Fluid automatic samplers and incubators

Abstract

The present invention provides an apparatus for performing automated analysis of one or more samples in a single-well or multi-well plate or vial, wherein the automated analysis process includes automated fluid flow, wherein the sample comprises a fluid or particles in a sample vessel, and wherein the apparatus includes an assembly of components for processing the sample for analytical evaluation by a fluid-based and / or particle-based instrument. The automated fluid flow may include a system for moving the sample, including a vacuum system, a pressure-based system, a pneumatic system, a pump, a peristaltic pump, a diaphragm, or a syringe. The apparatus may include an assembly of components for achieving movement in the X, Y, and Z directions, as well as switches, microfluidic tubing, a well plate, an electronic pressure controller, a pneumatic or fluid mixing device, components for fluid processing, a sampling vessel, and mechanical components for mobilizing or transporting system components.

Description

Technical Field

[0001] Specific embodiments of the present invention relate to devices and methods for using such devices that can automatically sample nanofluids, microfluids, or millifluids from one or more containers. The containers can range from single-well vials to multi-well plates. The devices provided by the present invention further enable mixing the contents of individual wells, creating sterile zones for biological research, and controlling parameters such as temperature, carbon dioxide concentration, and other cell growth conditions to achieve material stability and monitor cell-based processes. Background Art

[0002] The increasing use of automated systems for sample storage, handling, and analysis has led to extensive research in the field of automation. Existing sampling systems consist of robotic arms that can move samples from a storage area to a loading area for sampling, magnetic or mechanical stirrers that can keep the samples suspended, and heating or cooling zones that can regulate the sample storage or analysis temperature.

[0003] Existing technologies include several automated sampling devices covering various analytical research fields (such as liquid chromatography used in U.S. Patent No. 4,713,974), but they cannot be used for biological cell analysis using laser force cytometry (LFC) due to the lack of accurate and continuous nanoliter flow rate control, sufficient sample mixing, and reliable sample processing temperature control.

[0004] U.S. Patent No. 4,816,730 to Wilhelm et al. describes an apparatus for handling and moving multiple objects, comprising a robotic arm having a gripper mechanism driven by an electronically controlled stepper motor for holding a sample, capable of vertical, horizontal, and rotational movement. Meanwhile, U.S. Patent No. 6,872,362 to Schimidt et al. further describes the use of an electric autosampler having a vial cup adapted to include a magnetic stir bar driven by various methods of altering the magnetic field surrounding the vial cup. While such prior art describes various methods for moving and mixing samples, these methods are not sufficient for performing biological cell analysis using LFC instruments. What is needed is a better device that can store, mix, and sample containers ranging from single-well to multi-well plates, while maintaining biological cell integrity through adequate mixing and temperature maintenance using pneumatic-based non-contact mixing and temperature-controlled single-well or multi-well plates.

[0005] Existing automatic well plate stacking or retrieval systems with automatic mechanisms can be stacked or retrieved in a vertical or horizontal order (CN204136215U, US20040206419A1), loaded and unloaded using cassettes (US Patent No. 9,744,535), or stacked or retrieved in batches into magazines or tower racks (US Patent No. 6,086,319). In addition, previous designs can enable well plates to be loaded or removed from storage towers in a random (disordered) manner, regardless of the order in which they are stacked (US Patent No. 7,670,555). However, what is needed is to achieve specificity and automated detection of target well plates, selection, and the use of a magnetic interface to stack target well plates into or retrieve them from storage towers in a disordered manner, and to cultivate well plates in combination with a composite analysis method for automating analysis of such well plates. Summary of the Invention

[0006] Specific embodiments according to the present invention relate to devices and methods for using the same that can automatically perform nanofluidic / microfluidic / millifluidic sampling (and optional incubation) from single-well or multi-well plate vessels. The devices are further capable of mixing the contents of individual wells, creating sterile zones for biological research, and controlling parameters such as temperature, carbon dioxide concentration, and other cell growth conditions to achieve material stability and monitor cell-based processes.

[0007] More specifically, the novel device described in the present invention enables fluid monitoring, including but not limited to determining nanofluid / microfluid / millifluid flow rates and degassing by using an electronic pressure controller and / or regulator in conjunction with a flow meter. The novel device further enables sample mixing by injecting small bubbles into the sample well or vial or by drawing in or expelling the sample or air through a vacuum pump connected to an electronic pressure controller (EPC). In addition, the present invention has the ability to create a sterile zone around the needle and orifice plate area using means such as ultraviolet radiation to ensure that biological materials do not contaminate the instrument housing. The device is further designed to optionally control the sample temperature prior to sampling by using a thermoelectric cooling module to incubate the sample. By reversing the current passing through the module, the thermoelectric cooler can also change the temperature, heat or incubate the sample as needed. Mixing, sterilization and temperature control can be performed separately, sequentially and / or simultaneously based on the device settings. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 A schematic diagram showing a housing of a specific embodiment of an "autosampler" according to the present invention is provided;

[0009] Figure 2 A horizontal schematic diagram showing the inner workings of the autosampler and needle manifold assembly is provided;

[0010] Figure 3 Provides a top view illustrating the various stages of movement within the autosampler;

[0011] Figure 4 Provided is a display of an automatic sampler and a laser force cytology instrument (Radiance TM ) between the interface diagram;

[0012] Figure 5 is a graphical diagram showing the process for automated mixing. Figure 5A is a schematic diagram showing a specific embodiment of a microfluidic needle design, Figure 5B Details of the automated sampling and mixing manifold are provided;

[0013] Figure 6A-1 , A-2, B, and C provide schematic diagrams showing representative UV sterile zones. Figure 6A-1 Provides a front view, Figure 6A-2 A side view is provided. Figure 6B Provides a system for sterile mixing and sample transport, Figure 6C Details of sterile tubes are provided;

[0014] Figure 7A A schematic diagram showing a specific embodiment of a single well plate incubator is provided, and Figure 7B Individual well or vial incubators are shown;

[0015] Figure 8 Specific embodiments of composite transport structures are provided;

[0016] Figure 9A Provides schematics for multi-board automated handlers. Figure 9B A specific embodiment of a multi-board automatic handler is provided, in which the boards are connected during normal operation and disconnected when they need to be moved;

[0017] Figure 10 provides a schematic diagram illustrating the use of a specific embodiment of an automatic sampler for cell detachment. Figure 10(a) shows cells being placed on the bottom of a vessel / vial, and Figure 10(b) shows cells having left or detached from the bottom of the vessel / vial;

[0018] Figure 11 A schematic diagram illustrating a specific embodiment of a reagent delivery system is provided;

[0019] Figure 12 Schematic diagrams are provided showing specific embodiments of cell detachment methods that include the use of a scraping head / scraping adapter. DETAILED DESCRIPTION

[0020] The present invention is described in conjunction with specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations may be made in implementing the present invention without departing from the scope or purpose of the invention. It will be recognized by those skilled in the art that these features may be used alone or in any combination based on the requirements and specifications of a given application or design. It will be recognized by those skilled in the art that systems and devices according to embodiments of the present invention may be used in conjunction with any method according to the present invention, and that methods according to the present invention may be implemented using systems and devices according to the present invention. Embodiments that include various features may also consist of or consist essentially of the various features described. From the description and practice of the present invention, other embodiments according to the present invention will be apparent to those skilled in the art. The description provided herein is merely illustrative and, therefore, variations that do not depart from the essence of the invention fall within the scope of the present invention.

[0021] Before describing in detail at least one embodiment of the present invention, it should be understood that the present invention is not limited to its application in the detailed construction and arrangement of components listed in the following description or illustrated in the accompanying drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways. Furthermore, it should be understood that the terms and expressions used herein are for illustrative purposes only and are not to be construed as limiting.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present technology and methods belong.

[0023] The text and references mentioned in this application (including U.S. Provisional Patent Application Serial No. 62 / 654,335 filed on April 7, 2013) are incorporated into this application in their entirety.

[0024] The present invention provides a novel device for automating sample analysis, wherein the sample is placed in a vial, vessel, well, multi-well plate and similar container. The present invention also provides a method for using such a device. The device is referred to as an "autosampler" in the present invention. In some embodiments, the autosampler can be used to obtain a desired amount and / or a predetermined amount of sample for machine analysis or manual analysis. For example, the autosampler can be used to retrieve nanofluid / microfluid / millifluid samples from a multi-well plate, vial or other vessel, such samples can have various amounts and can be composed of various cells or particles. The autosampler then presents the sample to an appropriate medium / structure / vessel so that the liquid, particles or cells are analyzed by a fluid-based instrument or system (e.g., an instrument using laser force cytometry (LFC)).

[0025] See attached figure, Figure 1A schematic overview of an embodiment of the exterior of the autosampler device (110) is provided, wherein single or multiple multiwell plates or vials may be loaded into the plate block when the bifurcated door (120) is opened (see FIG. Figure 2 216). In other embodiments, the door may remain open or consist of a single retractable or collapsible door or other opening arrangement. 140 is the entry and exit point for sample fluid moving from 110 to 410 or other LFC-based or optical techniques.

[0026] like Figure 2-3 As shown, in a specific embodiment, two motors (200) driven by a controller board (for opening and closing the door of the automatic sampler) are used to open Figure 1 The motor rotates a ring gear (210) that meshes with a toothed door frame (212) to slide open a door in a low-friction channel (214). The sample is loaded into a well plate mounted on a temperature-controlled block (216) that can be heated or cooled as needed.

[0027] The needle and needle manifold assembly (226) comprises a suitable material such as a flexible polymer, plastic, silane, carbon or metal substrate (any alternative material serving the same purpose may be used) that holds the needle (298) and pneumatic connection (294), a sealing surface for sealing the vial or well plate (230), and a spring-loaded support structure (232) to assist in achieving a better seal. The needle may be a multi-tube design in which dedicated sample and mixing tubes are housed within the outer structure of a larger diameter needle. In certain embodiments, the needle may include additional modifications such as a sharpened tip to pierce the plate seal.

[0028] In some embodiments, the motion track (228) is positioned to allow the internal workings to move along the X-axis and Y-axis, and the cable carrier (296) is mounted to ensure that the cable can move safely during movement. The X-axis 296 is not shown. An additional motor or motor group (295) can provide Z-dimension movement by rotating the lead screw, thereby moving the block (216).

[0029] In some embodiments, the temperature control of 216 can be achieved by using a temperature control plate (310, see Figure 3) is achieved by a thermoelectric cooling (TEC) module and a thermistor connected to the sample. Temperature control can include heating and cooling the sample. A fan (218) runs constantly or as needed to remove heat from the heat sink (220). 216 and subsequent components are connected to a mobile platform, where 216 can be transported in the X, Y and Z axes within the confines of the automatic sampler. Movement is controlled by a combination of an industrial computer (320) and a motor (340). For movement in the X and Y axes, the industrial computer-controlled 340 rotates a timing pulley (360), which in turn drives a belt to move the gantry plate along its Y axis (380) and X axis (382). For movement in the Z axis, the industrial computer-driven 340 includes a lead screw (222). 222 is used to generate upward and downward Z axis movement by utilizing a Delrin moving nut (224). Any moving nut with the same purpose can be used. Motion control on any axis can be with or without position feedback.

[0030] Figure 4 The interface between 110 and the Radiance™ instrument (410) is illustrated as a specific embodiment in which 110 can be applied. Alternatively, 110 can be modified to be applied to any LFC, optical force, microfluidic or other instrument. In a specific embodiment, the interface between 110 and other instruments is as follows: Figure 4 As shown, it allows the transfer of information between the two instruments, including but not limited to fluids, samples and electronic signals, communications or other information.

[0031] Figure 5 Specific embodiments of an automated sampling and mixing system are illustrated. To maintain sample integrity while mixing, the Z-axis distance (550) between the bottom of the sampling line (530) and the bottom of the sampling vessel (such as the multi-well plate shown) can be adjusted to fine-tune the pneumatic mixing system for individual samples. Sampling is accomplished by physically moving or pressing the vial or well plate (700) toward the needle manifold assembly (226). A sealing surface or gasket (230) is used to create a seal between the needle manifold assembly and the well plate (700) or vial. The inflow or outflow rate of the vial or sampling well can then be adjusted by controlling the pressure of the head space (560) of the vial or sampling well. This is achieved by Figure 5B This is achieved by the inner chamber (570) shown in detail. Figure 5BAs shown in the side view of the figure, the inner chamber (570) has openings at two locations at the top and one location at the bottom. The two locations at the top are where the manifold is connected to the outer tube (540) and the fitting (580) in an airtight manner. The location at the bottom is an additional opening location connected to the inner chamber on the bottom of the manifold (226), which is shown in the manifold bottom detail diagram. The outer tube (540) passes through the inner chamber (570) and protrudes through the manifold (226) and extends out as shown. This allows the outer tube (540) to be immersed in the sample during sampling. However, the outer diameter of the outer tube (540) is smaller than the diameter of the inner chamber (570), so that an airtight seal can be created when the orifice plate or vial is pressed against the sealing surface (230). There is a pipeline (590) that is connected to the top of the manifold (226) in an airtight manner through the fitting (580). The pipeline (590) is connected to an electronic pressure controller or some other pressure or vacuum source (595) as shown. By adjusting the pressure of the source (595), the pressure in the headspace 560 can be precisely controlled. Please note that this is a specific embodiment and other embodiments can have a greater number of openings in the manifold as needed. Separate tubes (500 and 530, respectively) are used for both mixing and sampling, which share a larger outer tube or needle (540). The inner tubes (500 and 530) can have different or the same diameter to improve or change the desired mixing or sample transport. The materials of the inner and outer tubes can be different and can be composed of metal, plastic, ceramic, composite, glass / capillary, carbon fiber, composite or other suitable materials. The inner tube can be connected to the outer tube using a variety of connectors. These connectors between the inner and outer tubes can be reusable, such as joints, sheaths or other housings, or can be permanent, such as adhesives or epoxy resins. The tubes can also be constructed using a single material or multiple materials using 3D printing or other manufacturing techniques. Figure 5A An example design is shown. In a specific embodiment, a vacuum-driven electronic pressure controller (EPC) (520) is connected to a line that can be directly connected to a manifold (510), which contains a vacuum trap (512) for preventing fluid from entering 514 and 520. The manifold 510 and the vacuum trap 512 are connected to form an airtight seal, thereby achieving a pneumatic connection between the line 514 and the line 505. The line 505 is connected to a valve so that the vacuum source can be isolated from the line 500 on the other side of the valve and the sample contained in the orifice plate 700 or sample vial. The valve can be a solenoid valve, a pinch valve, a rotary valve or a ball valve, but is not limited to this. The lines are connected on both sides of the valve to achieve an airtight seal and a liquidtight seal.

[0032] To mix the contents of the sample, the vacuum-driven EPC (520) first applies negative pressure to a portion of the tubing (514). At a similar time (earlier, simultaneously, or later), valve 516 is opened to allow negative pressure to be transmitted through 512, 505, 516, and 500, causing fluid to be pumped from the sample vessel into the mixing tubing. After a set time, the EPC switches from the vacuum solution (P EPC <P 大气 )Switch to positive pressure solution ((P EPC >P 大气 ) to reverse the direction of fluid flow and return it to the sample well. This process can be repeated one or more times to properly mix the sample. Once the sample is thoroughly mixed, it is withdrawn by the LFC or other instrument 140. The sample can be removed using a headspace pressure-based sampling method or by vacuum within the instrument.

[0033] FIG6A illustrates a method for creating a sterile zone within a 110 or other autosampler system using an ultraviolet (UV) germicidal lamp, light emitting diode (LED) or other light source (610) mounted in a single compartment, double compartment or other compartment arrangement, positioned at the rear, front, side, top or bottom of the space to be sterilized and attached to or attached to or adjacent to a reflective shield at a desired intensity and distance. A front view of one embodiment is shown. Figure 6A-1 , while the top and side views are shown in Figure 6A-2 The area to be sterilized can be defined by continuously or purposefully laying polished aluminum or any other sufficiently reflective material (620). The shield can also be used to protect the wiring and other components from repeated UV exposure, while the area to be sterilized is fully exposed to the UV light. The shield can also be a mirror, lens or other optical element 640 designed to focus and / or reflect UV light to a specific location. The UV source 610 can be mounted to a static component of the automatic sampler 110, or can be mounted to the same plate or other location of the orifice plate 700 so that the UV source can move relative to the needle assembly 226. In addition, the orifice plate 700 can be moved along the Z axis to provide a favorable position for sterilization. The UV source can also be directly integrated into the orifice plate 216 to sterilize the needle assembly 226. The orifice plate will be moved into a position below the needle assembly so that the UV source in the block can sterilize the needle assembly. Additional structure or support 630 can be integrated to assist in positioning the UV source. The required intensity and time are determined by calculating the energy required to achieve the kill factor of the target to be eliminated.

[0034] Figure 6BThe system is used for aseptic mixing and sample transport. In order to maintain the sterility of the sample during movement from the incubator, biosafety box, laminar flow hood or other device to the automatic sampler 110, an airtight seal 632 can be used to cover the orifice plate. The seal can be made of rubber, polymer, silicon, Viton™ (fluororubber), plastic or any other suitable material. Once in the incubator, the sample remains sealed during movement as the needle assembly 226 is placed above the orifice plate. During the sampling and mixing process, the plate is moved vertically along the Z axis so that the needle assembly pierces the seal 632 of the plate to obtain the sample (sampling position). Figure 6C Further details of a specific embodiment are shown. As shown in the cross-sectional view, the outer tube 650 includes three inner tubes for mixing (680), sample transport (670) and pressure regulation (660). The bottom of the outer tube can be blunt, cut at an angle as shown, or otherwise configured to easily pierce the seal 632. The inner tubes can have different or identical diameters to improve or change mixing, sample transport, or pressure regulation as needed. The materials of the inner and outer tubes can be different, consisting of metal, plastic, ceramic, composite, glass / capillary, or other materials. The inner tubes can be connected to the outer tube using a variety of connectors. These connectors can be reusable, such as connectors, sleeves, or other housings, or they can be permanent, such as adhesives or epoxy resins. This connection between the inner and outer tubes creates an airtight seal shown as 655. The pressure regulating tube (660) is connected to an electronic pressure controller that can adjust the air pressure in the tube. There is also a hole or notch (657) above the bottom of the tube to allow air and pressure changes to pass between the inside and outside of the outer tube. In the sampling position (the sampling position is when the base of the needle is immersed in the fluid and the hermetic seal has been pierced), the needle assembly pierces the seal 632, so that the mixing tube 680 and the sample delivery tube 670 are immersed, but the recess 657 is above the surface of the fluid but below the seal. This creates a closed space 675, the pressure of which can be controlled by the EPC connected to the pressure regulating tube 660. This causes the sample to be pushed upward into the sampling tube 670.

[0035] The ability to continuously incubate a single plate under ideal culture conditions requires the inclusion of a separate incubation chamber that can be used at the desired sampling time. Figure 7AAs shown in 110. This design can be adapted to multiple orifice plates of various sizes, wherein the X-axis and Y-axis can allow the plate to move around 226. The incubation chamber (720) is connected to the top of the orifice plate (700) and can be made of plastic, plexiglass, glass or other suitable materials. The top of the incubation chamber can have a seal (710) that can be pierced by a sampling needle but still remain airtight. The incubation chamber can also have a seal around the bottom edge (715) to create a seal along the edge of the orifice plate. In another embodiment, the orifice plate 700 and the incubation chamber can be combined into a whole that still includes the top seal 710 but does not require the bottom seal 715. This will create a head space 725 that can be controlled to create suitable conditions for cell growth. Temperature control of each chamber can be achieved by adjusting the temperature of 216, and carbon dioxide is pumped into the chamber through a tube or hose (730) installed on 720, which is adapted to maintain sterility through a commercially available HEPA filter (740). Carbon dioxide and oxygen can be provided by a source (such as a compressed gas tank 750). The system can be equipped with additional sensing nodes to monitor pH, dissolved gases (oxygen, carbon dioxide, etc.) metabolites or any other detectable requirements. Cells will be grown in the chamber and sampled periodically by piercing the top seal with a needle assembly. Cells are removed from the sampling hole by aspirating (sip) the cells from the hole by increasing the total pressure of the head space with a vacuum-based system, or other means.

[0036] The sample contents can be mixed by mechanical, magnetic, pneumatic, fluidic or other means for suspension cell growth (or other objectives). Specific examples include, but are not limited to, a magnetic bed in each well or vial, a magnetic stir bar, or an impeller activated from below, above, or from the side.

[0037] In such Figure 7B In another embodiment shown, the wells can be replaced by discrete vessels, each having a separate chamber 720 as shown. Gas will still be provided and temperature controlled to achieve ideal conditions for cell growth. Cells will be harvested in the same manner as described above.

[0038] To create a multi-channel system where multiple boards can be automatically acquired, such as Figure 8One or more carriage systems are provided to support substrates for an XY-array (820) in a circular (800) or square / rectangular format. Multiplexed arrays can include multiple incubation chambers or large incubation chambers where all samples are connected in parallel or in series. Circular 720 and 710 are provided in conjunction with 800. A pivot rod (810) connected to 216 enables the well plate to undergo circular rotation when moved by mechanical force. These multiple trays can be moved in the X, Y, and Z axes to access the sampling needle.

[0039] Figure 9A A multi-plate storage tower (900) is illustrated, which is separated by fixed layers and can be used with an automated handler. The robotic arm can be programmed to retrieve plates from any space in the tower (e.g., positions 1-8 in the figure, where the number of trays can be increased or decreased based on design requirements). The system can be integrated into the automated sampler as part of the automated sampler, located directly below or adjacent to the automated sampler. The robotic arm can use any number of methods (e.g., mechanical, magnetic (including electromagnetic), electronic, or other means) to remove any plate. The individual well plates used in the system are designed in such a way that they can be used in conjunction with a robotic system. In one embodiment, for example, Figure 9B As shown, the plate has magnets on two adjacent sides. During normal operation, magnets 910 help keep the plate in place. In order to move the plate, the robotic arm 920 provides enough force to break the magnetic connection that holds the plate in the rack, thereby removing the plate as shown. The plate can then be sent to an automatic sampler or other device for sampling. In one embodiment, the cells can also be returned to the tower after sampling. In order to accommodate cells that grow in an adherent manner, the automatic sampler will have the ability to detach the cells from the surface of the growth substrate. Figure 10 shows several embodiments of this cell detachment method. The system includes some or all of the following components. Dedicated tubing or removable tips can be used to remove fluid from the wells or add fluid to the wells. Once the cells have received the reagent (such as trypsin, TrypLE TM Pancreatic enzyme substitutes, Accutase TM If the cells are treated with a cell digestion solution or other cell detachment reagent, they can be completely removed and suspended by physical scraping using a soft plastic scraper, a thin metal blade, or a plastic blade attached to a tip. Alternatively, they can be suspended using a liquid flow, making them suitable for analysis involving the above-mentioned mixing system, which can provide the necessary liquid flow to remove and resuspend the cells. Fluids can be removed by the mixing system ( Figure 5), then new fluid is added from the reagent addition system ( FIG. 10 ) to resuspend the cells and wash away debris. Temperature control of the well plate 216 can also be used to assist in detaching or washing suspended cells. Reagents that can be added include PBS or other buffers, trypsin or other cell detachment solutions, culture medium, fixatives, samples (e.g., cells or culture medium), EDTA, or any other relevant fluids. Figure 11 The actual delivery system is shown. This is achieved using any electronic pressure controller to pressurize the headspace above the fluid reagent to be dispensed into the vial containing the sample. Alternatively, a peristaltic syringe or other pump can be used to drive the reagent flow to the sample vial or rack. Figure 12 shows the physical cell detachment method, which can be performed at different locations within the autosampler. Once the sample is ready for analysis, the vial or plate will be Figure 12 Physical scraping is performed by moving the well plate or bottle block in three dimensions against a fixed soft plastic, rubber or polymer scraper, a thin metal blade, or a plastic blade connected to a tip that can be used to completely remove cells and suspend them.

[0040] In a specific embodiment, the present invention provides an apparatus for automated analysis of one or more samples, wherein the automated analytical processing comprises automated flow, wherein the sample comprises a fluid or particles in a sample vessel, and wherein the apparatus comprises an assembly of components capable of processing the sample (or samples) for analytical evaluation by a fluid-based and / or particle-based instrument. The apparatus may be referred to as an autosampler. Methods for using such an apparatus are also provided.

[0041] The automated flow in the device may include systems for moving samples, including vacuum systems, pressure-based systems, pneumatic systems, pumps, peristaltic pumps, diaphragms, or syringes. In specific embodiments, automated flow, such as pneumatic flow, can be customized based on analysis parameters, which include but are not limited to the properties of the sample being analyzed, the number of samples being analyzed, and / or the type of fluid-based and / or particle-based instrument used to perform analytical evaluation. In specific embodiments, the flow rate of the sample from the source to the delivery point (i.e., transported to the device, instrument, or optical force-based instrument such as Radiance™) can be from 0.1 to 100 L / min. In other specific embodiments, the flow rate can be from 0.1 to 100 L / min, 0.5 to 500 L / min, or 2 to 2000 L / min. The flow rate of the sample can be individually customized and adjusted to obtain optimal efficiency and consistency and to assist in rapid and accurate analytical evaluation in conjunction with the reader instrument.

[0042] In a specific embodiment, the samples analyzed by the automatic sampler instrument of the present invention may include, but are not limited to, polymer, metal, glass or alloy-based particles, biological cells, plant cells (algae cells or other cells), prokaryotic cells (bacteria), eukaryotic cells, yeast, fungi, mold cells, red blood cells, neurons, egg cells (eggs), sperm, leukocytes, basophils, neutrophils, eosinophils, monocytes, lymphocytes, macrophages, platelets, blisters, exosomes, stromal cells, multicellular structures (such as spheroids), mesenchymal cells and induced pluripotent stem cells (iPSCs) and subcellular components (including cell nuclei, mitochondria or chloroplasts). The sample can be generated synthetically or obtained from a natural source. The sample can be obtained from a body fluid or body substance, including but not limited to tears, saliva, sputum, blood, plasma, lymph, urine, sweat, pus, nasal discharge or semen.

[0043] In specific embodiments, analytical assessments performed by fluidic and / or particle-based instruments include, but are not limited to, measuring optical forces, laser force cytology, automated microscopy, capillary electrophoresis, single-cell droplet microfluidics, single-cell genomics, sequencing devices, mass spectrometry, and nucleic acid or protein analysis, amplification, and modification.

[0044] In specific embodiments, the component assembly may include, but is not limited to, motors in the X, Y, and Z directions, limit switches, microfluidic tubing, orifice plates, electronic pressure controllers for controlling the pressure of the headspace on the fluid to achieve flow, pneumatic or fluid mixing devices with or without temperature control, components for fluid handling, sampling vessels with or without temperature control, and mechanical components for translating sampling vessels or other system components. In some specific embodiments, the sample (or multiple samples) may be present in a single well, in a single bottle, or in a multi-well plate. The component assembly, such as the sampling tube and / or the orifice plate, can be sterilized. In some specific embodiments, the device further includes components for piercing the seal to access the sample headspace and fluid.

[0045] In a specific embodiment, processing of a sample (or multiple samples) includes operations selected from the group consisting of: selecting a flow rate for a fluid or particle to be transported to a fluid-based and / or particle-based instrument; mixing the contents of the sample; incubating the sample; heating the sample; cooling the sample; sterilizing the sample; creating a seal for a vial or well plate containing the sample; and adding reagents (e.g., biochemicals) or other biological components (e.g., cells) to the sample during a predetermined period.

[0046] In some embodiments, the components for handling fluids in the device include an outer tube that is mounted within the sample vessel, one or more dispersed inner tubes that are mounted within the diameter of the outer tube, a connector to one or more target vessels that are fluidically connected to the sampling vessel, and one or more separate systems for moving fluids into or out of the sampling vessel in a controlled manner. In some embodiments, valves can be used to preferentially drive fluid into one or more of the inner tubes or prevent fluid from entering one or more of the inner tubes, and the system for moving fluids can include a vacuum system, a pressure-based system, or a pump (such as a peristaltic pump), a diaphragm, a syringe, or other system. In some embodiments, the fluid handling device is located within a manifold that maintains an airtight seal relative to the sampling vessel or its separately isolated portion. The outer tube can be made of metal, plastic, ceramic, composite, glass / capillary, or other materials; the tube can be made of metal, plastic, ceramic, composite, glass / capillary, or other materials. In some embodiments, the inner tube is reversibly connected to the outer tube using a connector (such as a joint, sleeve, hoop structure or other housing), or can be permanently connected to the outer tube using adhesives, epoxy resins, cement or other adhesives. In some embodiments, the outer tube and the inner tube are made into a single workpiece having one or more material types using additive manufacturing techniques including 3D printing (such as stereolithography, digital light processing, fused deposition modeling, selective laser sintering, selective laser melting, electron beam melting, layered solid manufacturing, spray adhesive powder forming, material spray forming or other techniques). In addition, the outer tube and the inner tube can be made of glass using laser mapping and hydrofluoric acid (HF) or potassium hydroxide (KOH) etching and bonding methods. In additional embodiments, the inner tube of the device is connected to one or more fluid storage devices, which can be transported to sampling vessels or other vessels within the system. These fluids can be transported to detach adherent cells growing in the sample device. Sample vessels may include vials or well plates comprising 6, 12, 24, 48, 96, 192, 288, 384, 1536, or any custom number of wells.

[0047] In a specific embodiment, the outer tube is capable of mechanically scraping cells from the bottom surface of the sample vessel (see Figure 12 For example, the outer tube may have a wedge, tab, tongue, or other shaped connector made of hard or soft plastic, metal, or ceramic that can mechanically scrape cells off the bottom surface of the sample vessel.

[0048] In specific embodiments, the inner tube is connected to a reservoir having a maximum pressure sufficient to push air bubbles out of the system tubing into one or more vessels or fluid reservoirs.

[0049] In certain embodiments, the pneumatic pressure used by the automatic sampler according to the present invention can be adjusted according to the needs of the user. For example, in certain embodiments, the pressure can be 0-200 psig, 0-150 psig, 1-100 psig, 1-50 psig, or 50 psig. In certain embodiments, the flow of liquid through one or more of the inner tubes can be monitored using a flow meter.

[0050] In a specific embodiment, the device further includes a mechanism for monitoring liquid flow, such as a flow meter. The flow meter can be used to calculate the amount of fluid removed from or delivered to a sample vessel or any other vessel or container connected to the system.

[0051] In a specific embodiment, the activity of sterilizing the sample includes creating a sterile zone within a sampling device that includes one or more light sources capable of sterilizing a surface by generating ultraviolet light. The light source can be an ultraviolet (UV) germicidal lamp, UV-A, UV-B, UV-C, a light emitting diode (LED), a laser, or other ultraviolet or other broad or narrow wavelength light sources. In a specific embodiment, the device further includes a structure or surface that contains, directs, or reflects light to one or more specific areas or surfaces. The light source can be mounted to a static component of the sampling device, or can be mounted to a moving component within the sampling device.

[0052] In certain embodiments, the seal of the sample-containing vial or well plate comprises an airtight seal made of rubber, a polymer, silicone, Viton™ (fluoroelastomer), plastic, or any other suitable material. The outer tube of the fluid handling device can be capable of piercing the airtight seal. The outer tube in contact with the seal can be blunt, angled, or otherwise configured to facilitate piercing the seal. In certain embodiments, the seal is constructed so that it remains airtight even after being pierced by the outer tube of the fluid handling device.

[0053] In particular embodiments, a hole or notch may be located above the bottom of the outer tube, above the fluid surface but below the seal, thereby allowing air and pressure changes to pass between the inside and outside of the outer tube.

[0054] In a specific embodiment, the sample vessel or a portion thereof has an airtight seal that creates a headspace above the sample fluid and can be continuously incubated under controlled temperature, headspace gas concentration and sample mixing conditions. The outer tube of the fluid handling device is capable of piercing the airtight seal and the end of the outer tube that contacts the seal can be blunt, angled or have other structures that can easily pierce the seal. In a specific embodiment, the hole or notch can be located above the bottom of the outer tube, above the fluid surface but below the seal, so as to allow air and pressure changes to pass between the inside and outside of the outer tube. The seal is constructed in such a way that it remains airtight after being pierced by the outer tube of the fluid handling device.

[0055] In a specific embodiment, the Figure 7A and B), the gas pumped into the headspace can be sterile.

[0056] In specific embodiments, sensors are used to measure temperature, pH, gas concentration, or other parameters of the headspace or sample volume.

[0057] In certain embodiments, suspension cells may be cultured in one or more sample vessels.

[0058] In specific embodiments, adherent cells can be cultured on microcarriers, fiber-based membranes, discs, or other structures, or other suitable growth substrates that can be mixed with or perfused with culture medium.

[0059] In specific embodiments, cells can be mixed by mechanical, magnetic, pneumatic, fluidic or other means.

[0060] In specific embodiments, a sample vessel may be composed of a plurality of dispersion sub-vessels.

[0061] In a specific embodiment, each sub-vessel has its own headspace, controls, and sensor array.

[0062] In certain embodiments, an autosampler device according to the present invention further comprises features for delivering one or more reagents to a sample reservoir for mixing and subsequent analysis.

[0063] In specific embodiments, multiple plates are placed or configured in a multiplex system and accessed by an automated sampling device as needed. These plates can be incubated, sterilized, etc. as needed. In specific embodiments, the device can be further customized to allow multiple different plates to be loaded into the system in circular, rectangular, or other configurations, well plates to be loaded into a multi-plate storage device by an automated system, and / or well plates to be connected to the tower by mechanical, magnetic, electromagnetic, or other means.

Claims

1. A device for automatically analyzing one or more samples, characterized in that The device comprises: a sample vessel configured to hold one or more liquid or particulate samples, and a component assembly in fluid communication with the sample vessel, capable of processing the one or more liquid or particulate samples for analytical evaluation using a fluid-based and / or particle-based instrument, wherein the component assembly includes components for fluid processing, comprising i) an outer tube (650) housed within the sample vessel, and ii) one or more inner tubes (680) disposed within the outer tube and configured to transport the one or more liquid or particulate samples; wherein mixing occurs in a tube other than the tube used for sample transport, and wherein the inner tube is reversibly maintained in fluid communication with the outer tube using a connector comprising a fitting, sleeve, or ferrule structure, or is permanently maintained in fluid communication with the outer tube through the use of an adhesive or epoxy; and A system for automated flow of the one or more liquid or particulate samples, wherein the system is in fluid communication with at least the sample vessel, the system comprising a vacuum system, a pressure-based system, or a pneumatic system.

2. The device according to claim 1, characterized in that The system used for automated flow is a vacuum system.

3. The device according to claim 1, characterized in that The sample comprises polymer, metal, glass or alloy-based particles.

4. The device according to claim 1, characterized in that The sample includes biological cells.

5. The device according to claim 1, characterized in that Such analytical assessments performed by fluidic and / or particle-based instruments include optical force measurements, laser force cytometry, automated microscopy, capillary electrophoresis, single-cell droplet microfluidics, single-cell genomics, sequencing devices, mass spectrometry, and nucleic acid or protein analysis, amplification, and modification.

6. The device according to claim 1, characterized in that The component assembly further includes motors in the X, Y and Z directions, limit switches, microfluidic pipelines, orifice plates, electronic pressure controllers, pneumatic or fluid mixing devices with or without temperature control, sampling vessels with or without temperature control, or mechanical components for mobilizing or transporting sampling vessels.

7. The device according to claim 1, characterized in that The sample is present in a single well, a single bottle, or a multi-well plate.

8. The device according to claim 1, characterized in that Processing of a sample includes activities selected from the group consisting of: selecting a flow rate for a liquid or particles to be transported to a fluid and / or particle-based instrument; mixing the contents of the sample; incubating the sample; heating the sample; cooling the sample; sterilizing the sample; creating a seal for a vial or well plate containing the sample; and adding reagents to the sample during a predetermined period.

9. The device according to claim 1, characterized in that The device further includes means for piercing the seal to access the sample headspace and liquid.

10. The device according to claim 6, characterized in that At least one of the sampling vessel or the well plate is sterilized.

11. The device according to claim 6, characterized in that Valves are used to preferentially drive fluid into one or more of the inner tubes or to prevent fluid from entering one or more of the inner tubes.

12. The device according to claim 6, characterized in that The system for automated flow is a pressure based system.

13. The device according to claim 6, characterized in that The outer tube comprises metal, plastic, ceramic or glass.

14. The device according to claim 6, characterized in that The inner tube is made of metal.

15. The device according to claim 6, characterized in that The outer tube and the inner tube are made into a single workpiece having one or more types of materials by utilizing additive manufacturing techniques including 3D printing, wherein the 3D printing includes stereolithography, digital light processing, fused deposition modeling, selective laser sintering, selective laser melting, electron beam melting, layered solid manufacturing, powder bonding forming or material spraying forming.

16. The device according to claim 6, characterized in that The outer tube and the inner tube are made of glass using laser patterning and hydrofluoric acid or potassium hydroxide etching and bonding methods.

17. The device according to claim 6, characterized in that The inner tube is connected to one or more reservoirs of fluid that can be transported to the sampling vessel.

18. The device according to claim 16, characterized in that The transported fluid is used to separate adherent cells growing in the sample device.

19. The device according to claim 6, characterized in that The sample vessels include vials or well plates including 6, 12, 24, 48, 96, 192, 288, 384, 1536 or any custom number of wells.

20. The device according to claim 6, characterized in that The outer tube is capable of mechanically scraping cells from the bottom surface of the sample vessel.

21. The device according to claim 6, characterized in that The outer tube has a wedge, a blade or a tongue made of hard or soft plastic, metal, ceramic, which can mechanically scrape cells from the bottom surface of the sample vessel.

22. The device according to claim 6, characterized in that One or more of the inner tubes are connected to a reservoir, and the maximum pressure of the reservoir is high enough to push air bubbles out of the system pipeline into one or more vessels or fluid reservoirs.

23. The device according to claim 6, characterized in that The apparatus further comprises means for monitoring the flow of liquid.

24. The device according to claim 23, characterized in that The mechanism for monitoring liquid flow is a flow meter and is used to calculate the amount of fluid removed from or delivered into the sample vessel.

25. The device according to claim 8, characterized in that The activity of sterilizing the sample includes creating a sterile zone within a sampling device that includes one or more light sources capable of sterilizing surfaces by generating ultraviolet light.

26. The device according to claim 25, characterized in that The light source is a germicidal lamp.

27. The device according to claim 25, characterized in that Structures or surfaces are used to contain, direct, or reflect light to one or more specific areas or surfaces.

28. The device according to claim 25, characterized in that The light source is mounted to a static component of the sampling device.

29. The device according to claim 25, characterized in that The light source is mounted to a moving part within the sampling device.

30. The device according to claim 25, wherein The seal of the vial or well plate containing the sample comprises an airtight seal.

31. The device according to claim 30, characterized in that The outer tube of the component for fluid handling is capable of piercing the airtight seal.

32. The device according to claim 31, characterized in that The end of the outer tube that contacts the seal is blunt or cut at an angle.

33. The device according to claim 31, characterized in that There is a hole or notch above the bottom of the outer tube, above the surface of the liquid but below the seal, to allow air and pressure changes to pass between the inside and outside of the outer tube.

34. The device according to claim 31, characterized in that The seal is constructed in such a way that it remains airtight even after being pierced by the outer tube of the component for fluid handling.

35. The device according to claim 1, wherein The sample vessel, or a component thereof, has a hermetic seal that creates a headspace above the sample fluid and is continuously incubated under controlled temperature, headspace gas concentration, and sample mixing conditions.

36. The device according to claim 35, characterized in that The outer tube of the component for fluid handling is capable of piercing the airtight seal.

37. The device according to claim 36, characterized in that The end of the outer tube that contacts the seal is blunt or cut at an angle.

38. The device according to claim 36, characterized in that There is an upper hole or notch in the bottom of the outer tube, above the surface of the liquid but below the seal, to allow air and pressure changes to pass between the inside and outside of the outer tube.

39. The device according to claim 36, characterized in that The seal is constructed in such a way that it remains airtight even after being pierced by the outer tube of the component for fluid handling.

40. The device according to claim 35, characterized in that The gas pumped into the headspace is sterile by filtration.

41. The device according to claim 35, characterized in that Sensors are used to measure temperature, pH or gas concentration.

42. The device according to claim 35, characterized in that Suspension cells are cultured in one or more sample vessels.

43. The device according to claim 35, characterized in that Adherent cells are cultured in one or more sample vessels attached to microcarriers, fiber-based membranes, or disks.

44. The device according to claim 35, characterized in that Cells are mixed by mechanical, magnetic, pneumatic, or fluidic means.

45. The device according to claim 35, characterized in that The sample vessel is composed of a plurality of dispersion sub-vessels.

46. ​​The device according to claim 45, characterized in that Each sub-vessel has its own headspace, controls, and sensor array.

47. The device according to claim 4, characterized in that The biological cells include eukaryotic cells.

48. The device according to claim 1, wherein The sample includes subcellular components.

49. The device according to claim 48, characterized in that The subcellular components include nuclei, mitochondria or chloroplasts.

50. The device according to claim 6, characterized in that The outer tube includes a capillary tube.

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