a tube or channel, which can be assisted or regulated by any suitable pump, valve, channel, conduit, solenoid, liquid reservoir, etc. Generally, fluidly coupled components share a bounding physical pathway, e.g., a channel or tube, that defines a fluid pathway.

[0028] As used herein, the terms "fluid" and "liquid" are used interchangeably.

[0029] As used herein, the term "cell" refers to any "biological cell." Non-limiting examples of biological cells include animal cells such as eukaryotic cells, plant cells, mammalian cells, insect cells, avian cells, and fish cells; prokaryotic cells, bacterial cells, fungal cells, and protozoan cells; cells dissociated from tissues such as muscle, cartilage, fat, skin, liver, lung, and nervous tissue; immune cells such as T cells, B cells, natural killer cells, and macrophages; embryos (e.g., zygotes); oocytes; sperm cells; hybridomas; cultured cells; cells from cell lines; cancer cells; infected cells; transfected and / or transformed cells; reporter cells; and the like. Mammalian cells can be derived from any mammal, e.g., human, mouse, rat, horse, goat, sheep, cow, primate, and the like.

[0030] As used herein, the term "sample" refers to any liquid composition that is the subject of analysis or contains components that are the subject of analysis. The nature of the sample is not particularly limited. For example, a liquid containing cells that are the subject of full-length transcriptome analysis can be a sample. In other embodiments, a sample contains one or fewer cells or one or fewer cell nuclei. In other embodiments, a sample can contain cell nuclei. The cells or cell nuclei in a sample can be fixed or unfixed. In other embodiments, a sample can contain nucleic acids of any nature, such as DNA (genomic DNA, cfDNA), RNA (e.g., but not limited to, mRNA, rRNA, cfRNA), or hybrid DNA / RNA molecules. A sample can include nucleic acids extracted from tissues or cells, or a sample can be a tissue or cell lysate, or any material derived therefrom.

[0031] As used herein, a "biological sample" is a substance or material obtained from any biological source for study, including the body of a subject. A "biological sample" can be derived from any part of an organism, such as an organ or tissue or cell.

[0032] Sources of samples include blood or any blood component; body fluids; solid tissues from fresh, frozen, and / or preserved organ or tissue samples or biopsies or aspirates; and cells from any time point in a subject's pregnancy or development. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, ocular fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, urine, cerebrospinal fluid (CSF), saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, as well as tissue extracts, such as homogenized tissues, tumor tissues, and cell extracts, cell lysates, and the like. Samples also include biological samples that have been manipulated in some way after their acquisition, for example, by treatment with reagents to solubilize or enrich for certain components, such as proteins or nucleic acids, or embedded in a semi-solid or solid matrix for sectioning purposes, e.g., thin sections of tissue or cells in histological samples, such as FFPE samples.

[0033] As used herein, "reagent" or "reagent mixture" or "reagents" or similar expressions refer to any material, reagent, substance, or molecule (biological or chemical) used, alone or in combination, to analyze or react in combination with a sample. In some aspects, a reagent is any substance that participates in or is used to perform any type of test or analysis, but is not itself the subject of the analysis. Reagents can be used in chemical reactions to detect or measure one or more analytes in a sample or to generate other substances from components in the sample. Reagents can include positive control analytes, such as nucleic acid templates or cell extracts, that can act as positive controls for chemical or biological reactions.

[0034] Various embodiments of the present disclosure are described in further detail in the following paragraphs.

[0035] Nanosyringe In some aspects, the present disclosure provides liquid handling devices capable of aspirating and / or dispensing from the same orifice; these devices, referred to herein as "nanosyringe" devices, have various advantages over the prior art. The present invention eliminates the complexity of current devices known in the art and is capable of manipulating very small amounts of liquid without requiring large dead volumes of either the working fluid or the fluid researchers wish to dispense to achieve the aspiration and dispensing of the desired fluid.

[0036] In its broadest sense, the present disclosure provides nanosyringe devices and apparatuses comprising nanosyringe devices that can be used to dispense small volumes of liquid. In some embodiments, the liquid dispensing volumes used in the devices of the present disclosure are nanoscale volumes, i.e., from (1) nanoliter (nL) to 999 nL. In some embodiments, the devices utilize dispensing volumes ranging from 10 nL to about 300 nL.

[0037] In some embodiments, the aspiration volume is the same as the dispensed volume. In other embodiments, the aspiration volume is larger than a single dispense volume. This is useful for performing multiple dispense events after a single larger aspiration step. The aspiration volume is not particularly limited and is a function of the holding capacity of the nanosyringe's cylindrical components. In some embodiments, the nanosyringe's aspiration volume is about 12,000 nL (i.e., 12 μL). In other embodiments, the nanosyringe has a larger volume, including, for example, an aspiration volume of about 15 μL, about 20 μL, about 25 μL, about 30 μL, about 40 μL, or about 50 μL.

[0038] The nanosyringe used herein essentially comprises three components, which can be understood from the schematic diagram in Figure 1. These are: (i) An actuator, for example a linear actuator 110 . Actuators, such as linear actuators, are generally known in the art and include a motor 112 (e.g., a stepper motor) and a movable shaft 114 driven by the motor, and in some cases, for example, when the actuator is a linear actuator, the shaft moves along a single axis, i.e., in a straight line. (ii) an elongated body, such as a cylinder 120; The elongate body is characterized by a central channel 124, and the distal end of the elongate body includes an orifice 126. In some embodiments, the orifice at the distal end of the elongate body is characterized by a diameter smaller than the diameter of the central channel. A variety of orifice and orifice plate sizes and geometries can be used. (iii) Nanosyringe plunger 150. A nanosyringe plunger resides within and is movably positioned within the central channel 124 and is mechanically coupled (i.e., operatively connected) to the linear actuator shaft 114 by any suitable coupling 130. The plunger 150 is characterized by an outer diameter that is only slightly smaller than the diameter of the central channel 124, for example, by a few thousandths of an inch (referred to as a "mil" or "thou") or less, to provide a seal between the central channel sidewall 122 and the plunger 150.

[0039] The nanosyringe described herein offers advantages over prior art, such as microsolenoid nanofluidic devices (MSNDs), as shown in the schematic diagram provided in FIG. 15. Current dispensing systems often require a large reservoir to supply the dispensing mechanism because small volumes of liquid are dispensed, creating a "dead volume" of liquid supplied to the dispensing mechanism, where fluid is wasted and not actually dispensed. A prior art configuration is shown in FIG. 15. As shown in the diagram, there are two fluid paths: one for dispensing and one for aspiration. For dispensing, a hybrid valve connects the pressure reservoir to the dispensing tip. A microsolenoid valve acts as a gate for the fluid, which can be opened for a long period to flush the dispensing tip or for a short period to dispense. One advantage of the nanosyringe of the present invention, as shown in FIG. 1, is that it is a displacement technology, and there is a linear relationship between the volume manipulated and the movement of the linear actuator.

[0040] For aspiration in the MSND, a hybrid valve switches to connect the dispensing tip to a syringe pump. The syringe moves the plunger downward, creating negative pressure and drawing fluid through the dispensing tip. Mixing the working fluid with the desired dispensed fluid is one of the biggest challenges in the prior art, as containing the fluid requires either aspirating a very large volume of the desired fluid or incorporating an air gap, which adds undesirable springiness to the system and negatively impacts dispense quality. The nanosyringe described herein offers the advantage of significantly reducing the amount of excess sample and reagent aspirated, since it does not require an actuating fluid and therefore does not need to keep the dispensed fluid and the working fluid separate. The nanosyringe described herein requires minimal dead volume compared to prior art devices.

[0041] Prior art systems require a large volume of working fluid to supply the dispensing mechanism. It is important that this working fluid does not mix with the dispensed fluid. It is also important to degas the working fluid before use. Degassing the working fluid in the fluid reservoir requires a series of steps, including opening a vent valve to a helium source. Helium is forced through an air stone positioned at the bottom of the tank to degas the working fluid, after which the valve is returned to its original position for dispensing. The nanosyringe has the advantage that no part of the system needs to be degassed due to the absence of working fluid.

[0042] These types of prior art dispensing systems, such as that shown in FIG. 15, have significant dead volume and can be prohibitively expensive when the liquid being dispensed includes, for example, reagents (e.g., enzymes) that are expensive or in limited supply. Furthermore, these types of prior art systems require a large amount of hardware, connections, and lines that require maintenance and are subject to potential failure. The nanosyringe described herein minimizes fluid paths and reduces the relative number of connections and lines required to operate the delivery system compared to the prior art system shown in FIG. 15.

[0043] The nanosyringe described in the present disclosure provides a solution to these problems as well as other limitations of prior art devices. Still further advantages of the nanosyringe of the present disclosure will be apparent to those skilled in the art upon reading this specification.

[0044] In some embodiments, the nanosyringe devices described by the present disclosure further include a support structure that holds the nanosyringe device components in proper alignment, resulting in a single rigid unit.

[0045] In some embodiments, the dispense event is a non-contact dispense event. In other embodiments, the dispense event is a contact dispense event.

[0046] Typical operation of a nanosyringe device begins with the distal end of the nanosyringe's elongated body, e.g., a cylinder, containing an orifice that is immersed in a reservoir of fluid to be dispensed. This aspect of operation is illustrated in FIGS. 5A and 5B. The nanosyringe's plunger 510 is first fully inserted into the elongated body 530 (FIG. 5A), e.g., a cylinder, and in some embodiments, it contacts an orifice plate or other distal structure covering the distal end of the elongated body, e.g., a cylinder. The plunger 510 is then retracted by a linear actuator (FIG. 5B), thereby drawing liquid through the orifice 540 and into the central channel of the nanosyringe. In some embodiments, the plunger 510 is retracted approximately 90% of the length of the elongated body, e.g., a cylinder, thereby utilizing nearly the entire volume of the nanosyringe's central channel. As illustrated in FIG. 5B, the plunger 510 is withdrawn only to a point before being completely removed from the central channel. This action draws fluid through orifice 540 and into the elongated body, e.g., a cylinder. The extent to which the plunger is retracted from the elongated body, e.g., a cylinder, is not limited and can be retracted to any extent. In some embodiments, the plunger is retracted a small distance (e.g., less than 90% of the length of the elongated body, e.g., a distance between 5% and 50%) to aspirate a small amount.

[0047] After aspirating the liquid, the distal end of the elongate body, e.g., a cylinder, is removed from the dispense fluid reservoir (i.e., source reservoir) and placed on a container, liquid container, well, glass slide, or other flat surface or other target where the fluid is to be dispensed. The liquid in the elongate body, e.g., a cylinder, now acts as a dispense fluid reservoir, and the nanosyringe device acts as a positive displacement pump. See FIG. 6. A plunger 606 moves within the central channel toward an orifice in the distal end of the elongate body, e.g., a cylinder, of the nanosyringe, expelling a volume equal to the desired dispense volume. In some embodiments, a single aspirated volume of the elongate body, e.g., a cylinder, results in multiple dispense cycles.

[0048] In some embodiments, as shown in FIG. 6 , the linear actuator shaft, and consequently the plunger 606, moves at a velocity V1. While the movement of the plunger can be characterized by velocity V1, the velocity of the plunger is not limited in any respect, and one of ordinary skill in the art can readily determine the velocity or range of velocities that are optimized for a particular application utilizing the nanosyringe. It is important to note that in some aspects, the velocity of the linear actuator is set by the user using software controlling the nanosyringe device. However, because the actual velocity of movement of the linear actuator shaft (and consequently the plunger) can differ from the programmed velocity, measuring the actual velocity of the shaft is a preferred method for system optimization.

[0049] For example, in some embodiments, V1 can be any value between 10 mm / sec and 1,000 mm / sec. For example, in some embodiments, V1 can be any value between 50 mm / sec and 150 mm / sec. For example, in some embodiments, V1 can be any value between 75 mm / sec and 125 mm / sec. In one embodiment, V1 can be approximately 100 mm / sec. A velocity of 100 mm / sec was the actual plunger velocity measured in a successful implementation of the nanosyringe liquid handler described herein. This velocity was experimentally measured using an ImageXpert Inc.® JetXpert Jr™ (also known as JetXpert OEM) in-line droplet visualization and measurement platform.

[0050] As an example, as used in the work of Example 8, the programmed velocity was actually determined by a series of parameters entered by the user. In this example, the velocity was initially programmed Vstart, followed by V1, and finally reached a Vmax setting of 66.5 mm / s, with an acceleration A1 of 4,768 mm / s. 2, the motor current setting is 900mA, and the motor microstep mode is 6400 microsteps / rev.

[0051] The movement of the plunger is also characterized by an acceleration, which is not limited in any way and any suitable value can be used. In various embodiments, the minimum acceleration of the plunger is 500 mm / sec. 2 and the maximum acceleration of the plunger is 50,000 / s 2 In some embodiments, the plunger may rotate at a speed of, for example, 2,000 mm / sec. 2 ~15,000mm / sec 2 , e.g., 4,000 mm / sec. 2 ~10,000mm / sec 2 etc., 1,000 mm / sec. 2 ~20,000mm / sec 2 In some embodiments, the plunger reaches its maximum speed about 1 millisecond (ms) after the plunger begins to move, although faster and slower accelerations are also used in the present invention. For example, the plunger's maximum speed can be reached anywhere between about 0.1 ms and 10 ms.

[0052] Those skilled in the art will recognize that a range of velocity and acceleration values ​​may be used with the liquid handler devices described herein, and that additional velocities and accelerations may also be used.

[0053] The velocity and acceleration of the plunger forces the dispensed fluid through the orifice. Because orifice 614 has a diameter D2 that is smaller than the diameter D1 of the chamber of the elongated body, e.g., a cylinder, this results in an increased velocity (V2) of the expelled liquid 622 compared to the velocity of plunger 606. This results in a precise amount of fluid being expelled from the orifice of the elongated body of the nanosyringe, e.g., a cylinder, into or onto whatever device is being used to capture it. The dispense cycle can be repeated until the original volume of aspirated liquid is consumed by repeated dispense cycles. At this point, if more dispense cycles are desired, the aspirating of fluid into the central channel of the elongated body, e.g., a cylinder, may be repeated. The linear actuator and the programmability of its control enable the precise aspirating and dispensing of very small amounts of dispensed fluid. This is a significant advantage over the current state of the art, which requires many more components (e.g., valves, pumps, and reservoirs) to accomplish this same task.

[0054] In other embodiments of the nanosyringe devices described herein, a port can be located on the side of the elongate body, e.g., a cylinder, to add additional benefits and functionality to the nanosyringe devices described herein. This configuration is shown in the schematic diagrams of FIGS. 20A and 20B. Plunger 550 and seal 552 are shown. Port 554 can be located within the elongate body, e.g., a cylinder, at a location closer to the proximal end of the elongate body, e.g., a cylinder 558, than to the distal end of the elongate body, e.g., a cylinder, where the proximal end of the elongate body, e.g., a cylinder, is closest to the linear actuator shaft, and the distal end of the elongate body, e.g., a cylinder, is the end containing orifice 556. Note that this schematic diagram does not reflect the relative positions of the distal or proximal ends of the elongate body, e.g., a cylinder, to scale. The inner diameter of the port can be the same diameter as the inner diameter of the elongate body, e.g., a cylinder, of the nanosyringe (i.e., the central channel of the nanosyringe), or it can be smaller or larger than the central channel of the nanosyringe.

[0055] This port enhances the functionality of the nanosyringe device, allowing the device to function as a valve as well as a dispensing device. When the distal end of the plunger extends below and covers the port (as shown in FIG. 20B), the device operates as described above without the port injecting liquid into the central channel 560. When the distal end of the plunger is above the level of the port (as shown in FIG. 20A), the port can now be used to direct fluid flow 562 into the elongated body, e.g., a cylinder, as indicated by the arrow in FIG. 20A, thereby facilitating the flushing and rinsing of contaminants or air from the elongated body, e.g., a cylinder. In some embodiments, a vacuum can also be applied to the port to facilitate the removal of air trapped in the central channel of the elongated body, e.g., a cylinder.

[0056] As described above, in a nanosyringe device, no actuating fluid exists between the pump force (i.e., the linear actuator shaft) and the elongated body of the nanosyringe device, e.g., a cylinder. As a result, only the experimental fluid of interest is drawn into the nanosyringe central channel of the elongated body, e.g., a cylinder, and dispensed therefrom. This configuration has the advantage that there is no risk of the desired experimental fluid of interest mixing with the actuating fluid. Another advantage is that the amount of fluid dispensed is directly related to the amount displaced by the plunger, allowing for more precise control of the dispensed volume.

[0057] Non-limiting embodiments of the liquid handling devices described herein are illustrated in the accompanying drawings. In the following description, one embodiment of a nanosyringe is described in which the elongate body is a cylinder and the actuator is a linear actuator, although the invention is not so limited, for example, as described in more detail below.

[0058] 1 provides a schematic diagram of one embodiment of a nanosyringe 100 of the present disclosure, showing a coaxial arrangement of a linear actuator shaft 114 and a nanosyringe cylinder plunger 150. In this configuration, the linear actuator shaft 114 and the nanosyringe plunger 150 share the same axis, i.e., the axis of the linear actuator shaft 114 is the same as the axis of the nanosyringe cylinder plunger 150. The nanosyringe 100 of FIG. 1 is operated by a linear actuator motor 112 that is electronically coupled to a user interface that allows a user to program and control the operation of the linear actuator 110. The linear actuator shaft 114 is operably coupled to the nanosyringe plunger 150 by a coupling 130. In this embodiment, the coupling 130 can take any form and is not particularly limited in terms of design or material; the only requirement of the coupling is that it provides a physical connection between the linear actuator shaft 114 and the nanosyringe plunger 150 such that powered movement of the actuator shaft 114 results in corresponding movement of the nanosyringe plunger 150.

[0059] 1 , a nanosyringe plunger 150 travels within a nanosyringe channel 124 inside a nanosyringe cylinder 120. The nanosyringe plunger 150 and nanosyringe sidewall 122 are manufactured to close tolerances such that the plunger 150 controls the aspiration and dispensing of liquid into the nanosyringe cylinder channel 124 through a bottom orifice 126, which is characterized by a given opening size. In some embodiments, the nanosyringe 100 incorporates a seal 140 that surrounds the junction between the nanosyringe cylinder plunger 150 and the nanosyringe cylinder 120. The seal 140 can be any suitable material, including, but not limited to, Teflon™ (i.e., polytetrafluoroethylene [PTFE]), Delrin® plastic, polyether ether ketone (PEEK), high-strength slippery PEEK, Slippery Delrin Acetal AF resin (Teflon blended with Delrin), or other suitable materials known to those skilled in the art. The seal can be in the form of an O-ring or any other suitable shape that encompasses the interface between the proximal end of the cylinder 120 and the plunger. The seal 140 provides a barrier to prevent leakage of liquid from the nanosyringe cylinder 120. See, e.g., FIGS. 5A and 5B.

[0060] FIG. 2 shows an enlarged view 200 of region 102 of FIG. 1. As shown in FIG. 2, a linear actuator shaft 214 is coupled to a nanosyringe plunger 250 by a coupling 230. The nanosyringe plunger 250 moves within a nanosyringe channel 224 inside a nanosyringe cylinder 220. The nanosyringe plunger 250 and nanosyringe sidewall 222 are fabricated with a suitable diameter so that the plunger 250 controls the aspiration and dispensing of liquid into and from the nanosyringe cylinder channel 224 through a bottom orifice 226, which is characterized by a given opening size. In some embodiments, the nanosyringe incorporates a seal 240 that surrounds the junction between the nanosyringe plunger 250 and the nanosyringe cylinder 220. The seal can be any suitable material, such as, for example, Teflon™, although any suitable material can be used as described herein.

[0061] FIG. 3 illustrates an alternative embodiment of a nanosyringe 300 of the present disclosure, in which the linear actuator shaft 314 and nanosyringe cylinder plunger 350 are not coaxial (i.e., non-coaxial) in their arrangement. That is, the linear actuator 314 and nanosyringe cylinder plunger 350 do not have the same axis of movement. The nanosyringe 300 is operated by a linear actuator motor 312 that is electronically coupled to a user interface that allows a user to control the operation of the linear actuator 310. The linear actuator shaft 314 is coupled to the nanosyringe plunger 350 by a coupling 330. A dogleg coupling allows for a non-coaxial arrangement between the linear actuator 310 and the nanosyringe plunger 350 and cylinder 320. This configuration is useful because it allows for a compact arrangement of multiple nanosyringe cylinder units to form a nanosyringe array, which is used for simultaneous fluid manipulation in multi-well systems.

[0062] 3, nanosyringe plunger 350 moves within nanosyringe channel 324 inside nanosyringe cylinder 320. Nanosyringe plunger 350 and nanosyringe sidewall 322 are in contact and form a seal such that plunger 350 controls the aspiration and dispensing of liquid into nanosyringe cylinder channel 324 through bottom orifice 326, which is characterized by a given opening size. In some embodiments, nanosyringe 300 incorporates seal 340 that surrounds the junction between nanosyringe plunger 350 and nanosyringe cylinder 320. The seal can be any suitable material, including, but not limited to, Teflon, Delrin® plastic, or any other suitable material described herein.

[0063] 4 illustrates another embodiment of a nanosyringe in which a nanosyringe plunger 450 employs a plunger seal 460 at the distal end of the plunger 450. In this embodiment, a linear actuator shaft 414 is coupled to the nanosyringe plunger 450 by a coupling 430. The nanosyringe plunger 450 moves within a nanosyringe channel 424 inside a nanosyringe cylinder 420. The nanosyringe plunger 450 and the nanosyringe sidewall 422 are fabricated with dimensions such that the diameter D3 of the plunger 450 is smaller than the diameter D1 of the nanosyringe cylinder channel 424. A plunger seal 460 is installed at the distal end of the plunger 450, and the plunger seal 460 and the wall of the nanosyringe channel 422 form a tight seal, e.g., the diameter of the plunger seal 460 is less than or equal to 1 mil greater than the diameter of the cylinder channel 424. In this embodiment, the plunger is capable of aspirating and dispensing liquid through an orifice 426 having a diameter D2, and movement of the plunger 450 controls the movement of liquid through the orifice 426.

[0064] 5A and 5B illustrate one embodiment of the operation of a nanosyringe plunger. In these figures, nanosyringe plunger 510 forms a tight bond with the sidewall of nanosyringe sidewall 530, e.g., a bond of about 1 / 1000 of an inch or less. The portion of the structure where plunger 510 enters the nanosyringe cylinder is surrounded by seal 520, which aligns plunger 510 with the nanosyringe cylinder and also prevents leakage of liquid that may leak from the plunger / nanosyringe cylinder junction. FIG. 5A shows plunger 510 in an extended position, where liquid has been dispensed from the nanosyringe cylinder through orifice 540. FIG. 5B shows plunger 510 in a retracted position, leaving a void within the nanosyringe cylinder, where liquid can be aspirated through orifice 540 to occupy the nanosyringe channel. In some embodiments, the seal can take the form of a cap, as shown in structure 520. In other embodiments, the seal can take any suitable form, such as a smaller structure similar to an O-ring, hi still other embodiments, the nanosyringe does not use any seal around the plunger / cylinder junction where the plunger is inserted into the cylinder.

[0065] 6 shows one embodiment of a nanosyringe described herein. In this illustration, plunger 606 is intimately associated with cylinder sidewall 612. For example, in some embodiments, the plunger is about 1 mil or less in diameter than the cylinder in which it resides. Having such a close association between plunger 606 and sidewall 612 has the added benefit of reducing the dead volume of the nanosyringe device.

[0066] 6 illustrates the relationship between the relative velocity of plunger 606 (having velocity V1) and liquid 622 (having velocity V2) exiting orifice 614, and the effect of the difference between the diameter of nanosyringe channel 620, characterized by diameter D1, and the diameter of orifice 614, characterized by diameter D2. Generally, V2 can be characterized by the ratio of D1:D2, with a higher ratio indicating a faster V2. That is, the smaller D2 is relative to D1, the greater the value of V2.

[0067] The nanosyringe can be designed to have any desired liquid dispensing speed, V2. In some embodiments, preferred dispensing speeds are from about 3 m / s to about 5 m / s. In other embodiments, preferred dispensing speeds range from about 1 m / s to about 10 m / s.

[0068] A preferred liquid dispense rate V2 for non-contact dispensing is one that is fast enough to cleanly deliver the dispensed liquid to the receiving well without leaving hanging droplets or other liquid amounts near the orifice or otherwise adhering to the outer surface of the nanosyringe cylinder, i.e., has a minimum dispense rate. A preferred dispense rate for non-contact dispensing is one that is not so fast that the dispensed liquid sprays from the orifice or splashes around or out of the receiving well, contaminating surrounding wells.

[0069] The diameter D1 of the nanosyringe channel is larger than the diameter D2 of the orifice, thereby accelerating the velocity of the liquid dispensed through the orifice 614 compared to the downward velocity of the nanosyringe plunger. That is, the liquid dispensing velocity V2 is always greater than the velocity V1 of the downward plunger movement. The liquid dispensing velocity V2 can be adjusted by changing the ratio of the width D2 of the orifice 614 to the width D1 of the cylinder channel 620 and / or by changing the velocity V1 of the downward movement of the plunger 606.

[0070] 7A-7H illustrate embodiments of designs for the distal end of a nanosyringe cylinder used in the devices described herein. Various configurations for the distal-most end of the nanosyringe cylinder containing the orifice can be used. In some aspects, the orifice is machined out of the nanosyringe cylinder material, e.g., stainless steel, such that the orifice is an integral component of and formed from the cylinder. Examples of such integral orifice configurations are shown, for example, in FIGS. 7B, 7C, 7E, and 7G.

[0071] In other embodiments, the distal end of the nanosyringe cylinder is capped by a secondary structure containing an orifice. As used herein, the term "orifice plate" refers to a structure attached to the distal-most portion of the nanosyringe cylinder, and in those embodiments, the orifice resides within the orifice plate. Non-limiting examples of orifice plates are shown, for example, in Figures 7A, 7D, 7F, and 7H.

[0072] The orifice plate can be attached into or onto the nanosyringe cylinder by any suitable means, such as by press-fitting, pressure deformation, welding, machining, or attachment with an epoxy adhesive or any other suitable adhesive.

[0073] FIG. 8 shows a schematic diagram illustrating one embodiment of a system comprising an apparatus for liquid handling described herein, capable of both liquid aspiration and liquid dispensing. The relative positions and sizes of the various components as shown in FIG. 8 are not fixed and are not to scale, and are intended only to provide a generalized scheme of one embodiment of a liquid handling device. Those skilled in the art will recognize that the components of apparatus 800 described herein can assume any of a variety of configurations. Furthermore, those skilled in the art will also recognize that not all of the components shown in embodiment apparatus 800 are required to construct an operable apparatus for liquid dispensing, and that in other embodiments, additional components not shown in FIG. 8 can be incorporated into the device. Apparatus 800 shown in FIG. 8 is not intended to be limiting in any way, except for the presence of at least one, and preferably multiple, nanosyringes as described herein.

[0074] Examples of this device consistent with this schematic diagram in FIG. 8 have been constructed, and these devices have been tested as described herein and have functioned successfully.

[0075] At a minimum, liquid dispensing device 800, as described herein, incorporates at least one, and preferably a plurality of, nanosyringes, referred to as a nanosyringe array 810. Various embodiments of the nanosyringe array incorporate any plurality of nanosyringes, for example, at least 4, 8, 10, 12, 20, 24, or 48 individual nanosyringes. Those skilled in the art will recognize that the nanosyringes in a nanosyringe array require a stable framework to secure and properly align the nanosyringes within the device. Examples of such frameworks can be seen, for example, in FIGS. 10, 11, and 12. Those skilled in the art will recognize that any suitable framework can be constructed to house and support the plurality of nanosyringes in a nanosyringe array, and further recognize that the present invention is not limited to any particular design for the supporting structural framework. The device also incorporates a power supply 850.

[0076] The liquid handling devices described herein are designed to aspirate and dispense liquid samples in a highly parallel and automated manner, and optionally incorporate optical imaging hardware to facilitate highly parallel and automated processing of multiple samples, as desired, for example, in highly parallel processing of single cell samples, or highly parallel biochemical reactions such as highly parallel nucleic acid analysis, including nucleic acid cloning, amplification and sequencing.

[0077] In some embodiments, the nanosyringe array 810 is fixed in one location in the x / y plane within the liquid dispensing device 800, and the various source plates 876, waste or wash stations 874, and dispense targets 875 reside on a stage 870 that moves in the x / y plane below the nanosyringe array 810. The nanosyringe array 810 is movable in the Z dimension (i.e., vertically) so that the distal ends of the nanosyringe cylinders can be raised and lowered within the various liquids to aspirate liquid from the various sample source plates, clean the rinsing troughs, and set the appropriate height for dispensing.

[0078] In some embodiments, liquid treatment device 800 includes a computer control system that further includes a user interface 802. The computer control system may be part of the liquid treatment device or may be an external computer. This computer control system may house any software needed to operate the liquid dispensing device and any of the hardware associated with the device, such as camera assembly 830, cooling unit 806, humidifier 808, and motor 872 that moves movable stage 870 beneath nanosyringe array 810 in the x / y plane.

[0079] In some embodiments, the computer module 802 containing the software and user interface may be integrated into the device or into a dedicated workstation, for example, such that the user interface is displayed on or within the device housing 820 or is otherwise attached to the housing 820. In some embodiments, the device 800 comprises one or more internal computer modules 804 that control or distribute signals to the device components.

[0080] The software controlling the liquid handling device allows the user to program any desired sequence and pattern of liquid handling, including aspiration, dispensing, washing and rinsing steps, any of which can be programmed to occur at any addressable location on a target surface, for example within the wells of a multi-well device such as a multi-well chip or source-reservoir plate.

[0081] In some embodiments, the liquid treatment device comprises a housing 820 in which all or a subset of the device's components are contained. The housing can be of any shape, design, or material, without limitation. In some embodiments, the housing functions to form a sufficiently sealed chamber so that the environment within the housing can be controlled, for example, by temperature, humidity, or gas content. That is, the housing can create an environmentally controlled environment.

[0082] The apparatus includes a target for liquid delivery, which in some embodiments is most often a multi-well device, such as a plate or chip, that includes fluidically separated, i.e., physically separated, wells or other chambers, each of which can contain a separate volume of liquid that does not mix with any other wells on the plate or chip.

[0083] In some embodiments, the device includes a cooling / heating unit 806, e.g., a thermoelectric cooling unit (TEC), i.e., a solid-state heat pump, that requires a heat exchanger to dissipate heat using the Peltier effect. In some embodiments, the TEC can reside below the multi-well device 875 (e.g., a plate or chip). In other embodiments, the TEC can reside below or be integrated within the movable stage 870, thereby cooling not only the receiving multi-well device, e.g., a chip, but also any source plate, e.g., a source reservoir plate, or other reagents located on the movable stage 870. In other embodiments, the cooling mechanism can be other than a TEC heat exchange unit and can be located at any other location on the device. The device can have one or more TEC units, as desired by the user.

[0084] In some embodiments, the device comprises a humidifier 808 that humidifies the interior of the device when a sealed environment is formed by a suitable housing 820. The humidifier may be operatively and fluidly coupled to a humidifier water supply 809. Humidifying the interior of the device is advantageous to minimize evaporative loss of the very small amounts of liquid that are dispensed by the nanosyringe into any suitable well within a multi-well device, e.g., a plate or chip.

[0085] In some embodiments, the device incorporates a camera assembly 830 for imaging a plate or chip contained in the device. The camera assembly minimally includes a camera 831, and may variously further include at least one objective lens 832 (e.g., a 2x objective lens), an absorption filter 833, a dichroic filter 834, a secondary focus objective lens 835 (e.g., a 4x objective lens), an excitation filter 836 (e.g., an epifluorescence filter set), a focusing lens 837, and a light source (e.g., an LED light source) 838.

[0086] As shown in FIG. 8 , in some embodiments, the device can also incorporate one or more fluid reservoirs, such as a first fluid reservoir 861 and a second fluid reservoir 862. The fluid reservoirs can contain liquids used in the cleaning / rinsing and disinfecting steps, for example, liquids such as water, 0.2% bleach or alcohol solution, or similar liquids used by the nanosyringes for cleaning and disinfecting. The fluid reservoirs can be fluidly coupled to a washing station 874, where liquid from fluid reservoirs 861 / 862 is delivered and continuously supplied to washing station 874 as needed. Nanosyringes in nanosyringe array 810 can aspirate cleaning or disinfecting solution from washing station 874 and dispense the liquid into a suitable waste station before proceeding to the next sample delivery or source plate. In some embodiments, the liquid in fluid reservoirs 861 / 862 is delivered to washing station 874 through suitable tubing, with the flow of liquid being driven by any type of suitable pump, for example, a peristaltic pump.

[0087] 9A provides an engineering schematic of a fully operational model of a liquid treatment device 900 according to one embodiment described in this disclosure. Components of this engineering schematic include, for example, a housing 920, a nanosyringe array 910 and a structural framework surrounding the nanosyringe array, a movable stage 970 with integrated Peltier controllers for heating and cooling, a humidifier water supply 909, a camera assembly 930, and first and second fluid reservoirs 961 / 962 for delivering fluids such as water, bleach solution (e.g., 0.2% bleach), or alcohol to the appropriate washing stations. The schematic also shows a peristaltic pump 932 for delivering fluids from the reservoirs 961 / 962 to the appropriate washing stations. A cooling unit 906, such as a thermoelectric cooling unit (TEC), is also shown.

[0088] 9B provides an engineering schematic of a fully operational alternative model of a liquid treatment device 900B according to one embodiment described in this disclosure. Components of this engineering schematic include a housing 920B, a nanosyringe array 910B and a structural framework surrounding the nanosyringe array, a movable stage 970B with integrated Peltier controllers for heating and cooling, a humidifier water supply 909B, a camera assembly 930B, and first and second fluid reservoirs 961B / 962B for delivering fluids, such as water, bleach solution (e.g., 0.2% bleach), or alcohol, to the appropriate wash stations. The schematic also shows a peristaltic pump 932B for delivering fluids from the reservoirs 961B / 962B to the appropriate wash stations and for the humidifier water supply 909B. In this embodiment, the cooling unit has been replaced with a dehumidifier 906B.

[0089] Figure 10 provides an engineering schematic illustrating a nanosyringe array 10 for use in a liquid treatment device as described in this disclosure. The structural scaffolding that secures and aligns the nanosyringes to the device is also shown in this schematic. The nanosyringe array shown in this particular embodiment incorporates eight individual nanosyringes. Figure 10 shows the components: linear actuator motor 15, linear actuator shaft 11, coupling 12, which in this embodiment is a dogleg coupling, seal 13 (e.g., a Teflon seal) surrounding the junction of the nanosyringe cylinder and plunger, and nanosyringe cylinder 14.

[0090] Figure 11 provides an engineering schematic diagram of a portion of a nanosyringe array such as that used in a liquid treatment device as described in this disclosure. The structural scaffolding that secures and aligns the nanosyringes to the device can also be seen in this schematic. The nanosyringe array shown in this particular embodiment incorporates eight individual nanosyringes. Figure 11 shows the linear actuator shaft 20, the coupling 21, which in this embodiment is a dogleg coupling, the seal 22 surrounding the junction of the nanosyringe cylinder and plunger, the nanosyringe cylinder 23, and the distal end 24 of the nanosyringe cylinder.

[0091] Figure 12 provides another engineering schematic diagram showing a nanosyringe array and other components as used in a liquid treatment device as described in this disclosure. The structural scaffolding that secures and aligns the nanosyringes to the device is also visible. The nanosyringe array shown in this particular embodiment incorporates eight individual nanosyringes. The linear actuator motor 30, linear actuator shaft 31, dogleg coupling 32, and nanosyringe cylinder 33 are shown in the figure. The camera assembly 34 is also visible.

[0092] FIG. 13 provides an engineering schematic diagram of a portion of an apparatus for liquid handling as described in this disclosure. This diagram shows a movable stage 40 and components coupled to the movable stage. In some embodiments, the movable stage includes an integrated Peltier controller for heating and cooling. A multi-well device, e.g., a chip 41, is shown, into which samples and / or reagents are delivered by nanosyringes. A first source plate 42 and a second source plate 43 are shown. In some embodiments, the source plates contain 384 wells, which can function as either sample or reagent source plates. These source plates may be referred to as source reservoir plates, each containing 384 reservoirs. In some embodiments, reagents from the source plates are delivered to wells of a multi-well device, e.g., a chip. A wash trough 45 is shown, which can contain, for example, water, bleach, or alcohol, and can be used to clean and / or disinfect the nanosyringes. A waste trough 44 is shown for dispensing unused reagents from the nanosyringes and / or for draining cleaning or disinfecting solutions after the cleaning process.

[0093] FIG. 14 shows a detailed rendering of a single nanosyringe used in a nanosyringe array used in a liquid handling device described herein. A linear actuator 50 is shown, consisting of a linear actuator motor 51 and an actuator shaft 52. The linear actuator shaft is coupled to a nanosyringe plunger 54 by a dog-leg coupling 53. The dog-leg coupling 53 causes the axis of the linear actuator shaft and the axis of the nanosyringe cylinder 56 (and the nanosyringe plunger located within the cylinder) to be laterally offset and therefore not coaxially aligned. However, the linear actuator shaft 52 and the nanosyringe plunger 54 are mechanically coupled, such that vertical motion of the linear actuator shaft 52 is immediately translated into motion of the nanosyringe plunger 54, which moves in a different axis of motion but is equal in magnitude to the motion of the linear actuator shaft 52.

[0094] Aspects of the nanosyringe are described below.

[0095] I. Actuator The nanosyringe devices of the present disclosure incorporate the actuators described herein. The type and model of actuator used in the devices described herein is not particularly limited. An actuator is a component configured to move a plunger, for example, within a central channel, in a manner sufficient to aspirate and / or dispense liquid therefrom, as desired. Actuators are known in the art, and one of skill in the art will recognize that many different actuators can be used in the devices described herein.

[0096] In some embodiments, the actuator is a linear actuator. A linear actuator includes a motor and a movable shaft driven by the motor, where the shaft moves along a single axis, i.e., in a straight line. Various types of linear actuators are known in the art, including, but not limited to, mechanical actuators, including screw-type actuators. In some embodiments, the linear actuator is a stepper motor that incorporates a lead screw as a rotor that converts motor torque into linear thrust.

[0097] While some mechanical linear actuators are only capable of pulling or only of pushing, the linear actuators used in the devices described herein are capable of generating forces in both directions.

[0098] Other types of linear actuators that can be used in the nanosyringes described herein include, but are not limited to, hydraulic actuators, pneumatic actuators, piezoelectric actuators, electromechanical actuators, telescoping linear actuators, and linear motor actuators.

[0099] In some embodiments, the actuators used in the nanosyringe devices of the present invention, such as linear actuators, are capable of high speed and high acceleration, with motion resolution in the micron range. The actuators can be driven by stepping motors, as in this embodiment, or servo-driven by DC motors or voice coil motors. High speed and high acceleration are required to provide non-contact dispensing and precise motion resolution, as well as to provide accurate dispense volumes in the nanoliter range.

[0100] In one embodiment, nanosyringe arrays were fabricated using eight NEMA size 8 hybrid stepper motor linear actuators (DINGS' MOTION USA™, Morgan Hill, CA), which were successfully utilized in the device examples. These linear actuators have a compact footprint of 20 mm, a travel range of 25.4 mm, and a maximum speed of 100 mm / s.

[0101] The velocity of the linear actuator shaft, and consequently the velocity of the plunger moving within the cylinder channel, is controllable and can be selected from a variety of values. Furthermore, in some embodiments, the velocity of the plunger can be adjusted using a multi-phase profile during different phases of the aspiration or dispensing phase. For example, as used in the nanosyringe described by the present disclosure, the velocity and acceleration of the linear actuator shaft (and plunger) are controlled by a TRINAMIC (Hamburg, Germany) SixPoint™ motion controller ramp generator, which provides faster mechanical motion compared to classical linear acceleration ramp cycles. The SixPoint™ ramp generator allows the acceleration ramp to be matched to the torque curve of the stepper motor. Two different acceleration settings are used for the acceleration phase, and two different deceleration settings are used for the deceleration phase. Start and stop speeds greater than zero can also be used.

[0102] An aspect of this speed control program is shown in Figure 22. The six-point ramp program begins using a starting speed VSTART (which can be 0). The motor is then accelerated to a speed V1 using an acceleration value A1. Once speed V1 is reached, the motor is further accelerated using an acceleration value A2 until it reaches a speed VMAX. A deceleration phase begins using a deceleration value D2. After speed V1 is again reached, deceleration value D1 is used to decelerate to a stop speed VSTOP (which can be 0).

[0103] II. Nanosyringe Elongated Body and Plunger The nanosyringe devices described herein incorporate an elongate nanosyringe body. The elongate body includes a central channel configured to accommodate a volume of liquid to be aspirated and / or dispensed. The elongate body is a structure having a length greater than its width, and the dimensions of the elongate body can vary. In some cases, the length of the elongate body can range from 10 to 100 mm, e.g., 20 to 50 mm, and the outer width or diameter of the elongate body can range from 0.5 to 10 mm, e.g., 1 to 5 mm. The elongate body can have any convenient configuration, including, but not limited to, a circular cross-sectional shape, e.g., circular, elliptical, etc. In some embodiments, the elongate body can be configured to have a polygonal cross-sectional shape, e.g., rectangular, square, etc. The elongate body can have a uniform or varying external configuration. Thus, in some cases, the elongate body can have a constant width along its length. In other cases, the width of the elongate body can vary along its length. Thus, in some instances, the width is referred to as the outer diameter (e.g., when the elongate body is configured as a cylinder), and the outer diameter of the elongate body may be constant along the length of the elongate body, while in other instances, the outer diameter may vary along the length of the elongate body.

[0104] In some cases, the elongate body is a cylinder. See Figure 2. Cylinder 220 includes nanosyringe cylinder sidewalls 222 and a central channel 224 that runs the length of the cylinder. At the distal end of the cylinder is an orifice 226 through which aspirated / dispensed liquid passes.

[0105] In some embodiments, the cylinder is typically a stainless steel hypodermic tubing or a glass capillary, while in other embodiments, the cylinder is machined, i.e., milled, from a stainless steel cylinder to form the steel cylinder used in the nanosyringe.

[0106] In some embodiments, the cylinder has an initial inner diameter that is less than the desired target inner diameter, for example, 0.5 mils less than the desired target inner diameter. As an example, if the target inner diameter is 34 mils, a cylinder having an inner diameter of approximately 33.5 mils is obtained. The inner diameter is then expanded to the desired target inner diameter, for example, by using a reamer.

[0107] In some embodiments, the inner diameter of the cylinder channel is approximately 0.8 mm, and the length limit of the cylinder is determined by the stroke length of the linear actuator used, which in some embodiments is approximately 25 mm.

[0108] In the nanosyringes described herein, a plunger 250 is located within the nanosyringe cylinder's channel 224. In some embodiments, the plunger is a stainless steel rod with a diameter approximately the same as the inner diameter of the cylinder channel (e.g., about 1 mil or less smaller than the cylinder channel diameter) to ensure a tight fit between the plunger and the cylinder sidewall 222.

[0109] In some embodiments, the plunger is longer than the cylinder so that the orifice reaches the distal end of the cylinder. See FIG. 5A. In other embodiments, the opposite end (i.e., the proximal end) of the plunger forms a mechanical / physical connection that is then coupled to the shaft of the linear actuator. The coupling between the linear actuator shaft and the plunger can utilize any suitable mechanical / physical coupling, and there is no particular limitation as to how the mechanical / physical coupling is implemented.

[0110] In some embodiments, the linear actuator shaft and the nanosyringe cylinder are aligned along the same axis in a coaxial configuration, i.e., collinear. See, e.g., FIG. 1 . In other embodiments, the linear actuator shaft and the nanosyringe cylinder are laterally offset, such that the linear actuator shaft and the nanosyringe plunger are not coaxial in configuration. See, e.g., FIG. 3 . In this configuration, the linear actuator shaft and the nanosyringe plunger have different axes of movement, but they are synchronized in their vertical motion via coupling 330. As used herein, an offset coupling, i.e., a coupling that creates a lateral offset between two adjacent portions, is referred to as a “dog-leg coupling.”

[0111] In other embodiments, additional configurations for the coupling between the linear actuator shaft and the plunger are contemplated, as shown in FIGS. 21A and 21B. In these embodiments, the linear actuator shaft 260 and plunger 264 are not permanently physically coupled, but rather a coupling is used that allows transient contact while the linear actuator 260 is retracting (aspiration, FIG. 21B) or extending (dispensing, FIG. 21A). In one embodiment, using this configuration, the plunger 264 also includes a lip or overhang structure 263 at its proximal end, which allows the linear actuator shaft 260 to grip and retract the plunger 264 from the nanosyringe cylinder channel. Using this configuration, the coupling 262 can function as a hammer on top of the plunger. This configuration has the advantage of allowing the plunger to accelerate much faster during the dispensing process, thereby reducing the likelihood of liquid drooling or sticking after the dispensing event. In some aspects, this configuration may have the additional advantage of relaxing the stringent alignment requirements between the linear actuator shaft, plunger, and nanosyringe cylinder.

[0112] In one embodiment, eight nanosyringes successfully utilized in the nanosyringe device examples were constructed from stainless steel tubing with an orifice at one end. The plungers used for these cylinders were stainless steel rods. The nanosyringe cylinders had a volume capacity of 12 μL (i.e., 12,000 nL). The stroke length was 24 mm, and the cylinders had a dead volume of less than 0.4 μL (i.e., 400 nL). As used herein, this dead volume refers to the amount of fluid remaining inside the cylinder when the plunger is depressed to its lowest position (touching the distal end of the cylinder). This includes the orifice, the distal end of the cylinder, and the amount of liquid remaining between the inner wall of the cylinder and the OD of the plunger. A range of plunger diameters (e.g., 31-34 mils) and cylinder channel diameters (i.e., cylinder inner diameter) have been successfully tested. In some embodiments, the plunger diameter and cylinder channel dimensions are paired such that there is a maximum 1 mil difference between the diameter of the nanosyringe cylinder channel and the diameter of the nanosyringe plunger, although one skilled in the art will recognize that the liquid handler designs described herein are not limited to these dimensions.

[0113] 4, the nanosyringe cylinder and plunger can be configured such that plunger 450, which moves freely within a nanosyringe cylinder channel having a diameter D1, has a shaft diameter D3, and the plunger includes a distal flexible seal 460 at its outer diameter, the diameter of which is approximately the same as nanosyringe cylinder channel diameter D1. For example, if cylinder diameter D1 is approximately 33 mils, plunger shaft 450 connected to plunger seal 460 can be approximately 25 mils, although any value smaller than the diameter of the plunger seal can be used, for example, between approximately 25 and 29 mils.

[0114] The distal plunger seal 460 can be made of any suitable material capable of forming a tight bond with the sidewall of the nanosyringe cylinder. Suitable materials include, but are not limited to, Teflon, Delrin plastic, PEEK, and several composite materials, such as high-strength, slippery PEEK (containing Teflon, graphite, and carbon) and slippery Delrin Acetal Resin (Teflon blended with Delrin). Ceramic or sapphire plunger seals are also used in the inventive devices described herein.

[0115] III. Seal The devices described herein can optionally include a seal surrounding the junction between the nanosyringe cylinder and the nanosyringe plunger, which can prevent or reduce the loss of any liquid aspirated or dispensed from the nanosyringe.

[0116] One embodiment of such a seal is shown in Figure 2. As shown in this figure, seal 240 surrounds the junction between nanosyringe cylinder 220 and syringe plunger 250. Figures 5A and 5B show an alternative embodiment of seal 520 that can be used to create a leak-proof junction or reduce fluid loss between nanosyringe cylinder 530 and plunger 510.

[0117] The length of the seal is not limited in any way and can encompass either a smaller or larger portion of the cylinder / plunger junction. In some embodiments, the seal can have an O-ring type structure or be a longer structure that encompasses a larger area. In some embodiments, it is advantageous to have a smaller seal as opposed to a larger seal in that a larger seal can create more friction between the plunger and the surrounding seal material, which can affect the accuracy of the dispensing or aspiration event.

[0118] The seals may be of any suitable material, including, but not limited to, Teflon™, Delrin® plastic, polyetheretherketone (PEEK), and some composite materials such as high-strength, slippery PEEK (containing Teflon™, graphite, and carbon) and slippery Delrin Acetal Resin (Teflon™ compounded with Delrin®).

[0119] In some embodiments, no seal is required at the junction between the nanosyringe cylinder and the plunger.

[0120] IV. Elongated Body Geometry, Orifice, and Orifice Plate In some embodiments, the orifice dimensions and the configuration of the distal end of the elongated body, such as a cylinder, are important in achieving accurate and clean dispensing events from the nanosyringe. To this end, various features can be optimized. As shown in FIG. 6 , a preferred minimum liquid dispensing speed, V2, for non-contact dispensing is fast enough to cleanly deliver dispensed liquid 622 into the receiving well without leaving hanging droplets near orifice 614. Conversely, a preferred maximum dispensing speed, V2, should be a speed that does not result in spraying of the dispensed liquid from the orifice or splashing of the liquid, which could result in contamination of the surrounding well. In some embodiments, without limitation, a preferred speed, V2, is approximately 3-5 m / s, although one skilled in the art will recognize that a much wider range of dispensing speeds can be used with the devices described herein.

[0121] In some embodiments, the distal end of the elongate body, e.g., a cylinder, in a nanosyringe is capped with an orifice plate. As used herein, the term "orifice plate" refers to a separate part that is machined with an orifice and joined to the cylinder in a separate step. In various embodiments, the orifice plate is attached to the cylinder by welding, or alternatively by pressure deformation, press fit, or adhesive. In some embodiments, the devices described herein do not use an orifice plate when the elongate body, e.g., a cylinder, including the orifice, is machined entirely from one piece of material (e.g., by drilling a blind hole followed by drilling the orifice).

[0122] Figure 7 provides some non-limiting examples of distal tip configurations that may be used in the nanosyringes described herein. In some embodiments, the orifice plate may be formed from the same starting material used to construct the cylinder; for example, the orifice may be molded at the same time the cylinder is machined from stainless steel starting material; see, e.g., Figures 7B, 7C, 7E, and 7G. In other embodiments, the final structure of the nanosyringe cylinder, including the orifice plate, is added after the cylinder is manufactured and can be made from any material, e.g., sapphire, stainless steel, glass, or other suitable material, depending on the application. In various embodiments, the orifice plate may be formed, press-fit, pressure-deformed, welded, machined, or attached with an epoxy adhesive to the cylinder.

[0123] In some embodiments, the orifice plate is circular with an outer diameter the same as the inner diameter of the cylinder (see, e.g., FIG. 7A) and has a thickness of about 0.3 mm, although any other suitable thickness, e.g., about 0.1-1.0 mm, can be used.

[0124] In some embodiments, the orifice plate is circular and has an outer diameter the same diameter as the outer diameter of the cylinder (see, e.g., FIG. 7D), which is advantageous in that it is easier to manufacture than some other embodiments because the orifice plate can be sized precisely after installation.

[0125] In some embodiments, the diameter of the orifices in the orifice plate is approximately 0.12-0.13 mm (120-130 microns). In various embodiments, the orifice diameter can range from about 100 microns to about 150 microns in diameter. In some embodiments, the orifices are about 125 microns in diameter.

[0126] Figures 7E and 7F provide advantageous embodiments of distal geometries in which the orifice channels are elongated and result in laminar flow as liquid is dispensed, thereby minimizing spraying and cross-contamination of adjacent wells, for example, in a multi-well chip. Two independent features are shown that provide advantages.

[0127] (1) Longer orifice channel It was observed that liquid dispensed from a longer orifice channel tends to flow more linearly and is less likely to deflect in undesired directions than liquid dispensed from a naosilicate ring with a shorter orifice channel length. The flow rate also increases, resulting in better dispensing at lower volumes.

[0128] (2) Tapered shape of the end of the nanosyringe cylinder The tapered shape prevents liquid from adhering to the bottom of the distal end of the cylinder. It has been observed that with flat-bottom cylinders or flat-bottom orifice plates, a significant amount of liquid can adhere to the distal end of the syringe cylinder or the bottom of the orifice plate when the cylinder or plate retracts from the pool of liquid. Additionally, if the dispensed liquid rate is slow, liquid can accumulate at the bottom of the distal end of the cylinder over multiple dispense events. The tapered shape reduces the hanging drop volume in both of these cases. A final advantage of this geometry is that the outer dimensions of the distal orifice are smaller than the diameter of the target well and can be lowered into the well for more precise dispensing.

[0129] In still other embodiments, other distal cylinder geometries and orifice plates are also used in the devices described herein.

[0130] 7A shows a sapphire orifice plate. In some embodiments, the sapphire orifice plate is press-fit and glued, or alternatively, is pressure-deformed. In some embodiments, the sapphire plate is used in a cylinder with a notch in the cylinder tube to provide a step that acts as a hard stop for the jewel, and the end of the cylinder tube is pressure-deformed around the end of the jewel to hold the jewel in place.

[0131] FIG. 7B shows an orifice machined from a single piece of starting material, for example stainless steel.

[0132] FIG. 7C shows an orifice with an expanding tapered design that can be manufactured by machining.

[0133] FIG. 7D shows the orifice plate welded onto the end of the tube.

[0134] FIG. 7E shows an orifice manufactured by machining, which takes advantage of the tapered shape.

[0135] 7F shows a tapered orifice plate insert that can be fabricated, for example, using a sapphire jewel insert. This configuration has certain advantages for manufacturing, including: (i) it is easy to process the interior of the nanosyringe cylinder (e.g., polishing the interior surface to make the nanosyringe cylinder smoother), (ii) it is easy to keep the entire cylinder straight, and (iii) this design has consistent taper / orifice dimensions, thereby facilitating reproducible manufacturing.

[0136] FIG. 7G shows a nanosyringe cylinder design with an orifice used for contact dispensing.

[0137] FIG. 7H shows a nanosyringe cylinder design incorporating an orifice plate with an orifice through the plate, which has the advantage of a longer orifice channel.

[0138] Fabrication of nanosyringe cylinders and orifice plates with various orifice geometries and made from different materials (e.g., stainless steel or sapphire) is advantageous because the cylinder end or orifice plate can be designed to have various desired characteristics. For example, a distal end or orifice plate with a longer channel and / or in combination with a narrower opening channel can increase the velocity of liquid exiting the nanosyringe. Conversely, a distal end of a nanosyringe cylinder with a larger opening or a shorter exit channel can generate a slower liquid velocity exiting the nanosyringe. Those skilled in the art will recognize that various fabrication techniques can be used to custom-fabricate the distal end of a nanosyringe cylinder or orifice plate of any desired configuration. In other embodiments, a suitable orifice size can be selected based on the optimal size for aspirating and dispensing the particular cells of interest, with larger cells being optimally processed using larger orifice sizes to avoid shear damage to the cells.

[0139] V. Contactless and Contact Dispensing In some embodiments, the geometry of the distal end of the cylinder or the orifice plate can be optimized for either non-contact liquid dispensing or contact dispensing. The examples described herein are configured for non-contact dispensing. That is, the dispensing conditions for non-contact dispensing eject fluid at a suitable high velocity so that the liquid separates from the cylinder or orifice plate and travels through the air into the receiving well. However, those skilled in the art will recognize that these devices and methods are readily adapted for contact dispensing.

[0140] Contact dispensing is accomplished by slowly dispensing the fluid so that it creates a small accumulation of fluid at the bottom of the cylinder or orifice plate, and then lowering the distal end of the cylinder until the fluid comes into contact with the dispensing surface. The surface tension of the fluid causes the fluid to adhere, and when the cylinder is retracted, some or most of the fluid remains on the dispensing surface.

[0141] For contact dispensing, the cylinder or orifice plate is designed so that a ball of fluid reproducibly forms at the end of the cylinder or orifice plate. In some embodiments for contact printing applications, the cylinder or orifice plate is designed so that the distal end is not excessively sharp or tapered, and similarly, is not excessively large or flat so that a droplet does not have difficulty detaching from the distal end of the cylinder or orifice plate. In some embodiments, consistent with the present description, cylinders and orifices consistent with this design, such as, but not limited to, those shown in FIG. 7G, can be used for contact dispensing.

[0142] Those skilled in the art will recognize the variables and considerations when designing a cylinder end or orifice plate optimized for either non-contact or contact dispensing. See, e.g., Bammesberger et al., "A Calibration-Free, Noncontact, Disposable Liquid Dispensing Cartridge Featuring an Online Process Control," Journal of Laboratory Automation, Vol. 19(4) 394-402 (2014); and Kong et al., "Automatic Liquid Handling for Life Science: A Critical Review of the Current State of the Art," Journal of Laboratory Automation, 17(3) 169-185 (2012).

[0143] VI. Dispense Volume and Receiving Wells The nanosyringe devices described herein are optimally configured for dispensing nanoscale liquid volumes, for example, from 1 nanoliter (nL) to 999 nL. The nanosyringe devices described herein can also be used to aspirate and dispense larger volumes.

[0144] In some embodiments, the nanosyringes described herein are capable of reproducibly dispensing volumes of liquid as small as 5 nL, 10 nL, 15 nL, 20 nL, 25 nL, 30 nL, 35 nL, 40 nL, 50 nL, 60 nL, 70 nL, 80 nL, 90 nL, or 100 nL.

[0145] In some embodiments, the nanosyringes described herein are capable of reproducibly dispensing 50 nL ± 3 nL. In some embodiments, the nanosyringes described herein are capable of reproducibly dispensing 35 nL. In other embodiments, the nanosyringes described herein are capable of reproducibly dispensing 20 nL.

[0146] As used herein, the phrase "reproducibly dispenses" and similar phrases refer to a liquid dispensing operation of a device in which repeated dispenses of a given volume result in actual dispenses that differ from each other by no more than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%.

[0147] The configuration of the receiving well that receives the liquid dispensed from the nanosyringe is not particularly limited and varies based on the intended use of the system. In some embodiments, a multi-well plate called a chip is used in the system. For example, a chip containing wells with a 72 x 72 well configuration, totaling 5,184 wells, is particularly used in the system described herein. For example, such a chip can be the ICELL8® 5,184-well chip manufactured by Takara Bio USA, Inc.

[0148] VII.Suction amount The actual aspiration volume and aspiration capacity of a nanosyringe is not particularly limited and is a function of the holding capacity of the nanosyringe's cylinder component, which is determined by the cylinder diameter and the cylinder's usable height. Furthermore, these same metrics are reflected in the width and length of the plunger (in configurations where the plunger forms a tight bond with the inner wall of the cylinder).

[0149] The aspiration volume is not particularly limited and is a function of the holding capacity of the nanosyringe's cylindrical component. In some embodiments, the nanosyringe's aspiration volume is about 12,000 nL (i.e., 12 μL). In other embodiments, the nanosyringe has a larger volume, including, for example, aspiration volumes of about 15 μL, about 20 μL, about 25 μL, about 30 μL, about 40 μL, or about 50 μL.

[0150] In some embodiments, the aspiration volume is larger than a single dispense volume, which is beneficial for performing multiple dispense events after a single larger aspiration step.

[0151] Liquid processing method and use In some aspects, the present disclosure provides methods for liquid handling that find a wide variety of applications readily apparent to those of skill in the art. In one aspect, the present disclosure provides methods for dispensing liquid into wells of a multiwell device. In some aspects, the multiwell device is a multiwell plate or multiwell chip of any suitable design. In some aspects, the multiwell device contains 5,184 fluidly separated wells, such as, for example, the ICELL8® 5,184-well chip manufactured by Takara Bio USA, Inc. Although the following description is provided with respect to an embodiment in which the actuator is a linear actuator and the elongated body is a cylinder, the invention is not so limited, for example, as noted above.

[0152] In some aspects, the method of dispensing a liquid includes both aspirating the liquid and dispensing the liquid. In some aspects, the method includes providing an apparatus, the apparatus comprising: (i) a linear actuator having a movable shaft; (ii) a cylinder characterized by a central channel, a proximal end, and a distal end, the distal end comprising an orifice; (iii) A plunger characterized by a proximal end and a distal end, (A) The proximal end is mechanically coupled to a movable shaft; (B) a plunger, at least a portion of a distal end of the plunger being movably positioned within the central channel of the cylinder; Equipped with.

[0153] Part of this system includes, but is not limited to, any suitable receiving vessel, tube, dish, plate, multi-well dish, or chip. In some embodiments where a multi-well device is used, the multiple wells are fluidically separated. The multi-well device can be positioned to receive liquid dispensed from a nanosyringe. In some embodiments, the multi-well device is on a movable stage that can be repositioned in the x / y plane to position any given well or group (e.g., subset) of wells under the nanosyringe or nanosyringe array.

[0154] The first step of the method is to position the distal end of the cylinder in a source plate well containing the liquid to be dispensed. The liquid is aspirated into the syringe by retracting the linear actuator shaft, thereby retracting the plunger in the cylinder channel, and thereby aspirating the liquid into the cylinder channel. The nanosyringe is then withdrawn from the source plate well, and the distal end of the cylinder is repositioned over the well of the multi-well device receiving vessel. Dispensing is achieved by extending the linear actuator shaft, thereby extending the plunger in the central channel, thereby forcing the liquid from the cylinder channel into the well of the multi-well device.

[0155] In some aspects, the multi-well device or any other container is associated with a heating element that is capable of heating and / or cooling the multi-well device and maintaining it at a suitable temperature. In some embodiments, the heating device is capable of maintaining at least two different temperatures that can be selected by a user. In some embodiments, the heating element has the ability to heat the multi-well device as a thermal cycler, functioning as a thermal cycling device used in polymerase chain reaction.

[0156] The amount of liquid dispensed by these methods is not particularly limited. For example, in some embodiments, the liquid is dispensed in an amount of about 10 nanoliters (nL) to 300 nL. In one aspect, the amount dispensed is a reproducible amount, i.e., the amount is the same or nearly the same within some acceptable margin of variation between multiple dispensing events. In some aspects, the amount of liquid aspirated is the same as the amount of liquid dispensed.

[0157] In other aspects, the amount of liquid aspirated is greater than the amount of liquid dispensed, which is advantageous because it allows multiple dispense events to occur after a single aspiration event.

[0158] The nature of the liquid aspirated and dispensed is not particularly limited. Those skilled in the art will recognize the wide variety of liquids and useful applications of the liquid handler devices and apparatus described herein.

[0159] In some embodiments, the liquid is a liquid reagent or reagent mixture. The components of the reagent solution or reagent mixture often modify or react with components of the sample prior to some type of analysis. The nature and composition of the reagent solution will vary widely depending on the intended use.

[0160] In some aspects, the reagent fluid is a biochemical reaction mixture. For example, in some embodiments, the reagent fluid is a reaction mixture such as a polymerase chain reaction master mix, which can include any type of suitable enzyme, probe, primer, or other reagent that may be included in such a reaction. In some embodiments, the reaction mixture includes one or more of, but is not limited to, a buffer, an enzyme, a dNTP, a primer, a template switch oligonucleotide, or any combination thereof. In some embodiments, the reagent mixture can also include a mock sample, e.g., a nucleic acid template, which can act as a positive control for the components of the reagent mixture. The reaction mixture can be a next-generation sequencing (NGS) library preparation reaction mixture, and further, the methods described herein can include performing an NGS reaction. The range of useful reagent mixtures for biochemical reactions is broad and includes, but is not limited to, PCR reagent mixtures, RT reagent mixtures, reagent mixtures for any type of nucleic acid sequencing, including, but not limited to, next-generation sequencing or single-molecule sequencing, and reagent mixtures for any type of cloning.

[0161] In other aspects, the liquid manipulated by the liquid handling devices described herein is a liquid sample.

[0162] In various embodiments, the sample and reagents are combined, i.e., mixed, at some point. In some embodiments, this mixing step occurs in the wells of a multi-well device. In some embodiments, a fluid handling device described herein, e.g., a nanosyringe, delivers both sample and reagent fluids to the wells of a multi-well device in two separate processing steps. In some embodiments, either the sample or the reagent mixture is pre-dispensed into the wells of the chip prior to the addition of the other, such that the fluid handling device of the present disclosure dispenses only either the sample or the reagent mixture into the wells of a multi-well device.

[0163] In some embodiments where the liquid handling device delivers both sample and reagent mixtures to the wells of a multi-well chip, the order of addition is not limited, as the sample or reagents can be added in any order.

[0164] In some embodiments, the devices and systems described herein can include cameras and associated components, such as light sources, lenses, and filters, for imaging the contents of individual wells of a multiwell device, such as a multiwell plate or chip. These cameras are useful for a variety of purposes, including, in some embodiments, verifying proper delivery of sample or reagent fluids to individual wells. In some embodiments, the cameras are capable of imaging, quantifying, or real-time monitoring the accumulation of products produced by a biochemical reaction, such as amplicons produced by a PCR reaction. In some aspects, the biochemical reaction is a PCR reaction, and the camera imaging is quantitative imaging that measures the accumulation of PCR product amplicons produced by the reaction. In other embodiments, the cameras described herein can be used to align a multiwell chip, read barcodes or other identifying information that may be present on the multiwell chip or source plate, or image, e.g., count, cells in the wells of a multiwell chip.

[0165] Computer Control System Aspects of the present disclosure further include systems, such as computer control systems, for carrying out the above-described method embodiments. In some cases, the system further includes one or more computers for full or partial automation of the methods described herein. In some embodiments, the system includes a computer having a computer-readable storage medium having a computer program stored thereon.

[0166] In some embodiments, the system includes an input module, a processing module, and an output module. The subject systems may include both hardware and software components, where the hardware components may take the form of one or more platforms, e.g., in the form of servers, such that the functional elements, i.e., elements of the system that perform specific tasks of the system (e.g., managing information input and output, processing information, etc.), may be performed by executing software applications on or across one or more computer platforms representative of the system.

[0167] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, a barcode reader, etc. The processing module includes a processor that accesses a memory in which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are or become available. The processor executes an operating system, which interfaces with firmware and hardware in well-known ways to facilitate the processor's coordination and execution of the functions of various computer programs, which may be written in various programming languages, such as Java, Perl, C++, Python, JavaScript, C#, Go, R, Swift, PHP, or other high-level or low-level languages, and combinations thereof, as known in the art. The operating system typically cooperates with the processor to coordinate and execute the functions of the other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. The processor may be any suitable analog or digital system, hi some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0168] System memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-and-write compact disk, flash memory devices, or other memory storage devices. Memory storage devices may also be any of a variety of known or future devices, including compact disk drives, tape drives, removable hard disk drives, or diskette drives. Such types of memory storage devices typically read from and / or write to program storage media such as compact disks, magnetic tapes, removable hard disks, or floppy diskettes, respectively. Any of these program storage media, or others now in use or that may later be developed, may be considered computer program products. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0169] In some embodiments, a computer program product is described that includes a computer usable medium having stored thereon control logic (a computer software program including program code). The control logic, when executed by a processor, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementing a hardware state machine to perform the functions described herein will be apparent to one skilled in the art.

[0170] The processor may include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming may be provided remotely to the processor via a communications channel or may be pre-stored on a computer program product, such as a memory or some other portable or non-removable computer-readable storage medium using any of these devices in conjunction with the memory. For example, a magnetic or optical disk may carry the program and be readable by a disk writer / reader. The inventive system also includes programming, e.g., in the form of a computer program product, algorithms for use in implementing the above-described methods. Programming according to the present invention may be recorded on a computer-readable medium, e.g., any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; optical storage media such as CD-ROMs; electrical storage media such as RAM, ROM; portable flash drives; and hybrids of these categories, such as magnetic / optical storage media.

[0171] The processor may also have access to a communication channel for communicating with a user in a remote location, meaning that the user is not in direct contact with the system but instead relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), a telephone network, a satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0172] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications (e.g., radio frequency identification (RFID), Zigbee communications protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0173] In some embodiments, the communications interface is configured to include one or more communications ports, e.g., a physical port or interface such as a USB port, an RS-232 port, or any other suitable electrical connection port that allows data communication between the subject system and other external devices, such as computer terminals (e.g., in a doctor's office or hospital environment) configured for similar complementary data communication.

[0174] In some embodiments, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the target system to communicate with computer terminals and / or other devices such as networks, communication-enabled mobile phones, personal digital assistants, bar code readers, or any other communication device that a user can integrate and use.

[0175] In some embodiments, the communication interface is configured to provide a connection for data transfer using Internet Protocol (IP) over a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) in a local area network (LAN) connected to the Internet, or a WiFi connection to the Internet at a WiFi hotspot.

[0176] In one embodiment, the target system is configured to wirelessly communicate with a server device via a communications interface using common standards such as, for example, 802.11 or Bluetooth® RF protocols, or the IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0177] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in the target system, e.g., any data storage unit, with a network or server device using one or more of the above communication protocols and / or mechanisms.

[0178] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of graphical elements. A graphical user interface (GUI) controller provides a graphical input / output interface between the system and the user and may include any of a variety of known or future software programs for processing user input. The functional elements of a computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing modules to a remote user, for example, via the Internet, telephone, or satellite network, according to known techniques. Presentation of data by the output manager may be performed according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, CSV files, emails, or other files, or other formats of data. The data may also include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, CSV, or other documents or data from remote sources. The one or more platforms present in the subject system may be any type of known or future developed computer platform, such as a server. However, they may also be mainframe computers, workstations, or other computer types. They may be connected via any known or future type of cabling or other communication system, including wireless systems, and may or may not be networked. They may be co-located or physically separated. In some cases, various operating systems may be employed on any computer platform, depending on the type and / or manufacturer of the computer platform selected.Suitable operating systems include Windows, iOS, Oracle Solaris, Linux, IBM i, Unix, and others.

[0179] Aspects of the present disclosure further include non-transitory computer-readable storage media having instructions for implementing the subject methods. The computer-readable storage media may be used on one or more computers for fully or partially automating systems for implementing the methods described herein. In certain embodiments, instructions according to the methods described herein may be encoded on a computer-readable medium in the form of "programming," and the term "computer-readable medium" as used herein refers to any non-transitory storage medium involved in providing instructions and data to a computer for execution and processing. Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state disks, and network-attached storage (NAS), regardless of whether such devices are internal or external to the computer. Files containing information may be "stored" on a computer-readable medium, where "storing" means recording information so that it can be accessed and retrieved at a later date by a computer.

[0180] The non-transitory computer-readable storage medium may be used in one or more computer systems having a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, a barcode reader, etc. The processing module includes a processor that accesses a memory in which instructions for performing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices, and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors that are or become available. [Example]

[0181] It will be understood that the structures, materials, compositions, and methods described herein are intended to be representative examples of the present disclosure, and that the scope of the present disclosure is not limited by the scope of the examples. Those skilled in the art will recognize that the present disclosure can be practiced with variations on the disclosed structures, materials, compositions, and methods, and that such variations are considered to be within the scope of the present disclosure.

[0182] Example 1 Nanoliter dispensing over a range of volumes Tests were conducted to observe the dispensing accuracy of the nanosyringe of the present disclosure, using water as the dispensing liquid over a range of 30 nL to 300 nL, and the results are shown in FIG.

[0183] Consistent with this specification, experiments used a nanosyringe with a stepper motor linear actuator coupled to a plunger residing within a channel of a stainless steel cylinder. This configuration used a cylinder with a sapphire orifice plate having a design similar to that shown in FIG. 7A. The sapphire orifice plate incorporated an orifice approximately 125 microns in diameter. The plunger diameter was approximately 32 mils, and the cylinder channel diameter was approximately 33 mils. In this test, the plunger speed was programmed to 66.5 mm / s.

[0184] The nanosyringe was programmed to dispense water in 10 nL increments from 30 nL to 100 nL and in 20 nL increments from 120 nL to 300 nL. The actual volume dispensed was determined gravimetrically using a microbalance. Twenty dispenses were made onto a glass slide at each dispense increment, and the results were averaged. A cover slide was used to prevent evaporation during the measurements. The actual dispensed volume versus the programmed volume is shown in Figure 16. As can be seen in Figure 16, the correlation between the programmed and actual dispensed volumes is extremely good across the range of interest.

[0185] Example 2 Reproducibility of 35nL and 100nL liquid dispenses from a nanosyringe Tests were conducted to verify the reproducibility of dispensing test volumes of 35 nL to 100 nL from a nanosyringe as described in this disclosure, and the results are shown in Figures 17A and 17B.

[0186] Consistent with this specification, experiments used a nanosyringe with a stepper motor linear actuator coupled to a plunger residing within a stainless steel cylindrical channel. This configuration used a cylinder with an orifice plate welded and then drilled in a configuration similar to that shown in Figure 7A. The orifice plate incorporated an orifice approximately 125 microns in diameter. The plunger diameter was approximately 32 mils, and the cylindrical channel diameter was approximately 33 mils. For these tests, the plunger speed was programmed to 66.5 mm / s.

[0187] Multiple dispenses were performed for each dispense volume tested, with 20 repeats of the programmed 35 nL dispense and 7 repeats of the programmed 100 nL dispense. The actual dispense volume was estimated by taking the total cumulative weight of the dispense events; repeat dispense events were required to bring the total dispense weight within the range of the microbalance. After dispensing, multiple dispenses were immediately covered and weighed to prevent evaporation. The cumulative weight of the dispensed fluid was converted to a volume estimate based on fluid density and number of dispenses and then plotted against the programmed dispense volume. These results are shown in Figures 17A and 17B. These two graphs show good correlation between the programmed and actual dispense volumes for both the 35 nL and 100 nL volume tests.

[0188] Example 3 Reproducibility of liquid dispensing volumes from nanosyringes in the range of 20nL to 50nL A study was conducted to test the repeatability of dispensing liquid from a nanosyringe as described in this disclosure over a volume range of 20 nL to 50 nL. This experiment was performed by measuring the volume of liquid dispensed from the nanosyringe device using an ImageXpert Inc® JetXpert Jr™ (formerly known as JetXpert OEM) in-line droplet visualization and measurement platform.

[0189] A nanosyringe was constructed according to the teachings of the present disclosure. A nanosyringe was used with a stainless steel cylinder having an internal channel diameter of 0.86 mm, and an orifice plate with a circular orifice having a diameter of 125 microns was fitted to the distal end of the cylinder. The orifice plate had a configuration similar to that shown in FIG. 7A. A stainless steel plunger having a diameter of 0.84 mm was located within the cylinder. A seal was fitted between the proximal end of the cylinder and the plunger shaft residing within the cylinder channel. The seal was made of polyetheretherketone (PEEK) composite plastic.

[0190] The maximum plunger stroke length was 24 mm, resulting in a total nanosyringe volume of approximately 12 μL. Aspiration of the nanosyringe was achieved by retracting the plunger from the distal end of the cylinder, and dispensing was achieved by extending the plunger toward the distal end of the cylinder.

[0191] Dispensing consisted of an initial starting velocity followed by two acceleration stages, the fastest of which was 4,768 mm / s, until a maximum velocity of 66.5 mm / s was reached. 2 The motor current setting was 700 mA, and the motor microstepping mode was 6400 microsteps / rev.

[0192] Data acquisition during the programmed water dispense was performed using the ImageXpert Inc® JetXpert Jr™ (formerly known as JetXpert OEM) in-line droplet visualization and measurement platform. The JetXpert Jr camera was synchronized with the nanosyringe controller to capture images of the dispensed fluid stream throughout its entirety. The JetXpert Jr software can perform image processing on the captured images to estimate the total volume of fluid.

[0193] The nanosyringe assembly was positioned so that the distal end of the cylinder of a single nanosyringe channel was directly above the field of view of the JetXpert Jr. The nanosyringe was manually filled by submerging the distal end of the cylinder in a disposal tube with deionized water and then aspirating 12 μL of water. The nanosyringe was programmed to dispense 200 target volumes of 50 nL with a 50 ms pre-dispense delay and a 50 ms post-dispense delay. JetXpert captured every fourth image and recorded the resulting data. This experiment was repeated for dispense volumes of 45 nL, 40 nL, 35 nL, 30 nL, 25 nL, and 20 nL. A total of 50 images were acquired for each dispense volume. The results are shown in the table below.

[0194] [Table 1] CV, or coefficient of variation, is the ratio of the standard deviation to the mean (STDV / mean)

[0195] These results are presented graphically in Figure 18. As can be seen in the table above and in Figure 18, there was good linearity between the programmed target volume and the measured volume (slope of 1.0034), the dispense CV was less than 15%, and the dispense resolution was 5 nl. A minimum dispense volume of 20 nl was also demonstrated.

[0196] Example 4 Accuracy of liquid dispensing onto nanowell arrays Tests were performed to verify the integrity of liquid dispensing from the nanosyringe as described in this disclosure, and the results are shown in FIG.

[0197] An experiment was designed to demonstrate the accuracy of the nanosyringe to dispense precise liquid volumes into the nanowells of an ICELL8® 5,184-well chip manufactured by Takara Bio USA, Inc. The chip was placed on the chip platform of a liquid dispensing device incorporating the nanosyringe of the present disclosure.

[0198] In separate experiments, an apparatus consisting of a 2 x 4 array of nanosyringes was programmed to deliver 35 nL or 50 nL liquid volumes from a 384-well source plate to predetermined well addresses on a multiwell chip. The nanosyringes were configured and used dispensing parameters according to the teachings of the present disclosure and as described in Example 3.

[0199] The dispensed liquid contained a UV dye. The liquid was delivered to designated wells in a repeating grid pattern over four aspirate-dispense-wash cycles. For the dispense portion of each cycle, a 2x4 array of nanosyringes simultaneously deposited liquid into 32 wells in the grid pattern, such that each liquid-containing well was surrounded by three to four empty wells. This allowed for inspection of empty wells as a visual indication of cross-contamination. After liquid delivery, the chip was removed from the device and imaged on a Takara Bio USA SmartChip® Real-Time PCR System.

[0200] An image from a representative experiment dispensing 35 nL is shown in Figure 19. Similar results were obtained for a 50 nL dispense volume. The image demonstrates accurate dispensing of liquid volumes to programmed, addressable locations on a 5,184-well chip, with no cross-contamination (i.e., no dispensing or contamination of unprogrammed well locations), and shows a nearly uniform dispense volume delivered to each programmed well based on dye intensity. The image demonstrates accurate programmed dispenses from the nanosyringe using test volumes of 35 nL or 50 nL.

[0201] Example 5 Assessment of contamination in liquids dispensed from nanosyringes A study was conducted to verify the reproducibility and integrity of nanosyringe dispensing as described in this disclosure. Positive and negative control reagent mixtures for PCR amplification reactions were dispensed into wells of an ICELL8® 5,184 nanowell chip (Takara Bio USA, San Jose, CA), and the amplified products were quantified by qPCR reactions. This study evaluated the extent of cross-contamination and carryover contamination in the nanosyringe dispensing system.

[0202] As used herein, the phrase "cross-contamination" refers to contamination occurring outside of the nanosyringe cylinder as a result of an improper dispensing event, for example, where satellite droplets from the distal end of the nanosyringe cylinder may land in adjacent wells. Another example of cross-contamination refers to a scenario where there is a large defect in the liquid that bridges between the distal end of the cylinder and the wells of the chip, resulting in the liquid dispensed from the distal end of the nanosyringe cylinder being dragged across multiple wells.

[0203] As used herein, the term "carryover contamination" refers to contamination that occurs inside the nanosyringe cylinder, for example, when the cylinder cleaning step is insufficient, resulting in some of the previous liquid mixing with the newly aspirated liquid.

[0204] To check for carryover and cross-contamination, the nanosyringes were tested using an interdigitated pattern of positive and negative control reagent solutions dispensed onto a 5,184 nanowell plate.

[0205] Nanosyringes were constructed according to the teachings of the present disclosure. Each syringe used a stainless steel cylinder with an internal channel diameter of 0.86 mm, and the distal end of the cylinder was fitted with an orifice plate with a circular orifice with a diameter of 125 microns. The orifice plate used a configuration similar to that shown in FIG. 7A. Within the cylinder was a stainless steel plunger with a diameter of 0.84 mm. The plunger had a maximum stroke length of 24 mm, resulting in a total nanosyringe volume of approximately 12 μL. A seal was used to fit the proximal end of the cylinder and the plunger shaft residing within the cylinder channel. The seal was made of Delrin® plastic.

[0206] A test solution containing qPCR reagents containing a positive control template expected to produce amplicons was dispensed into preprogrammed locations on a 5,184-well chip. This test was conducted to evaluate the cleaning effect of switching between the solution containing the qPCR reagents containing the positive control template and the wash solution, and to test how effectively the nanosyringe could be washed between qPCR reagent dispense steps.

[0207] The first part of the experiment was to examine the level of cross-contamination while dispensing into the nanowell chip. A qPCR positive reagent mix lacking template nucleic acid and a PCR negative control reagent mix were prepared and placed into wells at either end of a 384-well source plate, which was individually sealed at each end. The source plate and an empty 5,184-well chip were placed in the chamber of the disclosed liquid handler system. The source plate was oriented so that the negative qPCR reagent mix could be dispensed. A predetermined "negative grid" dispense pattern was initiated by the control software, and 35 nL of the negative qPCR reagent mix was delivered using the nanosyringe. This was accomplished over 10 aspirate-dispense-wash cycles. Each wash cycle was programmed with a Vmax of 10 mm / s and a flow rate of 40 mm / s from fluids supplied from bleach or water troughs. 2The maximum stroke length suction sequence was followed by a programmed Vmax of 300 mm / s and a Vmax of 5,000 mm / s. 2 At A1, the contents were purged into a waste trough.

[0208] The negative grid pattern fills half of the chip with a negative control qPCR mix (called an NTC, or no template control), while the other side is filled in grid format only. After dispensing was completed, the chip was aspirated to remove the liquid on top of the chip, and the source plate was inverted so that the positive qPCR reagent mix could be dispensed. A predefined "positive grid" pattern was initiated by the software, and a nanosyringe was used to deliver 35 nL of positive qPCR reagent mix only to the grid side of the chip, alternating with the negative qPCR mix within the wells. This was achieved over eight aspirate-dispense-wash cycles. Surrounding positive wells with negative wells is a way to assess the level of cross-contamination; if a significant amount of positive reagent mix enters an adjacent negative control well, amplification events will be observed in the negative control well.

[0209] After dispensing of the positive qPCR reagents was completed, the chip was blotted again to remove the liquid on top of the chip, and the chip was removed, sealed, centrifuged, and then thermal cycled and imaged on a SmartChip cycler (Takara Bio USA, Inc., San Jose, California).

[0210] To check for carryover contamination, a second, complementary experiment was also performed: the same qPCR positive and negative control reagent mixes were prepared and the experiment described above was repeated, except that the positive wells were dispensed before the negative wells.

[0211] For experiments looking at cross-contamination, Figure 24A provides a photograph of the nanowell chip imaged after amplification. Wells in which amplification occurred appear lighter in color than the negative wells, which appear black in the image. The right side of Figure 24A contains the negative control reagent mix dispensed into each well. This right side was used to calculate the natural background level of contamination. The positive and negative grid wells are shown on the left side of Figure 24A. The data collected from Figure 24A is summarized in the table below. The % delta true contamination is the % contamination on the left side minus the % natural background contamination from the right side. The black wells in the figure represent wells with a Ct value greater than 6 from the average Ct value of the true positive wells from the grid pattern on the left side. t These wells are not counted.

[0212] [Table 2]

[0213] The expected lot Ct (or threshold cycle) metric is used to confirm that there are no significant errors in the thermal cycling or preparation of the qPCR positive control reagent mix. The Ct SD provides an indication of the uniformity of the dispense and the inherent uniformity of the environmental conditions experienced by the nanowells. The % natural background is the number of wells on the right side of the chip with a Ct within 6 Ct of the true mean positive well divided by the total number of wells on this side (i.e., 2,592 wells). This percentage is used as the cutoff for what is defined as a "significant" level of contamination. This number is used as a baseline for the contamination level of the nanowell chip. The % delta true contamination represents the percentage of expected negative wells on the left side of the nanowell chip with a Ct within 6 Ct of the true mean positive well Ct value, minus the % natural background contamination. As shown in the table above, all metrics passed the pre-established criteria for successful dispense with only minor, i.e., experimentally acceptable, levels of cross-contamination.

[0214] The results for the complementary carryover contamination experiment are shown in Figure 24B and presented in the table below.

[0215] [Table 3]

[0216] As shown in the table above, all metrics passed the pre-established criteria for successful dispensing with only negligible, i.e., experimentally acceptable, levels of carryover contamination. Thus, the nanosyringe cylinder cleaning protocol using 0.2% bleach was effective in cleaning the interior of the cylinder to remove, or at least denature or inactivate, any carryover contaminating material.

[0217] Summarizing the results of the experiments shown in Figures 24A and 24B, it was found that there were no significant levels of cross- or carryover contamination (i.e., there was no amplification in negative control wells that were within 6 Ct of the mean Ct value of the true positive wells).

[0218] Example 6 Nanosyringe-to-nanowell chip dispensing in a temperature- and humidity-controlled environment A study was conducted to determine the effect of dew point control on the reproducibility of nanosyringe liquid dispensing into wells of an ICELL8® 5,184 nanowell chip (Takara Bio USA, San Jose, CA). To determine the effect of dew point control on the precision of nanosyringe dispensing characteristics into the wells of a 5,184 nanowell chip, reagents containing a positive control template for a PCR amplification reaction were dispensed into two identical chips. One chip was processed without a dew point controlled environment (i.e., no temperature or humidity control), and the second chip was processed in a dew point controlled environment (i.e., temperature and humidity control).

[0219] The nanosyringe device configuration used to dispense qPCR reagents, including the positive control template, into the chip nanowells included a pair of nanosyringes, each with a 33 mil internal cylinder diameter and a 32 mil plunger diameter. The distal end of each cylinder in the device was similar to the design shown in Figure 7D. The syringes were driven by a plunger V of 66.5 mm / sec. max The seal material was Teflon™.

[0220] The qPCR reagent mix containing the positive control template was dispensed from two nanosyringes into an 8 x 72-well block, starting at one side of the chip and progressing systematically across the chip. After dispensing, the cylinders were washed, and the process was repeated for a total of four cycles to fill the chips. The two chips were subjected to amplification temperature cycling, and the amplified products were quantified.

[0221] During dispensing, on a chip without temperature control, the first three dispense cycles were observed to have major failures in the qPCR chemistry, while the last two sectors that received qPCR reagents on that same chip performed much better. This indicates that reactions in the wells of the first three dispense sectors were adversely affected by evaporation, while the last two were not as severely affected.

[0222] Chips treated with temperature and humidity control during the dispensing process showed a significant improvement in the rate of successful qPCR reactions, with successful and uniform amplification reactions across all sectors of the chip. The improved results were a result of dew point control.

[0223] Therefore, the dew point was achieved by controlling both the chip temperature and the dispense chamber humidity. The dew point controller sets the chip temperature by adjusting the temperature of the mounting chuck under the chip. In this example, the chip was maintained at a temperature between evaporation and condensation, so the chip temperature was adjusted to bring the system to the target dew point. The dispense chamber was a sealed environment, and a humidifier module was included as part of the system, successfully controlling the temperature via under-chip temperature control. The system included a humidity / temperature sensor that monitored the environmental conditions inside the dispense chamber and calculated the desired dew point temperature for controlling the chip. This temperature and humidity control presumably optimized chemical performance by minimizing evaporation losses and condensation.

[0224] Example 7 Assessment of cell viability after cell dispensing from a nanosyringe into a nanowell chip Objective: A study was performed to measure cell viability after aspirating and dispensing mammalian cells from a nanosyringe into the wells of a 384-well plate.

[0225] Materials and Methods: Human lymphocyte K562 cells were counted using ORFLO® Moxi Flow (ORFLO Technologies, Ketchum, ID), diluted to 50,000 cells / mL, and counted again to confirm the starting concentration. Three samples were made from this solution for testing. (i) Control sample Control samples were placed in a 384-well source plate and subjected to the same environmental conditions as the two dispense systems (ii) and (iii), but were not dispensed. (ii) Sample dispensed using a nanosyringe Samples were dispensed using eight nanosyringes described in this disclosure using a cylinder distal end style similar to that shown in Figure 7A. Cells were aspirated from a 2 x 4 grid of wells on a 384-well source plate and dispensed into pools in new wells on the same source plate. Dispensing used pulses that mimic the dispensing of liquid volumes into individual nanowells on the ICELL8 chip. (iii) Samples dispensed using the Takara Bio ICELL8 cx system Samples were dispensed using a Takara Bio ICELL8 cx system. Cells were aspirated from a 2 x 4 grid of wells on a 384-well source plate and dispensed into a pool in a new well on the same source plate. Dispensing used pulses that mimicked the dispensing of liquid volumes into individual nanowells on an ICELL8 chip.

[0226] Approximately 5,000 dispenses were performed using methods (ii) and (iii). Dispensations from eight nanosyringes were pooled for each system and stained with propidium iodide, along with controls for each system. Cell viability was measured for each of the three samples using Moxi Flow as a percentage of all cells in the sample. Moxi Flow uses the Coulter Effect to count cells; as each cell passes through the orifice used to count in the flow cell, this activates optics to take a fluorescence measurement and excite PI to check for a death stain signal.

[0227] This experiment was repeated three times using the nanosyringe system and three times using the Takara Bio ICELL8 cx system. Cell viability was measured with an ORFLO® Moxi flow cassette-based flow cytometer.

[0228] Results / Data: The following cell viabilities were obtained:

[0229] [Table 4]

[0230] [Table 5]

[0231] Cell viability measurements were similar across control and experimental systems.

[0232] Based on the Moxi Flow plot data, there was no obvious damage to the cells caused by the nanosyringe, indicating no shift in the plot distribution after nanosyringe dispensing. The cluster locations in the dispensed and control populations appear relatively similar across all three experiments. Dispensing with the nanosyringe tip style shown in Figure 7A also had no significant effect on cell viability of human lymphocyte K562 cells.

[0233] Example 8 Successful use of SSPro in a 5,184-well chip using nanosyringe liquid handling A study was conducted to demonstrate the effectiveness of the nanosyringe as described herein to enable single-cell full-length transcriptome analysis using the SMART-Seq® Pro application protocol by Takara Bio USA, Inc. for single-cell transcriptome analysis. The analysis was performed on an ICELL8® 5,184-well chip manufactured by Takara Bio USA, Inc. Human peripheral blood mononuclear cells (PBMCs) were used for the analysis.

[0234] The SMART-Seq® Pro Application Kit (Takara Bio USA, San Jose, CA) implements a full-length transcriptome library preparation workflow on the ICELL8® cx Single-Cell System (Takara Bio USA, San Jose, CA), enabling automated preparation of full-length single-cell mRNA libraries from more than 1,000 single cells in a single sample run. This tool has many valuable applications, including alternative splicing studies, biomarker discovery, such as cancer biomarker discovery, and the discovery of rare biological events (e.g., gene fusions, isoforms). In conjunction with Cogent™ NGS bioinformatics tools, SMART-Seq® Pro creates an end-to-end automated solution for biomarker discovery, providing an efficient way to accelerate translational research by providing insights into key biomarkers in single-cell samples.

[0235] The workflow of the SMART-Seq® Pro Application Kit is shown in Figure 23. First, single cells were dispensed into individual wells of a 5,184-well chip. Wells containing viable single-cell candidates were identified by fluorescent imaging and selected for downstream reagent dispensing. The chip underwent one freeze-thaw cycle at -80°C to lyse the cells before reagent dispensing. A total of five reagent dispensing steps were performed to complete the SMART-Seq® protocol (reverse transcription, PCR cDNA amplification, tagmentation, i5 index, and i7 index), with chip centrifugation and thermal cycling between each dispensing step to generate the corresponding product. After cDNA library synthesis and barcoding for i5 and i7 indexes, the resulting NGS library was recovered from the chip into a 1.5 mL Eppendorf tube, and off-chip extraction, amplification, purification, and quantification processes were performed before the library was loaded onto a sequencer (Illumina NextSeq500, Illumina Inc, San Diego, CA).

[0236] This experiment was performed in parallel using two different liquid handling systems. In one experiment, cell and reagent dispensing was performed on a Takara Bio USA ICELL8® cx Single-Cell System for single-cell manipulation. In the other experiment, cell and reagent dispensing was performed using a liquid handling system incorporating the nanosyringe device described herein. The two experiments used identical samples, reagents, and workflow.

[0237] Experiments were performed using a Takara Bio USA ICELL8® cx Single-Cell System following the manufacturer's recommended protocols for both Takara Bio USA ICELL8® cx Single-Cell Systems and the Takara Bio USA, Inc. SMART-Seq® Pro application protocol for single-cell transcriptome analysis.

[0238] Experiments using a liquid handling system with the disclosed design incorporating an array of nanosyringe devices are described in detail below. This design for the liquid handling system accommodated a 2 x 4 array of individually controllable nanosyringes for depositing samples and reagents. The nanosyringe assembly was mounted on a linear Z-stage, which was enclosed in an environmentally controlled chamber. The chamber contained sensors for monitoring the chamber's internal temperature and humidity, and a humidifier module for increasing the chamber's humidity. The chamber's working platform, located on a movable XY stage, had a dedicated location for a 384-well source plate and a temperature-controlled location for a 5,184-well nanowell chip, i.e., Takara Bio USA, Inc., ICELL8® 5,184-well chip.

[0239] While the system is actively dispensing, the dew point control subsystem monitors the chamber temperature and humidity and controls the chamber humidity and nanowell chip temperature to maintain the nanowell chip at the dew point, a temperature between evaporation and condensation. The dew point control capability of the liquid handling system is important to ensure there is no significant evaporation or condensation during the dispensing period, which in the case of the SMART-Seq Pro protocol can vary between 10 and 30 minutes per sample or reagent dispensing step. The working platform also includes a three-well wash station, consisting of a bleach trough, a water trough, and a waste trough, which is used to clean the interior and exterior of the nanosyringe cylinder and orifice at the end of each aspirate-dispense cycle.

[0240] A nanosyringe cylinder with a geometry similar to that shown in Figure 7A was constructed, comprising a stainless steel cylinder with an internal channel diameter of 0.86 mm. The distal end of the cylinder was fitted with an orifice plate with a circular orifice with a diameter of 125 microns. Within the cylinder was a stainless steel plunger with a diameter of 0.84 mm. A seal was fabricated from Delrin® plastic, fitting between the proximal end of the cylinder and the plunger shaft residing within the cylinder channel. The maximum stroke length of the plunger was 24 mm, resulting in a total nanosyringe volume of approximately 12 μL. Generally speaking, aspiration of the nanosyringe was achieved by retracting the plunger from the distal end of the cylinder, and dispensing was achieved by extending the plunger downward toward the distal end of the cylinder.

[0241] The stepper motors driving the nanosyringe and liquid handler's XYZ stages are driven by a TRINAMIC Motion Control TMCM-6212 motion controller board (Hamburg, Germany), which supports a TRINAMIC SixPoint™ ramp generator. The six-point ramp features two different acceleration settings for the acceleration phase and two different deceleration settings for the deceleration phase, with the option to use non-zero start and stop speeds. See FIG. 22. The cycle begins with a start speed Vstart, then accelerates using acceleration value A1 until the motor reaches V1. The motor is then further accelerated using acceleration value A2 until it reaches speed Vmax. Similarly, for the deceleration phase, the motor begins deceleration using deceleration value D2, and after the motor reaches speed V1, it is decelerated by value D1 until it reaches a stop speed Vstop.

[0242] The linear stepper motor used for the nanosyringe was obtained from DINGS MOTION USA™ (Morgan Hill, CA) and had 200 steps per revolution. In this experiment, the motor was operated in 32 microstepping modes, or a total of 6400 microsteps per revolution. The maximum current used by the motor during motion was 900 mA, with a holding current of 100 mA.

[0243] The first step in the protocol was for a nanosyringe to dispense isolated cells into a Takara Bio USA, ICELL8® 5,184-well chip. Human peripheral blood mononuclear cells, also known as PBMCs, were counted on an ORFLOW Moxi flow cytometer, diluted to 50,000 cells / mL, and stained with Hoechst and propidium iodide (PI) dyes. The stained cells were then placed into 2 x 4 sets of wells on a 384-well source plate (positions A1-D2, 80 μL per well). A 25 μL volume of negative control reagent mixture (containing buffer only) was added to position A24, and 25 μL of positive control reagent mixture (buffer and control RNA) was added to position P24. The source plate and empty ICELL8® 350 nL 5,184-well chip were placed in the chamber of a liquid handling system. The cell dispensing process was initiated by the control computer software, which initiated the dew point control system, dynamically calculated all aspiration and dispense volumes required for each step, and determined the optimal dispense path to be employed. The aspiration volume was a function of the number of target wells, the target dispense volume per well, and the addition of a 1 µL overhead to facilitate dispensing of the final volume from the syringe. The dispense path optimization algorithm attempted to minimize XY stage movement and maximize the number of times the nanosyringe could dispense simultaneously, thereby minimizing overall dispense time.

[0244] Before starting the dispense, an initial cylinder wash cycle was performed with a programmed Vmax of 10 mm / s and a Vmax of 40 mm / s from fluid supplied from the bleach or water trough. 2 A series of maximum stroke length aspirations at A1 acceleration of 300 mm / s was performed, followed by a programmed Vmax of 300 mm / s and a Vmax of 5,000 mm / s. 2At A1, the contents were purged into a waste trough. This experiment involved one 0.2% bleach aspirate-purge cycle followed by seven water aspirate-purge cycles. At the end of the cylinder wash, the plunger of the nanosyringe cylinder remained in its lowest position to ensure it was in the correct position for aspiration (i.e., the plunger extended as far into the cylinder channel as possible, with the distal end of the plunger as close as possible to the orifice at the distal end of the cylinder).

[0245] At this point, a specific aspirate-dispense-wash cycle can begin. Generally speaking, a dispensing process involves multiple aspirate-dispense-wash cycles. In this experiment, the 35 nL cell dispensing process consisted of five cycles: a negative control dispense, a first pass of cell dispensing, a second pass of cell dispensing, a third pass of cell dispensing, and finally a positive control dispense. The aspirate-dispense-wash cycles are described in detail below.

[0246] To aspirate, the movable stage positioned the target well of the 384-well source plate below the nanosyringe cylinder and lowered the nanosyringe assembly to a position such that the nanosyringe cylinder was 0.5 mm above the bottom of the source well. The default Vmax for all aspirations used in this experiment was 1 mm / s (although other speed settings, such as 10 mm / s or any value between 1 and 10 mm / s, could be used). This relatively low aspiration speed was used to minimize the shear forces experienced by cells as they passed through the 125 micron orifice and maximize cell viability, although higher speeds can be used for reagent aspiration. After aspiration was completed, the nanosyringe cylinder was raised above the source plate, and the movable stage positioned the nanosyringe cylinder 450 microns above the nanowell chip well across the top surface of the nanowell chip. Dispensing was performed with Vstart, V1, and Vmax of 66.5 mm / s and 4,768 mm / s. 2The A1 was programmed with a motor current setting of 900 mA and a motor microstep mode of 6,400 microsteps per revolution. After the dispense was completed, the remaining 1 µL of fluid was discarded into the waste trough and a cylinder / orifice wash sequence was performed to reset the cylinder state. For the 35 nL cell dispense step, this cycle was repeated five times to fill the nanowell chip with cells and a predetermined pattern of positive and negative controls.

[0247] After the 35 nL cell dispensing step was completed, the nanowell chip was blotted with blotting paper to remove any small droplets that may have landed on the top surface, sealed with RC sealing film (Takara Bio USA, Inc., San Jose, CA), and the chip was spun down in a centrifuge at 300 × g for 5 minutes at 4 ° C. The chip was placed back onto the working platform of the liquid handler.

[0248] Cell scanning was initiated by the control software using the onboard camera system. The camera optics first scanned the entire chip using a UV LED (365 nm) to excite the Hoechst viability stain. The camera's field of view spanned a grid of 6 x 6 nanowells, and at each position, a series of seven images was acquired at a depth of 2.2 mm from the top surface of the chip, then stepped up by 0.1 mm per image. This was intended to capture images of cells that were not at the bottom of the well but may have attached to the sidewalls. The optics were then switched to a blue LED (460 nm) to excite the propidium iodide (PI) stain, collectively distinguishing live from dead cells.

[0249] After image acquisition was complete, the chip was removed from the liquid handler, resealed, and frozen at -20 °C for a minimum of 1 hour to allow cells to lyse. At this point, software was used to process the images and determine which nanowells contained single viable cells. The algorithm begins by flattening a stack of Z images (images taken at different Z-axis planes) into a single image and then determining how many cell-shaped objects were present in each well. If a cell-shaped object appeared in both the Hoechst and PI channels and those objects were located in the same location, the cell was considered dead. Similarly, if a cell-shaped object appeared in the Hoechst channel but not the PI channel, it was considered a viable cell. Dispensing the cell-containing liquid results in a Poisson distribution of cell numbers per well. The software then generated a "filter file" representing the locations of all wells containing a single viable cell, also known as a "candidate." In this experiment, 1601 single viable PBMC dispense candidates were identified on chips dispensed using the nanosyringe and 1539 candidates were identified on chips dispensed using the ICELL8® cx system. For each subsequent dispense, the target wells were limited to those specified in the filter file.

[0250] At this point, first-strand cDNA synthesis by reverse transcriptase was performed on each well containing a single cell. The nanowell chips were removed and thawed, and the RT mix was prepared and placed in a new source plate according to the SMART-Seq® Pro protocol. The source plate and nanowell chips were returned to the liquid handler, and dispensing was initiated. The RT dispense step was initiated in the software, and the liquid handler dispensed 35 nL of RT mix into the wells specified by the filter file in the same manner as above. The chips were then aspirated, sealed, and spun down in a centrifuge at 3,220 x g (minimum 2,600 x g) for 3 minutes at 4 °C. Three additional cylinder wash steps were performed in the liquid handler while the chips were centrifuged. After centrifugation, the chips were removed and placed in a Bio-Rad Laboratories T-100 thermocycler (Bio-Rad Laboratories, Hercules, CA).

[0251] The following steps were then followed according to the manufacturer's protocol, including the necessary dispensing and thermal cycling. (i) Full-length cDNA amplification by PCR1 (ii) Dispense index primer set 1 (reverse index primer) (iii) Tagmentation (iv) Dispense index primer set 2 (forward index primer) and PCR2 (v) Extract the resulting NGS library from the chip into a single collection tube (following the ICELL8® cx Single-Cell System recommended protocol, including centrifuging the chip to collect the liquid volume from each nanowell).

[0252] The extracted library material was then purified and sequenced on an Illumina® NextSeq® 500 sequencing system with a read length of 2 × 75 bp. Sequencing data were separated and analyzed using the Cogent AP and DS bioinformatics packages (Takara Bio USA, Inc., San Jose, CA).

[0253] We identified an average of 127,115 barcoded reads and 2,953 genes for each single cell, and extracted seven unique cell clusters based on UMAP analysis. The library also demonstrated remarkable metrics: 81.84% of reads mapped uniquely to the human reference genome, with exonic reads greater than 52%, and minimal contamination from intergenic (5%), mitochondrial (7.28%), and ribosomal (4.87%) reads.

[0254] Parallel experiments performed with the ICELL8®cx library using the same PBMCs showed comparable results. A total of 1,539 wells containing single-cell candidates were identified. The average sequencing depth was 148,382 barcoded reads per cell, identifying an average of 3,487 genes. Based on UMAP analysis, the same number of unique cell clusters (i.e., 7) were extracted. This ICELL8®cx library had 79.48% uniquely mapped reads, 51.80% exonic reads, and minimal contamination from intergenic reads (4.33%), mitochondrial reads (7.99%), and ribosomal reads (5.57%). Reagent dispensing times for each step were faster with the new nanosyringe system compared to the ICELL8®cx library.

[0255] In summary, this SMART-Seq® Pro protocol for transcriptome analysis of single PBMC cells can be effectively implemented using the nanosyringe for liquid handling described in this disclosure. Use of the nanosyringe described herein enables full-length transcriptome analysis of single cells in conjunction with Takara Bio USA, Inc., ICELL8® 5,184-well chips, and further enables the use of the SMART-Seq® Pro protocol, along with the ICELL8 cx Single-Cell System liquid handler.

[0256] Example 9 Evaluation of qPCR detection linearity using nanosyringes A study was conducted to compare the qPCR detection linearity of the nanosyringe dispensing system as described in this disclosure with a state-of-the-art microsolenoid nanofluidic device (MSND, Takara Bio, USA Inc., FIG. 15). A set of TaqMan® assays for 24 gastrointestinal infection targets is provided in the table below.

[0257] [Table 6]

[0258] We used 5x PrimePath qPCR Mix (Takarabio catalog no. 638347) with 400 nM ROX spike-in as a passive reference. The target probe was FAM-labeled and used at a concentration of 250 nM, with the forward and reverse primers each at a concentration of 900 nM. The template was a plasmid containing the amplicon of interest, which was serially diluted 1:2 with EASY Dilution (for real-time PCR) solution (Takarabio catalog no. 9160). Using both the nanosyringe device of the present disclosure and a conventional device (MSND), we first dispensed a 50 nL mixture of assay and reagents into each nanowell, followed by dispensing another aliquot containing 50 nL of sample into each well, for a total reaction volume of 100 nL. All assays were dispensed in triplicate. Experiments were performed using one MSND device of the present disclosure and two nanosyringe devices. After dispensing, the reactions were cycled for qPCR, and the fluorescent signal in each well was detected using a SmartChip Real-Time PCR Cycler (Cat. No. 640023, Takara Bio, USA Inc.). Figure 25 shows the results. Ct values ​​were plotted on the Y-axis, and the log concentration of template copies per reaction was plotted on the X-axis. The results show that the nanosyringe device of the present disclosure and the prior art device perform with comparable detection linearity, as the regression fit of the curves to the MSND curve was at or close to 1.0.

[0259] Example 10 Using nanosyringe liquid handling for combined indexing workflows Tests were conducted to demonstrate the performance of the nanosyringe liquid handling system of the present disclosure in implementing a single-cell combinatorial indexing protocol for high-throughput single-cell full-length transcriptome analysis, for example, as described in International Application No. PCT / US2022 / 053883, published as WO 2023 / 122309, the disclosure of which is incorporated herein by reference.

[0260] Human K562 and mouse 3T3 cell lines were used for the analysis. One million K562 cells and one million 3T3 cells were mixed and washed with PBS. The resulting cell mixture was then fixed by incubation in an appropriate fixative solution (e.g., 1% paraformaldehyde) for 15 minutes. Cell fixation was stopped by adding a quenching solution (1 M Tris-Cl, pH 8), and the cells were washed with PBS. The fixed cells were aliquoted into a 96-well plate and mixed with a fragmentation buffer containing 125 mM Tris (pH 8.8), 250 mM KOAc, 50 mM MgCl, DTT, MgCl, dNTPs, and random ATP. Cellular RNA was fragmented by heating to 85°C for 6 min, and then a reverse transcription master mix containing digitonin, RNase inhibitor, reverse transcriptase (200 μL / μL), and indexed template switching oligo (TSO) was added to the heat-treated cells. RT reactions were performed by incubating at 10°C for 10 min, 15°C for 10 min, 20°C for 10 min, 25°C for 10 min, 30°C for 10 min, 35°C for 10 min, 42°C for 70 min, and holding at 4°C. A first well-specific barcode (BC1) was added during the in situ reverse transcription (RT) reaction using template switching oligos (TSO), each with a unique first well-specific barcode (BC1).

[0261] After RT, the cells were pooled, washed, and then split again by cell dispensing using a nanosyringe liquid handling system. 50 nL droplets containing an average of five cells, each with a different first well-specific barcode, were dispensed into each nanowell of a 5184-nanowell ICELL8 chip (Takara Bio USA, Inc., San Jose, CA). Subsequently, a 50 nL droplet of the first partial second-round well-specific barcode (BC2a) was deposited into the cell-containing well. A second 50 nL droplet containing the second partial second-round well-specific barcode (BC2b) was then added to the same well. Finally, a 50 nL droplet of PCR master mix containing SeqAmp DNA polymerase and 2x CB buffer was added to each cell-containing well using a nanosyringe liquid handling system.

[0262] The next PCR reaction was performed in nanowells of an ICELL8 chip using the following five-cycle program: 94°C for 1 minute, 100°C for 15 seconds, 49.3°C for 5 seconds, 54.5°C for 10 seconds, 72.2°C for 9 seconds, 67.9°C for 31 seconds, 67.9°C for 2 minutes, and a 4°C hold. Each nanowell contained a unique combination of BC2a and BC2b, which together generated a second well-specific barcode that was incorporated into the final library DNA during PCR. The final library DNA from each individual cell had a unique combination of the first and second well-specific barcodes, which, when combined, created a unique cell source-specific barcode for each cell. After the PCR reaction, the libraries were pooled and washed with 0.7x magnetic beads. The beads were eluted with 56 μL of HO. Four ZapR™ (Takara Bio USA, Inc., San Jose, CA) reactions were set up to remove rRNA from the libraries. Each ZapR reaction contained 2.2 μL of 10x ZapR buffer, 2.8 μL of scZapR, 2.1 μL of heated probe, and 13.7 μL of purified PCR product. The ZapR reaction was run at 37°C for 1 hour and 72°C for 10 minutes. After ZapR was completed, a second PCR reaction was performed to amplify the library by adding 80 μL of PCR mixture (2 μL SeqAmp DNA polymerase, 2 μL PCR2 primer, 50 μL of 2x CB buffer, and 26 μL of nuclease-free water) to each tube containing 22 μL of ZapR product under the following program: 94°C for 1 minute, 98°C for 15 seconds, 55°C for 15 seconds, 68°C for 30 seconds, 10 cycles, and a 4°C hold. After the second PCR reaction, the products were purified using 0.7x magnetic beads and quantified using Qubit BioAnalyzer and qPCR. Based on the quantification, 1.5 pM library DNA and 1% PhiX were loaded onto a Nextseq500 with a medium output 150 cycle (2 × 75 bp) kit for sequencing. Sequencing reads were analyzed using the Cogent NGS Analysis Pipeline (Takara Bio USA, Inc., San Jose, CA) and mapped to both the human and mouse genomes.

[0263] As shown in the table below, 92.8% of the sequencing reads were successfully barcoded, and only 7.2% of the total reads were undetermined, indicating that combinatorial indexing was successfully achieved to barcode individual single cells.

[0264] The above results demonstrate that the liquid dispensing system of the present disclosure can be used to perform barcoding applications, as described in International Application No. PCT / US2022 / 053883, published as WO 2023 / 122309, the disclosure of which is incorporated herein by reference.

[0265] Demultiplexing results of sequencing data Leads Percentage Barcoded 164,547,548 92.8% Undecided 12,829,353 7.2%

[0266] As shown in the table below, a total of 4,271 human cells were analyzed with 14.8% unmapped reads and 85.2% mapped reads. Of the mapped reads, 95.5% were uniquely mapped and 4.5% were multiply mapped. Among the uniquely mapped reads, there were 44.6% exonic reads, 44.6% intronic reads, and 10.6% intergenic reads. There were 0.8% mitochondrial reads and 2.6% ribosomal reads. An average of 3,242 genes were detected per cell, with an average sequencing progress of 12,658 reads per cell. The doublet rate per 1,000 cells was 0.8%.

[0267] In summary, this combinatorial indexing protocol for high-throughput single-cell transcriptome analysis has been shown to be successfully performed using the nanosyringe liquid handling system described herein in conjunction with Takara Bio USA Inc., ICELL8® 5,184-well chips.

[0268] Human Cell Mapping Metrics Metrics Cells for analysis # 4,173 % Unmapped Reads 14.7% % mapped reads 85.3% Uniquely Mapping % 95.5% Multiple Mapping % 4.5% Exon read % 44.6% Intronic reads 44.6% Intergenic reads % 10.6% Mitochondrial lead% q0.8% Ribosomal reads % 2.6% No gene 3242 Seq progress (reads / cell) 12,658 Doublet rate per 1000 cells: 0.8%

[0269] Further Considerations The above description has been provided to enable one skilled in the art to practice the various configurations described herein. While the present technology has been specifically described with reference to various figures and configurations, it should be understood that these are for illustrative purposes only and should not be construed as limiting the scope of the present technology.

[0270] There may be many other ways to implement the present technology. The various functions and elements described herein may be divided differently than shown without departing from the scope of the subject technology. Various modifications to these configurations will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other configurations. Accordingly, many changes and modifications may be made to the present technology by those skilled in the art without departing from the scope of the technology.

[0271] It is understood that the specific order or hierarchy of steps in the processes disclosed is an illustration of example approaches. Based on design preferences, it is understood that the specific order or hierarchy of steps in the processes may be rearranged. Some of the steps may even be performed simultaneously. The accompanying method claims present elements of the various steps in a sample order, and are not meant to be limited to the specific order or hierarchy presented.

[0272] While the detailed description contains many details, these should not be construed as limiting the scope of the subject technology, but merely as illustrating different examples and aspects of the subject technology. It should be understood that the scope of the subject technology includes other embodiments not discussed in detail above. Various other modifications, changes, and variations can be made in the arrangement, operation, and details of the methods and apparatus of the subject technology disclosed herein without departing from the scope of the present disclosure. Unless otherwise specified, reference to an element in the singular is not intended to mean "one and only one," but rather "one or more," unless explicitly stated. Furthermore, it is not necessary for a device or method to address every problem that can be solved (or have every advantage that can be achieved) by different embodiments of the present disclosure to be encompassed within the scope of the present disclosure.

[0273] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 119, this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 428,509, filed November 29, 2022, the entire disclosure of which is incorporated herein by reference.

Claims

1. A liquid treatment device comprising: (a) an actuator having a movable shaft; (b) an elongate body including a central channel, a proximal end, and a distal end, the distal end comprising an orifice; (c) a plunger including a proximal end and a distal end, (i) the proximal end is mechanically coupled to the movable shaft; (ii) a plunger, wherein at least a portion of the distal end of the plunger is movably positioned within the central channel; and A liquid treatment device comprising:

2. The device of claim 1 , further comprising a support structure physically coupled to and stabilizing the actuator, the elongate body, and the plunger.

3. 3. The device of claim 1, wherein the actuator is a mechanical actuator.

4. The device of claim 3 , wherein the mechanical actuator comprises a stepper motor.

5. The device according to any one of claims 1 to 4, wherein the actuator is a linear actuator.

6. A device according to any preceding claim, wherein the device is configured to aspirate and dispense liquid through the orifice.

7. The device of any one of claims 1 to 6, wherein the elongate body comprises a cylinder.

8. The device of any one of claims 1 to 7, wherein the central channel has an inner diameter in the range of 0.7 mm to 1.0 mm, and in some cases in the range of 0.8 mm to 0.9 mm.

9. The device of any one of claims 1 to 8, wherein the elongate body further comprises a port located on a side of the elongate body and fluidly coupled to the central channel.

10. The device of any one of claims 1 to 9, wherein the orifice has a diameter smaller than the inner diameter of the central channel.

11. The device of any one of claims 1 to 10, wherein the orifice has a diameter in the range of 100 microns (micrometers; μm) to 150 microns.

12. The device of claim 11 , wherein the orifice has a diameter of 125 microns.

13. The device of any one of claims 1 to 12, wherein the elongate body and the orifice are machined from a cylindrical rod.

14. The device of any one of claims 1 to 13, wherein the orifice is part of an orifice plate coupled to the distal end of the elongate body.

15. The device of claim 14 , wherein the orifice plate is a pressure-deformed orifice plate.

16. 15. The device of claim 14, wherein the orifice plate is press fit into the cylinder.

17. 15. The device of claim 14, wherein the orifice plate is welded to the cylinder.

18. 15. The device of claim 14, wherein the orifice plate is epoxied to the cylinder.

19. The device of any one of claims 14 to 18, wherein the orifice plate is a sapphire orifice plate, a glass orifice plate, or a stainless steel orifice plate.

20. A device according to any preceding claim, wherein the plunger is a stainless steel plunger.

21. 21. The device of any one of claims 1 to 20, wherein the plunger has an outer diameter in the range of 31 to 34 mils (thousandths of an inch).

22. A device according to any preceding claim, wherein the outer diameter of the plunger is smaller than the inner diameter of the central channel.

23. 23. The device of claim 22, wherein the difference between the outer diameter of the plunger and the inner diameter of the central channel is less than or equal to one thousandth of an inch.

24. The device of any preceding claim, wherein the distal end of the plunger further comprises a plunger seal.

25. The device of any one of claims 1 to 24, further comprising a seal surrounding the junction between the proximal end of the elongate body and a plunger.

26. 26. The device of claim 1, further comprising a seal surrounding the proximal end of the elongate body, the seal surrounding an outer surface of at least a portion of the proximal end of the elongate body and surrounding the plunger at a position where the plunger exits the proximal end of the elongate body.

27. 27. The device of claim 26, wherein the seal comprises Teflon™, Delrin® plastic, PEEK, high strength slippery PEEK and Slippery Delrin Acetal AF resin.

28. 28. The device of claim 26 or 27, wherein the seal is an O-ring seal.

29. A device according to any preceding claim, wherein the actuator is configured to move the moveable shaft at a maximum linear velocity of 100 mm / s.

30. 30. The device of any one of claims 1 to 29, wherein the device is capable of reproducibly dispensing liquid volumes in the range of 10 nanoliters (nL) to 300 nL.

31. 31. The device of claim 30, wherein the device is capable of reproducibly dispensing 10 nL of liquid volume.

32. 31. The device of claim 30, wherein the device is capable of reproducibly dispensing liquid volumes of 20 nL.

33. 31. The device of claim 30, wherein the device is capable of reproducibly dispensing liquid volumes of 35 nL.

34. 31. The device of claim 30, wherein the device is capable of reproducibly dispensing liquid volumes of 50 nL.

35. 1. A system for simultaneous processing of multiple liquid samples, comprising: (i) a nanosyringe array comprising a plurality of nanosyringes, each nanosyringe comprising: (a) an actuator having a movable shaft; (b) an elongate body including a central channel, a proximal end, and a distal end, the distal end comprising an orifice; (c) a plunger including a proximal end and a distal end, the proximal end mechanically coupled to the movable shaft and at least a portion of the distal end of the plunger movably positioned within the central channel; and (ii) a computer control system for controlling said actuator. A system comprising:

36. 36. The system of claim 35, wherein the system is configured to house a multi-well device in liquid-receiving relationship with the nanosyringe array.

37. 37. The system of claim 35 or 36, wherein the nanosyringe array comprises eight nanosyringes.

38. The system of any one of claims 35 to 37, further comprising a housing containing the nanosyringe array.

39. 40. The system of claim 38, wherein the housing forms an environmentally controllable chamber.

40. 40. The system of claim 39, wherein the chamber is capable of being humidified.

41. 41. The system of claim 39 or 40, wherein the chamber is capable of being set to a temperature above the dew point of the chamber.

42. 42. The system of any one of claims 35 to 41, further comprising at least one component selected from a power supply, a heater unit, a camera for imaging the multi-well device, a cooling unit, a humidifier, a humidifier water dispenser, a wash trough, and a waste trough.

43. 43. The system of any one of claims 35 to 42, wherein the system is configured to accommodate at least one multi-well source plate.

44. The system of any one of claims 1 to 43, wherein the system further comprises a movable stage.

45. 45. The system of claim 44, wherein the movable stage is configured to accommodate a multi-well device and a source reservoir plate.

46. 46. ​​The system of claim 45, wherein the movable stage is configured to position the multi-well device to receive liquid dispensed from the nanosyringe or to position the nanosyringe to aspirate liquid from the source reservoir plate.

47. 47. The system of any one of claims 36 to 46, wherein the multi-well device comprises 5,184 fluidically isolated wells.

48. 48. The system of any one of claims 35 to 47, wherein each nanosyringe is capable of reproducibly dispensing liquid volumes in the range of 10 nanoliters (nL) to 300 nL.

49. The system of any one of claims 35 to 48, further comprising a heating element.

50. 1. A method for dispensing liquid into wells of a multi-well device, comprising: (a) providing: (i) a nanosyringe, an actuator having a movable shaft; an elongate body including a central channel, a proximal end, and a distal end, the distal end including an orifice; a plunger including a proximal end and a distal end, the proximal end mechanically coupled to the movable shaft and at least a portion of the distal end of the plunger movably positioned within the central channel; A nanosyringe comprising: (ii) providing a multi-well device comprising a plurality of fluidically separated wells; (b) positioning the distal end of the cylinder into a source reservoir containing a liquid to be dispensed; (c) retracting the movable shaft to retract the plunger in the central channel and aspirate the liquid from the source reservoir into the central channel; (d) positioning wells of the multi-well device beneath the distal end of the elongate body; (e) extending the movable shaft to extend the plunger in the central channel toward the distal end of the elongate body and dispense the liquid from the central channel into the wells of the multi-well device; A method comprising:

51. 51. The method of claim 50, wherein the multi-well device comprises 5,184 fluidically isolated wells.

52. 52. The method of claim 50 or 51, wherein the amount of liquid dispensed in step (e) is in the range of 10 nanoliters (nL) to 300 nL.

53. 53. The method of any one of claims 50 to 52, further comprising adjusting the temperature of the multi-well device.

54. 54. The method of claim 53, wherein adjusting the temperature comprises cooling the multi-well device.

55. 55. The method of any one of claims 50 to 54, wherein the amount of liquid aspirated is the same as the amount of liquid dispensed.

56. 56. The method of any one of claims 50 to 55, wherein the amount of liquid aspirated is greater than the amount of liquid dispensed.

57. 57. The method of any one of claims 50 to 56, wherein steps (d) and (e) are repeated to dispense a quantity of liquid into multiple wells of the multi-well device.

58. 58. The method of any one of claims 50 to 57, wherein the liquid is a liquid sample.

59. 59. The method of claim 58, wherein the liquid sample comprises cells or isolated cell nuclei.

60. 60. The method of claim 59, wherein the liquid sample comprises at least one cell or at least one isolated cell nucleus.

61. 61. The method of claim 60, wherein the liquid sample comprises no more than one cell or no more than one cell nucleus.

62. 59. The method of claim 58, wherein the liquid sample comprises nucleic acids.

63. 63. The method of claim 62, wherein the nucleic acid is selected from DNA and RNA.

64. 64. The method of any one of claims 58 to 63, wherein the method further comprises imaging the well after dispensing the liquid sample into the well.

65. 58. The method of any one of claims 50 to 57, wherein the liquid comprises a biochemical reaction mixture.

66. 66. The method of Claim 65, wherein the biochemical reaction mixture comprises one or more of a buffer, an enzyme, a dNTP, a primer, a template switch oligonucleotide, or any combination thereof.

67. 67. The method of claim 66, wherein the biochemical reaction mixture comprises a next generation sequencing (NGS) library preparation reaction mixture.

68. 68. The method of claim 67, further comprising performing an NGS library preparation reaction.

69. 67. The method of claim 66, wherein the biochemical reaction mixture comprises a PCR reaction mixture.

70. 70. The method of any one of claims 65 to 69, wherein the method further comprises dispensing a liquid sample into the well containing the biochemical reaction mixture.

71. 71. The method of claim 70, further comprising imaging the contents of the well containing the biochemical reaction mixture.

72. 72. The method of claim 71, further comprising determining the progress of a biochemical reaction in the imaged well.

73. 73. The method of claim 71 or 72, further comprising determining whether cells are present in the imaged well.

74. 73. The method of claim 71 or 72, wherein the biochemical reaction is a PCR reaction and the imaging is quantitative imaging that measures the accumulation of PCR products generated by the reaction.

Images