Fluidic autosampler and incubator

Abstract

To provide devices and methods of using the devices that enable automated nano / micro / millifluidic sampling from one or more containers.SOLUTION: A process of automated analysis comprises an automated flow, where samples comprise liquid or particles in a sample vessel, and where devices comprise an assembly of components that enable processing of a sample for analytical assessment by fluidic and / or particle-based instruments. The automated flow may comprise systems for moving samples including vacuum systems, pressure-based systems, pneumatic systems, pumps, peristaltic pumps, diaphragms, or syringes. The devices may comprise an assembly of components that enable movement in X, Y and Z dimensions, as well as switches, microfluidic tubing, well plate blocks, electronic pressure controllers, pneumatic or fluidic mixing devices, components for fluid handling, sampling vessels, and mechanical components for translating or transporting system components.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Embodiments of the present disclosure relate to an apparatus that enables automated nano / micro / milli-fluid sample collection from one or more containers, and a method of using the apparatus. The containers may include containers ranging from one well or vial to multiple multi-well plates. Further, the apparatus provided herein can mix the contents of individual wells, create a sterile field for biological investigations, and control parameters such as temperature, carbon dioxide concentration, and other cell growth conditions for monitoring the stability of materials and cell-based transitions.

Background Art

[0002] The increased use of automated systems for sample storage, processing, and analysis has led to extensive research in the field of automation. Current sample collection systems include a robotic arm that can move samples from a storage zone to a loading zone for sample collection, a magnetic or mechanical stirring member that can keep the sample in a suspended state, and a heating zone or a cooling zone that can adjust the sample storage temperature or the analysis temperature.

[0003] The prior art includes some automated sample collection devices (e.g., Patent Document 1 (U.S. Patent No. 4,713,974) for use in liquid chromatography) that cover various fields of analytical research. However, due to the lack of accurate and consistent nanoliter flow rate control, sufficient sample mixing, and reliable temperature control for sample processing, the prior art cannot be used for biological cell analysis using laser force cytometry (LFC).

[0004] Patent Document 2 of Wilhelm et al. (U.S. Patent No. 4,816,730) describes the use of an apparatus comprising a gripping mechanism for sample holding driven by an electronically controlled stepping motor and consisting of a robotic arm capable of vertical movement, horizontal movement, and rotational movement, for processing and moving a plurality of objects. Patent Document 3 of Schmidt et al. (U.S. Patent No. 6,872,362) further describes the use of a powered autosampler having a vial cup configured to include a magnetic stir bar driven by various different methods of changing the magnetic field around the vial cup. Such prior art describes methods for moving and mixing samples, but such methods are not sufficient for biological cell analysis using LFC equipment. What is needed is an improved apparatus that enables storage, mixing, and sampling of containers ranging from single wells to multiple multi-well plates while maintaining the integrity of biological cells through non-contact mixing based on air pressure and sufficient mixing and temperature maintenance by using a temperature-controlled single-well or multi-well plate block.

[0005] A system for automatically stacking or retrieving well plates with a robotic mechanism is available that enables a series of stacking or retrieval operations to be performed vertically or horizontally (Patent Document 4 (Chinese Utility Model Patent No. 204136215 Specification) and Patent Document 5 (US Patent Application Publication No. 2004 / 0206419 Specification)), or utilizes cassettes for loading and unloading (Patent Document 6 (US Patent No. 9,744,535 Specification)), or performs simultaneous stacking or retrieval of an entire lot of plates into a magazine or tower rack (Patent Document 7 (US Patent No. 6,086,319 Specification)). Further, in previous designs, well plates could be loaded into or removed from a storage tower in a random (non-sequential) manner regardless of the stacking order (Patent Document 8 (US Patent No. 7,670,555 Specification)). However, what is needed is a non-sequential method that enables well plate culturing using a magnetic interface and can be used in combination with a multiplex analysis method for automatic sampling of such well plates, to achieve the functions of specifically detecting, selecting a desired well plate, stacking the desired well plate onto a storage tower, or retrieving it from the same tower.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Summary of the Invention

[0007] Embodiments of the present disclosure relate to an apparatus that enables automated (and optionally cultured) nano / micro / milli-fluid sample collection from containers ranging from a single well or vial to multiple multi-well plates, and a method of using the same apparatus. Further, the apparatus can mix the contents of individual wells, create a sterile field for biological investigations, and control cell growth conditions such as temperature and carbon dioxide concentration for monitoring the stability of materials and cell-based transitions.

[0008] More specifically, the novel apparatus described herein, through the use of an electronic pressure controller and / or regulator used in cooperation with a flow meter, in addition to determining milli / micro / nano-fluid flow rates, achieves fluid monitoring including, but not limited to, degassing. The novel apparatus further enables sample mixing by injecting small air bubbles into the sample well or vial or by aspirating and dispensing the sample or air using a vacuum pump coupled to an electronic pressure controller (EPC). Further, the present invention has the ability to create a sterile field around the needle and well plate area using means such as ultraviolet radiation to ensure that biological materials do not contaminate the housing of the instrument. The apparatus is further designed to optionally culture the sample prior to sample collection by controlling the temperature of the sample using a thermoelectric cooling module. By reversing the current between the modules, the thermoelectric cooler can also change the temperature and heat or culture the sample as needed. Mixing, sterilization, and temperature control can be performed separately, sequentially, and / or simultaneously depending on the settings of the apparatus.

Brief Description of the Drawings

[0009]

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[0010] The present invention will be described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and changes can be made in the practice of the present invention without departing from the scope or spirit of the present invention. Those skilled in the art will recognize that such features can be used alone or in any combination based on a given application or design requirements and specifications. Those skilled in the art will recognize that the systems and devices of the embodiments of the present invention can be used by any of the methods of the present invention, and that any method of the present invention can be implemented using any of the systems and devices of the present invention. Embodiments including various features can additionally be composed of or essentially composed of such various features. Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the present invention. The description of the present invention provided is exemplary in nature, and thus it is intended that variations that do not depart from the essence of the present invention are within the scope of the present invention.

[0011] Before describing in detail at least one embodiment of the present invention, it is to be understood that the present invention is not limited in its application to the details of construction and arrangement of components set forth in the following description or shown in the drawings. The present invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting.

[0012] 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 this technology and methodology belong and would be used by those skilled in the art.

[0013] The text and references described herein, including U.S. Provisional Patent Application No. 62 / 654,335, filed Apr. 7, 2018, are incorporated herein by reference in their entirety.

[0014] A novel apparatus for automating the analysis of samples is provided herein. In this apparatus, the sample is present in vials, containers, wells, multi-well plates, etc. In addition, a method for using such an apparatus is provided. The apparatus may be referred to herein as an “auto-sampler”. In certain embodiments, the auto-sampler may be used to obtain a desired volume and / or a predetermined volume of sample for mechanical or manual analysis. For example, the auto-sampler may be used to retrieve nano / micro / milli-fluidic samples from multi-well plates, vials or other containers, such samples being of various volumes and possibly composed of various cells or particles. Next, the auto-sampler presents the sample to a fluid-based instrument or system for liquids, particles or cells, such as an instrument using Laser Force Cytometry (LFC), in a suitable medium / configuration / container for analysis.

[0015] Turning to the drawings, Figure 1 provides a schematic diagram of an exterior embodiment of an autosampler device (110). In this device, single or multiple multi-well plates or vials can be loaded into a plate block (216, see Figure 2) when a two-part door (120) opens. In alternative embodiments, the door may remain open or consist of a single retractable or folding door or other variations of opening design. 140 is the entry and exit point for sample fluid to transition from 110 to 410 or other LFC or optically based techniques.

[0016] As shown in Figures 2 and 3, in one embodiment, 120 of Figure 1 opens utilizing two motors (200) driven by a control board (for opening and closing the autosampler door). The motors rotate a ring gear (210) that meshes with a toothed door rack (212) to slide open the door of a low friction channel (214). Samples are loaded into a well plate mounted in a temperature control block (216) that can be heated or cooled as needed.

[0017] The needle and needle manifold assembly (226) is constructed of a suitable material such as a flexible polymer, plastic, silicone, carbon or metal base that holds the needle (298) and pneumatic fitting (294) (any alternative material would work depending on the purpose), a sealing surface (230) for sealing against a vial or well plate, and a spring-loaded support structure (232) to facilitate a good seal. The needle may be constructed of a multi-tube design where dedicated sample and mixing tubes are housed within the outer structure of the larger diameter needle. In certain embodiments, the needle may include additional modifications such as a sharpened end for penetrating plate seals.

[0018] In certain embodiments, the travel rail (228) is positioned to allow movement of internal mechanisms along the X and Y axes, and the cable carrier (296) is attached to ensure safe movement of the cable during movement. The X axis is not depicted for 296. An additional motor or set of motors (295) enables movement in the Z dimension by turning a lead screw to move the block (216).

[0019] In certain embodiments, temperature control of 216 may be achieved using a thermoelectric cooling (TEC) module and a thermistor connected to a temperature control panel (310 (see FIG. 3)). Temperature control may include heating or cooling of the sample. A fan (218) operates either constantly or as needed to remove heat from the heat sink (220). Components after 216 can be coupled to a moving platform and 216 can be transported within the range of an autosampler on the X, Y, and Z axes. This movement is controlled by a combination of an industrial computer (320) and a motor (340). For movement along the X and Y axes, the industrially computer-controlled 340 rotates a timing pulley (360) and drives a belt to move the gantry plate along its Y axis (380) and X axis (382). For movement along the Z axis, the industrially computer-driven 340 includes both lead screws (222). The 222 is used to generate up and down Z-axis movement using a Delrin moving nut (224). Any travel nut can be utilized for the purpose. There may or may not be position feedback for the operation control of any axis.

[0020] FIG. 4 shows the junction between 110 and a Radiance (trademark) instrument (410) which is an available embodiment for the application of 110. Separately, 110 can be modified for application to any LFC instrument, optical force instrument, microfluidic instrument, or other instrument. In one embodiment, the junction between 110 and another instrument is as shown in FIG. 4 and enables transfer between two instruments including, but not limited to, fluids, samples, and electronic signals, communications, or other information.

[0021] Figure 5 shows an embodiment of an automated sample collection and mixing system. To facilitate the integrity of the sample during mixing, the Z distance (550) between the bottom of the sample collection tube (530) and the bottom of the sample collection container (shown as a multi-well plate) can be adjusted to finely tune the pneumatic mixing system for each sample. Sample collection is achieved by physically moving or pressing a vial or well plate (700) against 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. Next, by controlling the pressure within the headspace (560) of the vial or sample collection well, the flow rate into and out of the vial or sample collection well can be adjusted. This is made possible by an internal chamber (570), as shown in detail in Figure 5B. As shown in the side view of 5B, the internal chamber (570) is open at two locations at the top and one location at the bottom. The two locations at the top are where the manifold makes airtight connections to the outer tube (540) and the joint (580), respectively. The one location at the bottom is an additional open location connected to the internal chamber at the bottom of the manifold (226), as shown in the detailed view of the bottom of the manifold. The outer tube (540) passes through the internal chamber (570) and extends protruding from the manifold (226) as shown. This allows the outer tube (540) to be submerged into the sample during sample collection. However, since the outer diameter of the outer tube (540) is smaller than the diameter of the internal chamber (570), an airtight seal can be implemented when the well plate or vial is pressed against the sealing surface (230). A tubing (590) is airtightly attached to the top of the manifold (226) via the joint (580). As shown, the tubing (590) is connected to an electronic pressure controller or some alternative pressure source or vacuum source (595). Next, by adjusting the pressure of the source (595), the pressure within the headspace 560 can be accurately controlled. This is one particular embodiment, and it should be noted that in other embodiments, there may be more openings within the manifold as needed. For both mixing and sample collection, separate tubes (500 and 530 respectively) that share a larger outer tube or needle (540) are used.The inner tubes (500 and 530) may be composed of different diameters or the same diameter, if necessary, to improve or correct mixing or sample delivery. The materials of the inner tube and the outer tube may be different and may be composed of metal, plastic, ceramic, composite material, glass / capillary, carbon fiber, composite material or other suitable materials. The inner tube may be connected to the outer tube by various connectors. Such connectors between the inner tube and the outer tube can be reusable ones such as joints, sheaths or other housings, or permanent ones such as adhesives or epoxies. The tubes may also be composed of a single material or multiple materials using 3D printing or other manufacturing techniques. A design example is shown in FIG. 5A. In one embodiment, a vacuum-compatible electronic pressure controller (EPC) (520) is connected to a pipe that can be directly connected to a manifold (510) containing a vacuum trap (512) that helps prevent liquid from entering 514 and 520. The manifold 510 and the vacuum trap 512 are connected to form an airtight seal and enable a pneumatic connection between the pipe 514 and the pipe 505. The pipe 505 is connected to a valve so that a vacuum source can be isolated from the O pipe 500 on the opposite side of the valve and a sample contained in the well plate 700 or the sample vial. The valve can be an electromagnetic valve, a pinch valve, a rotary valve or a ball valve, but is not limited to those listed here. The pipes are connected on both sides of the valve to create an airtight and liquidtight seal.

[0022] To mix the contents of the sample, a vacuum-compatible EPC (520) first applies a negative pressure to a length of tubing (514). Almost simultaneously (before, simultaneously, or after), valve 516 opens, allowing the negative pressure to propagate through 512, 505, 516, and 500, enabling liquid to be drawn from the sample container into the mixing tubing. After a set time has elapsed, the EPC switches from a vacuum regime (PEPC < Patmospheric) to a positive pressure regime (PEPC > Patmospheric) to reverse the flow of the liquid back into the sample well. This cycle may be performed one or more times to properly mix the sample. When the sample is fully mixed, the sample is removed by the LFC or other instrument 140. The sample can be taken using a headspace pressure-based sampling method or by the vacuum within the instrument.

[0023] Figure 6A shows a method of creating a sterilization field within an autosampler 110 or other autosampler system by using an ultraviolet (UV) germicidal lamp, a light emitting diode (LED), or other light source (610) at a desired power and at a desired distance. This light source is attached at a predetermined interval, twice the predetermined interval, or various other intervals, and is disposed on the back, front, side, top, or bottom of the sterilization target space that is attached to or fixed to the reflective shield or in the vicinity of the sterilization target space. A front view of one embodiment is shown in FIG. 6A-1, and a top view and a side view are shown in FIG. 6A-2. The sterilization target area can be enclosed by a continuous arrangement or a defined arrangement of polished aluminum or other sufficiently reflective material (620). This shield also serves the purpose of protecting components such as internal wiring from repeated exposure to ultraviolet light while the sterilization target area is sufficiently exposed to ultraviolet light. The shield may also be in the form of mirrors, lenses, or other optical elements 640 designed to focus and / or reflect ultraviolet light at specific locations. The ultraviolet light source 610 may be attached to a static component of the autosampler 110, or may be attached to the same plate as the well plate 700 or other location to enable movement of the ultraviolet light source relative to the needle assembly 226. Further, the well plate 700 may move along the Z-axis to provide a preferred position for sterilization. It cannot be ruled out that the ultraviolet light source may also be directly incorporated into the well plate block 216 to sterilize the needle assembly 226. The well plate block will move to a position below the needle assembly such that the ultraviolet light source incorporated within the block sterilizes the needle assembly. Additional structures or support elements 630 may be integrated to assist in positioning the ultraviolet light source. The power and time required for sterilization are determined by calculating the energy required to achieve the kill factor for the target to be removed.

[0024] Figure 6B shows a system for sterile mixing and sample delivery. To maintain the sterility of the sample during transfer from an incubator, biological safety cabinet, laminar flow hood, or other device to the autosampler 110, the well plate may be covered with an airtight seal 632. This seal may be made of rubber, polymer, silicon, Viton™, plastic, or any other suitable material. The sample remains sealed during transfer as the needle assembly 226 is positioned over the well plate when it enters the incubator. During sample collection and mixing, the plate moves vertically in the Z dimension (sample collection position) so that the needle assembly penetrates the seal 632 of the plate to access the sample. Another detail of one embodiment is shown in Figure 6C. As seen in the cross-sectional view, the outer tube 650 includes three inner tubes for mixing (680), sample delivery (670), and pressure regulation (660). The outer tube may be configured to not be pointed at the bottom, be cut obliquely as shown in the figure, or otherwise be able to easily penetrate the seal 632. The inner tubes may be configured with different diameters or the same diameter to improve or modify mixing, sample delivery, or pressure regulation as needed. The materials of the inner and outer tubes may be different and may be made of metal, plastic, ceramic, composite material, glass / capillary, or other materials. The inner tubes may be connected to the outer tube by various connectors. Such connectors may be reusable, such as fittings, sheaths, or other housings, or may be permanent, such as adhesives or epoxies. This connection between the inner and outer tubes creates an airtight seal as shown at 655. The pressure regulation tube (660) is connected to an electronic pressure controller that can regulate the pressure of the air inside the tube. Additionally, above the bottom of the tube, there is a hole or notch (657) that allows changes in air and pressure to pass between the inside and outside of the outer tube. At the sample collection position (the sample collection position is the position where the bottom of the needle is submerged below the liquid and pierces the airtight seal), the needle assembly pierces the seal 632 so that the mixing tube 680 and sample delivery tube 670 are submerged, but the notch 657 is above the surface of the liquid and below the seal.This creates a sealed volume 675, the pressure of which can be controlled by an EPC connected to the pressure regulating tube 660. This allows the sample to be pushed up into the sample collection tube 670.

[0025] The ability to continuously culture a single plate under ideal culture conditions requires the incorporation of a self - contained culture chamber that can exist as shown in Figure 7A at 110 during the desired sample collection time. This design may be adapted to multiple well plates of various sizes where the X and Y axes can accommodate plate movement around 226. A culture chamber (720), which may be made of plastic, plexiglass, glass, or other suitable materials, is attached to the top of the well plate (700). The top of the chamber will have a seal (710) that can be pierced by a sample collection needle but maintains airtightness. The chamber may also have a seal around the bottom edge (715) to seal along the edge of the well plate. In an alternative embodiment, the well plate 700 and the culture chamber may be combined into one integral part that still includes the top seal 710 but may not require the bottom seal 715. This will create a headspace 725 that can be controlled to create appropriate conditions for cell growth. Temperature control of the chamber is made possible by regulating the temperature at 216, while CO2 is pumped into the chamber via tubing or a hose (730) that fits within 720 and is adapted to a commercially available HEPA filter (740) to maintain sterility. CO2 and O2 may be supplied from a source such as a compressed gas cylinder 750. The system may have additional sensing nodes attached to monitor pH, dissolved gases (O2, CO2, etc.), metabolites, or any other detectable requirements. Cells will grow within the chamber and be sampled periodically by piercing the top seal with a needle assembly. The cells may be transferred from the sample collection well by using a vacuum - based system to suck the cells out of the well or by raising the overall pressure in the headspace using some alternative means.

[0026] Mixing of the sample contents to enable floating cell growth (or any other optional goal) may be achieved by mechanical means, magnetic means, pneumatic means, fluid means or other means. Specific examples include, but are not limited to, magnetic beds within each well or vial, magnetic stir bars or impellers operating from below, above or the sides.

[0027] In an alternative embodiment, as shown in FIG. 7B, the wells may be replaced as shown by separate containers each having its own individual chamber 720. Gas will continue to be supplied and the temperature will be controlled to achieve ideal conditions for cell growth. Sampling of cells may occur in the same manner as described above.

[0028] To create a multiplexing system capable of automatically sampling multiple plates, one or more transport systems can be used to support a base plate in either a circular (800) or square / rectangular X - Y array (820) as shown in FIG. 8. The multiplexed array may include multiple self - culturing chambers or may incorporate a larger culture chamber in which all samples are adjacent in parallel or in series. Rings 720 and 710 can be used to incorporate 800. A rotating rod (810) attached to 216 will enable circular rotation of the well plate when moved via mechanical power. Such multiple trays may be movable in the X, Y and Z axes to access the sampling needles.

[0029] Figure 9A shows a multi-plate storage tower (900) divided by a fixed height and can be used in combination with a robot handler. The robot arm can be programmed to retrieve plates in any space within the tower (e.g., at positions 1-8 in the figure, the number of trays can be increased or decreased according to the design requirements). This system may be integrated as part of an autosampler, located directly below the autosampler, or positioned adjacent to the autosampler. The robot arm may use any number of methods for removing any of the plates, such as mechanical means, magnetic means (including electromagnets), electronic means, or other means. The individual well plates used in this system may be designed to function in cooperation with the robot system. For example, in one embodiment shown in Figure 9B, the plate has magnets on two adjacent sides. During normal operation, the magnet 910 helps hold the plate in place. To move the plate, the robot arm 920 provides sufficient force to break the magnetic coupling holding the plate in the rack and removes the plate as shown. The plate can then be transferred to an autosampler or other device for sample collection. In one embodiment, cells may also return to the tower after sample collection. To accommodate cells that grow concomitantly, the autosampler will be equipped with a function to separate cells from the surface of the growth substrate. Some embodiments of this cell separation method are shown in Figure 10. The system may be composed of some or all of the following components. Liquid can be introduced into and removed from the wells using dedicated tubing or removable tips. When the cells are treated with a reagent such as trypsin, TrypLE™, Accutase™, or other cell separation reagents, they are physically scraped off with either a flexible plastic scraper, a thin metal blade, or a plastic blade attached to the tip to completely remove and suspend the cells. Alternatively, the flow of liquid can be used to suspend the cells and supply the flow necessary to remove and resuspend the cells so that the cells can be utilized in an analysis including the above mixing system.The liquid can be removed by a mixing system (Figure 5), and then fresh liquid (fresh PBS) can be added from a reagent addition system (Figure 10) to resuspend the cells and wash away debris. In addition to temperature control of the well plate block 216, it can also be used to assist in separating or resuspending cells. Reagents that may be added include PBS or other buffers, trypsin or other cell separation solutions, media, fixatives, samples (such as cells and media), EDTA, or any other liquid for any purpose. Figure 11 shows a reagent delivery system. This is achieved by pressurizing the headspace above the liquid reagent dispensed into the vial containing the sample using any electronic pressure controller. Alternatively, a peristaltic pump, syringe pump, or other pump may be used to drive the flow of the reagent into the sample vial or receptacle. A physical cell separation method located at another location within the autosampler is shown in Figure 12. When the sample is ready for analysis, the vial or well plate will be moved back and forth as a whole relative to the scraper shown in Figure 12. The cells can be completely removed and resuspended using any of the physical scraping methods achieved by moving the well plate or vial block three-dimensionally relative to a fixed soft plastic, rubber, or polymer scraper, a thin metal blade, or a plastic blade attached to the tip.

[0030] In one embodiment, an apparatus for the automated analysis of one or more samples is provided herein, the automated analysis process includes an automated flow, the sample includes a liquid or particles within a sample container, and the apparatus includes an assembly of components that enables the processing of one (or more) samples for analysis evaluation by liquid and / or particle-based instruments. The apparatus may be referred to as an autosampler. In addition, a method for using such an apparatus is provided.

[0031] The automatic flow within the device may include a system for moving the sample, such as a vacuum system, a pressure-based system, a pneumatic system, a pump, a peristaltic pump, a diaphragm, or a syringe. In one embodiment, the automatic flow, such as a pneumatic flow, may be customized based on analysis parameters including, but not limited to, the nature of the sample to be analyzed, the number of samples to be analyzed, and / or the type of fluid and / or particle-based equipment used to perform the analysis evaluation. In one embodiment, the flow rate of the sample from the source to the delivery, i.e., in the case of delivery to an optical force-based instrument such as a device, instrument, or Radiance (trademark), may range from 0.01 to 50 μL / min. In an alternative embodiment, the flow rate may range from 0.1 to 100 μL / min, 0.5 to 500 μL / min, or 2 to 2000 μL / min. The flow rate of the sample may be customized and adjusted uniquely for optimal efficiency and consistency, and in conjunction with the reading instrument, may facilitate a rapid and accurate analysis evaluation.

[0032] In one embodiment, the sample analyzed by the autosampler device described herein may include, but is not limited to, polymers, metals, glass or alloy-based particles, biological cells, plant cells (such as algal cells), prokaryotic cells (bacteria), eukaryotic cells, yeast, fungi, mold cells, red blood cells, neurons, egg cells (ova), sperm, white blood cells, basophils, neutrophils, eosinophils, monocytes, lymphocytes, macrophages, platelets, vesicles, exosomes, stromal cells, spheroids and other multicellular constructs, mesenchymal cells, and induced pluripotent stem cells (iPSCs), as well as subcellular components such as nuclei, mitochondria, or chloroplasts. The sample may be synthetically produced or obtained from natural resources. The sample may be obtained from body fluids or bodily substances including, but not limited to, tears, saliva, sputum, blood, plasma, lymph, urine, sweat, pus, nasal discharge, or sperm.

[0033] In one embodiment, the analytical evaluation by fluid and / or particle-based instruments includes, but is not limited to, measurement of optical forces, laser force cytometry, automated microscopy, capillary electrophoresis, single cell droplet microfluidics, single cell genomics, sequencing devices, mass spectrometry, and analysis, amplification or modification of nucleic acids or proteins.

[0034] In one embodiment, the assembly of components may include motors in the X, Y, and Z dimensions, limit switches, microfluidic tubing, well plate blocks, an electronic pressure controller for controlling the headspace pressure across the fluid to achieve flow, a pneumatic or fluid mixing device that may or may not have temperature control, components for fluid processing, a sample collection container that may or may not have temperature control, and mechanical components for converting the sample collection container or other system components, but is not limited to those listed here. In certain embodiments, one (or more) samples may be present in a single well, single vial, or multi-well plate. The assembly of components such as sample collection tubes and / or well plates may be sterilized. In certain embodiments, the device further includes components that penetrate seals to access the headspace and liquid of the sample.

[0035] In one embodiment, the processing of one (or more) samples includes activities selected from the group consisting of selecting the flow rate of the liquid or particles delivered to fluid and / or particle-based instruments, mixing the contents of the sample, culturing the sample, heating the sample, cooling the sample, sterilizing the sample, creating a seal for the vial or well plate containing the sample, and adding reagents such as biochemicals or other biological components such as cells to the sample for a certain period of time.

[0036] In certain embodiments, the components for fluid processing within the apparatus include an outer tube that fits within the sample container, one or more individual inner tubes that fit within the diameter of the outer tube, connectors to one or more destination containers that are fluidly connected to the sample collection container, and one or more separate systems for moving fluid into and out of the sample collection container in a controlled manner. In some embodiments, valves may be used to preferentially drive fluid into one or more inner tubes or prevent fluid from entering one or more inner tubes, and the system for moving fluid may include a vacuum system, a pressure-based system, or pumps such as peristaltic pumps, diaphragms, syringes. In certain embodiments, the fluid processing apparatus is located within a manifold that creates a hermetic seal with respect to the sample collection container or a compartment thereof. The outer tube may be composed of metal, plastic, ceramic, composite material, glass / capillary, or other materials. The tubes may be composed of metal, plastic, ceramic, composite material, glass / capillary, or other materials. In some embodiments, the inner tubes are reversibly connected to the outer tube using connectors such as joints, sheaths, caps, or other housings, or are permanently connected to the outer tube using epoxy, cement, or other adhesives. In some embodiments, the outer tube and the inner tubes are manufactured as single parts of one or more material types through the use of additive manufacturing techniques such as stereolithography, digital light processing, fused deposition modeling, selective laser sintering, selective laser melting, electron beam melting, laminated object manufacturing, binder jetting, material jetting, or other techniques. Additionally, the outer tube and the inner tubes may be manufactured from glass using laser patterning and etching and bonding processes with hydrofluoric acid (HF) or potassium hydroxide (KOH). In additional embodiments, the inner tubes of the apparatus are connected to one or more reservoirs of fluid that can be delivered to the sample collection container or other containers within the system. The fluid may be delivered to separate adherent cells growing within the sample apparatus. The sample container may include a vial or a well plate containing 6, 12, 24, 48, 96, 192, 288, 384, 1536, or any custom number of wells.

[0037] In one embodiment, the outer tube can mechanically scrape cells from the bottom surface of the sample container (see, for example, FIG. 12). For example, the outer tube may have a wedge-shaped, wafer-shaped, tongue-shaped or other shaped fixture made of hard or soft plastic, metal, ceramic, and may be able to mechanically scrape cells from the bottom surface of the sample container.

[0038] In one embodiment, the inner tube is connected to the reservoir at a maximum pressure sufficient to extrude air bubbles from the system tubing into one or more containers or fluid reservoirs.

[0039] In one embodiment, the air pressure utilized by the autosampler of the present invention may be adjusted according to the needs of the user. For example, in certain embodiments, the pressure may be 0 - 200 psig, 0 - 150 psig, 1 - 100 psig, 1 - 50 psig, or 50 psig. In certain embodiments, the flow through one or more inner tubes is monitored using a flow meter.

[0040] In one embodiment, the apparatus further comprises a mechanism for monitoring the flow, such as a flow meter. The flow meter may be used to calculate the amount of fluid entering or exiting a sample container or any other container or receptacle connected to the system.

[0041] In one embodiment, the activity of sterilizing the sample includes creating a sterilization field within a sample collection device comprising one or more light sources capable of sterilizing the surface through the generation of ultraviolet light. The light source may be an ultraviolet (UV) germicidal lamp, UV - A, UV - B, UV - C, light - emitting diode (LED), laser, or a light source of ultraviolet or other broad or narrow wavelengths. In one embodiment, the apparatus further comprises a structure or surface for containing, directing, or reflecting light to one or more specific regions or surfaces. The light source may be attached to a static component within the sample collection device or to a movable component within the sample collection device.

[0042] In certain embodiments, seals for vials or well plates containing samples include airtight seals made from rubber, polymer, silicon, Viton™, plastic, or any other suitable material. The outer tube of the fluid processing device may be capable of piercing the airtight seal. The outer tube that contacts the seal may not be sharp, may be cut at an angle, or may be configured in another way to easily penetrate the seal. In one embodiment, the seal is configured to maintain airtightness after being pierced by the outer tube of the fluid processing device.

[0043] In one embodiment, a hole or notch is positioned above the bottom of the outer tube that is located above the surface of the liquid and below the seal so that changes in air and pressure can pass between the inside and outside of the outer tube.

[0044] In one embodiment, the sample container or its components may have an airtight seal that creates a headspace above the sample fluid and may be continuously cultured under controlled temperature, headspace gas concentration, and sample mixing. The outer tube of the fluid processing device may be able to pierce the airtight seal, and the end of the outer tube that contacts the seal may not be sharp, may be cut at an angle, or may be configured in another way to allow easy penetration of the seal. In one embodiment, a hole or notch is positioned above the bottom of the outer tube that is located above the surface of the liquid and below the seal so that changes in air and pressure can pass between the inside and outside of the outer tube. The seal may be configured to maintain airtightness after being pierced by the outer tube of the fluid processing device.

[0045] In one embodiment, the gas pumped into the headspace may be sterile as a result of filtration or other suitable means (Figures 7A and 7B).

[0046] In one embodiment, a sensor is implemented to measure the temperature, pH, gas concentration, or other parameters of the headspace or sample volume.

[0047] In one embodiment, floating cells may be grown in one or more sample containers.

[0048] In one embodiment, adherent cells may be grown in one or more sample containers attached to a microcarrier, a fiber-based membrane, a disk or other structure, or other suitable growth substrate that can be mixed with or perfused with the culture medium.

[0049] In one embodiment, the cells may be mixed by mechanical means, magnetic means, pneumatic means, fluid means or other means.

[0050] In one embodiment, the sample container may be composed of a plurality of separate auxiliary containers.

[0051] In one embodiment, each auxiliary container has its own headspace, control and sensor array.

[0052] In certain embodiments, the autosampler device of the present invention further comprises the feature of delivering one or more reagents to the sample storage section for mixing and subsequent analysis.

[0053] In one embodiment, a plurality of plates are placed or configured in a multiplexing system and accessed by the autosampler device as needed. Culturing, sterilization, etc. may be performed on the plates as needed. In one embodiment, the device may be further customized to accommodate loading a plurality of separate plates into a circular, rectangular or other shaped system. The well plate can be loaded into a multi-plate storage device by an automated system and / or the well plate can be attached to a tower via mechanical means, magnetic means, electromagnetic means or other means.

Claims

1. 1. An apparatus for automated analysis of one or more samples, said apparatus comprising: a vacuum system, a pressure-based system, a pneumatic system, a pump, a peristaltic pump, a diaphragm, or a syringe configured to automate the flow of a liquid or particle-containing sample; a sample container configured to incubate the sample; an assembly of components including components for fluid handling, including an outer tube (650) including three inner tubes for mixing (680), sample delivery (670) and pressure regulation (660), wherein valves are configured to preferentially direct fluid into one or more of the inner tubes or to prevent fluid from entering one or more of the inner tubes.

2. The apparatus of claim 1, wherein the one or more separate systems for moving fluids are vacuum systems, pressure-based systems or pumps such as peristaltic pumps, diaphragm, syringe or other types.

3. The apparatus of claim 1, wherein the fluid processing components are located within a manifold that creates an airtight seal against the sample container or its compartment.

4. The device described in claim 1, wherein the outer tube is constructed from metal, plastic, ceramic, composite material, glass / capillary or other material.

5. The device described in claim 1, wherein the inner tube is made from metal, plastic, ceramic, composite material, glass / capillary or other material.

6. The device described in claim 1, wherein the inner tube is connected to the outer tube in a reversible manner using a connector such as a fitting, sheath, nozzle or other housing, or in a permanent manner through the use of glue, epoxy, cement or other adhesive.

7. The apparatus of claim 1, wherein the outer tube and the inner tube are manufactured as a single part of one or more material types by 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, laminated object manufacturing, binder jetting, material jetting or other techniques.

8. The apparatus of claim 1, wherein the outer tube and the inner tube are fabricated from glass using laser patterning and hydrofluoric acid (HF) or potassium hydroxide (KOH) etching and bonding processes.

9. The apparatus of claim 1, wherein the sample comprises polymer, metal, glass or alloy based particles, biological cells, plant cells (such as algal cells), prokaryotic cells (bacteria), eukaryotic cells, yeast, fungus, mold cells, red blood cells, neurons, egg cells (ova), sperm, leukocytes, basophils, neutrophils, eosinophils, monocytes, lymphocytes, macrophages, platelets, vesicles, exosomes, interstitial cells, multicellular entities such as spheroids, mesenchymal cells and induced pluripotent stem cells (iPSCs), as well as intracellular components including nuclei, mitochondria or chloroplasts.

10. The device of claim 1, wherein the automated analysis is performed by equipment configured to perform optical force-based measurements, laser force cytology, automated microscopy, capillary electrophoresis, single cell droplet microfluidics, single cell genomics, sequencing, mass spectrometry and analysis, amplification or modification of nucleic acids or proteins.

11. The apparatus of claim 1, wherein the assembly of components includes motors in X, Y and Z dimensions, switches to control the application of vacuum and positive pressure, microfluidic tubing, a well plate block, an electronic pressure controller, a pneumatic or fluid mixing device with or without temperature control, a temperature control unit for the sample container, and mechanical components for translating or transporting the sample container or other system components.

12. The device described in claim 1, wherein the sample is present in a single well, a single vial, or a multi-well plate.

13. The apparatus of claim 1, further comprising a sealing surface or gasket configured to form a seal against the sample container and a component that penetrates the seal to access the headspace and liquid of the sample.

14. The device of claim 1, wherein the assembly of components comprises a light source configured to sterilize microfluidic tubing or well plates.

15. The fluid processing component further comprising: one or more destination container connectors fluidly connected to the sample container; and one or more separate systems for moving fluid into and out of the sample vessel in a controlled manner.

16. An apparatus for automated analysis of said one or more liquid or particulate samples, comprising: a sample vessel configured to contain the one or more liquid or particle samples; an assembly of components that enables the one or more liquid or particle samples to be processed for analytical evaluation by a fluid- or particle-based instrument; The assembly of components comprises a fluid handling component configured to mix and deliver the one or more liquid or particle samples, the fluid handling component comprising: an outer tube that fits into the sample container; one or more inner tubes disposed in the outer tube; The one or more inner tubes are configured to deliver a sample from within the one or more liquid or particle samples, and mixing occurs outside the one or more inner tubes for sample delivery.

17. The apparatus of claim 16, further comprising means for generating an automated flow of the one or more liquid or particle samples.

18. The apparatus of claim 17, wherein the means for generating an automated flow of the one or more liquid or particle samples includes a vacuum system, a pressure-based system, a pneumatic system, a pump, a peristaltic pump, a diaphragm, or a syringe.

19. The apparatus of claim 16, wherein the one or more liquid or particle samples comprise polymer particles, metal particles, glass particles, alloy particles, biological cells, plant cells, algal cells, prokaryotic cells, bacteria, eukaryotic cells, yeast cells, fungi, mold cells, red blood cells, neurons, egg cells, sperm, leukocytes, basophils, neutrophils, eosinophils, monocytes, lymphocytes, macrophages, platelets, vesicles, exosomes, interstitial cells, multicellular entities, spheroids, mesenchymal cells, induced pluripotent stem cells, intracellular components, nuclei, mitochondria, chloroplasts, or combinations thereof.

20. The apparatus of claim 16, wherein the assembly of components further comprises an X-dimensional motor, a Y-dimensional motor, a Z-dimensional motor, a limit switch, microfluidic tubing, a well plate block, an electronic pressure controller, a temperature controlled pneumatic mixer, a non-temperature controlled pneumatic mixer, a temperature controlled fluid mixer, a non-temperature controlled fluid mixer, a temperature controller for a sample container, a mechanical component for translating a component of the assembly of components, a mechanical component for translating the sample container, or a combination thereof.

21. The device of claim 16, wherein the sample container is a single well, a single vial, or a well in a multi-well plate.

22. The apparatus of claim 16, further comprising a component that penetrates the seal to access the one or more liquid or particulate samples, the headspace above the samples, or a combination thereof.

23. The apparatus of claim 16, wherein the fluid processing component is configured to remove one or more liquid or particle samples from the sample container, deliver the one or more liquid or particle samples to the sample container, or a combination thereof.

24. The fluid processing component comprising: To pump fluid into a separate inner tube, to prevent fluid from entering the separate inner tube, or 17. The device of claim 16, further comprising a valve configured to effect the combination.

25. The device described in claim 16, wherein the one or more inner tubes are reversibly connected to the outer tube by a connector, fitting, sheath, nozzle, housing, or combination thereof.

26. The device described in claim 16, wherein the one or more inner tubes are irreversibly connected to the outer tube by adhesive.

27. ​​The device described in claim 16, wherein the one or more inner tubes are connected to a fluid reservoir, and the device is configured to deliver fluid from the fluid reservoir to the sample container or another container within the device.

28. The device described in claim 27, wherein the fluid is configured to separate adherent cells growing within the device.

29. The apparatus of claim 16, further comprising a flow meter.

30. The apparatus of claim 16, wherein the apparatus is configured to select a flow rate of the one or more liquid or particle samples to be delivered to a fluid- or particle-based device, mix the contents of the one or more liquid or particle samples, incubate the one or more liquid or particle samples, heat the one or more liquid or particle samples, cool the one or more liquid or particle samples, sterilize the one or more liquid or particle samples, create a seal on the sample container, add a reagent to the one or more liquid or particle samples, add cells to the one or more liquid or particle samples, or a combination thereof.

31. The device of claim 30, wherein the device is configured to create a sterile field with ultraviolet light.

32. The apparatus of claim 16, wherein the apparatus comprises a plurality of sample containers configured to accommodate a plurality of samples from the one or more liquid or particulate samples.

33. A method for automated analysis of a liquid or particulate sample using the apparatus of claim 16, comprising: moving the liquid or particle sample using an automated flow; processing the one or more liquid or particle samples for analytical evaluation by the fluid- or particle-based device; A method comprising:

34. The method of claim 33, wherein the automated flow is performed by a vacuum system, a pressure-based system, a pneumatic system, a pump, a peristaltic pump, a diaphragm, a syringe, or a combination thereof.

35. The method of claim 33, wherein the processing comprises selecting a flow rate for the one or more liquid or particle samples, delivering the one or more liquid or particle samples to the fluid- or particle-based device, mixing the contents of the one or more liquid or particle samples, incubating the one or more liquid or particle samples, heating the one or more liquid or particle samples, cooling the one or more liquid or particle samples, sterilizing the one or more liquid or particle samples, creating a seal on the sample container, adding a reagent to the one or more liquid or particle samples, adding cells to the one or more liquid or particle samples, or a combination thereof.

36. The device described in claim 16, wherein the one or more inner tubes have a diameter to improve or modify mixing or sample delivery.

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