Systems, apparatus and methods for microfluidic fluid analysis

Abstract

Systems, apparatus, and methods for high-throughput and automated separation of extracellular matrix bodies (ECMB) using microfluidic chips are described herein. A system for separating extracellular matrix bodies (ECMB) from a biological fluid may include: a holder configured to contain 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 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 between about 10 mm HG and about 760 mm HG to the at least one microfluidic chip.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 608,790, filed December 11, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The apparatus, systems and methods described herein relate to the isolation and / or analysis of extracellular matrix bodies (ECMB) from biological fluids. Background Technology

[0004] Conventional methods for disease diagnosis and prognosis include the isolation and analysis of microfractions of biological samples (e.g., biofluids), where, for example, individual cells, extracellular matrix, extracellular vesicles, or soluble tissue microenvironments, proteins, and nucleic acid molecules can be detected and analyzed. For example, the inventors have previously described methods using microfluidic chips and positive pressure to detect and analyze molecules. However, disadvantages of such positive pressure systems include low throughput, limited scalability, and reproducibility. Additionally, prior systems often require excessive manual labor. For example, the user needs to pipette or manually inject each biological sample and / or reagent set into the corresponding inlet of the microfluidic chip using a syringe. Furthermore, positive pressure systems rely on numerous tubing channels to deliver samples to the microfluidic chip, and therefore, the liquid volume per microfluidic chip may be inconsistent. Moreover, these systems tend to suffer from high contamination rates because they require time-intensive manual labor for setup and cleaning. Other conventional methods for disease diagnosis and prognosis include the isolation and analysis of intact organ tissues, whole cells, biomarkers (e.g., extracellular vesicles such as exosomes), etc. However, a drawback of such methods is that they cannot detect or characterize disease when the separated structures do not readily reflect the disease state. For example, biomarkers are often inherently limited by not being directly related to the pathology of interest. Therefore, alternative systems, devices, and methods are needed for the separation and analysis of biological fluids. Summary of the Invention

[0005] Systems, apparatus, and methods for the automated and high-throughput separation of extracellular matrix bodies (ECMB) from biofluids or indirectly from tissues or gels are described herein. In this manner, ECMB can be used, for example, for the diagnosis, prognosis, and / or treatment of subjects using histochemical staining techniques, immunohistochemistry, or nucleic acid hybridization and analysis. Generally, the systems described herein for separating extracellular matrix bodies from biofluids may include: a holder configured to contain the biofluid; a robot configured to transfer the biofluid 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 between about 10 mm HG and about 760 mm HG to at least one microfluidic chip.

[0006] In some variations, the chip connector may include a base and a cap, the base being configured to contact a bottom portion of at least one microfluidic chip, and the cap being configured to contact a top portion of at least one microfluidic chip. In some variations, the chip connector may be configured to distribute compressive force applied by the negative pressure to the periphery of the microfluidic chip. In some variations, the bottom portion may comprise the periphery of at least one microfluidic chip. In some variations, the cap may define a plurality of holes. In some variations, the base may include a first fastener, and the cap may include a second fastener. The first and second fasteners 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 cap and the at least one microfluidic chip.

[0007] In some variations, at least one outlet connector may include an elongated body defining a lumen and including multiple steps along the length of the elongated body. In some variations, at least one outlet connector may include an elongated body defining a lumen whose inner diameter decreases in a distal direction. In some variations, one or more of the microfluidic chips, the inlet connector, and the outlet connector may include disposable components. In some variations, the chip connector may include durable components.

[0008] In some variations, the holder may be configured to contain one or more reagents, and the robot may be 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. In some variations, 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 microfluidic chips.

[0009] In some variations, at least one microfluidic chip may include at least one confining channel fluidly coupled between an inlet and an outlet of the microfluidic chip. In some variations, the at least one confining channel may include at least one obstruction. In some variations, the at least one confining channel may have a length between about 5 mm and about 30 mm. In some variations, each channel may have a cross-sectional dimension between about 5 µm and about 30 µm. In some variations, at least one microfluidic chip may include at least one obstruction configured to confine fluid flow. In some variations, at least one obstruction may include a strut.

[0010] In some variations, the at least one microfluidic chip may include a confined region configured to hold a first fraction of the biofluid and allow fluid flow in a second fraction of the biofluid. In some variations, the first fraction may include the ECMB.

[0011] In some variations, the confined region may include a plurality of barriers configured to hold the first segment. In some variations, the spacing between the plurality of barriers in the confined region decreases along the length of the microfluidic chip from the inlet to the outlet of the confined region. In some variations, the spacing between the plurality of barriers in the confined region is between about 100 µm and about 4 µm. In some variations, each of the plurality of barriers has a diameter between about 50 µm and about 1 mm.

[0012] A method for isolating extracellular matrix (ECMB) from a biofluid is also described herein. Generally, the method comprises transferring the biofluid to an inlet reservoir of a microfluidic chip and applying a negative pressure to the microfluidic chip. The microfluidic chip may include at least one confining channel having an inlet and an outlet. The inlet reservoir may be fluidly coupled to the inlet of the at least one confining channel, as well as at least one support and an outlet reservoir. A negative pressure between about 10 mm HG and about 760 mm HG may be applied to the outlet reservoir of the microfluidic chip, wherein the ECMB remains in the microfluidic chip after the biofluid has been removed from the microfluidic chip.

[0013] In some variations, the compressive force applied by the negative pressure can be distributed from the outlet reservoir to the periphery of the microfluidic chip. In some variations, the at least one confining channel can include the at least one strut. In some variations, the at least one confining channel can have a length between about 5 mm and about 30 mm. In some variations, the at least one confining channel can have a cross-sectional dimension between about 5 µm and about 30 µm. In some variations, the at least one microfluidic chip can include at least one obstruction configured to restrict fluid flow. In some variations, the at least one microfluidic chip can include a confined region configured to hold a first fraction of the biofluid and allow fluid flow in a second fraction of the biofluid. In some variations, the confined region can include a plurality of obstructions configured to hold the first fraction. In some variations, the spacing between the plurality of obstructions in the confined region can decrease along the length of the microfluidic chip from the inlet to the outlet of the confined region. In some variations, the spacing between the plurality of obstacles in the confined area may be between about 100 µm and about 4 µm. In some variations, each of the plurality of obstacles may have a diameter between about 50 µm and about 1 mm.

[0014] Once ECMBs are isolated from the biofluid, other methods can be used to analyze them. For example, in some variations, one or more of the following can be applied to the ECMBs in the microfluidic chip: histochemical staining (including immunohistochemical (IHC) staining and multiplex IHC staining), protein staining, nucleic acid staining, chemical fixation, and protease inhibitors. In some variations, one or more biomarkers of the biofluid and the ECMBs can be measured by one or more of the following: immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, and UV-VIS.

[0015] In some variations, one or more of the following can be used to analyze the biofluid removed from the microfluidic chip: microscopy, microfluidic devices, mass spectrometry, microarrays, nucleic acid amplification, hybridization, proteomic analysis, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next-generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, fluorescence in situ hybridization (FISH), chelation complexation, quantitative HPLC, spectrophotometry, antibody arrays, Western blotting, immunoassay, immunoprecipitation, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS / MS, RT-PCR, and quantitative PCR.

[0016] In some variations, one or more of the following can be used to process the biofluid removed from the microfluidic chip: microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, sieve filtration, percolation, tangential flow filtration, membrane filtration, immunoaffinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC electrokinetics.

[0017] In some variations, the biological fluid may comprise one or more of the following: whole blood, plasma, serum, cerebrospinal fluid, intrathecal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspiration, semen, amniotic fluid, vitreous humor, aqueous humor, lymph, bile, cerumen, chyle, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, pus, inflammatory secretions, sebum, serous fluid, smegma, phlegm, synovial fluid, vaginal secretions, menstrual discharge, and vomitus, as well as fluids that pass through one or more of tissues and gels. Attached Figure Description

[0018] Figure 1 It is a block diagram illustrating a variant of the system.

[0019] Figure 2A-2C A perspective view depicting an illustrative variation of the system is provided. Figure 2D Depicting Figure 2C The system shown is a side view.

[0020] Figure 3A A plan view depicting an illustrative variant of a microfluidic chip. Figure 3B Depicting Figure 3A The image shows a cross-sectional side view of the microfluidic chip.

[0021] Figure 3CA plan view depicting an illustrative variation of a microfluidic chip array.

[0022] Figure 4A A perspective view depicting an illustrative variation of the chip connector is provided. Figure 4B Depicting Figure 4A An exploded perspective view of the cap and microfluidic chip shown in the image. Figure 4C Depicting Figure 4A The image shows an exploded top perspective view of the base and the microfluidic chip. Figure 4D Depicting Figure 4A The image shows an exploded bottom perspective view of the base and microfluidic chip.

[0023] Figures 5A-5E The illustrative variations of the export connector are depicted in the corresponding top perspective view, bottom perspective view, side view, top view, and bottom view.

[0024] Figures 6A-6D The illustration shows the corresponding top perspective view, bottom perspective view, top view, and bottom view of the export connector.

[0025] Figure 7 A flowchart depicting the illustrative changes in the separation of ECMB from biological fluids is provided.

[0026] Figure 8A and 8B This is an illustrative diagram of ECMB quantity based on microfluidic channel size.

[0027] Figure 8C-8E These are illustrative images of ECMB on microfluidic chips with different channel sizes.

[0028] Figure 9 This is an illustrative diagram showing the fluid flow rate through a microfluidic chip with and without a chip connector.

[0029] Figure 10A This is an illustrative diagram showing the setup times for various export connector configurations. Figure 10B This is an illustrative diagram showing the experimental duration of staining procedures for both positive and negative pressure systems. Figure 10C It is an illustrative diagram of the abnormal configurations of various export connectors.

[0030] Figure 11 This is an illustrative diagram showing the number of parallel experiments conducted on positive and negative pressure systems.

[0031] Figure 12A and 12B This is an illustrative image of biocontamination in a positive pressure microfluidic system. Figure 12C and 12D This is an illustrative image of biofouling using the negative pressure system described in this article. Figure 12E It is an illustrative diagram of biological contamination per unit area for positive and negative pressure systems.

[0032] Figure 13A and 13B This is an illustrative diagram of ECMB volume based on negative pressure. Figure 13C and 13D These are illustrative images of ECMB on a microfluidic chip under different negative pressures.

[0033] Figure 14A , 14C 14D, 14E, and 14I are illustrative diagrams of ECMB quantities based on manifold ports. Figure 14B and 14J This is an illustrative diagram based on the ECMB volume in the pillar region. Figure 14F , 14G 14H are illustrative images of ECMB on a microfluidic chip with different negative pressures applied.

[0034] Figure 15A This is an explanatory table of ECMB quantities based on systems with and without manifolds. Figure 15B This is an illustrative diagram based on the pillar region and the ECMB quantities of systems with and without manifolds.

[0035] Figure 16A and 16B This is an illustrative diagram of ECMB volume based on negative pressure.

[0036] Figure 17A This is an illustrative image of ECMB on a microfluidic chip filled with a sample of aqueous humor from a healthy (control) person.

[0037] Figure 17B This is an illustrative diagram of ECMB levels on a microfluidic chip that falls between the control sample and the primary open-angle glaucoma (POAG) sample.

[0038] Figure 18A A plan view depicting another illustrative variant of the microfluidic chip is shown. Figure 18B Depicting Figure 18A The image shows a perspective view of a microfluidic chip. Figure 18C Depicting Figure 18B The image shows a perspective view of the microfluidic chip coupled to the cap. Figure 18D Depicting Figure 18A The image shows a detailed plan view of the microfluidic chip.

[0039] Figure 19A A plan view depicting an illustrative variant of a multichannel microfluidic chip is shown. Figure 19B Depicting Figure 19A The image shows a detailed plan view of the microfluidic chip. Figure 19C A plan view depicting an illustrative variation of a multichannel microfluidic chip array.

[0040] Figure 20A A plan view depicting another illustrative variant of the microfluidic chip is shown. Figure 20B Depicting Figure 20A The image shows a detailed plan view of the microfluidic chip. Figure 20C A plan view depicting an illustrative variation of a microfluidic chip array. Figure 20D Depicting Figure 20A The image shows a perspective view of the microfluidic chip array coupled to the cap. Figure 20E Depicting Figure 20D The lid is shown in a plan view. Figure 20F Depicting Figure 20D The image shows a plan view of the microfluidic chip array and the cap.

[0041] Figure 21 A plan view depicting another illustrative variant of a multichannel microfluidic chip is shown.

[0042] Figure 22 A perspective view depicting another illustrative variation of the chip connector is shown.

[0043] Figure 23 A perspective view depicting an illustrative variation of a multi-chip connector.

[0044] Figures 24A-24D Perspective, top, bottom, and side views depicting an illustrative variation of the inlet connector.

[0045] Figures 25A-25D Perspective, top, bottom, and perspective side views depict another illustrative variation of the inlet connector.

[0046] Figures 26A-26D Perspective views, top views, bottom views, bottom perspective views, and side views of another illustrative variation of the inlet connector are depicted.

[0047] Figures 27A-27D Perspective, top, bottom, and side views depicting an illustrative variation of the export connector.

[0048] Figures 28A-28D Perspective, top, and bottom views depict another illustrative variation of the export connector.

[0049] Figure 29A It is an illustrative image of fluid flow through a single-channel microfluidic chip. Figure 29B This is an illustrative image of fluid flow through the channels of an 8-channel microfluidic chip.

[0050] Figure 30A This is an illustrative diagram of the non-laminar flow region based on single-channel and multi-channel microfluidic chips. Figure 30B This is an illustrative diagram of the clogging rate based on single-channel and multi-channel microfluidic chips. Figure 30C This is an illustrative diagram of background noise based on single-channel and multi-channel microfluidic chips.

[0051] Figure 31A This is an illustrative image of staining artifacts that may be caused by non-laminar flow passing through a single-channel microfluidic chip. Figure 31B This is an illustrative diagram of the artifact size based on single-channel and multi-channel microfluidic chips.

[0052] Figure 32 This is an illustrative diagram of ECMB quantities on microfluidic chips, based on inlet connectors with and without silicone. Detailed Implementation

[0053] Systems, apparatus, and methods for isolating ECMB from biofluids (e.g., for analysis, etc.) are described herein. For example, the systems, apparatus, and methods described herein can be used to diagnose, predict, and / or treat subjects by: isolating, enriching, extracting, and / or immobilizing predetermined fractions (e.g., ECMB, proteins, nucleic acids, particles, materials, molecules, extracellular vesicles, or soluble tissue microenvironments, proteins) from biofluids into biofluid fractions (e.g., isolate fractions) while preserving the fraction's composition and properties; increasing the yield of fractions with reduced contamination from biofluids to thereby increase the signal intensity of the pathology of interest; identifying one or more fractions within the fractions using disease-associated biomarkers (e.g., proteins, genes, ECMB components) for disease target identification; and predicting disease risk and / or medical symptoms based on analysis of multiple biofluid fractions.

[0054] Typically, the systems and devices described herein can separate ECMBs from biofluids in a manner that maintains the composition and properties of the ECMB, facilitating their use as biomarkers for disease diagnosis and / or monitoring of chemical or biological processes. For example, the systems and devices described herein can be further used for: high-throughput, scalable, and automated microfluidic processing of histological biofluids; reduced compression of microfluidic chips due to negative pressure applied based on dispensing pressure; reduced clogging and increased laminar flow, flow consistency, and signal-to-noise ratio based on microfluidic chip channel density; reduced risk of contamination, setup time, manual handling, and cleaning; visualization of ECMBs using fixation and staining (e.g., histochemistry, immunohistochemistry) for one or more spatial localization and spectral analyses (e.g., protein, gene); and facilitating the identification of diseased ECMBs and physiologically normal ECMBs. Therefore, biofluid separation and histochemical analysis as described herein can be performed faster, cheaper, and with higher throughput (e.g., volume) compared to positive pressure microfluidic systems.

[0055] Typically, the systems and apparatus described herein can include microfluidic systems configured for bioassays of ECMB in biological fluids. For example, a microfluidic system can include multiple disposable microfluidic chips (e.g., an array of microfluidic chips) configured to receive biological fluids (e.g., samples) from a robot, thereby facilitating automated and high-throughput processing. The system can be configured to separate ECMB from the biological fluid within the microfluidic chip using a negative pressure source coupled to the microfluidic chip. The separated ECMB can be exposed to one or more reagents and buffers for histochemical staining and subsequent imaging and / or analysis. Thus, the microfluidic system can process multiple biological samples in parallel.

[0056] For example, a system for separating extracellular matrix (ECMB) from a biofluid may include: a holder configured to contain the biofluid; one or more robots configured to transfer the biofluid 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 between about 10 mm HG and about 760 mm HG to the at least one microfluidic chip. In some variations, the system may include multiple holders, multiple chip connectors, multiple manifolds, multiple negative pressure sources, multiple reservoirs, multiple robots, etc.

[0057] The negative pressure system of the apparatus and system described herein facilitates high-throughput, low-contamination separation of ECMBs from biofluids. In this way, multiple samples can be processed (e.g., subjected to ECMB separation) and analyzed in parallel in an automated manner, thereby significantly reducing the time and effort associated with positive pressure microfluidic systems and methods. For example, negative pressure can be uniformly distributed in parallel across multiple microfluidic chips using manifolds to provide consistent fluid flow and reproducible results. Furthermore, the negative pressure applied at the outlet of the microfluidic chip makes the inlet of the microfluidic chip accessible for robotic fluid transfer, thereby improving throughput and consistency and reducing manual labor. However, modifying a positive pressure microfluidic system to apply negative pressure to the microfluidic chip and sample will impair the composition and properties of the biofluid, as well as the structural integrity of the microfluidic chip, thus hindering the separation, enrichment, and / or extraction of the biofluid into usable biofluid fractions. In other words, without the innovations described herein, applying negative pressure to positive pressure microfluidic chips and systems will produce unpredictable results.

[0058] Biological fluids may include one or more of the following: human or animal body fluids, tissues, cells, whole blood, plasma, serum, cerebrospinal fluid, intrathecal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric juice, peritoneal fluid, breast milk, nipple aspiration, semen, amniotic fluid, vitreous humor, aqueous humor, lymph, bile, cerumen, chyle, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, pus, inflammatory secretions, sebum, serous fluid, smegma, phlegm, synovial fluid, vaginal secretions, menstrual discharge, vomitus, tumors, carriers, reagents, solutions, binding components, etc.

[0059] Buffer solutions may contain one or more of the following: MES, HCl acidic buffer, acidic phthalate buffer, basic borate buffer, acetate buffer, ammonium acetate buffer, acetone buffer, ammonia buffer, barbiturate buffer, buffered copper sulfate solution, glycerol solution, glycine buffer, palladium chloride buffer, citrate Na2HPO4, citrate-sodium citrate buffer preparation, sodium acetate-acetate 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, etc.

[0060] International patent application No. PCT / US2021 / 023827, entitled “DEVICE AND METHODS FOR ISOLATING EXTRACELLULAR MATRIX BODIES”, filed on March 24, 2021, and international patent application No. PCT / US2019 / 052310, entitled “COMPOSITIONS AND METHODS FOR GLAUCOMA”, filed on September 21, 2019, describe some microfluidic chips and systems suitable for the systems described herein, each of which is hereby incorporated by reference in its entirety.

[0061] I. Systems and Devices

[0062] Typically, the systems and apparatus described herein can provide high-throughput separation of extracellular matrix bodies (ECMBs) from biofluids. The separated ECMBs and the remaining biofluid (or portions or fractions thereof) can then be used for diagnosis, prognosis, or to help determine treatment plans for subjects and the effectiveness of such plans. Figure 1A block diagram of an exemplary system 100 is depicted herein. System 100 may include one or more of the following: a holder 112, a storage device 113, a robot 114, at least one microfluidic chip 116, a chip connector 118, a manifold 120, a negative pressure source 122, one or more sensors 124, an input device 126, a processor 128, a memory 130, a communication device 132, and an output device 134, each of which is described in more detail herein.

[0063] In some variations, the holder 112 (e.g., a material storage device) may be configured to store one or more fluids (e.g., biofluids) for transfer to the microfluidic chip 116. The holder 112 may comprise one or more of a tray and a container. In some variations, the reservoir 113 may be configured to contain predetermined fractions of biofluids (e.g., waste liquids) from the microfluidic chip 116. For example, non-ECMB fractions of the biofluid may be transferred and stored in the reservoir 113 for further processing (e.g., separation, analysis) and / or disposal. Alternatively or additionally, the system 100 may include one or more pretreatment components, such as material separation assemblies, aliquot assemblies, biobanks, etc.

[0064] In some variations, robot 114 may be configured to transfer fluid (e.g., biofluid, reagent) from holder 112 to at least one microfluidic chip 116. In some variations, microfluidic chip 116 may be configured to contain and process biofluid. For example, microfluidic chip 116 may include channels (e.g., microfluidic channels) and at least one barrier (e.g., a strut) configured to separate and retain (e.g., trap) predetermined fractions of biofluid within microfluidic chip 116, while remaining fractions flow out of microfluidic chip 116. In some variations, chip connector 118 may be configured to hold at least one microfluidic chip 116. Microfluidic chip 116 may be releasably coupled to chip connector 118 to facilitate cleaning and help reduce setup time. For example, microfluidic chip 116 may be a disposable component, and chip connector 118 may be a durable component. In some variations, the chip connector 118 may include a disposable connector (e.g., an inlet connector, an outlet connector) configured to facilitate the application of negative pressure from the negative pressure source 122 to the microfluidic chip 116 in order to infuse and allow the biofluid to flow through the microfluidic chip 116.

[0065] In some variations, manifold 120 may be configured to be coupled to at least one microfluidic chip 116. In some variations, negative pressure source 122 may be configured to be coupled to manifold 120. In this way, a single manifold 120 can be fluidly coupled to multiple microfluidic chips 116 to facilitate the application of negative pressure (e.g., vacuum, suction) to multiple microfluidic chips 116 using a single negative pressure source 122.

[0066] In some variations, sensor 124 may include 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 biofluids and components of systems 100, 200. In some variations, input device 126 may be configured to generate input signals based on operator input. In some variations, processor 128 and memory 130 may be configured to control system 100, 200. In some variations, communication device 132 may be configured to communicate with one or more components of system 100, 200, as well as with networks and other computer systems. In some variations, output device 134 may be configured to output data corresponding to system 100, 200, such as images, flow rates, etc., of the biofluids within microfluidic chip 116.

[0067] In some variants, systems 100 and 200 can be configured to perform a variety of assays, such as ELISA, immunoassays, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, UV-VIS, Raman, NMR, mass spectrometry, NG sequencing, protein arrays, ribonucleic acid arrays, gene arrays, qPCR, RT-qPCR, and RT-PCR. In some variants, the microfluidic chip 116 can be analyzed by systems 100 and 200 and / or analyzed and removed using another device (e.g., a multi-well plate or microscope slide). Examples of imaging techniques include electron microscopy, stereomicroscopy, wide-field microscopy, bright-field microscopy, phase-contrast microscopy, polarization microscopy, phase-contrast microscopy, multiphoton microscopy, differential interference phase-contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, X-ray microscopy, ultrasound microscopy, positron emission tomography, computed tomography, and magnetic resonance imaging.

[0068] Figure 2A and 2BThis is a perspective view of system 200, which includes a holder 212, a reservoir 213, a robot 214, an end effector 215 (e.g., a pipette) coupled to the robot 214, multiple chip connectors 218, each holding multiple microfluidic chips 216, a manifold 220 including multiple fluid conduits 221a, 221b, and a negative pressure source 222. System 200 may include, as described above... Figure 1 The sensor, input device, processor, memory, communication device, and output device are described, but for clarity, they are not listed. Figure 2A-2D As shown in the diagram. In some variations, the end effector 215 may include multiple pipettes configured to transfer fluid stored in the retainer 212 to a microfluidic chip 216 retained in a corresponding chip connector 218. In some variations, the system 200 may include multiple robots 214. For example, a first robot may be configured to transfer biological fluids, a second robot (not shown for clarity) may be configured to transfer reagents, and a third robot (not shown for clarity) may be configured to releasably couple the microfluidic chip 216 to the corresponding chip connector 218. The third robot may be configured to assemble and disassemble disposable components including the microfluidic chip 216 from durable components of the system 200 (e.g., chip connector 218) to reduce manual labor and / or contamination. Figure 2A In this configuration, each microfluidic chip 216 is coupled to a corresponding fluid conduit 221a, and each of the fluid conduits is coupled to a manifold 220. A single fluid conduit 221b can couple the manifold 220 to a reservoir 213 and a negative pressure source 222.

[0069] In some variations, a robot 214 can be used to load one or more fluids from a holder 212 into corresponding inlets of multiple microfluidic chips 216. A negative pressure source 222 can apply suction to each microfluidic chip 216 via a manifold 220 and fluid conduits 221a, 221b. Fluid at the inlet (e.g., an inlet reservoir) can be drawn toward the corresponding outlet of the microfluidic chip 216, while ECMB remains within the microfluidic chip 216. Separated non-ECMB fluid is drawn through fluid conduits 221a, 221b and manifold 220 and contained in a reservoir 213 (e.g., waste disposal).

[0070] Figure 2C It is a detailed perspective view of System 200, and Figure 2D This is a detailed side view of the system, which includes an end effector 215 (e.g., a pipette), a microfluidic chip 216, a fluid conduit 221a, an inlet connector 240, an outlet connector 242, and optionally a clamp 250 and a funnel 260. For clarity, in Figure 2C and2D Chip connector 218 and robot 214 are not shown.

[0071] An inlet connector 240 may be coupled between the 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 a robot 214. For example, the funnel 260 may be configured to receive and / or guide fluid transferred from the end effector 215 to the microfluidic chip 216. An outlet connector 242 may be coupled between the outlet 232 of the microfluidic chip 216 and a fluid conduit 221a. In some variations, the fluid conduit 221a may be configured to receive fluid transferred from the microfluidic chip 216 to a reservoir 213 (e.g., by negative pressure).

[0072] In some variations, one or more grippers 250 may be configured to releasably couple (e.g., fix, hold) the inlet connector 240 and the outlet connector 242 to the microfluidic chip 216, respectively. For example, the gripper 250 may include one or more springs (not shown) and hinges 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 of the gripper 250 (e.g., an actuator) may be actuated (e.g., pushed downwards by a robot or operator) to release one or more of the inlet connector 240 and outlet connector 242 to facilitate removal of the microfluidic chip 216 from the chip connector 218.

[0073] A. Holder

[0074] The system described herein may include holders 112, 212 configured to contain one or more fluids. In some variations, holders 112, 212 may be configured to contain and store multiple fluids, including biological fluids (e.g., samples) and one or more reagents. Examples of reagents include buffers, lysis solutions, nucleic acid cleavage agents, cleavage inhibitors, precipitants, immobilization reagents, carrier fluids, biological fluids, water, purified water, saline solutions, organic solvents, gelling agents, surfactants, ligands for binding or associating with components of ECMB, combinations thereof, and reagents for interacting with biological components of the sample. In some variations, reagents may include one or more reagents for measuring the level or amount of a biomarker, or for comparing biomarker levels with controls. In some variations, holders 112, 212 may include multiple reservoirs to store each fluid separately without mixing. Fluids may include any suitable fluid, including, for example, biological fluids for sampling, and / or one or more fluids that can be used for analysis. Biological fluids used for sampling may include one or more of the following: human or animal body fluids, tissues, cells, whole blood, plasma, serum, cerebrospinal fluid, intrathecal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric juice, peritoneal fluid, breast milk, nipple aspiration, semen, amniotic fluid, vitreous humor, aqueous humor, lymph, bile, cerumen, chyle, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, pus, inflammatory secretions, sebum, serous fluid, smegma, phlegm, synovial fluid, vaginal secretions, menstrual discharge, vomitus, tumors, carriers, reagents, solutions, binding components, etc. Biological fluids that can be used for analysis may include carriers, reagents, binding components, etc. Holders 112 and 212 can be placed in locations accessible to robots 114 and 214 within systems 100 and 200. For example, holders 112, 212 can be configured to house multiple pipettes coupled to robots 114, 214 to transfer multiple fluids from holders 112, 212 to multiple microfluidic chips 116, 216.

[0075] B. Robot

[0076] The system described herein may include one or more robots 114, 214. Typically, robots 114, 214 may be configured to move and transfer one or more fluids (e.g., biological fluids, reagents, antibodies, buffers) between holders 112, 212 and at least one microfluidic chip 116, 216. For example, robots 114, 214 may be configured to automatically load fluids into at least one microfluidic chip 116, 216 without manual (e.g., human) intervention. In some variations, robots 114, 214 may be coupled to an end effector 215 comprising one or more pipettes (e.g., micropipettes, multichannel pipettes) configured to transfer fluids (e.g., biological fluids, reagents) from holders 112, 212 to at least one microfluidic chip 116, 216 (e.g., through an inlet connector 240 of chip connectors 118, 218). For example, robots 114, 214 can be used to first transfer biofluids from the subject's holders 112, 212 to multiple microfluidic chips 116, 216. Then, robots 114, 214 can be used to transfer different reagents from the holders 112, 212 to predetermined microfluidic chips 116, 216 for different treatments (e.g., different histochemical staining). Robots 114, 214 can be coupled to processor 128 and memory 130 to control the type and volume of fluid transferred using one or more pipettes. In some variations, end effector 215 can be configured to releasably couple disposable components to durable components of system 200 (e.g., assembly, disassembly). Alternatively or additionally, system 100 may include one or more material transport components, such as tracks and containers configured to translate along the tracks.

[0077] In some variations, robots 114, 214 can be configured to removably couple microfluidic chips 116, 216 to one or more of chip connectors 118, 218 and negative pressure sources 122, 222. For example, robots 114, 214 can be configured to couple microfluidic chips 116, 216 to chip connectors 118, 218 such that microfluidic chips 116, 216 are fluidly coupled to manifolds 120, 220 and negative pressure sources 122, 222. Furthermore, robots 114, 214 can be configured to releasably couple other components (e.g., inlet connectors, outlet connectors) to microfluidic chips 116, 216. Conversely, robots 114, 214 can be configured to decouple microfluidic chips 116, 216 from chip connectors 118, 218 for transfer to another device (e.g., microscope slides, imaging systems, analysis systems). In some variations, robots 114, 214 can be configured to remove one or more of holders 112, 212 and reservoirs 113, 213 from systems 100, 200 (e.g., replacing them with different holders 112, 212 and reservoirs 113, 213). Automated system setup, fluid transfer, and cleaning using robots 114, 214 can reduce contamination and human error, and increase consistency and throughput, among other benefits.

[0078] In some variations, robots 114 and 214 may be, for example, linear robotic arms, articulated robotic arms, and / or SCARA robotic arms. Robots 114 and 214 may comprise one or more segments coupled together by joints (e.g., shoulder, elbow, wrist) configured to provide a single degree of freedom. A joint is a mechanism that provides a single translational or rotational degree of freedom. For example, robots 114 and 214 may have six or more degrees of freedom. The set of Cartesian degrees of freedom can be represented by three translational (position) variables (e.g., sway, heave, roll) and three rotational (orientation) variables (e.g., roll, pitch, torsion). In some variations, robots 114 and 214 may have fewer than six degrees of freedom.

[0079] In some variations, robots 114, 214 can be configured to move over all areas of systems 100, 200 in up to three dimensions. Robots 114, 214 may include one or more motors configured to translate and / or rotate joints and move robots 114, 214 to a desired position and orientation. In some variations, the robot's position may be temporarily locked when fluid is delivered to a predetermined microfluidic chip, or when one or more microfluidic chips 116, 216 are removed from chip connectors 118, 218 (e.g., for transfer to a microscope or other imaging system). Robots 114, 214 can be mounted to any suitable object, such as a platform (e.g., a table), wall, ceiling, or may be self-supporting (e.g., on the ground). Alternatively or additionally, robots 114, 214 can be configured for manual movement.

[0080] C. Microfluidic chip

[0081] The system described herein may include one or more microfluidic chips 116, 216. Typically, the microfluidic chips 116, 216 may be configured to contain and process one or more fluids. In some variations, the microfluidic chips 116, 216 may include an inlet reservoir, at least one channel (e.g., a confining channel, a uniform flow channel), at least one obstruction configured to restrict fluid flow (e.g., a support), and an outlet reservoir. The confining channel may include an inlet and an outlet. The inlet reservoir may be fluidly coupled to the inlet of the confining channel, and the outlet of the confining channel may be fluidly coupled to the outlet reservoir. The inlet reservoir may be configured to contain and store fluid transferred from the robot 114, 214. The microfluidic chips 116, 216 may be configured to process biofluids flowing through the channels while receiving negative pressure from the outlet of the microfluidic chips 116, 216 (e.g., the outlet reservoir).

[0082] Figure 3A and 3B These are corresponding plan views and cross-sectional side views of a microfluidic chip 316, which includes an inlet reservoir 310, an outlet reservoir 330, and a confined region 320 (e.g., a filtration region) in fluid communication with the inlet reservoir 310 and the outlet reservoir 330. The inlet reservoir 310 may include an inlet 312 (e.g., an opening) and at least one obstruction 340 (e.g., a support). Similarly, the outlet reservoir 330 may include an outlet 332 (e.g., an opening) and at least one obstruction 340. The size, shape, and spacing of the obstructions 340 in the inlet reservoir 310 and the outlet reservoir 330 may be the same or different. In some variations, fluid may be contained at the inlet 312 and flow through the confined region 320 toward the outlet 332. Coupled to a manifold and a negative pressure source ( Figures 3A-3CA fluid conduit (not shown) can be coupled to outlet 332. The suction applied by a negative pressure source through outlet 332 can draw the fluid contained at inlet 312 through inlet reservoir 310, restricted area 320 and outlet reservoir 330.

[0083] In some variations, the confined region 320 can be configured to restrict fluid flow so as to separate and retain (e.g., trap) a first fraction of fluid within the confined region 320 of the microfluidic chip 316, while allowing a second fraction of the remaining fluid to flow out of the microfluidic chip 316 by applying negative pressure to the microfluidic chip 316. In some variations, the first fraction of fluid may include an ECMB (e.g., an ECMB). Figure 3A As shown, the confined region 320 may include a flow barrier 322 defining a confining channel 324 (e.g., a uniform flow channel) and a plurality of obstacles 340 (e.g., struts) having a plurality of spacings 326. The confining channel 324 may be linear and its length may be at least equal to the length of the confined region 320. In some variations, one or more of laminar flow and flow consistency may be based on the length of the confining channel 324. For example, a confining channel 324 that does not extend into one or more of the inlet reservoir 310 and the outlet reservoir 330 may increase one or more of the laminar flow and flow consistency throughout the microfluidic chip (e.g., along the width and length of the microfluidic chip 316).

[0084] Multiple barriers 340 within the confined region 320 can be configured to restrict (e.g., impede) fluid flow and hold the first fraction of the biofluid. For example, the spacing between the multiple barriers 340 in the confined region 320 can decrease along the length of the microfluidic chip 316 from the inlet to the outlet of the confined region 320. In some variations, the spacing between the multiple barriers 340 in the confined region 320 can be between about 100 µm and about 4 µm. For example, the spacing between barriers 340 at the inlet of the confined region 320 can be about 100 µm, while the spacing between barriers 340 at the outlet of the confined region 320 can be about 4 µm. In some variations, the spacing can vary in a progressively increasing manner. For example, the spacing can decrease in the order from about 100 µm to about 50 µm to about 25 µm to about 15 µm and to about 4 µm. Alternatively or additionally, the spacing between the barriers 340 can vary continuously along the length of the confined region 320. In some variations, a larger proportion of ECMB can be captured within a smaller-spaced portion of the restricted region 320.

[0085] The length of the limiting channel 324 may be between approximately 5 mm and approximately 30 mm, between approximately 10 mm and approximately 30 mm, between approximately 15 mm and approximately 30 mm, between approximately 20 mm and approximately 30 mm, between approximately 5 mm and approximately 25 mm, between approximately 5 mm and approximately 20 mm, between approximately 15 mm, approximately 20 mm, approximately 25 mm, and approximately 30 mm, including all ranges and sub-values ​​therein. The cross-sectional dimensions of the limiting channel 324 may be between approximately 5 µm and approximately 30 µm, between approximately 10 µm and approximately 30 µm, between approximately 15 µm and approximately 30 µm, between approximately 20 µm and approximately 30 µm, between approximately 5 µm and approximately 25 µm, between approximately 5 µm and approximately 20 µm, between approximately 15 µm, approximately 20 µm, approximately 25 µm, and approximately 30 µm, including all ranges and sub-values ​​therein. In some variations, the restrictive channel 324 may include at least one obstacle. Multiple obstacles 340 may have diameters of approximately 50 µm and approximately 1 mm.

[0086] like Figure 3B As shown in the cross-sectional side view, the microfluidic chip 316 may include a substrate 350 coupled to a cap 360 (e.g., a top plate, a glass slide). The cap 360, positioned above and on top of the substrate 350, facilitates fluid processing through the microfluidic chip 316. For example, in addition to one or more inlets 312 and one or more outlets 332, the cap 360 may be configured to enclose the substrate 350 (e.g., including an inlet reservoir 310, a confined region 320, and an outlet reservoir 330). In some variations, the substrate 350 may include multiple microfluidic chips 316. For example, Figure 3C An array of eight microfluidic chips 316 arranged in parallel on a substrate 350 is shown. In this manner, a single microfluidic chip array 370 can be used to process multiple fluid samples separately (e.g., individually), thereby increasing throughput and efficiency, and reducing system size. For example, a microfluidic chip array 370 with eight microfluidic chips 316 enables independent ECMB separation of eight fluid samples using any combination of histological staining, immunohistochemical (IHC) staining, reagents, etc. In some variations, the substrate 350 may comprise suitable materials, including one or more silicon-based polymers (e.g., polydimethylsiloxane (PDMS)), other polymers, thermoplastics, thermoplastic elastomers, hydrogels, paper, and glass.

[0087] In some variations, the restricted channel can extend beyond the length of the restricted area. For example, Figure 18A and 18BCorresponding plan and perspective views of a microfluidic chip 1800 are depicted, the microfluidic chip including an inlet reservoir 1810, an outlet reservoir 1830, and a confined region 1820 (e.g., a filtration region) in fluid communication between the inlet reservoir 1810 and the outlet reservoir 1830. The inlet reservoir 1810 may include at least one inlet obstruction 1841, and the outlet reservoir 1830 may include at least one outlet obstruction 1843. The inlet reservoir 1810 and the outlet reservoir 1830 may have a generally circular shape. Figure 18C A microfluidic chip 1800 coupled to a cap 1802 is depicted. In some variations, the cap 1802 may include an inlet 1812 in fluid communication with an inlet reservoir 1810 of the microfluidic chip 1800 and an outlet 1832 in fluid communication with an outlet reservoir 1830. Multiple barriers 1840 may be positioned between the inlet reservoir 1810 and the outlet reservoir 1830. Similar to microfluidic chip 316, a confined region 1820 may be configured to restrict fluid flow to separate and retain (e.g., trap) a first fraction of fluid within the confined region 1820 of the microfluidic chip 1800, while allowing a second fraction of the remaining fluid to flow out of the microfluidic chip 1800 by a negative pressure applied to the microfluidic chip 1800. In some variations, the first fraction of fluid may include an ECMB. (See below for details.) Figure 18D In more detail, the confined region 1820 may include a flow barrier 1822 defining a confining channel 1824 (e.g., a uniform flow channel) and a plurality of obstacles 1840 (e.g., struts) having a plurality of spacings 1826. The confining channel 1824 may be linear and its length may be greater than the length of the confined region 1820. The confining channel 1824 may be configured to facilitate fluid flow through the microfluidic chip 1800 when a negative pressure is applied to the outlet 1832 of the microfluidic chip 1800. For example, the confining channel 1824 may reduce bubble formation in the microfluidic chip 1800.

[0088] Multiple barriers 1840 within the confined region 1820 can be configured to restrict (e.g., impede) fluid flow and impound the first fraction of the biological fluid for analysis (e.g., image analysis). For example, Figure 18DA detailed view of a confined region 1820 of a microfluidic chip 1800 is provided, the microfluidic chip including a fluid barrier 1822, a confining channel 1824, and a plurality of spacings 1821-1829. In some variations, the spacing between the plurality of barriers 1840 in the confined region 1820 may decrease along the length of the microfluidic chip 1800 from the inlet 1818 to the outlet 1819 of the confined region 1820. In some variations, the spacing between the plurality of barriers 1840 in the confined region 1820 may be between approximately 100 µm and approximately 4 µm. For example, the first spacing 1821 between barriers 1840 located at the inlet 1818 of the confined region 1820 can be approximately 100 µm, followed by a second spacing 1823 of approximately 50 µm, a third spacing 1825 of approximately 25 µm, a fourth spacing 1827 of approximately 15 µm, and a fifth spacing 1829 of approximately 4 µm located at the outlet of the confined region 1820. The length of each spacing and the diameter of the barriers 1840 can be the same or different. In some variations, the confined region 1820 can extend across the width of the microfluidic chip 1800, in addition to the fluid barrier 1822 and the confining channel 1824.

[0089] In some variations, microfluidic chips may include multiple confined channels to facilitate fluid flow and reduce bubble formation and clogging within the microfluidic chip. For example, Figure 19A and 19B A plan view of a multi-channel confined microfluidic chip 1900 is depicted, the multi-channel microfluidic chip including an inlet reservoir 1910, an outlet reservoir 1930, and a confined region 1920 (e.g., a filtration region) in fluid communication between the inlet reservoir 1910 and the outlet reservoir 1930. The inlet reservoir 1910 may include at least one inlet obstruction 1941, and the outlet reservoir 1930 may include at least one outlet obstruction 1943. The inlet reservoir 1910 and the outlet reservoir 1930 may have a generally circular shape. A plurality of obstructions 1940 may be disposed between the inlet reservoir 1910 and the outlet reservoir 1930. Similar to microfluidic chips 316 and 1800, the confined region 1920 can be configured to restrict fluid flow so as to separate and retain (e.g., trap) a first fraction of fluid within the confined region 1920 of the microfluidic chip 1900, while allowing a second fraction of the remaining fluid to flow out of the microfluidic chip 1900 by applying negative pressure to the microfluidic chip 1900. In some variations, the first fraction of fluid may include an ECMB (extracorporeal membrane oxygenation). Figures 19A-19C A microfluidic chip 1900 is depicted having multiple confinement channels 1924, 1926, 1928 (e.g., uniform flow channels), which may be spaced apart, linear, and parallel. For example, as per [reference to...] Figure 19BIn more detail, the confined region 1920 may include flow barriers 1922 defining corresponding confining channels 1924, 1926, 1928, and multiple obstacles 1940 (e.g., pillars) having multiple spacings 1921, 1923, 1925, 1927, 1929. The lengths of the confining channels 1924, 1926, 1928 may be greater than the length of the confined region 1920. The confining channels 1924, 1926, 1928 may have the same or different lengths relative to each other. Therefore, the multiple confining channels 1924, 1926, 1928 can be configured to facilitate fluid flow through the microfluidic chip 1800 while minimizing bubbles and blockage when a negative pressure is applied to the outlet reservoir 1930 of the microfluidic chip 1900. Furthermore, Figure 19C A microfluidic chip array 1902 comprising multiple multi-channel microfluidic chips 1900 is depicted. A single microfluidic chip array 1902 can be used to process multiple fluid samples separately (e.g., individually), thereby increasing throughput and efficiency, and reducing system size. In some variations, the microfluidic chip array may include multiple microfluidic chips with different configurations (e.g., including at least one microfluidic chip 316, at least one microfluidic chip 1800, at least one microfluidic chip 1900, etc.).

[0090] Multiple barriers 1940 within the confined area 1920 can be configured to restrict (e.g., impede) fluid flow and impound the first fraction of the biological fluid for analysis (e.g., image analysis). For example, Figure 19BA detailed view of a confined region 1920 of a microfluidic chip 1900 is provided, the microfluidic chip including a fluid barrier 1922, a first confining channel 1924, a second confining channel 1926, a third confining channel 1928, and a plurality of spacings 1921, 1923, 1925, 1927, and 1929. In some variations, the spacing between the plurality of barriers 1940 in the confined region 1920 may decrease along the length of the microfluidic chip 1900 from the inlet 1918 to the outlet 1919 of the confined region 1920. In some variations, the spacing between the plurality of barriers 1940 in the confined region 1920 may be between approximately 100 µm and approximately 4 µm. For example, the first spacing 1921 between barriers 1940 located at the inlet 1918 of the confined region 1920 can be approximately 100 µm, followed by a second spacing 1923 of approximately 50 µm, a third spacing 1925 of approximately 25 µm, a fourth spacing 1927 of approximately 15 µm, and a fifth spacing 1929 of approximately 4 µm located at the outlet 1919 of the confined region 1920. The length of each spacing and the diameter of the barriers 1840 can be the same or different. In some variations, the confined region 1920 can extend across the width of the microfluidic chip 1900, in addition to the fluid barrier 1922 and the confining channels 1924, 1926, 1928.

[0091] Figure 20A and 20B A plan view of a microfluidic chip 2000 is depicted, the microfluidic chip including an inlet reservoir 2010, an outlet reservoir 2030, and a confined region 2020 (e.g., a filtering region) in fluid communication between the inlet reservoir 2010 and the outlet reservoir 2030. The inlet reservoir 2010 and the outlet reservoir 2030 may include at least one inlet obstruction 2040. The inlet reservoir 2010 and the outlet reservoir 2030 may have a generally polygonal shape.

[0092] Multiple barriers 2040 may be positioned between the inlet reservoir 2010 and the outlet reservoir 2030. Similar to microfluidic chips 316, 1800, and 1900, a confined region 2020 may be configured to restrict fluid flow to separate and retain (e.g., trap) a first fraction of fluid within the confined region 2020 of the microfluidic chip 1800, while allowing a second fraction of the remaining fluid to flow out of the microfluidic chip 2000 by applying negative pressure. In some variations, the first fraction of fluid may include an ECMB. (See related information...) Figure 20BIn more detail, the confined region 2020 may include a flow barrier 2022 defining a confining channel 2024 (e.g., a uniform flow channel) and a plurality of obstacles 2040 (e.g., pillars) having a plurality of spacings 2021, 2023, 2025, 2027, 2029. The confining channel 2024 may be linear and its length may be at least equal to the length of the confined region 2020. The confining channel 2024 may be configured to facilitate fluid flow through the microfluidic chip 2000 when a negative pressure is applied to the outlet 2032 of the microfluidic chip 2000. For example, the confining channel 2024 may reduce bubble formation in the microfluidic chip 2000.

[0093] Multiple barriers 2040 within the confined area 2020 can be configured to restrict (e.g., impede) fluid flow and impound the first fraction of the biological fluid for analysis (e.g., image analysis). For example, Figure 20B A detailed view of a confined region 2020 of a microfluidic chip 2000 is provided, the microfluidic chip including a fluid barrier 2022, a confining channel 2024, and multiple spacings 2021, 2023, 2025, 2027, and 2029. In some variations, the spacing between the multiple barriers 2040 in the confined region 2020 may decrease along the length of the microfluidic chip 2000 from the inlet 2018 to the outlet 2019 of the confined region 2020. In some variations, the spacing between the multiple barriers 2040 in the confined region 2020 may be between approximately 100 µm and approximately 4 µm. For example, the first spacing 2021 between barriers 2040 located at the entrance 2018 of the confined region 2020 can be approximately 100 µm, followed by a second spacing 2023 of approximately 50 µm, a third spacing 2025 of approximately 25 µm, a fourth spacing 2027 of approximately 15 µm, and a fifth spacing 2029 of approximately 4 µm located at the exit of the confined region 2020. The length of each spacing and the diameter of the barriers 2040 can be the same or different. In some variations, the confined region 2020 can extend across the width of the microfluidic chip 2000, in addition to the fluid barrier 2022 and the confining channel 2024.

[0094] Figure 20C A plan view of a microfluidic chip array 2002 comprising multiple microfluidic chips 2000 is depicted. A single microfluidic chip array 2002 can be used to process multiple fluid samples separately (e.g., individually), thereby increasing throughput and efficiency, and reducing system size. Figure 20D A microfluidic chip array 2002 coupled to a cover 2004 is depicted. In some variations, the cover 2004 may include an inlet 2012 in fluid communication with an inlet reservoir 2010 of a corresponding microfluidic chip 2000 and an outlet 2032 in fluid communication with an outlet reservoir 2030. Figure 20E Depicting Figure 20D The diagram shows a plan view of the cap 2004. For example, the inlet 2012 and outlet 2032 of the cap 2004 may correspond to each corresponding microfluidic chip 2000 of the microfluidic chip array 2002. In some variations, the distance between adjacent inlets 2012 may be based on the diameter of the end effector (e.g., a pipette). For example, the distance between adjacent inlets 2012 may be between about 5 mm and about 15 mm, between about 7 mm and about 13 mm, between about 8 mm and about 12 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, and all ranges and sub-values ​​therein. Figure 20F Depicting Figure 20D The image shows a plan view of the microfluidic chip array 2002 and the cover 2004.

[0095] The microfluidic chips described herein can comprise a single channel (e.g., microfluidic chips 1800, 1900, 2000) or multiple channels (e.g., microfluidic chip array 2002). In some variations, unexpected, surprising, and significant improvements can be found in one or more of laminar flow, flow consistency, congestion, signal-to-noise ratio, and artifacts, based on the channel density of the microfluidic chip (e.g., a single-channel microfluidic chip compared to a multi-channel microfluidic chip array). For example, Figure 29A It is a fluorescence image of fluid flow through a single-channel microfluidic chip 2900, and Figure 29B These are fluorescence images corresponding to the fluid flow through the channels of the 8-channel microfluidic chip array 2950. Specifically, Figure 29A and 29B Corresponding confined regions 2020 and 2070, including multiple barriers 2940, confining channels 2924 and 2974, and fluid barriers 2940 and 2990, are depicted. For example, secondary antibodies (e.g., B100) are perfused into each of the microfluidic chip 2900 and the microfluidic chip array 2950 at approximately 100 mmHg and approximately 75 mmHg, respectively, until steady-state fluid flow is achieved. Comparing the single-channel microfluidic chip 2900 with the multi-channel microfluidic chip array 2950, ​​the dead zone 2910 of the microfluidic chip array 2950 is significantly smaller than that of the microfluidic chip 2900. Figure 29AThe dead zone 2910 in the microfluidic chip array 2950 is characterized by relatively slow and / or turbulent (e.g., non-laminar) fluid flow, which manifests as a white line across the length of the confined region 2020. In contrast, the fluid flow across the width and length of the channels in the microfluidic chip array 2950 is laminar. The fluid flow through the obstacle 2940 and confined channel 2974 in the microfluidic chip array 2950 is more uniform (e.g., equal) than the fluid flow through the obstacle 2940 and confined channel 2924 in the microfluidic chip 2900.

[0096] like Figures 30A-30C As shown in the figure, the laminar flow difference between the single-channel microfluidic chip 2900 and the multi-channel microfluidic chip array 2950 is unexpected, surprising, and significant. Figure 30A Figure 3000 shows the non-laminar flow regions (e.g., the number of dead zones) based on single-channel microfluidic chips and multi-channel microfluidic chip arrays. For example, the number of dead zones in a single-channel microfluidic chip (e.g., microfluidic chip 2900) is compared to the number of dead zones in a single channel of an eight-channel microfluidic chip array (e.g., microfluidic chip array 2950). The error bars correspond to the standard deviation. The improved laminar flow through the multi-channel microfluidic chip array 2950 can be correlated with the reduced dead zone frequency. The data shows a significant increase in laminar flow interruptions (bars, left) in a single-channel microfluidic chip compared to flow interruptions in a single lane in an 8-channel microfluidic chip array. Surprisingly, fewer flow interruptions are observed in the 8-channel microfluidic chip array.

[0097] Figure 30B Figure 3010 shows the clogging rates based on single-channel microfluidic chips (e.g., microfluidic chip 2900) and multi-channel microfluidic chips (e.g., microfluidic chip 2950). Each chip 2900, 2950 was loaded with 200 µl of human plasma sample and stained, and was identified as clogged if there was essentially no fluid flow. Figure 30B The microfluidic chip choke rates are shown to be approximately 20% for a single-channel microfluidic chip and approximately 2% for an 8-channel microfluidic chip array. The improved laminar flow through the multi-channel microfluidic chip array 2950 corresponds to the reduced choke rate. Figure 30CFigure 3020 shows the background noise (e.g., average gray value) of stained fluid samples based on single-channel microfluidic chips (e.g., microfluidic chip 2900) and multi-channel microfluidic chips (e.g., microfluidic chip 2950). Lower background noise values ​​are observed in the multi-channel microfluidic chip array due to the improved fluid flow rate compared to the single-channel microfluidic chip. For example, Figure 3020 depicts a reduction in background noise of approximately 15% in the multi-channel microfluidic chip array compared to the single-channel microfluidic chip. The corresponding standard deviations observed for the single-channel microfluidic chip and the 8-channel microfluidic chip array are 0.83 and 0.49, respectively.

[0098] In some variations, reduced laminar flow in the channels of a microfluidic chip can correspond to increased artifacts and higher non-laminar flow. For example, Figure 31A This is a non-laminar image 3100 of a sample stained with eosin (e.g., SRX 1032) passing through a confined region 3120 of a single-channel microfluidic chip. Specifically, the boundary (e.g., edge, periphery) regions of the confined region 3120 may have higher artifact frequencies. For example, the top edge region 3110 of the confined region 3120 may include higher frequency artifacts 3130 (e.g., retained eosin-stained aggregates) corresponding to turbulent flow. Artifacts and ECMBs can be distinguished based on morphology. Figure 31B Figure 3140 shows the artifact sizes based on single-channel and multi-channel microfluidic chips. The difference in artifact sizes between single-channel and multi-channel microfluidic chip arrays is unexpected, surprising, and significant. For example, edge artifacts in single-channel microfluidic chips are quantified at a higher frequency because fluid flow through the microfluidic chip is disturbed relative to the microfluidic chip array. Specifically, no edge artifacts were observed in the 8-channel microfluidic chip array due to the laminar and consistent flow patterns through it. Error bars correspond to standard deviations.

[0099] Figure 21A plan view of a multi-channel confined microfluidic chip 2100 is depicted, the multi-channel microfluidic chip including an inlet reservoir 2110, an outlet reservoir 2130, and a confined region 2120 (e.g., a filtration region) in fluid communication between the inlet reservoir 2110 and the outlet reservoir 2130. The inlet reservoir 2110 and the outlet reservoir 2130 may include at least one obstruction 2140. The inlet reservoir 2110 and the outlet reservoir 2130 may have a generally polygonal shape. Multiple obstructions 2140 may be disposed between the inlet reservoir 2110 and the outlet reservoir 2130. Similar to microfluidic chips 316, 1800, 1900, and 2000, the confined region 2120 can be configured to restrict fluid flow to separate and retain (e.g., trap) a first fraction of fluid within the confined region 2120 of the microfluidic chip 2100, while allowing a second fraction of the remaining fluid to flow out of the microfluidic chip 2100 by applying negative pressure. In some variations, the first fraction of fluid may include an ECMB (electro-internal flow band). The microfluidic chip 2100 may include a plurality of confining channels 2124, 2126, and 2128 (e.g., uniform flow channels), which may be spaced apart, linear, and parallel. For example, the confined region 2120 may include flow barriers 2122 defining the respective confining channels 2124, 2126, and 2128 and a plurality of obstacles 2140 (e.g., struts) with multiple spacings. The lengths of the confining channels 2124, 2126, and 2128 may be greater than the length of the confined region 2120. The confinement channels 2124, 2126, and 2128 may have the same or different lengths relative to each other. Therefore, multiple confinement channels 2124, 2126, and 2128 can be configured to facilitate fluid flow through the microfluidic chip 1800 while minimizing bubbles and blockage when a negative pressure is applied to the outlet reservoir 2130 of the microfluidic chip 2100. In some variations, the microfluidic chip array may comprise multiple microfluidic chips 2100.

[0100] The microfluidic chips described herein can have various configurations and sizes. For example, the obstacles described herein can be one or more of the following: circular, spherical, triangular, square, polygonal, rhomboid, and fin-shaped. Obstacles can have the same or different shapes, sizes, and spacings. For example, the spacing between adjacent pillars of an obstacle can be between approximately 4 µm and approximately 100 µm, approximately 4 µm, approximately 15 µm, approximately 25 µm, approximately 50 µm, approximately 100 µm, and all ranges and sub-values ​​in between.

[0101] In some variations, microfluidic chips that apply negative pressure, based on the cross-sectional dimensions of the channels, can improve the yield (e.g., immobilization) of predetermined fractions of biofluids. For example, each channel may contain cross-sectional (e.g., height) dimensions 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, and about 15 µm, including all ranges and sub-values ​​therebetween.

[0102] In some variations, the ECMB capture volume using a negative pressure system as described herein can depend on the contents of the fluid (e.g., healthy control samples, positive samples). Figure 17A Images 1700 and 1702 show ECMBs captured in a confined area of ​​a microfluidic chip from aqueous humor samples from glaucoma patients after eosin-gamma staining. The magnified image 1702 depicts the truncated ECMB a1 wrapped around strut a2. The high abundance of the truncated, aggregated material in the microfluidic chip suggests that healthy aqueous humor is clogged with ECMBs in the trabecular meshwork, potentially leading to increased intraocular pressure. In contrast, healthy (control) aqueous humor samples show a relatively lower abundance of truncated ECMBs on the microfluidic chip compared to images 1700 and 1702. For example, Figure 17B Figure 1710 shows the variation of ECMB abundance (e.g., chip coverage %) with sample type (healthy control sample, primary open-angle glaucoma (POAG) sample). The amount of ECMB trapped in the microfluidic chip was significantly higher in the POAG sample than in the healthy control aqueous humor sample. Therefore, the data suggest that the increased ECMB in the POAG sample may be due to blockage and obstruction of ocular fluid outflow at the trabecular meshwork, and may also contribute to increased intraocular pressure in glaucoma.

[0103] In some variations, the ECMB capture amount using a negative pressure system as described herein can depend on the size of one or more microfluidic channels. For example, Figure 8A Figure 800 shows the variation of ECMB quantity with the cross-sectional channel size in a microfluidic chip. For example, Figure 800 compares the total amount of ECMB retained in the corresponding microfluidic channels at cross-sectional heights of 8 µm, 11 µm, and 15 µm. The normalized carboxyfluorescein succinimide (CFSE) signal intensity indicates the total amount of protein and is correlated with ECMB abundance, showing a linear relationship between channel size and ECMB abundance. Figure 8B Figure 810 shows the ECMB quantity as a function of the spacing between obstacles (e.g., pillars) in a microfluidic chip. For example, Figure 8BThe total amount of ECMB across strut zones with different height (cross-sectional) spacing was compared. For all channels with dimensions of 15 µm (dark gray stripe), 11 µm (light gray), and 8 µm (white), most ECMB was intercepted in the 100 µm, 50 µm, and 25 µm spacing zones.

[0104] Figure 8C-8E Illustrative figures 820, 830, and 840 are of ECMB on microfluidic chips with different channel cross-sectional (e.g., height) dimensions. Figure 8C Image 820 shows ECMBs a2 stained with CSFE with a channel size of 15 µm. Most of the ECMBs are truncated in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 822 depicts the truncated ECMBs a2 wrapped around strut a1. Figure 8D Image 830 shows ECMBs a2 stained with CSFE with a channel size of 11 µm. Most of the ECMBs are truncated in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 832 depicts the truncated ECMBs a2 wrapped around strut a1. Figure 8E Image 840 shows ECMBs a2 stained with CSFE with a channel cross-sectional (e.g., height) dimension of 8 µm. Most of the ECMBs are truncated in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 842 depicts the truncated ECMBs a2 wrapped around strut a1.

[0105] D. Chip connector

[0106] The system described herein may include a chip connector. Typically, chip connectors 118, 218 can be configured to hold one or more microfluidic chips 116, 216, 316, 1800, 1900, 2000, 2100 and improve the performance of systems 100, 200. Applying negative pressure to a microfluidic chip generates forces on the structure of the microfluidic chip itself, which can reduce and / or prevent the use of the microfluidic chip for processing biological fluids. For example, negative pressure applied to the channels (e.g., confined channels) of a microfluidic chip can generate forces at the outlet that can compress (and / or collapse) the outlet reservoir of the microfluidic chip, corresponding to unpredictable flow rates, bubbles, and uneven distribution of material within the microfluidic chip. In contrast, the chip connector described herein can be configured to hold the microfluidic chip and distribute the negative pressure applied by the negative pressure source more evenly across the entire microfluidic chip 116, 216, 316 to maintain the structural integrity of the microfluidic chips 116, 216, 316 and provide consistent results.

[0107] Figure 4A A perspective view of a chip connector 418 is depicted, which is configured to hold a plurality of microfluidic chips 416. Figure 4B An exploded perspective view depicting the chip connector 418 and the cover 420 of the microfluidic chip 416 is shown. Figures 4A-4D As shown, the chip connector 418 may include a cap 420 configured to contact the top portion of the microfluidic chip 416, and a base 430 configured to contact the bottom portion of the microfluidic chip 416. The cap 420 may define a plurality of holes 422 and include (e.g., overlap, cover) the periphery of the microfluidic chip 416. The plurality of holes 422 may be configured to overlap one or more of the inlet 450 and outlet 460 of the microfluidic chip 416. Thus, a fluid conduit (not shown) of the manifold may be coupled to the microfluidic chip 416 through the holes 422. In some variations, the inlet connector 440 may be coupled to the inlet 450 and configured to receive fluid from the robot 114. Similarly, the outlet connector 442 may be coupled between the fluid conduit (not shown for clarity) and the outlet 460 and configured to receive fluid transferred to the reservoir 113. The outlet connector 442 may be a connection interface between the fluid conduit and the outlet 460.

[0108] In some variations, the microfluidic chip 416 held between the cap 420 and the bottom portion 430 can distribute the negative pressure applied at the outlet 460 of the microfluidic chip 416 to the periphery of the microfluidic chip 416 (e.g., the non-channeled sides of the chip), thereby reducing compression at the outlet of the microfluidic chip and at non-peripheral portions of the microfluidic chip (e.g., where fluid flows), and thereby promoting a consistent flow rate and more equal material distribution through the microfluidic chip 416. For example, Figure 4C and 4D Exploded top and bottom perspective views of the base 430 of the chip connector 418 and the microfluidic chip 416 are depicted, with the periphery of the microfluidic chip 416 contacting the periphery of the base 430, such that the inner (e.g., non-peripheral) bottom portion of the microfluidic chip 416 does not contact the chip connector 418. This configuration allows the force applied by negative pressure at the outlet 460 (pushing the microfluidic chip 416 toward the base 430) to be distributed to the periphery of the microfluidic chip 416 (where fluid does not flow), reducing compression at the outlet 460 and the non-peripheral portion of the microfluidic chip 416 (where fluid flows). In this way, the chip connector 418 can be configured to distribute the compressive force applied by negative pressure to the microfluidic chip 416 to release pressure on the microfluidic chip 416 and hold the fluid channel open for fluid flow. Therefore, the chip connector promotes laminar fluid flow and consistent flow rate with minimal dead zones, blockages, background noise, and artifacts.

[0109] In some variations, the chip connector 418 may include one or more fasteners configured to couple the microfluidic chip 416 to the chip connector 418. For example, as Figure 4B As shown, the chip connector 418 may include a plurality of magnets 424 configured to securely hold the microfluidic chip 416 in a suitable position within the chip connector 418. For example, each of the base 430 and the cap 420 of the chip connector 418 may include a magnet 424 configured to align the microfluidic chip 418 in a predetermined orientation. Figure 4B As shown, the periphery (e.g., the side) of the cover 420 may include one or more magnets 424. In some variations, the magnets 424 may be configured to provide a reaction force to the negative pressure applied to the outlet 460 to further reduce compression at the outlet 460. In variations, the fastener may include one or more of the following: latches, strips, clips, mounting elements, snap rings, snap fasteners, clamps, closures, anchors, straps, adhesives, hook-and-loop fasteners, combinations thereof, etc.

[0110] Figure 22 An exploded perspective view of a chip connector 2200 configured to hold one or more microfluidic chips 2216 is depicted. The chip connector 2200 may include a base 2230, an outlet connector 2242, and a cap 2220. The base is configured to contact a bottom portion of the microfluidic chip (e.g., a portion facing the cap of the microfluidic chip), and the cap is configured to contact a top portion of the microfluidic chip 2216 (e.g., a portion facing the outlet connector 2242). The cap 2220 may define a plurality of orifices 2222 and include (e.g., overlap, cover) the periphery of the microfluidic chip 2216. The plurality of orifices 2222 may be configured to overlap one or more of the inlet 2250 and outlet 2260 of the microfluidic chip 2216. Therefore, a fluid conduit (not shown) of a manifold can be coupled to the microfluidic chip 2216 through the corresponding orifices 2222. In some variations, an inlet connector (not shown for clarity) may be coupled to inlet 2250 and configured to receive fluid from robot 114. Similarly, an outlet connector 2242 may be coupled between a fluid conduit (not shown for clarity) and outlet 2260 and configured to receive fluid transferred (e.g., aspirated) to reservoir 113. In some variations, outlet connector 2242 may be a connection interface between the fluid conduit and outlet 2260. For clarity, chip connector 2200 may further include... Figure 22 The base not shown in the image (e.g., base 430, Figure 4C and 4D The base can be configured to interact with... Figure 4C and 4DThe bottom portion of the microfluidic chip 2216 is shown and described in the same manner.

[0111] In some variations, the microfluidic chip 2216, held between the cap 2220 and the base 2230 of the chip connector 2200, can distribute the negative pressure applied at the outlet 2260 of the microfluidic chip 2216 to the periphery of the microfluidic chip 2216 (e.g., the channelless side of the chip), thereby reducing compression at the outlet 2260 of the microfluidic chip 2216 and thereby promoting a consistent flow rate and more equal distribution of material (e.g., first fraction, ECMB) through the microfluidic chip 2216. This configuration allows the force applied by the negative pressure at the outlet 2260 (pushing the microfluidic chip 2216 toward the base 2230) to be redistributed to the periphery of the microfluidic chip 2216 to reduce compression at the outlet 2260 of the microfluidic chip 2216. In this manner, the chip connector 2200 can be configured to distribute the compressive force applied by negative pressure to the microfluidic chip 2216 to release the pressure on the microfluidic chip 2216 and hold the fluid channel open for fluid flow. Therefore, the chip connector 2200 promotes laminar fluid flow and consistent flow rate, minimizing dead zones, blockages, background noise, and artifacts.

[0112] In some variations, such as Figure 22 As shown, the chip connector 2200 may include a plurality of magnets 2224 configured to securely hold the microfluidic chip 2216 in a suitable position within the chip connector 2200. For example, each of the base 2230 and the cap 2220 of the chip connector 2200 may include at least one corresponding magnet 2224 configured to align the microfluidic chip 2200 in a predetermined orientation. Figure 22 As shown, the periphery (e.g., side) of the cover 2220 and the inner periphery (e.g., sidewall) of the base 2230 may each contain one or more magnets 2224, which are configured to enclose the microfluidic chip 2216 in the chip connector 2200.

[0113] In some variations, chip connectors 118, 218 can be configured to hold one or more microfluidic chips 116, 216, 316, 1800, 1900, 2000, 2100 and / or microfluidic chip arrays 370, 2002 to increase throughput and / or reduce system size. Furthermore, the chip connectors can be configured to distribute the negative pressure applied by the negative pressure source more evenly across the multiple microfluidic chips to maintain structural integrity and provide consistent results in a compact space.

[0114] Figure 23An exploded perspective view of a chip connector 2300 configured to hold one or more microfluidic chip arrays 2314 is depicted. For example, the chip connector 2300 may include a cap 2320 and a base 2330, the cap being configured to contact a top portion of the microfluidic chip array 2314 (e.g., the portion facing inlet 2350 and outlet 2360), and the base being configured to contact a bottom portion of the microfluidic chip array 2314 (e.g., the portion facing away from inlet 2350 and outlet 2360). The cap 2320 may define a plurality of orifices 2322 and include (e.g., overlap, cover) the periphery of the microfluidic chip array 2314. The plurality of orifices 2322 may be configured to overlap one or more of the plurality of inlets 2350 and plurality of outlets 2360 of the microfluidic chip array 2314. Thus, a fluid conduit (not shown) of a manifold can be coupled to the microfluidic chip array 2314 through the orifices 2322. In some variations, the inlet connector 2340 of the chip connector 2300 can be coupled to multiple inlets 2350 of the microfluidic chip array 2314 and configured to receive fluid from the robot 114. Similarly, the outlet connector 2342 of the chip connector 2300 can be coupled between a fluid conduit (not shown for clarity) and multiple outlets 2360 of the microfluidic chip array 2314 and configured to receive fluid transferred (e.g., aspirated) into the reservoir 113. Therefore, the outlet connector 2342 can be a connection interface between the fluid conduit and the multiple outlets 2360. Regarding... Figures 27A-27D 28A-28D further describes the chip connector's entry connector, and regarding... Figures 24A-24D 25A-25D and 26A-26D further describe the chip connector's export connector.

[0115] In some variations, the microfluidic chip array 2314 held between the cap 2320 and the bottom portion 2330 of the chip connector 2300 can distribute the negative pressure applied at the multiple outlets 2360 of the microfluidic chip array 2314 to the periphery of the microfluidic chip array 2314 (e.g., the non-channel sides of the chip), thereby reducing compression at the outlets of the microfluidic chip array and thus promoting a consistent flow rate and more equal material distribution through the microfluidic chip array 2314. This configuration allows the force applied by the negative pressure at the multiple outlets 2360 (pushing the microfluidic chip array 2314 toward the base 2330) to be redistributed to the periphery of the microfluidic chip array 2314 to reduce compression at the multiple outlets 2360 of the microfluidic chip array 2314. In this manner, the chip connector 2300 can be configured to distribute the compressive force applied by negative pressure to the microfluidic chip array 2314 to relieve pressure on the microfluidic chip array 2314 and hold the fluid channels open for fluid flow. Therefore, the chip connector 2300 promotes laminar fluid flow and consistent flow rate, minimizing dead zones, blockages, background noise, and artifacts.

[0116] In some variations, such as Figure 23 As shown, the chip connector 2300 may include a plurality of magnets 2324 configured to securely hold the microfluidic chip array 2314 in a suitable location within the chip connector 2300. For example, each of the base 2330 and the cap 2320 of the chip connector 2300 may include at least one corresponding magnet 2324 configured to align the microfluidic chip 2300 with a predetermined orientation. Figure 23 As shown, the periphery (e.g., sides) of the cover 2320 and the inner periphery (e.g., sidewalls) of the base 2330 may each include one or more magnets 2324, which are configured to enclose the microfluidic chip array 2314 in the chip connector 2300. For clarity, the chip connector 2300 may further include Figure 23 The base not shown in the image (e.g., base 430, Figure 4C and 4D The base can be configured to interact with... Figure 4C and 4D The bottom portion of the microfluidic chip array 2314 is shown and described in the same manner.

[0117] In some variations, by holding the microfluidic chips 116, 216, 316, 416 in chip connectors 118, 218, 418, 2200, 2300, the fluid flow rate and flow consistency through the microfluidic chips 116, 216, 316, 416 under negative pressure can be improved. Figure 9Improvements in flow rate and flow consistency are demonstrated between microfluidic chips coupled to a chip connector as described herein and microfluidic chips without a chip connector. Specifically, Figure 9 Figure 900 shows the flow rates for three fluid flow processes (e.g., PBS pre-filling, BVH stained with CFSE, PBS washing) between system 910 (e.g., system 100, dark gray bar) using a microfluidic chip and chip connector, and system 920 (light gray bar) using a microfluidic chip without a chip connector, under a negative pressure of approximately 100 mmHg. Specifically, the PBS pre-filling flow rate is 10.4 ± 3.27 µl / min without the chip connector, and 11.4 ± 0.36 µl / min with the chip connector. The signal-to-noise ratio (SNR) (e.g., the mean standard deviation of the flow rate) increased by 9.8-fold, from 3.2 without the chip connector to 31.3 with the chip connector. For BVH stained with CFSE, the flow rate is 3.9 ± 1.42 µl / min without the chip connector, and 4.8 ± 0.10 µl / min with the chip connector. The signal-to-noise ratio (SNR) increased by 17.9-fold from 2.8 without the chip connector to 50.0 with the chip connector. The PBS flow rate for the washing reagent was 4.9 ± 0.79 µl / min without the chip connector and 6.7 ± 0.22 µl / min with the chip connector. The SNR also increased by 4.8-fold from 6.2 without the chip connector to 30.0 with the chip connector. For microfluidic chips without the chip connector, the standard deviation was consistently large. The pressure redistribution across the microfluidic chip via the chip connector described herein corresponds to a lower standard deviation, higher SNR, and predictable fluid flow, and further enables the automation of the system described herein.

[0118] In some variations, chip connectors 118, 218, 418 may include at least one inlet connector 440 and at least one outlet connector 442 disposed between cap 420 and microfluidic chip 416. In some variations, applying negative pressure to microfluidic chip 416 can generate force at the connection interface between microfluidic chip 416 and fluid conduit, which can reduce and / or prevent the use of microfluidic chip 416 for handling biological fluids. For example, outlet 460 of microfluidic chip 416 may be coupled to fluid conduit (e.g., vacuum tube) of manifolds 120, 220 and negative pressure sources 122, 222 (not shown). Negative pressure applied through fluid conduit and at outlet 460 of microfluidic chip 416 may otherwise displace the fluid connection between microfluidic chip 416 and manifolds 120, 220, resulting in leakage. 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 the sealing, connection strength, and alignment tolerance between the microfluidic chip 416 and the manifolds 120, 220, and to provide consistent flow (e.g., by preventing leakage) and equal material dispensing. In some variations, one or more of the inlet connector 440 and the outlet connector 442 may be disposable to reduce contamination and setup time.

[0119] Figures 5A-5E The diagram depicts corresponding top perspective, bottom perspective, side view, top view, and bottom view of the outlet connector 500. In some variations, the outlet connector 500 (e.g., a gasket) may comprise an elongated body defining a lumen 510 and including multiple steps 520 along the length of the elongated body. For example, the outlet connector 500 has four steps (e.g., layers) with different diameters, although any outlet connector described herein may have two, three, four, five, or more steps. The steps of the outlet connector 500 help increase the contact area between the fluid conduit and the microfluidic chip, thereby strengthening the connection interface.

[0120] Figures 6A-6D Corresponding top perspective view, bottom perspective view, top view, and bottom view of the outlet connector 600 are depicted. In some variations, the outlet connector 600 may include an elongated body defining a lumen 610, the inner diameter of which decreases in the distal direction. For example, the lumen 610 may be tapered, such that the inner diameter 614 at the proximal end of the outlet connector 600 is larger than the inner diameter 612 at the distal end of the outlet connector 600. For example, the lumen 610 may have a conical shape, and the elongated body may have a cylindrical shape. The lumen 610 with a varying inner diameter can provide increased tolerance for coupling a fluid conduit to the outlet connector 600, and thereby reduce setup time and leakage due to misalignment.

[0121] In some variations, a single inlet connector can be configured to transfer (e.g., contain) fluid from multiple microfluidic chips, thereby reducing the complexity of the chip connector and reducing setup and cleaning time. Figures 24A-24D Perspective views, top views, bottom views, and side perspective views of the inlet connector 2400 are depicted. Figures 25A-25D Perspective views, top views, bottom views, and side perspective views of another inlet connector 2500 are depicted. Figures 26A-26D Perspective views, top views, bottom views, and side perspective views of another inlet connector 2600 are depicted. In some variations, inlet connectors 2400, 2500, 2600 (e.g., gaskets) may comprise bodies 2420, 2520, 2620 defining multiple lumens 2410, 2510, 2610. The multiple lumens 2410, 2510, 2610 may be configured to fluidly communicate with corresponding multiple inlet reservoirs of a microfluidic chip array (e.g., multiple individual microfluidic chips). The bodies 2420, 2520, 2620 may include one or more collars 2424, 2524, 2624 configured to extend the length of the lumens 2410, 2510, 2610 and facilitate robust fluid communication between the end effector 215 (e.g., a pipette) and the corresponding microfluidic chip, thereby strengthening the connection interface.

[0122] In some variations, the inlet connectors 2400, 2500, and 2600 may include a body defining a plurality of lumens, each lumen defining a distal portion (e.g., a portion facing the inlet of the microfluidic chip) and a proximal portion (e.g., a portion approaching the collar 2424, 2524, and 2624). Each lumen 2410, 2510, and 2610 may have an increased inner diameter in one or more of the distal and proximal directions. For example, as... Figures 24A-24D As shown, the lumen 2410 can be tapered, increasing the inner diameter in both the distal and proximal directions. Specifically, the lumen 2410 can have an hourglass shape within the body 2420 of the inlet connector 2400 and a cylindrical shape through the collar 2424. In some variations, such as Figures 26A-26D As shown, the lumen 2610 can be tapered, such that the inner diameter increases from the distal end to the proximal end. For example, the lumen 2610 can have a conical shape within the body 2620 of the inlet connector 2600 and a cylindrical shape through the collar 2624. In some variations, the inlet connector described herein can surprisingly, unexpectedly, and significantly increase ECMB capture in microfluidic chips. For example, Figure 32Figure 3200 shows the amount of ECMB on a microfluidic chip (e.g., within a confined area) based on inlet connectors including silicone (e.g., inlet connectors 2400, 2500, 2600) and silicone-free inlet connectors (e.g., inlet connectors 2400, 2500, 2600). Specifically, homogenized bovine vitreous fluid is infused into the microfluidic chip through the corresponding 8-channel inlet connectors (e.g., inlet connectors 2400, 2500, 2600) and further stained with eosin. Inlet connectors with silicone (e.g., silicone surfaces) can reduce protein adhesion to the inlet connector surface and thereby increase the amount of ECMB captured in the microfluidic chip. Error bars correspond to standard deviations.

[0123] In some variations, a single outlet connector can be configured to transfer (e.g., extract) fluid from multiple microfluidic chips, thereby reducing chip connector complexity and minimizing setup and cleaning times. For example, a single outlet connector exhibits a lower flow standard deviation compared to a conventional connector. Figures 27A-27D Perspective views, top views, bottom views, and perspective side views of an export connector 2700 (e.g., a washer) are depicted. Figures 28A-28D Perspective views, top views, bottom views, and perspective side views of another outlet connector 2800 are depicted. In some variations, outlet connectors 2700, 2800 may include bodies 2730, 2830, which include one or more lumens 2740, 2840. Lumens 2740, 2840 may be configured to contain fluid from multiple outlets of the microfluidic chip for transfer through a single outlet of the outlet connector. Additionally, bodies 2730, 2830 and one or more lumens 2740, 2840 may be configured to redistribute the negative pressure applied to the microfluidic chip to reduce compression at the respective outlets of the multiple microfluidic chips, thereby improving flow consistency.

[0124] In some variations, lumens 2740, 2840 may define a distal portion (e.g., the portion facing the outlet of the microfluidic chip), a proximal portion (e.g., the portion adjacent to collar 2834), and an inner diameter. For example... Figure 27C and 28C As shown, lumens 2740 and 2840 may have a defined inner diameter in their proximal portions and may be tapered in their distal portions, such that the inner diameter increases distally. For example, lumens 2740 and 2840 may have a cylindrical shape in their proximal portions and a grooved shape in their distal portions. In some variations, such as Figures 28A-28DAs shown, the body 2830 of the outlet connector may include one or more collars 2834, which are configured to extend one or more lumens 2810 to facilitate fluid communication and contact between the fluid conduit and the microfluidic chip, thereby enhancing the connection interface.

[0125] In some variations, the inlet and outlet connectors may comprise suitable materials, including one or more silicone-based polymers, other polymers, thermoplastics, thermoplastic elastomers, etc. For example, one or more of the inlet and outlet connectors may comprise silicone-coated plastic. Silicone-containing inlet and outlet connectors can further improve flow consistency. Silicone may include liquid silicone rubber, high-consistency silicone, solid silicone rubber, room temperature vulcanizing (RTV) silicone, fluorosilicone, combinations thereof, etc.

[0126] In some variations, the geometry of the disposable inlet and outlet connectors described herein can improve operational aspects of the negative pressure system, such as reducing system setup time during assembly and cleaning. For example, Figure 10A Figure 1000 shows the setup time of a 16-microfluidic chip system using a conventional (e.g., tapered) outlet connector and the outlet connector described herein. The improved geometry (e.g., layered, tapered) of the disposable outlet connector, compared to the tapered outlet connector, improves the fluid seal between the chip connector and the manifold's fluid conduit and allows for easier manipulation, thus reducing setup time.

[0127] In some variations, using a negative pressure system, relative to a positive pressure system, can improve the overall experimental duration of the exemplary staining procedure. For example, Figure 10B Figure 1010 shows the total duration (e.g., including setup, experiment, and cleaning) of immunofluorescence (IF) and CSSE staining procedures using positive and negative pressure systems. Specifically, the average time for IF experiments using a negative pressure system is reduced by approximately 12 hours compared to a positive pressure system. The average time for CFSE staining using a negative pressure system is reduced by approximately 8.25 hours compared to a positive pressure system. This improvement in efficiency can be partly attributed to disposable outlet connectors and the negative pressure source, which eliminate the time-consuming process (e.g., up to approximately 8 hours) of rinsing the system to remove contaminants. For example, disposable outlet connectors can be replaced between experiments instead of being cleaned, thus reducing cleaning time and total experiment duration, and reducing variability between connectors due to cleaning variability.

[0128] In some variations, the improved fluid seals of the disposable inlet and outlet connectors described herein, compared to the reusable connectors used in positive pressure systems, can reduce contamination in microfluidic chips corresponding to anomalous morphologies. Figure 10CFigure 1020 shows the average frequency of anomalous BVH morphologies when using the tapered outlet connector and outlet connector described herein. For example, incomplete sealing (e.g., air leakage) at the connection interface between the fluid conduit, outlet connector, and microfluidic chip may correspond to an increase in anomalous morphologies. Incomplete sealing may be partly due to misalignment of the outlet connector with one or more of the fluid conduit and microfluidic chip. The improved geometry of the outlet connector described herein reduces setup time and the frequency of anomalous morphologies.

[0129] E. manifold

[0130] The system described herein may include manifolds 120 and 220. Typically, manifolds 120 and 220 may be configured to be coupled to a plurality of microfluidic chips 116, 216, and 316 and negative pressure sources 122 and 222. For example, manifolds 120 and 220 may be in fluid communication with a single negative pressure source 122 and 222 and each microfluidic chip 116, 216, and 316 held by chip connectors 118 and 218, such that a negative pressure generated by the negative pressure source 122 and 222 can be applied to each microfluidic chip 116, 216, and 316 coupled to manifolds 120 and 220. In some variations, manifolds 120 and 220 may include a plurality of fluid conduits (e.g., vacuum tubes, fluid lines) configured to be releasably coupled to each of the microfluidic chips 116, 216, and 316 and the negative pressure sources 122 and 222. Therefore, the manifold can reduce the size of the system by 100 because each microfluidic chip does not need its own negative pressure source.

[0131] In some variations, manifolds and negative pressure sources significantly increase throughput by scaling up and increasing efficiency without reducing consistency or flow rate. Figure 11 Figure 1100 shows the number of parallel (e.g., simultaneous) experiments performed on the positive and negative pressure systems described herein. For example, a manifold coupled to eight microfluidic chips, each with eight channels, enables 64 experiments to be run simultaneously, thereby significantly increasing the throughput of a positive pressure system without the use of negative pressure and a manifold. In contrast, positive pressure systems rely on pumps and sample injection mechanisms with limited connectivity.

[0132] The microfluidic system described in this paper, incorporating a manifold and a negative pressure source, not only contributes to improved efficiency and throughput but also achieves this in a relatively uniform manner, which would otherwise be impossible using conventional solutions. For example, Figure 14AFigure 1400 shows ECMB volumes based on a 3-port manifold under a negative pressure of 100 mmHg. As can be seen from the figure, there was no statistically significant difference in ECMB volumes retrieved from each port (Kruskal-Wallis, p = 0.863). However, even at higher negative pressures (e.g., 400 mmHg), ECMB capture volumes did not differ statistically. Figure 14I Figure 1480 shows ECMB measurements from a 3-port manifold under negative pressure of 400 mmHg. There was no statistically significant difference in ECMB measurements from each port (Fischer's, p = 0.481).

[0133] Figure 14B Figure 1410 shows the ECMB amounts across strut zones with different spacings for different ports of the manifold under a negative pressure of 100 mmHg. For port 1 (dark gray bar), port 2 (light gray bar), and port 3 (white bar), most of the ECMB was trapped in the 100 µm, 50 µm, and 25 µm spacing zones. As shown in the figure, there were no statistically significant differences in ECMB amounts between each strut zone across different ports (100 µm zone - Fisher, p = 0.744, 50 µm zone - Kruskal-Wallis, p = 0.882, 25 µm zone - Fisher, p = 0.912, 15 µm zone - Kruskal-Wallis, p = 0.490, 4 µm zone - Kruskal-Wallis, p = 0.230). Figure 14C Figure 1420 shows the ECMB volume based on an 8-port manifold. There was no statistically significant difference in ECMB volume across each port (p = 0.078). Therefore, the manifold distributes the negative vacuum pressure (e.g., 100 mmHg) to each port and the corresponding microfluidic chip in a substantially uniform manner. However, even at higher negative pressures (e.g., 400 mmHg), there was no statistically significant difference in ECMB capture. For example, Figure 14JFigure 1490 shows the ECMB amounts across strut zones with different spacings for different ports of the manifold under a negative pressure of 400 mmHg. For port 1 (dark gray bar), port 2 (light gray bar), and port 3 (white bar), most of the ECMB was trapped in the 100 µm, 50 µm, and 25 µm spacing zones. There were no statistically significant differences in ECMB amounts between the different ports in each strut zone (100 µm zone - Fisher, p = 0.160, 50 µm zone - Fisher, p = 0.745, 25 µm zone - Kruskal-Wallis, p = 0.475, 15 µm zone - Kruskal-Wallis, p = 0.393, 4 µm zone - Kruskal-Wallis, p = 0.114).

[0134] Similar to Figure 14A There was no statistically significant difference in the amount of ECMB retrieved from each port of the 8-port manifold. Figure 14D Figure 1430 shows the ECMB amount across strut regions with different spacing and an 8-port manifold. The microfluidic chip has strut spacing regions of 100 µm (very dark gray bar), 50 µm (dark gray bar), 25 µm (medium gray bar), 15 µm (light gray bar), and 4 µm (white bar), with most of the ECMB trapped in the 100 µm, 50 µm, and 25 µm spacing regions. There were no statistically significant differences in the ECMB amount between each strut spacing region across the different ports (100 µm region - p = 0.067, 50 µm region - p = 0.153, 25 µm region - p = 0.281, 15 µm region - p = 0.943, 4 µm region - p = 0.675).

[0135] Fluid flow through the microfluidic chip, where negative pressure is applied through an 8-port manifold, can be relatively uniform. For example, Figure 14E Figure 1440 shows the flow rates of three fluid flow processes (e.g., PBS prefilling, BVH stained with CFSE, and PBS washing) using an 8-port manifold at a negative pressure of approximately 100 mmHg. There were no statistically significant differences in flow rates between the different ports for PBS prefilling (dark gray bars), BVH and CFSE (medium gray bars), and PBS washing (light gray bars) (PBS prefilling - p = 0.133, BVH and CFSE loading - p = 0.348, PBS washing - p = 0.170). However, even at higher negative pressures (e.g., 400 mmHg), ECMB capture was not statistically different.

[0136] Figure 14F-14I This is an image showing the ECMB trapped in a microfluidic chip coupled to a manifold of a negative pressure system. For example, Figure 14F Image 1450 shows ECMBs a2 stained with CSFE in a microfluidic chip coupled to a three-port manifold under a negative pressure of 100 mmHg. Most of the ECMBs are trapped in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 1452 depicts the trapped ECMBs a2 wrapped around strut a1. Figure 14G Image 1460 shows ECMBs a2 stained with CSFE in a microfluidic chip coupled to an eight-port manifold under a negative pressure of 100 mmHg. Most of the ECMBs are trapped in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 1462 depicts the trapped ECMBs a2 wrapped around strut a1. Figure 14H Image 1470 shows ECMBs a2 stained with CSFE in a microfluidic chip coupled to a three-port manifold under a negative pressure of 400 mmHg. Most of the ECMBs are trapped in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 1472 depicts the trapped ECMBs a2 wrapped around strut a1.

[0137] Figure 15A Figure 1500 shows the ECMB quantities using systems with and without manifolds under a negative pressure of 400 mmHg. There was no statistically significant difference in ECMB quantities between microfluidic chips coupled to and uncoupled to a manifold (Welch's t, p = 0.400).

[0138] Figure 15B Figure 1510 shows the amount of ECMB across strut regions with different spacings, with and without manifolds, under a negative pressure of 400 mmHg. For a single microfluidic chip with and without a manifold (dark gray bar), most of the ECMB was trapped in the 100 µm, 50 µm, and 25 µm spacing regions. There was no statistically significant difference in the amount of ECMB between each strut region with and without a manifold (100 µm region - Welch's t, p = 0.220, 50 µm region - Student's t, p = 0.831, 25 µm region - Welch's t, p = 0.638, 15 µm region - Student's t, p = 0.198, 4 µm region - Student's t, p = 0.172).

[0139] F. Negative pressure source

[0140] The system described herein may include negative pressure sources 122, 222. Typically, negative pressure sources 122, 222 may be configured to be coupled to manifolds 120, 220 and at least one microfluidic chip 116, 216, 316. The application of negative pressure can help reduce contamination and increase throughput compared to conventional solutions such as positive pressure systems. In some variations, the negative pressure source may include a fluid pump. A processor 128 and a memory 130 coupled to the negative pressure sources 122, 222 may be configured to control fluid flow and negative pressure. In some variations, negative pressure sources 122, 222 may be configured to apply a negative pressure between approximately 10 mm HG and approximately 760 mm HG to each microfluidic chip 116, 216, 316 of systems 100, 200.

[0141] Figure 12A and 12B Images 1200 and 1200 show biocontamination in a positive pressure microfluidic system. These images were obtained after immunofluorescence staining with bovine vitreous fluid, staining with fibronectin, and imaging using a fluorescence microscope. Arrows indicate biocontaminants. The positive pressure system with a reusable conical outlet connector was cleaned using standard operating procedures, and a new microfluidic chip was placed in the system and rinsed with buffer. Figure 12C and 12D Images 1230 and 1240 show biocontamination using the system described herein, where the system was cleaned using standard operating procedures, a new microfluidic chip was placed in the chip connector, and buffer rinsing and a new outlet connector were provided. Figure 12E Figure 1250 shows the biofouling per unit area for positive and negative pressure systems. Compared to positive pressure systems, the system described herein, utilizing negative pressure and an outlet connector, offers a significantly reduced biofouling, thereby increasing the accuracy and reliability of the system described herein.

[0142] In some variations, the amount of ECMB retained in the microfluidic chip does not necessarily increase linearly with the applied negative pressure. For example, Figure 13A Figure 1300 shows the ECMB values ​​for negative pressures of 100 mmHg and 400 mmHg. There was no statistically significant difference in ECMB values ​​for each negative pressure (Studden's t, p = 0.988). Similarly, Figure 13BFigure 1310 shows the ECMB amounts across strut zones with different spacing for negative pressures of 100 mmHg and 400 mmHg. For a negative pressure of 100 mmHg (dark gray bars) and 400 mmHg (light gray bars), most of the ECMB was trapped in the 100 µm, 50 µm, and 25 µm spacing zones. There were no statistically significant differences in ECMB amounts between different negative pressures within each strut zone (100 µm zone - Welch t, ​​p = 0.211, 50 µm zone - Studden t, p = 0.738, 25 µm zone - Studden t, p = 0.335, 15 µm zone - Mann-Whitney U, p = 0.238, 4 µm zone - Mann-Whitney U, p = 0.657).

[0143] Figure 13C and 13D This is an image showing the truncated ECMB in a microfluidic chip coupled to a negative pressure system under different negative pressures. For example, Figure 13C Image 1320 shows ECMBs a2 stained with CSFE under a negative pressure of 100 mmHg. Most of the ECMBs are trapped in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 1322 depicts the trapped ECMBs a2 wrapped around strut a1. Figure 13D Image 1330 shows ECMBs a2 stained with CSFE under a negative pressure of 400 mmHg. Most of the ECMBs are trapped in strut a1 regions spaced at 100 µm, 50 µm, and 25 µm intervals. Enlarged image 1332 depicts the trapped ECMBs a2 wrapped around strut a1.

[0144] In some variations, applying a negative pressure between approximately 10 mm HG and approximately 760 mm HG to the microfluidic chip can effectively separate ECMB from the biofluid. For example, Figure 16A and 16B It uses a negative pressure between 10 mmHg and 760 mmHg and a corresponding conical outlet connector (e.g., Figures 6A-6D ) and layered export connectors (e.g., Figures 5A-5E Figures 1600 and 1610 show the corresponding amounts of ECMB retained in the microfluidic channels of a microfluidic chip. For Figure 16A Based on the t-test (Welch, p = 0.001), the amount of ECMBS retained at 5 mmHg was statistically significantly less than the amount retained at negative pressures between 10 mmHg and 760 mmHg. Similarly, for Figure 16BBased on the t-test (Studen's test, p < 0.001), the amount of ECMBS retained at 5 mmHg was statistically significantly less than the amount of ECMBS retained at negative pressures between 10 mmHg and 760 mmHg.

[0145] G. Sensor

[0146] The system described herein may include one or more sensors 124. Typically, sensor 124 may be configured to measure one or more characteristics corresponding to one or more of the following (e.g., pressure, flow rate, optical image, temperature, humidity): biofluid, retainers 112, 212, reservoir 113, 213, robot 114, 214, microfluidic chips 116, 216, 316, chip connector 118, 218, manifold 120, 220, and negative pressure source 122, 222. In this way, the system and fluid can be monitored during use. In some variations, the sensor may be coupled to any component of system 100 (e.g., inlet connector, chip connector, etc.) or integrated into any component of said system.

[0147] In some variations, the sensor may be an optical sensor coupled to any component of system 100, 200 (such as chip connectors 118, 218). The optical sensor may be configured to image one or more channels of microfluidic chips 116, 216, 316 for histochemical and morphological studies. The optical sensor may be used to receive light signals (e.g., light beams) reflected by fluids in microfluidic chips 116, 216, 316. The received light may be used to generate signal data, which may be processed by processor 128 and memory 130 to generate sample data. The optical sensor may also be configured to image one or more identifiers (e.g., tags, barcodes) and identifiers of microfluidic chips 116, 216, 316. In some variations, the optical sensor may include one or more of a lens, a camera, and measurement optics. For example, the optical sensor may include a charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) optical sensor and may be configured to generate image signals transmitted to output device 134 (e.g., a 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). Alternatively, in some variations, the sensor may be an ultrasonic sensor configured to generate an ultrasonic signal for determining fluid flow rate.

[0148] In some variations, systems 100, 200 may include a radiation source configured to emit light signals (e.g., illumination) directed at microfluidic chips 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 the following: light-emitting diodes, lasers, microscopes, optical sensors, lenses, and flashlights. For example, the radiation source may generate light that can be carried by an optical fiber cable, or one or more LEDs may be configured to provide illumination. In another example, a fiber optic mirror comprising a bundle of flexible optical fibers may be configured to receive and propagate light from an external light source.

[0149] H. Input device

[0150] Typically, the input device 126 of systems 100 and 200 can serve as a communication interface between the operator and systems 100 and 200. The input device 126 can be configured to receive input data and output data to one or more of the robot 114, sensors 124, and output devices 134. For example, operator control of the input device 126 (e.g., foot controller, joystick, keyboard, touchscreen) can be processed by the processor 128 and memory 130 so that the input device 126 outputs control signals to one or more of the robots 114 and 214, the negative pressure source, and sensors 124. As another example, images generated by sensors 124 can be processed by the processor 128 and memory 130 and displayed by the output device 134 (e.g., a display). Sensor data from one or more sensors 124 can be output by one or more output devices 134 in a visual, auditory, and / or tactile feedback manner.

[0151] Some variations of the input device may include at least one switch configured to generate control signals. The input device may be coupled to or separated from other components of the systems 100, 200. For example, the input device 126 may be located in a different room from the robots 114, 214 and the microfluidic chips 116, 216, 316 to reduce potential contamination. Control signals may include, for example, robot signals, negative pressure signals, sensor signals, and other signals. In some variations, the input device 126 may include a wired and / or wireless transmitter configured to transmit control signals to a wired and / or wireless receiver of the controller. Robot signals (e.g., for controlling motion, position, and orientation) may control the robot's articulation in at least four degrees of freedom and may include yaw and / or pitch rotation. For example, the input device 126 including a touch surface can be configured to detect contact and movement on the touch surface using any of a variety of touch-sensitive technologies, including capacitive technology, resistive technology, infrared technology, optical imaging technology, dispersive signal technology, acoustic pulse identification technology, and surface acoustic wave technology.

[0152] In a variation of input device 126 that includes at least one switch, the switch may include at least one of the following: a button (e.g., a hard key, a soft key), a touch surface, a keyboard, an analog stick (e.g., a joystick), a direction pad, a mouse, a trackball, a dial, a step switch, a rocker switch, a pointer device (e.g., a stylus), a motion sensor, an image sensor, and a microphone. The motion sensor may receive operator movement data from an optical sensor and classify operator gestures as control signals. The microphone may receive audio and recognize operator speech as control signals. In a variation of the system that includes multiple input devices, different input devices may generate different types of signals. For example, some input devices (e.g., buttons, analog sticks, direction pads, and keyboards) may be configured to generate robot signals, while other input devices (e.g., step switches, rocker switches) may be configured to control negative pressure sources 122, 222, and sensor 124.

[0153] I. Processor

[0154] like Figure 1As depicted, systems 100 and 200 may include a processor 128 and a machine-readable memory 130 (e.g., a controller) communicating with robots 114 and 214, negative pressure sources 122 and 222, and sensors 124. The processor 128 may be connected to systems 100 and 200 via wired or wireless communication channels. The processor 128 may be located in the same or a different room from the microfluidic chips 116, 216, and 316. The processor 128 may be configured to control one or more components of system 100 and 200, such as being configured to transfer fluid to the microfluidic chips via robots 114 and 214, or to visualize the separated ECMB using optical sensors of the microfluidic chips 116, 216, and 316.

[0155] Processor 128 can be implemented in accordance with many general-purpose or special-purpose computing systems or configurations. Various exemplary computing systems, environments and / or configurations suitable for use with the systems and apparatuses disclosed herein may include, but are not limited to, software or other components within or embodied therein in personal computing devices, networked appliances, servers or server computing devices (such as routing / connectivity components, portable (e.g., handheld) or laptop devices, multiprocessor systems, microprocessor-based systems and distributed computing networks).

[0156] Examples of portable computing devices include smartphones, personal digital assistants (PDAs), cellular phones, tablet PCs, phablets (personal computing devices larger than smartphones but smaller than tablets), wearable computers in the form of smartwatches, portable music devices, and portable or wearable augmented reality devices that interact with the operator's environment through sensors and can be visualized, have eyes tracked, and allow user input using head-mounted displays.

[0157] Processor 128 may incorporate data received from memory 130 and operator input to control one or more robots 114, 214 and negative pressure sources 122, 222. Memory 130 may further store instructions to cause processor 128 to execute modules, processes, and / or functions associated with systems 100, 200. Processor 128 may be any suitable processing device configured to run and / or execute instruction sets or code, and may include one or more data processors, image processors, graphics processing units, physical processing units, digital signal processors, and / or central processing units. Processor 128 may be, for example, a general-purpose processor, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC) configured to perform application processes, and / or other modules, processes, and / or functions associated with the system and / or its associated network. It can provide basic device technologies for various component types, such as metal-oxide-semiconductor field-effect transistor (MOSFET) technology like complementary metal-oxide-semiconductor (CMOS), bipolar technology like emitter-coupled logic (ECL), polymer technologies (e.g., silicon conjugated polymers and metal conjugated polymer-metal structures), hybrid analog and digital, and combinations thereof.

[0158] J. Memory

[0159] Some variations of the memory 130 described herein relate to computer storage products having a non-transitory computer-readable medium (also referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. A computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include transient propagation signals (e.g., propagation of electromagnetic waves carrying information on a transmission medium such as air or cable). The medium and the computer code (also referred to as code or algorithm) may be designed and constructed for one or more specific 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 tapes; optical storage media such as optical discs / digital video discs (CD / DVD), optical disc read-only memory (CD-ROM), and holographic devices; magneto-optical storage media such as optical discs; solid-state storage devices such as solid-state drives (SSDs) and solid-state hybrid drives (SSHDs); carrier signal processing modules; and hardware devices specifically 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 computer program products that may include, for example, the instructions and / or computer code disclosed herein.

[0160] The systems, apparatuses, and / or methods described herein can be executed by software (executing on hardware), hardware, or a combination thereof. Software modules (executing on hardware) can be expressed in various software languages ​​(e.g., computer code), including C, C++, Java®, Python, Ruby, Visual Basic®, and / or other object-oriented, procedural, or other programming languages ​​and development tools. Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions (such as those generated by a compiler), code for generating network services, and files containing higher-level instructions executed by a computer using an interpreter. Further examples of computer code include, but are not limited to, control signals, encryption code, and compression code.

[0161] K. Communication devices

[0162] In some variations, the systems 100 and 200 described herein can communicate with networks and computer systems via communication device 128. In some variations, systems 100 and 200 can communicate with other devices via one or more wired and / or wireless networks. A wireless network can refer to any type of digital network not connected by any type of cable. Examples of wireless communication in a wireless network include, but are not limited to, cellular, radio, satellite, and microwave communications. However, a wireless network can connect to a wired network to connect to the Internet, other carrier voice and data networks, commercial networks, and personal networks. Wired networks are typically carried by copper twisted-pair, coaxial, and / or fiber optic cables. Many different types of wired networks exist, including wide area networks (WANs), metropolitan area networks (MANs), local area networks (LANs), Internet Area Networks (IANs), Campus Area Networks (CANs), Global Area Networks (GANs) (like the Internet), and Virtual Private Networks (VPNs). In the following text, a network refers to any combination of wireless, wired, public, and private data networks typically interconnected via the Internet to provide a unified networking and information access system.

[0163] Cellular communication can 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 hybrid of cellular, Wi-Fi, and satellite communications. In some variations, communication device 132 may include a radio frequency receiver, a transmitter, and / or an optical (e.g., infrared) receiver and transmitter. Communication device 132 can communicate via wired and / or wireless means with one or more of the following: robots 114, 214; negative pressure sources 122, 222; sensors 124; input devices 126; output devices 134; networks; databases; servers; combinations thereof, etc.

[0164] L. Output device

[0165] The output device 134 of systems 100 and 200 can be configured to output data corresponding to the system and may include one or more of a display device, an audio device, and a tactile device. For example, the display device may allow an operator to view images of one or more microfluidic chips 116, 216, 316 and robots 114, 214. In some variations, the output device may include a display device comprising at least one of the following: light-emitting diodes (LEDs), liquid crystal displays (LCDs), electroluminescent displays (ELDs), plasma display panels (PDPs), thin-film transistors (TFTs), organic light-emitting diodes (OLEDs), electronic paper / electronic ink displays, laser displays, and / or holographic displays.

[0166] Audio devices can output fluid data, sensor data, system data, alarms, and / or warnings in an audible manner. For example, an audio device can output an audible warning when sensor data (e.g., pressure, flow rate) falls outside a predetermined range or when a malfunction is detected in the robot. As another example, audio can be output to prevent potential harm to the operator and / or the system (e.g., robot collision, excessive negative pressure) when operator input is overridden by the system. In some variations, the audio device may include at least one of a loudspeaker, a piezoelectric audio device, a magnetostrictive loudspeaker, and / or a digital loudspeaker. In some variations, the operator can use the audio device and a communication channel to communicate with other users. For example, the operator can establish an audio communication channel (e.g., VoIP call) with a remote operator and / or observer.

[0167] Tactile devices can be incorporated into one or more input and output devices to provide an additional sensory output (e.g., force feedback) to the operator. For example, a tactile device can generate a tactile response (e.g., vibration) to confirm operator input to an input device (e.g., a touch surface). Alternatively or additionally, tactile feedback can notify the operator that the input is being overridden by the system to prevent potential harm to the operator and / or the system (e.g., robot collision, excessive negative pressure).

[0168] II. Methods

[0169] Methods for isolating ECMB from biofluids are also described herein. The methods described herein can be used to provide high-throughput separation of ECMB and biofluids, enabling additional analyses, such as analysis of ECMB or biofluids (or portions or fractions thereof), to aid in diagnosis and / or development of treatment plans for subjects, and to monitor the effectiveness of treatment plans. For example, predetermined fractions of biofluids or ECMBs can be analyzed to determine the amount of disease-associated biomarkers, which can be used for diagnostics and target identification. Biomarkers can 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 safety and treatment signals in subjects after treatment initiation and during treatment. In some variations, pharmacodynamic biomarkers can serve as endpoints in clinical trials and can be surrogate endpoints.

[0170] Figure 7 This is a flowchart summarizing a method 700 for separating ECMB from a biological fluid using any of the systems and apparatus described herein. Method 700 may include transferring a biological fluid (e.g., about 400 µl of sample) to an inlet reservoir of a microfluidic chip 702. For example, the microfluidic chip may include at least one confined channel (e.g., a uniform flow channel) having an inlet and an outlet. The inlet reservoir may be fluidly coupled to the inlet of at least one confined channel and at least one obstruction (e.g., a strut) and an outlet reservoir. Optionally, the microfluidic chip may be pre-filled with a buffer solution before containing the biological fluid. A vacuum seal may be fabricated and maintained within the system to minimize air bubbles within the microfluidic chip.

[0171] In some variations, a negative pressure between approximately 10 mm HG and approximately 760 mm HG can be applied to the outlet reservoir of the microfluidic chip 704. The applied negative pressure can remove the non-ECMB portion of the biofluid from the microfluidic chip while the ECMB remains disposed within the microfluidic chip. For example, as fluid flows through the microfluidic chip, the shape of the ECMB within the fluid is altered by contact (e.g., bending) with obstacles (e.g., struts) disposed within the microfluidic chip, thereby facilitating ECMB fixation within the microfluidic chip (e.g., ECMB attachment to struts).

[0172] In some variations, the ECMB in the microfluidic chip can be processed 706. For example, one or more of the following can be applied to the ECMB in the microfluidic chip: histochemical staining, immunohistochemical (IHC) staining, multiplex IHC staining, multispectral imaging, protein staining, nucleic acid staining, chemical fixation, and protease inhibitors. For example, human plasma can be stained with hematoxylin and eosin. As described in more detail herein, histochemical staining can be used for any of the analytical methods described above.

[0173] In some variations, one or more predetermined antibodies (e.g., for extracellular matrix materials, extracellular vesicle markers), detergents, and reagents can be applied to the microfluidic chip to facilitate IHC staining. For example, multiple antibodies (e.g., extracellular matrix, cancer-related markers, extracellular vesicle markers) can be applied to microfluidic chip IHC for multispectral imaging.

[0174] In some variations, sequential staining of the fractions separated on the microfluidic chip can aid in the removal of material within the microfluidic chip. For example, antibody staining of a sample can create 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 result due to the movement of the sample within the microfluidic chip. Therefore, a useful comparison between the first and second spatial arrangements can be challenging. Thus, chemical immobilization (e.g., crosslinking) of biofluids (e.g., ECMBS) on the chip can be applied to immobilize the biofluids within the microfluidic chip. For example, the microfluidic chip can contain carbodiimide and aldehyde immobilizers configured to crosslink the biofluids to immobilize them on the microfluidic chip. In some variations, the inlet and outlet of the microfluidic chip can be sealed.

[0175] In some variants, biomarkers of biological fluids and ECMBs can be measured by one or more of the following: immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, and UV-VIS.

[0176] In some variations, the biofluid can be analyzed as a total biofluid fraction before transfer 708. In some variations, ECMB retained in the microfluidic chip can be analyzed as a separate fraction after applying negative pressure 710. In some variations, the biofluid removed from the microfluidic chip can be analyzed as an eluent fraction after applying negative pressure 712. In some variations, the biofluid removed from the microfluidic chip can be treated using one or more of the following: microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, sieve filtration, ultrafiltration, percolation, tangential flow filtration, membrane filtration, immunoaffinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC kinetics.

[0177] In some variations, one or more of the following can be used to analyze biofluids removed from the microfluidic chip (e.g., eluent fractions, total fluid, entrapped material): microscopy, microfluidic devices, mass spectrometry, microarrays, nucleic acid amplification, hybridization, proteomic analysis, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next-generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, fluorescence in situ hybridization (FISH), chelation complexation, quantitative HPLC, spectrophotometry, colorimetric assay, chemiluminescence assay, immunofluorescence assay, light scattering, antibody arrays, Western blotting, immunoassay, immunoprecipitation, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS / MS, RT-PCR, and quantitative PCR.

[0178] Although the foregoing variations have been described in considerable detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced, and such changes and modifications are intended to fall within the scope of the appended claims. Furthermore, it should be understood that the components and features of the systems and apparatuses described herein can be used in any combination. The description of certain elements or features with respect to the specific drawings is not intended to be limiting, nor should it be construed as implying that such elements cannot be used in combination with any other described elements. For all variations described herein, the steps of the method may not be performed sequentially. Some steps are optional, such that each step of the method may not be performed.

Claims

1. A system for isolating extracellular matrix bodies (ECMB) from a biological fluid, the system comprising: A retainer configured to contain the biofluid; A robot configured to transfer the biofluid from the holder to a microfluidic chip; A chip connector configured to hold at least one microfluidic chip; Manifold, the manifold being coupled to the at least one microfluidic chip; as well as A negative pressure source, coupled to the manifold, is configured to apply a negative pressure between approximately 10 mm HG and approximately 760 mm HG to the at least one microfluidic chip.

2. The system of claim 1, wherein the chip connector comprises a base and a cap, the base being configured to contact a bottom portion of the at least one microfluidic chip, and the cap being configured to contact a top portion of the at least one microfluidic chip.

3. The system according to claims 1 to 2, wherein the chip connector is configured to distribute the compressive force applied by the negative pressure to the periphery of the microfluidic chip.

4. The system according to any one of claims 1 to 3, wherein the bottom portion comprises the periphery of the at least one microfluidic chip.

5. The system according to any one of claims 1 to 4, wherein the cover defines a plurality of holes.

6. The system according to any one of claims 1 to 5, wherein the base includes a first fastener and the cover includes a second fastener, the first fastener and the second fastener being configured to align the microfluidic chip in a predetermined orientation.

7. The system according to any one of claims 1 to 6, further comprising at least one inlet connector and at least one outlet connector disposed between the cap and the at least one microfluidic chip.

8. The system according to any one of claims 1 to 7, wherein the at least one outlet connector comprises an elongated body defining a lumen and including a plurality of steps along the length of the elongated body.

9. The system according to any one of claims 1 to 8, wherein the at least one outlet connector comprises an elongated body defining a lumen whose inner diameter decreases in the distal direction.

10. The system according to any one of claims 1 to 9, wherein one or more of the microfluidic chips, the inlet connector, and the outlet connector comprise disposable components.

11. The system according to any one of claims 1 to 10, wherein the chip connector comprises a durable component.

12. The system according to any one of claims 1 to 11, wherein the holder is configured to contain one or more reagents, and the robot is configured to transfer the one or more reagents from the holder to the microfluidic chip.

13. The system according to any one of claims 1 to 12, further comprising a sensor coupled to the at least one inlet connector, the sensor being configured to measure one or more of flow rate and pressure.

14. The system according to any one of claims 1 to 13, further comprising an optical sensor coupled to the chip connector, the optical sensor being configured to image one or more of the microfluidic chips.

15. The system according to any one of claims 1 to 14, wherein the at least one microfluidic chip includes at least one confining channel, the at least one confining channel being fluidly coupled between the inlet and outlet of the microfluidic chip.

16. The system according to any one of claims 1 to 15, wherein the at least one restricted passage comprises at least one obstacle.

17. The system according to any one of claims 1 to 16, wherein the at least one limiting channel comprises a length between about 5 mm and about 30 mm.

18. The system according to any one of claims 1 to 17, wherein the at least one limiting channel comprises a cross-sectional dimension between about 5 µm and about 30 µm.

19. The system according to any one of claims 1 to 18, wherein the at least one microfluidic chip includes at least one obstruction configured to restrict fluid flow.

20. The system according to any one of claims 1 to 19, wherein the at least one obstacle comprises a support.

21. The system according to any one of claims 1 to 20, wherein the at least one microfluidic chip includes a confined region configured to hold a first fraction of the biofluid and allow fluid flow of a second fraction of the biofluid.

22. The system according to any one of claims 1 to 21, wherein the first segment comprises the ECMB.

23. The system according to any one of claims 1 to 22, wherein the restricted area comprises a plurality of obstacles configured to hold the first segment.

24. The system according to any one of claims 1 to 23, wherein the spacing between the plurality of obstacles in the confined region decreases along the length of the microfluidic chip from the entrance to the exit of the confined region.

25. The system according to any one of claims 1 to 24, wherein the spacing between the plurality of obstacles in the restricted area is between about 100 µm and about 4 µm.

26. The system according to any one of claims 1 to 25, wherein each of the plurality of obstacles comprises a diameter between about 50 µm and about 1 mm.

27. A method for isolating extracellular matrix bodies (ECMB) from a biological fluid, the method comprising: The biofluid is transferred to an inlet reservoir of a microfluidic chip, the microfluidic chip comprising: At least one limiting channel, the at least one limiting channel having an inlet and an outlet, wherein the inlet reservoir is fluidly coupled to the inlet of the at least one limiting channel, and At least one pillar, and Export storage; and A negative pressure between approximately 10 mm HG and approximately 760 mm HG is applied to the outlet reservoir of the microfluidic chip, wherein the ECMB remains in the microfluidic chip after the biofluid is removed from the microfluidic chip.

28. The method of claim 27, further comprising distributing the compressive force applied by the negative pressure from the outlet reservoir to the periphery of the microfluidic chip.

29. The method according to any one of claims 27 to 28, wherein the at least one limiting channel comprises the at least one pillar.

30. The method according to any one of claims 27 to 29, wherein the at least one limiting channel comprises a length between about 5 mm and about 30 mm.

31. The method according to any one of claims 27 to 30, wherein the at least one limiting channel comprises a cross-sectional dimension between about 5 µm and about 30 µm.

32. The method according to any one of claims 27 to 31, wherein the at least one microfluidic chip includes at least one obstruction configured to restrict fluid flow.

33. The method according to any one of claims 27 to 32, wherein the at least one microfluidic chip includes a confined region configured to hold a first fraction of the biofluid and allow fluid flow of a second fraction of the biofluid.

34. The method of any one of claims 27 to 33, wherein the restricted area comprises a plurality of obstacles configured to hold the first segment.

35. The method according to any one of claims 27 to 34, wherein the spacing between the plurality of obstacles in the confined region decreases along the length of the microfluidic chip from the entrance to the exit of the confined region.

36. The method according to any one of claims 27 to 35, wherein the spacing between the plurality of obstacles in the restricted area is between about 100 µm and about 4 µm.

37. The method according to any one of claims 27 to 36, wherein each of the plurality of obstacles comprises a diameter between about 50 µm and about 1 mm.

38. The method according to any one of claims 27 to 37, further comprising applying one or more of the following to the ECMB in the microfluidic chip: histochemical staining, immunohistochemical (IHC) staining, multiplex IHC staining, multispectral imaging, protein staining, nucleic acid staining, chemical fixation, and protease inhibitors.

39. The method according to any one of claims 27 to 38, further comprising measuring one or more biomarkers of the biological fluid and the ECMB by one or more of the following: immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, and UV-VIS.

40. The method according to any one of claims 27 to 39, further comprising using one or more of the following to analyze the biofluid removed from the microfluidic chip: microscopy, microfluidic device, mass spectrometry, microarray, nucleic acid amplification, hybridization, proteomic analysis, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next-generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, fluorescence in situ hybridization (FISH), chelation complexation, quantitative HPLC, spectrophotometry, antibody array, Western blotting, immunoassay, immunoprecipitation, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS / MS, RT-PCR, and quantitative PCR.

41. The method according to any one of claims 27 to 40, further comprising treating the biofluid removed from the microfluidic chip using one or more of the following: microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, sieve filtration, percolation, tangential flow filtration, membrane filtration, immunoaffinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC electrokinetics.

42. The method according to any one of claims 27 to 41, wherein the biological fluid comprises one or more of the following: whole blood, plasma, serum, cerebrospinal fluid, intrathecal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspiration, semen, amniotic fluid, vitreous humor, aqueous humor, lymph, bile, cerumen, chyle, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric juice, mucus, pericardial fluid, pus, inflammatory secretions, sebum, serous fluid, smegma, phlegm, synovial fluid, vaginal secretions, menstrual discharge, vomitus, and fluids passing through one or more of tissues and gels.

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