Patent Application
20250187003
The devices, systems, and methods herein relate to separating and/or analyzing extracellular matrix bodies (ECMBs) from a biological fluid.
Conventional methods for diagnosis and prognosis of disease include isolating and analyzing a tiny fraction of a biological sample (e.g., biological fluid) where, for example, individual cells, extracellular matrix, extracellular vesicles or soluble tissue microenvironments, proteins, and nucleic acid molecules may be detected and analyzed. For example, the inventors previously described a method of detecting and analyzing molecules using microfluidic chips and positive pressure. However, drawbacks of such positive pressure systems include low-throughput, limited scalability and reproducibility. In addition, previous systems tend to require over reliance on manual labor. For example, a user is needed to pipette or manually inject with a syringe each biological sample and/or set of reagents to a respective inlet port of a microfluidic chip. Moreover, a positive pressure system relies on a significant amount of tubing to deliver the sample to the microfluidic chip, and therefore, a volume of liquid for each microfluidic chip may be inconsistent. Furthermore, these systems tend to suffer from a higher rate of contamination, as they require time-intensive manual labor for set-up and cleaning. Other conventional methods for diagnosis and prognosis of disease include isolating and analyzing intact organ tissues, whole cells, biomarkers (e.g., extracellular vesicles such as exosomes), and the like. However, a drawback of such methods includes the inability to detect or characterize disease when these isolated structures do not readily reflect the disease state. For example, biomarkers are often inherently limited by not being related directly to a pathology of interest. Accordingly, additional systems, devices, and methods for separating and analyzing a biological fluid are desirable.
Described here are systems, devices, and methods useful for separating extracellular matrix bodies (ECMBs) from a biological fluid, or indirectly from tissue or a gel, in an automated and high-throughput manner. In this way, the ECMBs may be used for use in diagnosing, prognosing, and/or treating a subject, for example using histochemical staining techniques, immunohistochemistry or nucleic acid hybridization and analysis. In general, the systems described herein for separating extra-cellular matrix bodies from a biological fluid may comprise a holder configured to receive the biological fluid, a robot configured to transfer the biological fluid from the holder to a microfluidic chip, a chip connector configured to hold at least one microfluidic chip, a manifold coupled to at least one microfluidic chip, and a negative pressure source coupled to the manifold. The negative pressure source may be configured to apply a negative pressure of between about 10 mm HG and about 760 mm HG to at least one microfluidic chip.
In some variations, the chip connector may comprise a base configured to contact a bottom portion of at least one microfluidic chip, and a cover configured to contact a top portion of at least one microfluidic chip. In some variations, the chip connector may be configured to distribute a compression force applied by the negative pressure to a perimeter of the microfluidic chip. In some variations, the bottom portion may comprise a perimeter of at least one microfluidic chip. In some variations, the cover may define a plurality of apertures. In some variations, the base may comprise a first fastener and the cover may comprise a second fastener. The first fastener and the second fastener may be configured to align the microfluidic chip in a predetermined orientation. In some variations, at least one inlet connector and at least one outlet connector may be disposed between the cover and the at least one microfluidic chip.
In some variations, at least one outlet connector may comprise an elongate body defining a lumen and comprising a plurality of steps along a length of the elongate body. In some variations, at least one outlet connector may comprise an elongate body defining a lumen having an inner diameter decreasing in a distal direction. In some variations, one or more the microfluidic chip, the inlet connector, and the outlet connector may comprise a disposable component. In some variations, the chip connector may comprise a durable component.
In some variations, the holder may be configured to receive one or more reagents, and the robot is configured to transfer one or more reagents from the holder to the microfluidic chip. In some variations, a sensor may be coupled to at least one inlet connector. The sensor may be configured to measure one or more of flow rate and pressure. In some variations, an optical sensor may be coupled to the chip connector. The optical sensor may be configured to image one or more of the microfluidic chips.
In some variations, at least one microfluidic chip may comprise at least one restriction channel fluidically coupled between an inlet and an outlet of the microfluidic chip. In some variations, the at least one restriction channel may comprise at least one obstruction. In some variations, the at least one restriction channel may comprise a length of between about 5 mm and about 30 mm. In some variations, each channel may comprise a cross-sectional dimension of between about 5 μm and about 30 μm. In some variations, at least one microfluidic chip may comprise at least one obstruction configured to restrict fluid flow. In some variations, at least one obstruction may comprise a pillar.
In some variations, the at least one microfluidic chip may comprise a restricted region configured to hold a first fraction of the biological fluid and permit fluid flow of a second fraction of the biological fluid. In some variations, the first fraction may comprise the ECMBs.
In some variations, the restricted region may comprise a plurality of obstructions configured to hold the first fraction. In some variations, a spacing between the plurality of obstructions in the restricted region decreases along a length of the microfluidic chip from an inlet of the restricted region to an outlet of the restricted region. In some variations, the spacing between the plurality of obstructions in the restricted region is between about 100 μm and about 4 μm. In some variations, each obstruction of the plurality of obstructions comprise a diameter of between about 50 μm and about 1 mm.
Also described here are methods of separating extra-cellular matrix bodies (ECMBs) from a biological fluid. In general, the methods comprise transferring the biological fluid to an inlet reservoir of a microfluidic chip, and applying negative pressure to the microfluidic chip. The microfluidic chip may comprise at least one restriction channel having an inlet and an outlet. The inlet reservoir may be fluidically coupled to the inlet of the at least one restriction channel, and at least one pillar, and an outlet reservoir. Negative pressure of between about 10 mm HG and about 760 mm HG may be applied to the outlet reservoir of the microfluidic chip where the ECMBs remain in the microfluidic chip after removal of the biological fluid from the microfluidic chip.
In some variations, a compression force applied by the negative pressure may be distributed from the outlet reservoir to a perimeter of the microfluidic chip. In some variations, the at least one restriction channel may comprise the at least one pillar. In some variations, the at least one restriction channel may comprise a length of between about 5 mm and about 30 mm. In some variations, the at least one restriction channel may comprise a cross-sectional dimension of between about 5 μm and about 30 μm. In some variations, the at least one microfluidic chip may comprise at least one obstruction configured to restrict fluid flow. In some variations, the at least one microfluidic chip may comprise a restricted region configured to hold a first fraction of the biological fluid and permit fluid flow of a second fraction of the biological fluid. In some variations, the restricted region may comprise a plurality of obstructions configured to hold the first fraction. In some variations, a spacing between the plurality of obstructions in the restricted region may decrease along a length of the microfluidic chip from an inlet of the restricted region to an outlet of the restricted region. In some variations, the spacing between the plurality of obstructions in the restricted region may be between about 100 μm and about 4 μm. In some variations, each obstruction of the plurality of obstructions may comprise a diameter of between about 50 μm and about 1 mm.
Once the ECMBs have been separated from a biological fluid, additional methods may then be used to analyze those ECMBs. For example, in some variations, one or more of a histochemical stain (including immunohistochemical (IHC) stains and multiplex IHC stains), a protein stain, a nucleic acid stain, chemical fixation, and a protease inhibitor may be applied to the ECMBs in the microfluidic chip. In some variations, one or more biomarkers in one or more of the biological fluid and the ECMBs may be measured by one or more of immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, and UV-VIS.
In some variations, the biological fluid removed from the microfluidic chip may be analyzed using one or more of microscopy, microfluidic device, mass spectrometry, microarray, nucleic acid amplification, hybridization, proteomic profiling, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, fluorescent in situ hybridization (FISH), chelate complexation, quantitative HPLC, spectrophotometry, antibody array, Western blot, immunoassay, immunoprecipitation, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS/MS, RT-PCR, and quantitative PCR.
In some variations, processing the biological fluid removed from the microfluidic chip may use one or more of microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, mesh filtration, diafiltration, tangential flow filtration, membrane filtration, immuno-affinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC electrokinetics.
In some variations, the biological fluid may comprise one or more of whole blood, blood plasma, blood serum, cerebrospinal fluid, intrathecal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspirate, semen, amniotic fluid, vitreous, aqueous humor, lymph, bile, cerumen, chyle, chyme, endolymph, perilymph, exudates, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, pus, rheum, sebum, serous fluid, smegma, sputum, synovial fluid, vaginal secretion, menstrual effluent, and vomit, and fluids passed through one or more of tissues and gels.
Described here are systems, devices, and methods for separating ECMBs from a biological fluid (e.g., for analysis, etc.). For example, the systems, devices, and methods described herein may be useful for diagnosis, prognosis, and/or treating a subject by: separating, enriching, extracting, and/or immobilizing a predetermined fraction (e.g., ECMBs, proteins, nucleic acids, particles, materials, molecules, extracellular vesicles or soluble tissue microenvironments, proteins) from a biological fluid into a biological fluid fraction (e.g., isolate fraction) while preserving the fraction's composition and properties; improving a yield of a fraction from the biological fluid with reduced contamination to thereby improve a signal strength for a pathology of interest; identifying one or more of the fractions with a biomarker (e.g., proteins, genes, ECMB component) associated with a disease for disease target identification; and predicting a disease risk and/or medical condition based on an analysis of a plurality of biological fluid fractions.
Generally, the systems and devices described herein may separate ECMBs from a biological fluid in a manner that maintains their composition and properties to facilitate their use as biomarker(s) for disease diagnosis and/or monitoring of chemical or biological processes. For example, the systems and devices described herein may further be useful for: high-throughput, scalable, and automated microfluidic processing of biological fluids for histology; reduced compression of the microfluidic chip due to applied negative pressure based on distributed pressure; reduced clogging and increased laminar flow, flow rate consistency, and signal-to-noise ratio based on microfluidic chip channel density; reduced risk of contamination, set-up time, manual processing, and cleaning; visualization of ECMBs using immobilization and staining (e.g., histochemical, immunohistochemical) for one or more of spatial localization and profiling (e.g., protein, gene); and facilitating identification of disease ECMBs and physiologically normal ECMBs. Accordingly, biological fluid separation and histochemical analysis as described herein may be performed faster, cheaper, and with higher throughput (e.g., volume) than positive pressure microfluidic systems.
Generally, the systems and devices described herein may include microfluidic systems configured for biological assays of ECMBs of a biological fluid. For example, a microfluidic system may include a plurality of disposable microfluidic chips (e.g., microfluidic chip array) configured to receive a biological fluid (e.g., sample) from a robot that facilitates automation and high-throughput processing. The system may be configured to separate ECMBs from the biological fluid within the microfluidic chips using a negative pressure source coupled to the microfluidic chips. Separated ECMBs may be exposed to one or more reagents and buffers for histochemical staining and subsequent imaging and/or analysis. Accordingly, a plurality of biological samples may be processed in parallel by the microfluidic system.
For example, a system for separating extra-cellular matrix bodies (ECMBs) from a biological fluid may comprise a holder configured to receive the biological fluid, one or more robots configured to transfer the biological fluid from the holder to a microfluidic chip, a chip connector configured to hold at least one microfluidic chip, a manifold coupled to the at least one microfluidic chip, and a negative pressure source coupled to the manifold. The negative pressure source may be configured to apply a negative pressure of between about 10 mm HG and about 760 mm HG to the at least one microfluidic chip. In some variations, the system may comprise a plurality of holders, a plurality of chip connectors, a plurality of manifolds, a plurality of negative pressure sources, a plurality of reservoirs, a plurality of robots, and the like.
The negative pressure system of the devices and systems described herein help enable high-throughput, low contamination separation of ECMBs from biological fluid. In this way, a plurality of samples may be processed (e.g., undergo ECMB separation) and analyzed in an automated manner in parallel, thereby greatly reducing the time and effort associated with positive pressure microfluidic systems and methods. For example, negative pressure may be distributed evenly to a plurality of microfluidic chips in parallel using a manifold to provide consistent fluid flow and reproducible results. Furthermore, negative pressure applied at an outlet of a microfluidic chip enables an inlet of the microfluidic chip to be available for a robot to transfer fluids into such inlet, thereby increasing throughput and consistency, and reducing manual labor. However, modifying positive pressure microfluidic systems to apply a negative pressure to a microfluidic chip and sample would compromise the composition and properties of the biological fluid, as well as the structural integrity of the microfluidic chip, so as to hinder separation, enrichment, and/or extraction of the biological fluid into a useable biological fluid fraction. That is, applying negative pressure to positive pressure microfluidic chips and systems would generate unpredictable results without the innovations described herein.
A biological fluid may comprise one or more of human or animal bodily fluids, tissue, cells, whole blood, blood plasma, blood serum, cerebrospinal fluid, intrathecal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspirate, semen, amniotic fluid, vitreous, aqueous humor, lymph, bile, cerumen, chyle, chyme, endolymph, perilymph, exudates, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, pus, rheum, sebum, serous fluid, smegma, sputum, synovial fluid, vaginal secretion, menstrual effluent, vomit, tumors, carriers, reagents, solutions, binding moieties, and the like.
A buffer may comprise one or more of MES, HCL acid buffer, Acid Phthalate buffer, Alkaline borate buffer, Acetate buffer, Acetic ammonia buffer, acetone buffer, ammonia buffer, barbitone buffer, buffered copper sulfate solution, glycerin solution, glycine buffer solution, palladium chloride buffer solution, citric acid Na2HPO4, citric acid Sodium Citrate Buffer Preparation, Sodium Acetate Acetic Acid Buffer Preparation, Na2HPO4-NaH2PO4, Imidazole (glyoxaline), Sodium Carbonate, TBE, TAE, BIS-TRIS, Bis-Tris propane, Phosphate buffer, formic acid, Pyridine and conjugate acid, Ammonia and conjugate acid, Methylamine and conjugate acid, ADA, ACES, PIPES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Tris, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, and the like.
Some microfluidic chips and systems suitable for use in the systems here are described in International Patent Application No. PCT/US2021/023827, filed Mar. 24, 2021, and titled “DEVICE AND METHODS FOR ISOLATING EXTRACELLULAR MATRIX BODIES,” International Patent Application No. PCT/US2019/052310, filed Sep. 21, 2019, and titled “COMPOSITIONS AND METHODS FOR GLAUCOMA,” each of which is hereby incorporated by reference in its entirety.
Generally, the systems and devices described herein may provide high-throughput separation of extracellular matrix bodies from a biological fluid. The separated ECMBs and remaining biological fluid (or portions or fractions thereof) may then be used for diagnosing, prognosing, or in helping to determine a treatment plan for a subject, and the effectiveness of such treatment plan. A block diagram of an exemplary system 100 is depicted in
In some variations, the holder 112 (e.g., material storage) may be configured to store one or more fluids (e.g., biological fluid) for transfer to the microfluidic chip(s) 116. The holder 112 may comprise one or more of a tray and container. In some variations, the reservoir 113 may be configured to receive a predetermined fraction of the biological fluid (e.g., waste fluid) from the microfluidic chip 116. For example, a non-ECMB fraction of the biological fluid may be transferred to and stored in the reservoir 113 for further processing (e.g., separation, analysis) and/or disposal. Additionally or alternatively, the system 100 may comprise one or more preprocessing components such as a material separation assembly, aliquoting assembly, a biorepository, and the like.
In some variations, the robot 114 may be configured to transfer fluids (e.g., biological fluid, reagent) from holder 112 to at least one microfluidic chip 116. In some variations, the microfluidic chip 116 may be configured to receive and process a biological fluid. For example, the microfluidic chip 116 may include a channel (e.g., microfluidic channel) and at least one obstruction (e.g., pillar) configured to separate and hold (e.g., trap) a predetermined fraction of a biological fluid within the microfluidic chip 116 while the remaining fraction flows out of the microfluidic chip 116. In some variations, the chip connector 118 may be configured to hold at least one microfluidic chip 116. The microfluidic chip 116 may be releasably coupled to the chip connector 118 to facilitate cleaning and help reduce set-up time. For example, the microfluidic chip 116 may be a disposable component and the chip connector 118 may be a durable component. In some variations, the chip connector 118 may comprise disposable connectors (e.g., inlet connector, outlet connector) configured to facilitate negative pressure applied to the microfluidic chip 116 by the negative pressure source 122 in order to perfuse and flow the biological fluid through the microfluid chip 116.
In some variations, the manifold 120 may be configured to couple to at least one microfluidic chip 116. In some variations, the negative pressure source 122 may be configured to couple to the manifold 120. In this manner, a single manifold 120 may be fluidically coupled to a plurality of microfluidic chips 116 to facilitate application of negative pressure (e.g., vacuum, suction) to the plurality of microfluidic chips 116 using a single negative pressure source 122.
In some variations, the sensor 124 may comprise one or more sensors configured to measure one or more characteristics (e.g., pressure, flow rate, optical image, temperature, humidity) corresponding to one or more of the biological fluid and components of the system 100, 200. In some variations, the input device 126 may be configured to generate an input signal based on an operator input. In some variations, the processor 128 and memory 130 may be configured to control the system 100, 200. In some variations, the communication device 132 may be configured to communicate with one or more components of the system 100, 200 as well as with networks and other computer systems. In some variations, the output device 134 may be configured to output data corresponding to the system 100, 200 such as images of the biological fluid within the microfluidic chip 116, flow rates, and the like.
In some variations, the system 100, 200 may be configured to perform multiple assays such as ELISA, immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, UV-VIS, Raman, NMR, mass spectrometry, NG sequencing, protein array, ribonucleic acid array, gene array, qPCR, RT-qPCR, RT-PCR, and the like. In some variations, the microfluidic chip 116 may be analyzed by the system 100, 200 and/or removed and analyzed using another device (e.g., multi-well plate, microscope slide). Examples of imaging techniques include electron microscopy, stereoscopic microscopy, wide-field microscopy, bright-field microscopy, phase-contrast microscopy, polarizing microscopy, phase contrast microscopy, multiphoton microscopy, differential interference contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, ray microscopy, ultrasonic microscopy, positron emission tomography, computerized tomography, and magnetic resonance imaging.
In some variations, one or more fluids may be loaded from the holder 212 to a respective inlet of a plurality of microfluidic chips 216 using the robot 214. The negative pressure source 222 may apply suction to each of the microfluidic chips 216 via the manifold 220 and fluid conduits 221a, 221b. The fluid at the inlet (e.g., inlet reservoir) may be pulled toward the respective outlet of the microfluidic chip 216 while ECMBs remain in the microfluidic chip 216. The separated non-ECMB fluid is pulled through the fluid conduits 221a, 221b and manifold 220 and received at the reservoir 213 (e.g., waste disposal).
The inlet connector 240 may be coupled between an inlet 230 of the microfluidic chip 216 and one or more of the end effector 215 and the funnel 260. In some variations, the funnel 260 may be configured to receive the end effector 215 using the robot 214. For example, the funnel 260 may be configured to receive and/or guide fluid being transferred to the microfluidic chip 216 from the end effector 215. The outlet connector 242 may be coupled between an outlet 232 of the microfluidic chip 216 and the fluid conduit 221a. In some variations, the fluid conduit 221a may be configured to receive fluid being transferred from the microfluidic chip 216 to the reservoir 213 (e.g., via negative pressure).
In some variations, one or more clamps 250 may be configured to releasably couple (e.g., secure, hold) the inlet connector 240 and the outlet connector 242, respectively, to the microfluidic chip 216. For example, the clamp 250 may comprise one or more springs (not shown) and a hinge 252 configured to provide a predetermined force to hold the respective inlet connector 240 and outlet connector 242 in place relative to the microfluidic chip 216. In some variations, a portion 254 (e.g., actuator) of the clamp 250 may be actuated (e.g., pushed down by a robot or operator) to release one or more of the inlet connector 240 and the outlet connector 242 to facilitate removal of the microfluidic chip 216 from the chip connector 218.
The systems described herein may comprise a holder 112, 212 configured to receive one or more fluids. In some variations, the holder 112, 212 may be configured to receive and store a plurality of fluids including a biological fluid (e.g., sample) and one or more reagents. Examples of reagents include a buffer, a lysing solution, a nucleic acid cleavage agent, a cleavage inhibitor, a precipitation agent, a fixative reagent, a carrier fluid, a biofluid, water, purified water, a saline solution, an organic solvent, a gelling agent, a surfactant, a ligand for binding or associating with a component of an ECMB, combinations thereof, and reagents for interacting with biological components of the sample. In some variations, the reagent may include one or more reagents for measuring a biomarker level or quantity, or for comparing a biomarker level to a control. In some variations, the holder 112, 212 may comprise a plurality of reservoirs to separately store each fluid without mixing. Fluids may include any suitable fluids, including for example biological fluids for sampling, and/or one or more fluids useful in analysis. Biological fluids for sampling may include, for example, one or more of human or animal bodily fluids, tissue, cells, whole blood, blood plasma, blood serum, cerebrospinal fluid, intrathecal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspirate, semen, amniotic fluid, vitreous, aqueous humor, lymph, bile, cerumen, chyle, chyme, endolymph, perilymph, exudates, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, pus, rheum, sebum, serous fluid, smegma, sputum, synovial fluid, vaginal secretion, menstrual effluent, vomit, tumors, carriers, reagents, solutions, binding moieties, and the like. Biological fluids useful in analysis may include carriers, reagents, binding moieties, and the like. The holder 112, 212 may be disposed within the system 100, 200 at a location within reach of the robot 114, 214. For example, the holder 112, 212 may be configured to receive a plurality of pipettes coupled to the robot 114, 214 for transfer of a plurality of fluids from the holder 112, 212 to a plurality of microfluidic chips 116, 216.
The systems described herein may comprise one or more robots 114, 214. Generally, a robot 114, 214 may be configured to move and transfer one or more fluids (e.g., biological fluid, reagent, antibodies, buffer) between a holder 112, 212 and at least one microfluidic chip 116, 216. For example, the robot 114, 214 may be configured to automatically load at least one microfluidic chip 116, 216 with fluids absent manual (e.g., human) intervention. In some variations, the robot 114, 214 may be coupled to an end effector 215 comprising one or more pipettes (e.g., micro-pipette, multi-channel pipette) configured to transfer a fluid (e.g., biological fluid, reagent) from the holder 112, 212 to at least one microfluidic chip 116, 216 (e.g., through an inlet connector 240 of a chip connector 118, 218). For example, a biological fluid from a subject may be first transferred from the holder 112, 212 to a plurality of microfluidic chips 116, 216 using the robot 114, 214. Then, different reagents may be transferred from the holder 112, 212 to predetermined microfluidic chips 116, 216 using the robot 114, 214 for different processing (e.g., different histochemical stains). The robot 114, 214 may be coupled to the processor 128 and memory 130 to control a type and volume of fluid transferred using one or more of the pipettes. In some variations, the end effector 215 may be configured to releasably couple (e.g., assemble, disassemble) the disposable components from the durable components of the system 200. Additionally or alternatively, the system 100 may comprise one or more material transport components such as a track and container configured to translate along the track.
In some variations, the robot 114, 214 may be configured to removably couple a microfluidic chip 116, 216 to one or more of a chip connector 118, 218 and a negative pressure source 122, 222. For example, the robot 114, 214 may be configured to couple a microfluidic chip 116, 216 to a chip connector 118, 218 such that the microfluidic chip 116, 216 is fluidically coupled to the manifold 120, 220 and negative pressure source 122, 222. Furthermore, robot 114, 214 may be configured to releasably couple other components (e.g., inlet connector, outlet connector) to the microfluidic chip 116, 216. Conversely, the robot 114, 214 may be configured to de-couple the microfluidic chip 116, 216 from the chip connector 118, 218 for transfer to another device (e.g., microscope slide, imaging system, analysis system). In some variations, the robot 114, 214 may be configured to remove one or more of the holder 112, 212 and reservoir 113, 213 from the system 100, 200 (e.g., to replace with a different holder 112, 212 and reservoir 113, 213). Automated system set-up, fluid transfer, and cleaning using the robot 114, 214 may, among other benefits, reduce contamination and human error, and increase consistency and throughput.
In some variations, the robot 114, 214 may be, for example, a linear robot arm, an articulated robotic arm, and/or a SCARA robotic arm. The robot 114, 214 may comprise one or more segments coupled together by a joint (e.g., shoulder, elbow, wrist) configured to provide a single degree of freedom. Joints are mechanisms that provide a single translational or rotational degrees of freedom. For example, the robot 114, 214 may have six or more degrees of freedom. The set of Cartesian degrees of freedom may be represented by three translational (position) variables (e.g., surge, heave, sway) and by the three rotational (orientation) variables (e.g., roll, pitch, yaw). In some variations, the robot 114, 214 may have less than six degrees of freedom.
In some variations, the robot 114, 214 may be configured to move over all areas of a system 100, 200 in up to three dimensions. The robot 114, 214 may comprise one or more motors configured to translate and/or rotate the joints and move the robot 114, 214 to a desired location and orientation. In some variations, the position of the robot may be temporarily locked when delivering fluid to a predetermined microfluidic chip or when one or more microfluidic chips 116, 216 are being removed from a chip connector 118, 218 (e.g., for transfer to a microscope or other imaging system). The robot 114, 214 may be mounted to any suitable object, such as a platform (e.g., table), a wall, a ceiling, or may be self-standing (e.g., on the ground). Additionally or alternatively, the robot 114, 214 may be configured to be moved manually.
The systems described herein may comprise one or more microfluidic chips 116, 216. Generally, a microfluidic chip 116, 216 may be configured to receive and process one or more fluids. In some variations, a microfluidic chip 116, 216 may comprise an inlet reservoir, at least one channel (e.g., restriction channel, uniform flow channel), at least one obstruction (e.g., pillar) configured to restrict fluid flow, and an outlet reservoir. The restriction channel may include an inlet and an outlet. The inlet reservoir may be fluidically coupled to the inlet of the restriction channel, and the outlet of the restriction channel may be fluidically coupled to the outlet reservoir. The inlet reservoir may be configured to receive and store fluid transferred from robot 114, 214. The microfluidic chip 116, 216 may be configured to process a biological fluid through the channel while receiving negative pressure from an outlet (e.g., outlet reservoir) of the microfluidic chip 116, 216.
In some variations, the restricted region 320 may be configured to restrict fluid flow so as to separate and hold (e.g., trap) a first fraction of the fluid within the restricted region 320 of the microfluidic chip 316 while a remaining second fraction of the fluid is permitted to flow out of the microfluidic chip 316 via the negative pressure applied to the microfluidic chip 316. In some variations, the first fraction of the fluid may include ECMBs. As shown in
The plurality of obstructions 340 within the restricted region 320 may be configured to restrict (e.g., impede) fluid flow and hold the first fraction of the biological fluid. For example, a spacing between the plurality of obstructions 340 in the restricted region 320 may decrease along a length of the microfluidic chip 316 from an inlet of the restricted region 320 to an outlet of the restricted region 320. In some variations, the spacing between the plurality of obstructions 340 in the restricted region 320 may be between about 100 μm and about 4 μm. For example, the spacing between obstructions 340 at an inlet of the restricted region 320 may be about 100 μm whereas the spacing between obstructions 340 at an outlet of the restricted region 320 may be about 4 μm. In some variations, the spacing may vary in stepwise increments. For example, the spacing may decrease in order from about 100 μm to about 50 μm to about 25 μm to about 15 μm and to about 4 μm. Additionally or alternatively, the spacing between obstructions 340 may vary continuously along a length of the restricted region 320. In some variations, a larger proportion of ECMBs may be captured within the portions of the restricted region 320 having smaller spacing.
The restriction channel 324 may have a length of between about 5 mm and about 30 mm, between about 10 mm and about 30 mm, between about 15 mm and about 30 mm, between about 20 mm and about 30 mm, between about 5 mm and about 25 mm, between about 5 mm and about 20 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, including all ranges and sub-values in-between. The restriction channel 324 may have a cross-sectional dimension of between about 5 μm and about 30 μm, between about 10 μm and about 30 μm, between about 15 μm and about 30 μm, between about 20 μm and about 30 μm, between about 5 μm and about 25 μm, between about 5 μm and about 20 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, including all ranges and sub-values in-between. In some variations, the restriction channel 324 may comprise at least one obstruction. The plurality of obstructions 340 may comprise a diameter of about 50 μm and about 1 mm.
As shown in the cross-sectional side view of
In some variations, a restriction channel may extend beyond a length of a restricted region. For example,
The plurality of obstructions 1840 within the restricted region 1820 may be configured to restrict (e.g., impede) fluid flow and hold the first fraction of the biological fluid for analysis (e.g., image analysis). For example,
In some variations, a microfluidic chip may include a plurality of restriction channels to promote fluid flow and reduce formation of air bubbles and clogging within the microfluidic chip. For example,
The plurality of obstructions 1940 within the restricted region 1920 may be configured to restrict (e.g., impede) fluid flow and hold the first fraction of the biological fluid for analysis (e.g., image analysis). For example,
A plurality of obstructions 2040 may be disposed between the inlet reservoir 2010 and the outlet reservoir 2030. Similar to the microfluidic chips 316, 1800, 1900, the restricted region 2020 may be configured to restrict fluid flow so as to separate and hold (e.g., trap) a first fraction of the fluid within the restricted region 2020 of the microfluidic chip 1800 while a remaining second fraction of the fluid is permitted to flow out of the microfluidic chip 2000 via the negative pressure applied to the microfluidic chip 2000. In some variations, the first fraction of the fluid may include ECMBs. The restricted region 2020 may include flow barriers 2022 that define the restriction channel 2024 (e.g., uniform flow channel) and a plurality of obstructions 2040 (e.g., pillars) having a plurality of spacings 2021, 2023, 2025, 2027, 2029, as described in more detail with respect to
The plurality of obstructions 2040 within the restricted region 2020 may be configured to restrict (e.g., impede) fluid flow and hold the first fraction of the biological fluid for analysis (e.g., image analysis). For example,
The microfluidic chips described herein may comprise a single channel (e.g., microfluidic chip 1800, 1900, 2000) or a plurality of channels (e.g., microfluidic chip array 2002). In some variations, unexpected, surprising, and significant improvements may be found in one or more of laminar flow, flow rate consistency, clogging, signal-to-noise ratio, and artifacts based on a channel density a microfluidic chip (e.g., single channel microfluidic chip compared to a multi-channel microfluidic chip array). For example,
The difference in laminar flow between the single channel microfluidic chip 2900 and multi-channel microfluidic chip array 2950 was unexpected, surprising, and significant, as shown in the graphs in
In some variations, reduced laminar flow in a channel of a microfluidic chip may correspond to an increase in artifacts and higher non-laminar flow. For example,
The microfluidic chips described herein may have various configurations and dimensions. For example, the obstructions described herein may have one or more of a circular, a spherical, a triangular, a square, a polygonal, a diamond, and a fin-shape. The obstructions may have the same or different shapes, sizes, and spacings. For example, the obstructions may have a spacing between adjacent pillars of between about 4 μm and about 100 μm, about 4 μm, about 15 μm, about 25 μm, about 50 μm, about 100 μm, including all ranges and sub-values in-between.
In some variations, a microfluidic chip applied with negative pressure may improve a yield (e.g., immobilization) of a predetermined fraction of a biological fluid based on a cross-sectional dimension of a channel. For example, each channel may comprise a cross-sectional (e.g., height) dimension of between about 5 μm and about 30 μm, between about 5 μm and about 10 μm, between about 5 μm and about 20 μm, between about 5 μm and about 15 μm, between about 10 μm and about 20 μm, between about 15 μm and about 30 μm, about 8 μm, about 11 μm, about 15 μm, including all ranges and sub-values in-between.
In some variations, ECMB capture amounts using the negative pressure systems as described herein may depend on the contents of the fluid (e.g., healthy control sample, positive sample).
In some variations, ECMB capture amounts using the negative pressure systems as described herein may depend on one or more microfluidic channel dimensions. For example,
The systems described herein may comprise a chip connector. Generally, a chip connector 118, 218 may be configured to hold one or more microfluidic chips 116, 216, 316, 1800, 1900, 2000, 2100 and improve performance of the system 100, 200. Application of negative pressure to a microfluidic chip generates forces on the structure of the microfluidic chip itself that may reduce and/or prevent processing of a biological fluid using the microfluidic chip. For example, negative pressure applied to a channel (e.g., restriction channel) of a microfluidic chip may generate force at an outlet that may compress (and/or collapse) an outlet reservoir of the microfluidic chip corresponding to unpredictable flow rates, air bubbles, and unequal distribution of material within the microfluidic chip. By contrast, the chip connectors described herein may be configured to hold a microfluidic chip and distribute the negative pressure forces applied by a negative pressure source more evenly throughout a microfluidic chip 116, 216, 316 so as to maintain the structural integrity of the microfluidic chip 116, 216, 316 and provide consistent results.
In some variations, the microfluidic chip 416 held between the cover 420 and the bottom portion 430 may distribute the negative pressure forces applied at the outlet 460 of the microfluidic chip 416 to the perimeter of the microfluidic chip 416 (e.g., sides of the chip not having a channel), thereby reducing compression at an outlet of the microfluidic chip and non-perimeter portions of the microfluidic chip (e.g., were fluid flows), and thereby facilitate a consistent flow rate and more equal distribution of material through the microfluidic chip 416. For example,
In some variations, the chip connector 418 may comprise one or more fasteners configured to couple the microfluidic chip 416 to the chip connector 418. For example, as shown in
In some variations, the microfluidic chip 2216 held between the cover 2220 and the base 2230 of the chip connector 2200 may distribute the negative pressure forces applied at the outlet 2260 of the microfluidic chip 2216 to the perimeter of the microfluidic chip 2216 (e.g., sides of the chip not having a channel), thereby reducing compression at an outlet 2260 of the microfluidic chip 2216 and thereby facilitate a consistent flow rate and more equal distribution of material (e.g., first fraction, ECMBs) through the microfluidic chip 2216. This configuration enables the forces applied by negative pressure at an outlet 2260 (that push the microfluidic chip 2216 towards the base 2230) to be redistributed to the perimeter of the microfluidic chip 2216 to reduce compression at an outlet 2260 of the microfluidic chip 2216. In this manner, the chip connector 2200 may be configured to distribute a compressive force applied by the negative pressure to the microfluidic chip 2216 to relieve pressure on the microfluidic chip 2216 and keep a fluid channel open for fluid flow. Accordingly, the chip connector 2200 facilitates laminar fluid flow and consistent flow rates that minimize dead zones, clogging, background noise, and artifacts.
In some variations, as shown in
In some variations, the chip connector 118, 218 may be configured to hold one or more microfluidic chips 116, 216, 316, 1800, 1900, 2000, 2100 and/or microfluidic chip arrays 370, 2002 to improve the throughput and/or reduce the size of the system. Furthermore, a chip connector may be configured to distribute the negative pressure forces applied by a negative pressure source more evenly throughout a plurality of microfluidic chips to maintain structural integrity and provide consistent results in a compact space.
In some variations, the microfluidic chip array 2314 held between the cover 2320 and the bottom portion 2330 of the chip connector 2300 may distribute the negative pressure forces applied at the plurality of outlets 2360 of the microfluidic chip array 2314 to the perimeter of the microfluidic chip array 2314 (e.g., sides of the chip not having a channel), thereby reducing compression at the outlets of the microfluidic chip array and thereby facilitating a consistent flow rate and more equal distribution of material through the microfluidic chip array 2314. This configuration enables the forces applied by negative pressure at the plurality of outlets 2360 (that push the microfluidic chip array 2314 towards the base 2330) to be redistributed to the perimeter of the microfluidic chip array 2314 to reduce compression at the plurality of outlets 2360 of the microfluidic chip array 2314. In this manner, the chip connector 2300 may be configured to distribute a compressive force applied by the negative pressure to the microfluidic chip array 2314 to relieve pressure on the microfluidic chip array 2314 and keep a fluid channel open for fluid flow. Accordingly, the chip connector 2300 facilitates laminar fluid flow and consistent flow rates that minimize dead zones, clogging, background noise, and artifacts.
In some variations, as shown in
In some variations, a fluid flow rate and a flow rate consistency through a microfluidic chip 116, 216, 316, 416 being applied with negative pressure may be improved by holding the microfluidic chip 116, 216, 316, 416 in a chip connector 118, 218, 418, 2200, 2300.
In some variations, a chip connector 118, 218, 418 may include at least one inlet connector 440 and at least one outlet connector 442 disposed between the cover 420 and the microfluidic chip 416. In some variations, application of negative pressure to a microfluidic chip 416 may generate forces at a connection interface between the microfluidic chip 416 and a fluid conduit that may reduce and/or prevent processing of a biological fluid using the microfluidic chip 416. For example, the outlet 460 of the microfluidic chip 416 may be coupled to a fluid conduit (e.g., vacuum tube) of the manifold 120, 220 and negative pressure source 122, 222 (not shown). The negative pressure forces applied through the fluid conduit and at the outlet 460 of the microfluidic chip 416 may otherwise dislodge the fluid connection between the microfluidic chip 416 and the manifold 120, 220 so as to create a leak. In some variations, one or more of the connectors 440, 442 described herein may be coupled to the microfluidic chip 416 to improve one or more of a seal, connection strength, and alignment tolerance between the microfluidic chip 416 and the manifold 120, 220 and thereby provide a consistent flow rate (e.g., by preventing leaks) and equal distribution of material. In some variations, one or more of the inlet connectors 440 and outlet connectors 442 may be disposable to reduce contamination and set-up time.
In some variations, a single inlet connector may be configured to transfer (e.g., receive) fluid for a plurality of microfluidic chips, thereby reducing the complexity of the chip connector as well as reducing set-up and cleaning times.
In some variations, the inlet connector 2400, 2500, 2600 may comprise a body defining a plurality of lumens each defining a distal portion (e.g., portion facing an inlet of the microfluidic chip) and a proximal portion (e.g., portion approaching a collar 2424, 2524, 2624). Each lumen 2410, 2510, 2610 may have an inner diameter increasing in one or more of a distal and proximal direction. For example, as shown in
In some variations, a single outlet connector may be configured to transfer (e.g., withdraw) fluid from a plurality of microfluidic chips, thereby reducing the complexity of the chip connector as well as reducing set-up times and cleaning times. For example, the single outlet connector demonstrates a lower standard deviation for flow rates relative to conventional connectors. FIGS. 27A-27D depicts a perspective view, top view, bottom view, and perspective side view of an outlet connector 2700 (e.g., gasket).
In some variations, the lumen 2740, 2840 may define a distal portion (e.g., portion facing an outlet of the microfluidic chip), a proximal portion (e.g., portion approaching a collar 2834), and an inner diameter. As show in
In some variations, the inlet and outlet connectors may comprise suitable materials including one or more silicone-based polymers, other polymers, thermoplastics, thermoplastic elastomers, and the like. For example, one or more of the inlet and outlet connectors may comprise plastic coated with silicone. Inlet and outlet connectors comprising silicone may further improve flow rate consistency. Silicone may include liquid silicone rubber, high consistency silicone, solid silicone rubber, room temperature vulcanized (RTV) silicone, fluorosilicone, combinations thereof, and the like.
In some variations, a geometry of the disposable inlet and outlet connectors described herein may improve operational aspects of a negative pressure system such as assembly and cleaning that may reduce a set-up time of the system. For example,
In some variations, an overall experiment duration for exemplary staining procedures may be improved using a negative pressure system relative to a positive pressure system. For example,
In some variations, the improved fluid seal of the disposable inlet and outlet connectors described herein may reduce contamination in a microfluidic chip corresponding to abnormal morphology relative to a reusable connector used in a positive pressure system.
The systems described herein may comprise a manifold 120, 220. Generally, a manifold 120, 220 may be configured to couple to a plurality of microfluidic chips 116, 216, 316 and negative pressure source 122, 222. For example, the manifold 120, 220 may be in fluid communication with a single negative pressure source 122, 222 and each microfluidic chip 116, 216, 316 held by a chip connector 118, 218 such that a negative pressure generated by the negative pressure source 122, 222 may be applied to each microfluidic chip 116, 216, 316 coupled to the manifold 120, 220. In some variations, the manifold 120, 220 may comprise a plurality of fluid conduits (e.g., vacuum tubes, fluid lines) configured to be releasably coupled to each of the microfluidic chips 116, 216, 316 and the negative pressure source 122, 222. Accordingly, the manifold may reduce the size of the system 100 since each microfluidic chip does not need its own negative pressure source.
In some variations, the manifold and negative pressure source significantly increase throughput by scaling and increasing efficiency without a reduction in consistency or flowrate.
A microfluidic systems described herein comprising a manifold and negative pressure source help to not only increase efficiency and throughput, but do so in a relatively uniform manner otherwise unachievable using conventional solutions. For example,
Similar to
A fluid flow rate through a microfluidic chip being applied with negative pressure through an 8-port manifold may be relatively uniform. For example,
The systems described herein may comprise a negative pressure source 122, 222. Generally, a negative pressure source 122, 222 may be configured to couple to a manifold 120, 220 and at least one microfluidic chip 116, 216, 316. Application of negative pressure may help reduce contamination and increase processing throughput relative to conventional solutions such as positive pressure systems. In some variations, the negative pressure source may comprise a fluid pump. The processor 128 and memory 130 coupled to the negative pressure source 122, 222 may be configured to control the fluid flow rate and negative pressure. In some variations, the negative pressure source 122, 222 may be configured to apply a negative pressure of between about 10 mm HG and about 760 mm HG to each microfluidic chip 116, 216, 316 of the system 100, 200.
In some variations, a trapped ECMB amount in a microfluidic chip does not necessarily increase linearly with an amount of applied negative pressure. For example,
In some variations, a negative pressure of between about 10 mm HG and about 760 mm HG applied to a microfluidic chip may effectively separate ECMBs from a biological fluid. For example,
The systems described herein may comprise one or more sensors 124. Generally, a sensor 124 may be configured to measure one or more characteristics (e.g., pressure, flow rate, optical image, temperature, humidity) corresponding to one or more of the biological fluid, holder 112, 212, reservoir 113, 213, robot 114, 214, microfluidic chip 116, 216, 316, chip connector 118, 218, manifold 120, 220, and negative pressure source 122, 222. In this way, the system and the fluids may be monitored during use. In some variations, a sensor may be coupled to or integrated into any component of the system 100 such as the inlet connector, chip connector, etc.
In some variations, the sensor may be an optical sensor coupled to any component of the system 100, 200 such as the chip connector 118, 218. The optical sensor may be configured to image one or more channels of the microfluidic chips 116, 216, 316 for histochemical and morphological study. The optical sensor may be used to receive light signals (e.g., light beams) reflected by the fluid in the microfluidic chip 116, 216, 316. The received light may be used to generate signal data that may be processed by the processor 128 and memory 130 to generate sample data. The optical sensor may further be configured to image one or more identifiers (e.g., label, barcode) and identifiers of the microfluidic chip 116, 216, 316. In some variations, the optical sensor may include one or more of a lens, camera, and measurement optics. For example, the optical sensor may include a charged coupled device (CCD) or complementary metal-oxide semiconductor (CMOS) optical sensor and may be configured to generate an image signal that is transmitted to a output device 134 (e.g., display). For example, the optical sensor may include a camera with an image sensor (e.g., a CMOS or CCD array with or without a color filter array and associated processing circuitry). Additionally or alternatively, the sensor in some variations may be an ultrasound sensor configured to generate an ultrasound signal used to determine a fluid flow rate.
In some variations, the system 100, 200 may include a radiation source configured to emit a light signal (e.g., illumination) directed at the microfluidic chip 116, 216, 316 for visualization, imaging, and/or cleaning (e.g., UV light). In some variations, the radiation source may include one or more of a light emitting diode, laser, microscope, optical sensor, lens, and flash lamp. For example, the radiation source may generate light that may be carried by fiber optic cables or one or more LEDs may be configured to provide illumination. In another example, a fiberscope including a bundle of flexible optical fibers may be configured to receive and propagate light from an external light source.
Generally, an input device 126 of a system 100, 200 may serve as a communication interface between an operator and the system 100, 200. The input device 126 may be configured to receive input data and output data to one or more of the robot 114, sensor 124, and output device 134. For example, operator control of an input device 126 (e.g., foot controller, joystick, keyboard, touch screen) may be processed by processor 128 and memory 130 for input device 126 to output a control signal to one or more of robot 114, 214, negative pressure source, and sensor 124. As another example, images generated by sensor 124 may be processed by processor 128 and memory 130, and displayed by the output device 134 (e.g., display). Sensor data from one or more sensors 124 may be output visually, audibly, and/or through haptic feedback by one or more output devices 134.
Some variations of an input device may comprise at least one switch configured to generate a control signal. The input device may be coupled to or separate from other components of the system 100, 200. For example, the input device 126 may be disposed in a different room than the robot 114, 214 and microfluidic chip 116, 216, 316 to reduce potential contamination. The control signal may include, for example, a robot signal, a negative pressure signal, a sensor signal, and other signals. In some variations, the input device 126 may comprise a wired and/or wireless transmitter configured to transmit a control signal to a wired and/or wireless receiver of a controller. A robot signal (e.g., for the control of movement, position, and orientation) may control articulation of the robot in at least four degrees of freedom of motion, and may include yaw and/or pitch rotation. For example, an input device 126 comprising a touch surface may be configured to detect contact and movement on the touch surface using any of a plurality of touch sensitivity technologies including capacitive, resistive, infrared, optical imaging, dispersive signal, acoustic pulse recognition, and surface acoustic wave technologies.
In variations of an input device 126 comprising at least one switch, a switch may comprise, for example, at least one of a button (e.g., hard key, soft key), touch surface, keyboard, analog stick (e.g., joystick), directional pad, mouse, trackball, jog dial, step switch, rocker switch, pointer device (e.g., stylus), motion sensor, image sensor, and microphone. A motion sensor may receive operator movement data from an optical sensor and classify an operator gesture as a control signal. A microphone may receive audio and recognize an operator voice as a control signal. In variations of a system comprising a plurality of input devices, different input devices may generate different types of signals. For example, some input devices (e.g., button, analog stick, directional pad, and keyboard) may be configured to generate a robot signal while other input devices (e.g., step switch, rocker switch) may be configured to control a negative pressure source 122, 222 and the sensors 124.
A system 100, 200, as depicted in
The processor 128 may be implemented consistent with numerous general purpose or special purpose computing systems or configurations. Various exemplary computing systems, environments, and/or configurations that may be suitable for use with the systems and devices disclosed herein may include, but are not limited to software or other components within or embodied on personal computing devices, network appliances, servers or server computing devices such as routing/connectivity components, portable (e.g., hand-held) or laptop devices, multiprocessor systems, microprocessor-based systems, and distributed computing networks.
Examples of portable computing devices include smartphones, personal digital assistants (PDAs), cell phones, tablet PCs, phablets (personal computing devices that are larger than a smartphone, but smaller than a tablet), wearable computers taking the form of smartwatches, portable music devices, and the like, and portable or wearable augmented reality devices that interface with an operator's environment through sensors and may use head-mounted displays for visualization, eye gaze tracking, and user input.
The processor 128 may incorporate data received from memory 130 and operator input to control one or more robots 114, 214 and negative pressure source 122, 222. The memory 130 may further store instructions to cause the processor 128 to execute modules, processes, and/or functions associated with the system 100, 200. The processor 128 may be any suitable processing device configured to run and/or execute a set of instructions or code and may comprise one or more data processors, image processors, graphics processing units, physics processing units, digital signal processors, and/or central processing units. The processor 128 may be, for example, a general purpose processor, a Field Programmable Gate Array (FPGA), an Application Specific Integrated Circuit (ASIC), configured to execute application processes and/or other modules, processes, and/or functions associated with the system and/or a network associated therewith. The underlying device technologies may be provided in a variety of component types such as metal-oxide semiconductor field-effect transistor (MOSFET) technologies like complementary metal-oxide semiconductor (CMOS), bipolar technologies like emitter-coupled logic (ECL), polymer technologies (e.g., silicon-conjugated polymer and metal-conjugated polymer-metal structures), mixed analog and digital, combinations thereof, and the like.
Some variations of memory 130 described herein relate to a computer storage product with a non-transitory computer-readable medium (also may be referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include transitory propagating signals per se (e.g., a propagating electromagnetic wave carrying information on a transmission medium such as air or a cable). The media and computer code (also may be referred to as code or algorithm) may be those designed and constructed for a specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape; optical storage media such as Compact Disc/Digital Video Discs (CD/DVDs), Compact Disc-Read Only Memories (CD-ROMs), and holographic devices; magneto-optical storage media such as optical discs; solid state storage devices such as a solid state drive (SSD) and a solid state hybrid drive (SSHD); carrier wave signal processing modules; and hardware devices that are specially configured to store and execute program code such as Application-Specific Integrated Circuits (ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM), and Random-Access Memory (RAM) devices. Other variations described herein relate to a computer program product, which may include, for example, the instructions and/or computer code disclosed herein.
The systems, devices, and/or methods described herein may be performed by software (executed on hardware), hardware, or a combination thereof. Software modules (executed on hardware) may be expressed in a variety of software languages (e.g., computer code), including C, C++, Java®, Python, Ruby, Visual Basic®, and/or other object-oriented, procedural, or other programming language and development tools. Examples of computer code include, but are not limited to, micro-code or micro-instructions, machine instructions, such as produced by a compiler, code used to produce a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.
In some variations, systems 100, 200 described herein may communicate with networks and computer systems through a communication device 128. In some variations, the system 100, 200 may be in communication with other devices via one or more wired and/or wireless networks. A wireless network may refer to any type of digital network that is not connected by cables of any kind. Examples of wireless communication in a wireless network include, but are not limited to cellular, radio, satellite, and microwave communication. However, a wireless network may connect to a wired network in order to interface with the Internet, other carrier voice and data networks, business networks, and personal networks. A wired network is typically carried over copper twisted pair, coaxial cable and/or fiber optic cables. There are many different types of wired networks including wide area networks (WAN), metropolitan area networks (MAN), local area networks (LAN), Internet area networks (IAN), campus area networks (CAN), global area networks (GAN), like the Internet, and virtual private networks (VPN). Hereinafter, network refers to any combination of wireless, wired, public and private data networks that are typically interconnected through the Internet, to provide a unified networking and information access system.
Cellular communication may encompass technologies such as GSM, PCS, CDMA or GPRS, W-CDMA, EDGE or CDMA2000, LTE, WiMAX, and 5G networking standards. Some wireless network deployments combine networks from multiple cellular networks or use a mix of cellular, Wi-Fi, and satellite communication. In some variations, the communication device 132 may comprise a radiofrequency receiver, transmitter, and/or optical (e.g., infrared) receiver and transmitter. The communication device 132 may communicate by wires and/or wirelessly with one or more of the robot 114, 214, negative pressure source 122, 222, sensor 124, input device 126, output device 134, network, database, server, combinations thereof, and the like.
An output device 134 of a system 100, 200 may be configured to output data corresponding to a system, and may comprise one or more of a display device, audio device, and haptic device. For example, a display device may allow an operator to view images of one or more microfluidic chips 116, 216, 316 and the robot 114, 214. In some variations, an output device may comprise a display device including at least one of a light emitting diode (LED), liquid crystal display (LCD), electroluminescent display (ELD), plasma display panel (PDP), thin film transistor (TFT), organic light emitting diodes (OLED), electronic paper/e-ink display, laser display, and/or holographic display.
An audio device may audibly output fluid data, sensor data, system data, alarms and/or warnings. For example, the audio device may output an audible warning when sensor data (e.g., pressure, flow rate) falls outside a predetermined range or when a malfunction in a robot is detected. As another example, audio may be output when operator input is overridden by the system to prevent potential harm to the operator and/or system (e.g., robot collision, excessive negative pressure). In some variations, an audio device may comprise at least one of a speaker, piezoelectric audio device, magnetostrictive speaker, and/or digital speaker. In some variations, an operator may communicate to other users using the audio device and a communication channel. For example, the operator may form an audio communication channel (e.g., VoIP call) with a remote operator and/or observer.
A haptic device may be incorporated into one or more of the input and output devices to provide additional sensory output (e.g., force feedback) to the operator. For example, a haptic device may generate a tactile response (e.g., vibration) to confirm operator input to an input device (e.g., touch surface). Additionally or alternatively, haptic feedback may notify that an operator input is overridden by the system to prevent potential harm to the operator and/or system (e.g., robot collision, excessive negative pressure).
Also described here are methods for separating ECMBs from a biological fluid. The methods described herein may be useful in providing high-throughput separation of ECMBs and biological fluids so that additional analysis may be performed, for example analysis of the ECMBs or the biological fluids (or portions or fractions thereof) to help diagnose and/or develop a treatment plan for a subject, and to monitor the effectiveness of a treatment plan. For example, a predetermined fraction of the biological fluid or the ECMBs may be analyzed to determine an amount of a biomarker associated with a disease, which may be useful in diagnosis and target identification. Biomarkers may be used not only for disease screening and prediction, but also to help support disease prognosis, facilitate treatment selection, subtype patients for clinical trials, and/or monitor both the safety and therapeutic signals in subjects after treatment has begun, and during the course of treatment. In some variations, pharmacodynamic biomarkers may function as endpoints for clinical trials and may be surrogate endpoints.
In some variations, a negative pressure of between about 10 mm HG and about 760 mm HG may be applied to the outlet reservoir of the microfluidic chip 704. The applied negative pressure may remove the non-ECMB portion of the biological fluid from the microfluidic chip while the ECMBs remain disposed within the microfluidic chip. For example, as the fluid flows through the microfluidic chip, the shape of the ECMBs within the fluid is modified by contact (e.g., bending) with the obstructions (e.g., pillars) disposed within the microfluidic chip to facilitate immobilization of ECMBs within the microfluidic chip (e.g., attachment of ECMBs to the pillars).
In some variations, the ECMBs in the microfluidic chip may be processed 706. For example, the ECMBs in the microfluidic chip may be applied with one or more of a histochemical stain, an immunohistochemical (IHC) stain, a multiplex IHC stain, multi-spectral imaging, a protein stain, a nucleic acid stain, chemical fixation, and a protease inhibitor. For example, human plasma may be stained with hematoxylin and eosin. As described in more detail herein, histochemical staining may be used in any of the analytical methods described above.
In some variations, one or more predetermined antibodies (e.g., for extracellular matrix materials, extracellular vesicle markers), washes, and reagents may be applied to the microfluidic chip to help enable IHC staining. For example, a plurality of antibodies (e.g., extracellular matrix, cancer-related markers, extracellular vesicle markers) may be applied to the microfluidic chip IHC for multispectral imaging.
In some variations, successive staining of an isolate fraction on the microfluidic chip may help remove the material within the microfluidic chip. For example, antibody staining of a sample may generate a first spatial arrangement on the microfluidic chip. If the microfluidic chip is subsequently stained (e.g., with a different set of reagents), a second spatial arrangement different from the first spatial arrangement may be generated due to movement of the sample within the microfluidic chip. Thus, a useful comparison between the first and second spatial arrangements may be challenging. Accordingly, chemical fixation (e.g., crosslinking) of biological fluid (e.g., ECMBS) on the chip may be applied to immobilize the biological fluid in the microfluidic chip. For example, the microfluidic chip may receive a carbodiimide fixative and aldehyde fixative configured to crosslink the biological fluid to immobilize the biological fluid on the microfluidic chip. In some variations, an inlet and outlet of the microfluidic chip may be sealed.
In some variations, one or more biomarkers in one or more of the biological fluids and the ECMBs may be measured by one or more of immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, and UV-VIS.
In some variations, the biological fluid may be analyzed as a total biological fluid fraction prior to transferring the biological fluid 708. In some variations, the ECMBs remaining in the microfluidic chip may be analyzed as an isolate fraction after applying the negative pressure 710. In some variations, the biological fluid removed from the microfluidic chip may be analyzed as an eluate fraction after applying the negative pressure 712. In some variations, the biological fluid removed from the microfluidic chip may be processed using one or more of microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, mesh filtration, ultrafiltration, diafiltration, tangential flow filtration, membrane filtration, immuno-affinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC electrokinetics.
In some variations, the biological fluid removed from the microfluidic chip (e.g., eluate fraction, total fluid, trapped material) may be analyzed using one or more of microscopy, microfluidic device, mass spectrometry, microarray, nucleic acid amplification, hybridization, proteomic profiling, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, fluorescent in situ hybridization (FISH), chelate complexation, quantitative HPLC, spectrophotometry, colorimetric assay, chemiluminescence assay, immunofluorescence assay, light scattering, antibody array, Western blot, immunoassay, immunoprecipitation, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS/MS, RT-PCR, and quantitative PCR.
Although the foregoing variations have, for the purposes of clarity and understanding, been described in some detail by illustration and example, it will be apparent that certain changes and modifications may be practiced and are intended to fall within the scope of the appended claims. Additionally, it should be understood that the components and characteristics of the systems and devices described herein may be used in any combination. The description of certain elements or characteristics with respect to a specific figure are not intended to be limiting or nor should they be interpreted to suggest that the element cannot be used in combination with any of the other described elements. For all of the variations described herein, the steps of the methods may not be performed sequentially. Some steps are optional such that every step of the methods may not be performed.
This application claims the benefit of U.S. Provisional Application No. 63/608,790, filed Dec. 11, 2023, the content of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63608790 | Dec 2023 | US |
