Fluid autosampler and incubator

Abstract

An apparatus is provided for automated analysis of one or more samples in single-well or multiwell plates or containers. The process of automated analysis involves automated flow, where the sample comprises a liquid or particles in a sample container, and the apparatus includes an assembly of components that allows for processing of the sample for analytical evaluation by a liquid- and / or particle-based instrument. The automated 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 that allows for movement in the X, Y, and Z dimensions, as well as switches, microfluidic tubing, a well plate block, electronic pressure controllers, pneumatic or fluidic mixing devices, fluid handling components, sample collection containers, and mechanical components for translating or transporting the system components.

Description

[Technical field]

[0001] Embodiments of the present disclosure relate to devices and methods of using the devices that enable automated nano / micro / millifluidic sampling from one or more containers. The containers may range from a single well or vial to multiple multi-well plates. Additionally, the devices provided herein can mix the contents of individual wells, create a sterile field for biological interrogation, and control parameters such as temperature, carbon dioxide concentration, and other cell growth conditions for monitoring material stability and cell-based transitions. [Background technology]

[0002] The increasing use of automated systems for sample storage, processing and analysis has led to extensive research in the field of automation. Current sample collection systems consist of a robotic arm capable of moving samples from a storage zone to a loading zone for sampling, magnetic or mechanical stirring elements capable of keeping the samples in suspension, and heating or cooling zones capable of regulating the sample storage or analysis temperature.

[0003] The prior art includes several automated sample collection devices that encompass various areas of analytical research (e.g., U.S. Pat. No. 4,713,974 for use in liquid chromatography), but the lack of accurate and consistent nanoliter flow rate control, sufficient sample mixing, and reliable temperature control for sample processing precludes the use of the prior art for biological cell analysis using laser force cytology (LFC).

[0004] Wilhelm et al., U.S. Pat. No. 4,816,730, describes the use of an apparatus for handling and moving multiple objects, consisting of a robotic arm capable of vertical, horizontal and rotational movement with a gripping mechanism for holding samples driven by an electronically controlled stepping motor. Schmidt et al., U.S. Pat. No. 6,872,362, further describes the use of a powered autosampler having a vial cup configured with a magnetic stir bar driven by a variety of different methods of varying the magnetic field around the vial cup. While such prior art describes methods for moving and mixing samples, such methods are not sufficient for biological cell analysis using LFC instruments. What is needed is an improved apparatus that allows for the storage, mixing and sampling of containers ranging from single wells to multiple multiwell plates while maintaining the integrity of biological cells through non-contact mixing based on air pressure and sufficient mixing and temperature maintenance through the use of temperature-controlled single-well or multiwell plate blocks.

[0005] Automated well plate stacking or retrieval systems are available that include robotic mechanisms to allow for serial stacking or retrieval vertically or horizontally (Patent Document 4 (China Utility Model No. 204136215) and Patent Document 5 (US Patent Publication No. 2004 / 0206419)), utilize cassettes for loading and unloading (Patent Document 6 (U.S. Patent No. 9,744,535)), or perform simultaneous stacking or retrieval of an entire lot of plates into a magazine or tower rack (Patent Document 7 (U.S. Patent No. 6,086,319)). Additionally, previous designs allow well plates to be loaded or removed from a storage tower in a random (non-sequential) manner, regardless of the stacking order (Patent Document 8 (U.S. Patent No. 7,670,555)). However, what is needed is the ability to utilize a magnetic interface to provide specific automated detection, selection, stacking of the desired well-plate into or retrieval from a storage tower in a non-sequential manner that enables well-plate incubation that can be used in combination with multiple analytical methods for automated sampling of such well-plates. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Pat. No. 4,713,974 [Patent Document 2] U.S. Pat. No. 4,816,730 [Patent Document 3] U.S. Patent No. 6,872,362 [Patent Document 4] China Utility Model No. 204136215 Specification [Patent Document 5] US Patent Application Publication No. 2004 / 0206419 [Patent Document 6] U.S. Patent No. 9,744,535 [Patent Document 7] U.S. Patent No. 6,086,319 [Patent Document 8] U.S. Patent No. 7,670,555 Summary of the Invention

[0007] Embodiments of the present disclosure relate to devices and methods of using the devices that allow automated (and optionally cultured) nano / micro / millifluidic sampling from vessels ranging from a single well or vial to multiple multi-well plates. Additionally, the devices can mix the contents of individual wells, create sterile fields for biological interrogation, and control cell growth conditions including temperature, carbon dioxide concentration, for monitoring material stability and cell-based transitions.

[0008] More specifically, the novel device described herein achieves fluid monitoring including, but not limited to, determination of milli / micro / nano fluid flow rates as well as air bubble removal through the use of electronic pressure controllers and / or regulators used in concert with flow meters. The novel device further allows for mixing of samples by injecting small air bubbles into the sample wells or vials or aspirating and dispensing sample or air using a vacuum pump coupled to an electronic pressure controller (EPC). Additionally, 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 material does not contaminate the housing of the device. The device is further designed to optionally incubate the sample prior to sampling 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 incubate the sample as needed. Mixing, sterilization and temperature control can be performed separately, sequentially, and / or simultaneously depending on the device settings. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram showing the outer box of an "autosampler," an embodiment of the present invention. [Diagram 2]FIG. 2 is a horizontal schematic diagram showing the internal operation of the autosampler and needle manifold assembly. [Diagram 3] FIG. 3 is a top view showing the operating stages within the autosampler. [Figure 4] FIG. 4 is a schematic diagram showing the autosampler in conjunction with the Laser Force Cytology instrument (Radiance™). [Diagram 5] FIG. 5 is a schematic diagram showing an automatic mixing diagram. [Figure 5A] FIG. 5A is a schematic diagram illustrating an embodiment of a microfluidic needle design. [Figure 5B] FIG. 5B shows details of the autosampling and mixing manifold. [Figure 6A-1] FIG. 6A-1 is a front schematic diagram showing a typical UV sterilization field. [Figure 6A-2] FIG. 6A-2 is a side schematic diagram showing a representative UV sterilization field. [Figure 6B] FIG. 6B is a schematic diagram showing a representative ultraviolet sterilization field and provides a system for sterile mixing and sample delivery. [Figure 6C] FIG. 6C is a schematic diagram showing a typical UV sterilization field with details of the sterilization tube. [Figure 7A] FIG. 7A is a schematic diagram of an embodiment showing an individual well plate incubator. [Figure 7B] FIG. 7B shows an individual well or vial incubator. [Figure 8] FIG. 8 provides an embodiment of a transport structure for multiplexing. [Figure 9A] FIG. 9A is a schematic diagram of a multi-plate robotic handler. [Figure 9B] FIG. 9B provides an embodiment of a multi-plate robot handler showing plates loaded under normal operation and removed for transfer. [Figure 10A]FIG. 10A is a schematic diagram showing the use of an embodiment of an autosampler for cell separation, showing cells resting on the floor of a container / vial. [Figure 10B] FIG. 10B is a schematic diagram showing the use of an embodiment of an autosampler for cell detachment, showing cells removed / separated from the bed of a container / vial. [Figure 11] FIG. 11 is a schematic diagram illustrating an embodiment of a reagent delivery system. [Figure 12] FIG. 12 is a schematic diagram illustrating an embodiment of a cell separation method that includes the use of a stripping tip / adapter. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0011] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The 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 is 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 or used by one of ordinary skill in the art and methodology.

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

[0014] Provided herein is a novel device for automating the analysis of samples, where the samples are present in vials, containers, wells, multi-well plates, etc. Additionally, methods for using such devices are provided. The device may be referred to herein as an "autosampler." In certain embodiments, the autosampler may be used to obtain a desired and / or predetermined volume of sample for analysis by machine or manual. For example, the autosampler may be used to collect nano / micro / millifluidic samples from multi-well plates, vials, or other containers, where such samples may be of various volumes and comprise various cells or particles. The autosampler then presents the sample in an appropriate medium / composition / container for analysis by a fluid-based instrument or system for liquids, particles, or cells, such as an instrument using laser force cytology (LFC).

[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 a particular embodiment, running rails (228) are positioned to allow movement of the internal mechanism along the X and Y axes, and a cable carrier (296) is attached to ensure safe movement of the cables during movement. Not pictured is 296 for the X axis. 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 accomplished using a thermoelectric cooling (TEC) module and a thermistor connected to a temperature control board (310 (see FIG. 3)). Temperature control may include heating or cooling of the sample. A fan (218) operates constantly or as needed to remove heat from the heat sink (220). The components from 216 onwards are coupled to a moving platform that can transport 216 within the range of the 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 X and Y axis movement, the industrial computer control 340 rotates timing pulleys (360) and drives belts to move the gantry plate along its Y axis (380) and X axis (382). For Z axis movement, the industrial computer drive 340 contains both lead screws (222). 222 is used to generate up and down Z axis motion using a Delrin travel nut 224. Any travel nut can be utilized for the purpose. Any axis motion control may or may not have position feedback.

[0020] 4 shows an interface between 110 and a Radiance™ instrument (410), an embodiment that can be used for application of 110. Alternatively, 110 can be modified for application to any LFC, optical force, microfluidic, or other instrument. In one embodiment, the interface between 110 and the other instrument is as shown in FIG. 4 and allows for the transfer of fluids, samples, and electronic signals, communications, or other information between the two instruments, including but not limited to those listed here.

[0021] FIG. 5 shows an embodiment of an automated sampling and mixing system. To promote sample integrity during mixing, the Z-distance (550) between the bottom of the sampling tube (530) and the bottom of the sampling container (shown as a multi-well plate) can be adjusted to fine-tune the pneumatic mixing system for each sample. Sampling is accomplished by physically moving or pressing the 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. The flow rate into and out of the vial or sampling well can then be adjusted by controlling the pressure in the headspace (560) of the vial or sampling well. This is made possible by the internal chamber (570), as shown in detail in FIG. 5B. As shown in the side view of 5B, the internal chamber (570) is open in two places at the top and one place at the bottom. The two upper locations are where the manifold connects airtight to the outer tube (540) and fitting (580), respectively. The lower location is an additional open location connected to an internal chamber at the bottom of the manifold (226), shown in the bottom detail of the manifold. The outer tube (540) passes through the internal chamber (570) and extends out from the manifold (226) as shown. This allows the outer tube (540) to be submerged in the sample during sample collection. However, the outer diameter of the outer tube (540) is smaller than the diameter of the internal chamber (570) so that an airtight seal can be achieved when a well plate or vial is pressed against the sealing surface (230). Tubing (590) is airtightly attached to the top of the manifold (226) via fitting (580). As shown, the tubing (590) is connected to an electronic pressure controller or some alternative pressure or vacuum source (595). The pressure in the headspace 560 can then be precisely controlled by adjusting the pressure of the source (595). Note that this is one particular embodiment and other embodiments could have many more openings in the manifold, if desired. Both mixing and sampling use separate tubes (500 and 530 respectively) that share a larger outer tube or needle (540).The inner tubes (500 and 530) may be constructed of different or the same diameters to improve or modify mixing or sample delivery, as needed. The materials of the inner and outer tubes may be different and may be constructed of metal, plastic, ceramic, composite, glass / capillary, carbon fiber, composite, or other suitable materials. The inner tube may be connected to the outer tube by various connectors. Such connectors between the inner and outer tubes may be reusable, such as fittings, sheaths, or other housings, or permanent, such as glue or epoxy. The tubes may also be constructed of a single material or multiple materials using 3D printing or other manufacturing techniques. An example design is shown in FIG. 5A. In one embodiment, a vacuum-compatible electronic pressure controller (EPC) (520) connects to the tubing, which may be directly connected to a manifold (510) that contains a vacuum trap (512) that helps prevent liquid from entering 514 and 520. The manifold 510 and vacuum trap 512 are connected to form an airtight seal, allowing for a pneumatic connection between tubing 514 and tubing 505. The tubing 505 is connected to the valve such that the vacuum source can be isolated from the O tubing 500 on the other side of the valve and from the samples contained in the well plate 700 or sample vials. The valve can be, but is not limited to, a solenoid valve, a pinch valve, a rotary valve, or a ball valve. The tubing is connected on both sides of the valve to create an air-tight and liquid-tight 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] FIG. 6A illustrates how a sterile field can be created in 110 or other autosampler systems by utilizing ultraviolet (UV) germicidal lamps, light emitting diodes (LEDs) or other light sources (610) at desired power and spaced apart. The light sources can be mounted at predetermined intervals, twice predetermined intervals or other various intervals and positioned at the back, front, side, top or bottom of the space to be sterilized attached to or secured to a reflective shield or adjacent to a reflective shield. A front view of one embodiment is shown in FIG. 6A-1 and top and side views are shown in FIG. 6A-2. The area to be sterilized can be enclosed by a continuous or targeted arrangement of polished aluminum or other sufficiently reflective material (620). The shielding can also serve the purpose of protecting components, such as internal wiring, from repeated exposure to UV light while the area to be sterilized is fully exposed to UV light. The shielding can also be in the form of mirrors, lenses or other optical elements 640 designed to focus and / or reflect UV light to a specific location. The UV light source 610 may be mounted on a static component of the autosampler 110, or may be mounted on the same plate as the well plate 700 or elsewhere to allow movement of the UV light source relative to the needle assembly 226. Additionally, the well plate 700 may move along the Z axis to provide a preferred location for sterilization. An UV light source could also be integrated directly into the well plate block 216 to sterilize the needle assembly 226. The well plate block would move into position below the needle assembly such that a UV light source built into the block would sterilize the needle assembly. Additional structure or support elements 630 could be integrated to assist in positioning the UV light source. The power and time required for sterilization is determined by calculating the energy required to achieve the kill factor of the target to be removed.

[0024] FIG. 6B shows a system for sterile mixing and sample delivery. To maintain sterility of the samples during transfer from the 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, silicone, Viton™, plastic, or any other suitable material. Once inside the incubator, the needle assembly 226 is positioned above the well plate so that the samples remain sealed during transfer. During sampling and mixing, the plate moves vertically in the Z dimension such that the needle assembly penetrates the seal 632 of the plate to access the sample (sample position). Further details of one embodiment are shown in FIG. 6C. As seen in cross section, the outer tube 650 contains three inner tubes for mixing (680), sample delivery (670), and pressure regulation (660). The outer tubes may be blunt at the bottom, cut at an angle as shown, or otherwise configured to easily penetrate the seal 632. The inner tube may be constructed of different or the same diameter to improve or alter mixing, sample delivery or pressure regulation, as needed. The materials of the inner and outer tubes may be different and may be constructed of metal, plastic, ceramic, composite, glass / capillary or other materials. The inner tube 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 glue or epoxy. 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 in the tube. Additionally, there is a hole or notch (657) above the bottom of the tube that allows air and pressure changes to pass between the inside and outside of the outer tube. In the sample collection position (which is the position when the bottom of the needle is submerged under 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 whose pressure can be controlled by the EPC connected to the pressure regulation tube 660. This allows the sample to be forced up into the sampling 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 FIG. 7A at 110 for the desired sampling time. This design may be adapted for multiple well plates of various sizes where the X and Y axes can accommodate the movement of the plate around 226. On top of the well plate (700) is attached the culture chamber (720), which may be made from plastic, plexiglass, glass or other suitable material. The top of the chamber would have a seal (710) that can be pierced with a sampling needle but remain airtight. 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 culture chamber may be combined into one integral part that would still include the top seal 710 but would not require the bottom seal 715. This would create a headspace 725 that can be controlled to create the proper conditions for cell growth. Temperature control of the chamber is possible by adjusting the temperature of 216, while CO2 is pumped into the chamber through tubing or hose (730) housed within 720 and fitted with a commercially available HEPA filter (740) to maintain sterility. CO2 and O2 may be supplied from a source such as compressed gas cylinder 750. The system may be fitted with additional sensing nodes to monitor pH, dissolved gas (O2, CO2, etc.) metabolites or any other detectable requirements. Cells will be grown in the chamber and periodically sampled by piercing the top seal with a needle assembly. Cells may be displaced from the sampling well by increasing the overall pressure of the headspace using a vacuum-based system or some alternative means to suck the cells out of the well.

[0026] Mixing of the sample contents to allow for suspension cell growth (or any other goal) may be accomplished by mechanical, magnetic, pneumatic, fluidic or other means. Specific examples include, but are not limited to, magnetic beds within each well or vial, magnetic stir bars or impellers actuated from below, above or the side.

[0027] In an alternative embodiment, as shown in Figure 7B, the wells may be replaced as shown by separate containers, each with its own individual chamber 720. Gases would continue to be supplied and temperature controlled to achieve ideal conditions for cell growth. Cells may be sampled in the same manner as above.

[0028] To create a multiplexed system capable of automatically sampling multiple plates, one or more transport systems can be used to support base plates in either a circular (800) or square / rectangular XY array (820) as shown in FIG. 8. The multiplexed array may encompass multiple self-incubation chambers or incorporate even larger incubation chambers with all samples adjacent in parallel or series. Wheels 720 and 710 can be used to incorporate 800. A rotating rod (810) attached to 216 would allow circular rotation of the well plates when moved via mechanical power. Such multiple trays could move in the X, Y and Z axes to access the sampling needles.

[0029] FIG. 9A shows a multi-plate storage tower (900) that is divided by a fixed height and can be used in conjunction with a robotic handler. The robotic arm can be programmed to retrieve plates at any space in the tower (e.g., positions 1-8 in the figure, with more or fewer trays depending on the design requirements). The system may be integrated as part of an autosampler, located directly below the autosampler, or located adjacent to the autosampler. The robotic arm may use any number of methods to remove any of the plates, such as mechanical, magnetic (including electromagnets), electronic, or other means. Individual well plates used in this system may be designed to work in concert with the robotic system. For example, in one embodiment shown in FIG. 9B, the plate has magnets on two adjacent sides. During normal operation, magnets 910 help hold the plate in place. To move the plate, the robotic arm 920 provides sufficient force to break the magnetic coupling that holds the plate in the rack, removing the plate as shown. The plate can then be transferred to an autosampler or other device for sampling. In one embodiment, the cells may also be returned to the tower after sampling. To accommodate incidental cell growth, the autosampler will be equipped with the ability to separate cells from the surface of the growth substrate. Some embodiments of this cell separation method are shown in FIG. 10. The system may consist of some or all of the following components: Liquids can be moved into and out of the wells using dedicated tubing or removable tips. Once the cells have been treated with a reagent such as trypsin, TrypLE™, Accutase™ or other cell separation reagents, they are physically scraped off with either a soft plastic scraper, a thin metal blade or a plastic blade attached to a tip to completely remove and suspend the cells. Alternatively, a liquid flow may be used to suspend the cells, making them available for analysis including the mixing system described above, providing the flow necessary to remove and resuspend the cells.Liquid may be removed by a mixing system (FIG. 5) and then new liquid (fresh PBS) may be added from a reagent addition system (FIG. 10) to resuspend the cells and wash away debris. Temperature control of the well plate block 216 may also be used to assist in separating or resuspending the cells. Reagents that may be added include PBS or other buffer, trypsin or other cell separation solution, media, fixative, sample (such as cells and media), EDTA or any other liquid of interest. FIG. 11 shows a reagent delivery system. This is accomplished by using any electronic pressure controller to pressurize the headspace above the liquid reagents that are dispensed into the vial containing the sample. Alternatively, a peristaltic pump, syringe pump or other pump may be used to drive the flow of the reagents into the sample vial or container. A physical cell separation method that may be located elsewhere in the autosampler is shown in FIG. 12. When the sample is ready to be analyzed, the vial or well plate would generally shuttle against the scraper shown in FIG. 12. Cells can be completely removed and suspended using either a fixed soft plastic, rubber or polymer scraper, a thin metal blade, or physical scraping achieved by moving the well plate or vial block in three dimensions against a plastic blade attached to the tip.

[0030] In one embodiment, provided herein is an apparatus for automated analysis of one or more samples, the process of automated analysis including automated flow, the sample including liquid or particles in a sample container, the apparatus including an assembly of components that allows processing of the sample(s) for analytical evaluation by a liquid and / or particle-based instrument. The apparatus may be referred to as an autosampler. Additionally, methods for using such an apparatus are provided.

[0031] The automated flow within the device may include systems for moving the sample, including vacuum systems, pressure-based systems, pneumatic systems, pumps, peristaltic pumps, diaphragms, or syringes. In one embodiment, the automated flow, such as pneumatic flow, may be customized based on analytical parameters, including, but not limited to, the nature of the sample being analyzed, the number of samples being analyzed, and / or the type of fluid and / or particle-based instrument used to perform the assay. In one embodiment, the flow rate of the sample from source to delivery, i.e., the flow rate in the case of delivery to the device, instrument, or optical force-based instrument such as Radiance™, may range from 0.01 to 50 L / min. In alternative embodiments, the flow rate may range from 0.1 to 100 L / min, 0.5 to 500 L / min, or 2 to 2000 L / min. The sample flow rate may be uniquely customized and adjusted for optimal efficiency and consistency, and in conjunction with the reading instrument, facilitate rapid and accurate assay evaluation.

[0032] In one embodiment, samples analyzed by the autosampler device described herein may include, but are not limited to, polymer, metal, glass or alloy based particles, biological cells, plant cells (such as algae cells), prokaryotic cells (bacteria), eukaryotic cells, yeast, fungal, mold cells, red blood cells, neurons, egg cells (eggs), sperm, white blood cells, basophils, neutrophils, eosinophils, monocytes, lymphocytes, macrophages, platelets, vesicles, exosomes, interstitial cells, multicellular constructs such as spheroids, mesenchymal cells and induced pluripotent stem cells (iPSCs), as well as subcellular components such as nuclei, mitochondria or chloroplasts. Samples may be synthetically produced or obtained from natural sources. Samples may be obtained from bodily fluids or bodily substances including, but not limited to, tears, saliva, sputum, blood, plasma, lymph, urine, sweat, pus, nasal secretions or sperm.

[0033] In one embodiment, analytical assessments using fluid and / or particle based instruments include, but are not limited to, optical force measurements, laser force cytology, automated microscopy, capillary electrophoresis, single cell droplet microfluidics, single cell genomics, sequencing instruments, mass spectrometry and nucleic acid or protein analysis, amplification or modification.

[0034] In one embodiment, the assembly of components may include, but is not limited to, motors in X, Y and Z dimensions, limited switches, microfluidic tubing, well plate blocks, electronic pressure controllers to control headspace pressure throughout the fluid to achieve flow, pneumatic or fluid mixing devices with or without temperature control, components for fluid handling, sample collection containers with or without temperature control, mechanical components for transforming the sample collection containers or other system components. In certain embodiments, the sample(s) 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 the seal to access the headspace and liquid of the sample.

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

[0036] In certain embodiments, the components for fluid handling in the device include an outer tube that fits within the sample container, one or more separate inner tubes that fit within the diameter of the outer tube, a connector to one or more destination containers that are fluidly connected to the sample collection container, and one or more separate systems for moving fluids in and out of the sample collection container in a controlled manner. In some embodiments, valves may be used to preferentially drive fluids into one or more inner tubes or to prevent fluids from entering one or more inner tubes, and the systems for moving fluids may include vacuum systems, pressure-based systems, or pumps such as peristaltic pumps, diaphragms, syringes, etc. In certain embodiments, the fluid handling device is located within a manifold that creates an airtight seal against the sample collection container or a compartment thereof. The outer tube may be constructed of metal, plastic, ceramic, composite, glass / capillary, or other materials. The tubes may be constructed of metal, plastic, ceramic, composite, glass / capillary, or other materials. In some embodiments, the inner tube is reversibly connected to the outer tube using connectors such as fittings, sheaths, ferrules, or other housings, or permanently connected to the outer tube using epoxies, cements, or other adhesives. In some embodiments, the outer and inner tubes are fabricated as a single piece of one or more material types through the use of 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. Additionally, the outer and inner tubes may be fabricated from glass using laser patterning and hydrofluoric acid (HF) or potassium hydroxide (KOH) etching and bonding processes. In additional embodiments, the inner tube of the device is connected to one or more reservoirs of fluid that can be delivered to a sample collection container or other container in the system. Fluids may be delivered to separate adherent cells growing in the sample device. The sample containers may include vials or well plates 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 off the bottom surface of the sample vessel (see, e.g., FIG. 12). For example, the outer tube can have a wedge-shaped, wafer-shaped, tongue-shaped or other shaped attachment made from hard or soft plastic, metal, ceramic, and can mechanically scrape cells off the bottom surface of the sample vessel.

[0038] In one embodiment, the inner tube is connected to a reservoir with a maximum pressure high enough to force air bubbles out of the system tubing and 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 the inner tube or tubes is monitored using a flow meter.

[0040] In one embodiment, the device further comprises a mechanism for monitoring flow, such as a flow meter, which may be used to calculate the amount of fluid flowing into or out of a sample vessel or any other container or receptacle connected to the system.

[0041] In one embodiment, the activity of sterilizing the sample includes creating a sterile field within the sample-collecting device that includes one or more light sources capable of sterilizing a surface through the production of ultraviolet light. The light sources may be ultraviolet (UV) germicidal lamps, UV-A, UV-B, UV-C, light-emitting diodes (LEDs), lasers, or other sources of ultraviolet or other broad or narrow wavelength light. In one embodiment, the device further includes structures or surfaces for containing, directing, or reflecting the light to one or more specific areas or surfaces. The light sources may be attached to a static component within the sample-collecting device or to a movable component within the sample-collecting device.

[0042] In certain embodiments, the seal for the vial or well plate containing the sample includes an airtight seal made from rubber, polymer, silicone, Viton™, plastic, or any other suitable material. The outer tube of the fluid handling device may be capable of piercing the airtight seal. The outer tube that contacts the seal may be blunt, cut at an angle, or otherwise configured to easily pierce the seal. In one embodiment, the seal is configured to maintain airtightness after being pierced by the outer tube of the fluid handling device.

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

[0044] In one embodiment, the sample container or a component thereof has an airtight seal that creates a headspace above the sample fluid and may be continuously incubated under controlled temperature, headspace gas concentration and sample mixing. The outer tube of the fluid handling device may be capable of piercing the airtight seal, and the end of the outer tube that contacts the seal may be blunt, beveled, or otherwise configured to allow easy penetration of the seal. In one embodiment, a hole or notch is located above the bottom of the outer tube that is located above the surface of the liquid and below the seal to allow air and pressure changes to pass between the inside and outside of the outer tube. The seal may be configured to remain airtight after being pierced by the outer tube of the fluid handling device.

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

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

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

[0048] In one embodiment, adherent cells may be grown in one or more sample vessels attached to microcarriers, fiber-based membranes, discs or other structures, or other suitable growth substrates that can be mixed or perfused with culture medium.

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

[0050] In one embodiment, the sample container may be made up of multiple separate sub-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 a feature that delivers one or more reagents to the sample reservoir for mixing and subsequent analysis.

[0053] In one embodiment, multiple plates are placed or configured in a multiplexed system and accessed by an autosampler device as needed. Plates may be incubated, sterilized, etc. as needed. In one embodiment, the device may be further customized to accommodate loading multiple separate plates into a circular, rectangular, or other shaped system. Well plates may be loaded into a multi-plate storage device by an automated system and / or the well plates may be attached to a tower via mechanical, magnetic, electromagnetic, or other means.

Claims

1. 1. An apparatus for the 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 a particle-containing sample; a sample container configured to incubate the sample; and 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).

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

3. 10. The apparatus of claim 1, wherein the automated analysis is performed by an instrument configured to perform optical force based measurements, 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 or modification.

4. 10. The apparatus of claim 1, wherein the assembly of components includes motors in X, Y and Z dimensions, switches to control application of vacuum and positive pressure, microfluidic tubing, a well plate block, electronic pressure controllers, pneumatic or fluidic mixing devices with or without temperature control, a temperature control unit for the sample containers, and mechanical components for translating or transporting the sample containers or other system components.

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

6. 10. The apparatus of claim 1, further comprising a sealing surface or gasket configured to form a seal with the sample vessel and a component that penetrates the seal to access the headspace and liquid of the sample.

7. 10. The device of claim 1, wherein the fluid handling components comprise a light source configured to sterilize microfluidic tubing or well plates.

8. The fluid processing component further comprises: 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.

9. The apparatus of claim 1 , wherein 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.

10. 10. The apparatus of claim 8, wherein the one or more separate systems for moving fluids include a vacuum system, a pressure-based system or a pump such as a peristaltic, diaphragm, syringe or other type.

11. 11. The apparatus of claim 10, wherein the fluid handling components are located within a manifold that creates an air-tight seal with the sample vessel or compartment thereof.

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

13. The apparatus of claim 1 , wherein the inner tube is made from metal, plastic, ceramic, composite, glass / capillary or other material.

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

15. 10. The apparatus of claim 1, wherein the outer and inner tubes 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.

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

17. The apparatus of claim 1 , wherein the inner tube is connected to one or more reservoirs of fluid that can be delivered to the sample vessel or other vessel in the system.

18. 20. The device of claim 17, wherein the delivered fluid is used to detach adherent cells growing within the device.

19. 10. The apparatus of claim 1, wherein the sample container comprises a vial or a well plate having 6, 12, 24, 48, 96, 192, 288, 384, 1536, or any custom number of wells.

20. The device of claim 1 , wherein the outer tube is capable of mechanically scraping cells from a bottom surface of the sample vessel.

21. 10. The device of claim 1, wherein the outer tube has a wedge-shaped, wafer-shaped, tongue-shaped or other shaped fitting made of hard or soft plastic, metal, or ceramic that can mechanically scrape cells off the bottom surface of the sample container.

22. 11. The apparatus of claim 10, wherein one or more of the inner tubes are connected to a reservoir with a maximum pressure high enough to force air bubbles out of the system tubing and into one or more containers or fluid reservoirs.

23. 10. The apparatus of claim 8, further comprising a mechanism for monitoring flow, such as a flow meter.

24. 24. The apparatus of claim 23, wherein the mechanism for monitoring flow includes a flow meter used to calculate the amount of fluid being transferred into or out of the sample vessel or any other container or receptacle connected to the system.

25. 8. The device of claim 7, wherein sterilizing the sample comprises creating a sterile field within the device with ultraviolet light from one or more light sources.

26. 26. The apparatus of claim 25, wherein the one or more light sources include ultraviolet (UV) germicidal lamps, UV-A, UV-B, UV-C, light emitting diodes (LEDs), lasers, or other sources of ultraviolet or other broad or narrow wavelength light.

27. 26. The device of claim 25, wherein the structure or surface is used to contain, direct or reflect light to one or more specific areas or surfaces.

28. 26. The apparatus of claim 25, wherein the one or more light sources are mounted to a static component within the apparatus.

29. 26. The apparatus of claim 25, wherein the one or more light sources are mounted on a movable component within the apparatus.

30. The apparatus of claim 1, further comprising a sealing surface or gasket configured to form a seal against the sample container, the seal comprising an airtight seal made from rubber, polymer, silicone, Viton™, plastic or any other suitable material.

31. 31. The apparatus of claim 30, wherein the outer tube of the fluid handling component is capable of piercing the gas-tight seal.

32. 32. The apparatus of claim 31, wherein the end of the outer tube that contacts the seal is blunt, beveled, or otherwise configured to allow easy penetration of the seal.

33. 32. The device of claim 31, wherein there is a hole or notch on the bottom of the outer tube located above the surface of the liquid and below the seal to allow air and pressure changes to pass between the inside and outside of the outer tube.

34. 32. The apparatus of claim 31, wherein the seal is configured to maintain an airtight seal after being pierced by the outer tube of the fluid handling component.

35. 10. The device of claim 1, wherein the sample container or a component thereof has an airtight seal that creates a headspace above the sample, and the device is configured to selectively continuously incubate the sample under controlled temperature, headspace gas concentration, and sample mixing.

36. 36. The apparatus of claim 35, wherein the outer tube of the fluid handling component is capable of piercing the gas-tight seal.

37. 37. The apparatus of claim 36, wherein the end of the outer tube that contacts the seal is blunt, beveled, or otherwise configured to allow easy penetration of the seal.

38. 37. The device of claim 36, wherein there is a hole or notch on the bottom of the outer tube located above the surface of the liquid and below the seal to allow air and pressure changes to pass between the inside and outside of the outer tube.

39. 37. The apparatus of claim 36, wherein the seal is configured to maintain an airtight seal after being pierced by the outer tube of the fluid handling component.

40. 36. The apparatus of claim 35, wherein the gas pumped into the headspace is sterile as a result of filtration or other suitable means.

41. 36. The apparatus of claim 35, wherein sensors are implemented to measure temperature, pH, gas concentration or other parameters of the headspace or sample.

42. 36. The apparatus of claim 35, wherein suspension cells are grown in the sample vessel.

43. 36. The device of claim 35, wherein adherent cells are grown within the sample vessel attached to a microcarrier, fiber-based membrane, disc or other structure or other suitable growth substrate that can be mixed or perfused with culture medium.

44. The apparatus of claim 1 , wherein cells within the sample are mixed by mechanical, magnetic, pneumatic, fluidic or other means.

45. 36. The apparatus of claim 35, wherein the sample vessel is comprised of a plurality of separate auxiliary vessels.

46. 46. ​​The apparatus of claim 45, wherein each auxiliary container has its own headspace, control device and sensor array.

47. The apparatus of claim 1 , wherein the assembly of components is configured to add a reagent, such as a biochemical or other biological component, to the sample.

48. The assembly of components further comprises: i) an electronic pressure controller, regulator and / or flow meter configured to select the flow rate of the liquid or particles delivered to the device; ii) a mechanical, magnetic, pneumatic, or fluidic mixing system configured to mix the contents of the sample; iii) a temperature control configured to heat and / or cool the sample; iv) one or more light sources configured to sterilize the sample; v) a sealing surface or gasket configured to create a seal against the sample vessel; vi) a combination thereof.

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