System, device, and method for microfluidic fluid analysis

Abstract

The present invention describes a system, apparatus, and method useful for separating extracellular matrix from biological fluid in a high-throughput and automated manner using a microfluidic chip. A system for separating extracellular matrix from biological fluid may include a holder configured to receive biological fluid, a robot configured to transfer biological fluid from the holder to a microfluidic chip, a chip connector configured to secure at least one microfluidic chip, a manifold connected to at least one microfluidic chip, and a negative pressure source connected to the manifold. The negative pressure source may be configured to apply a negative pressure of about 10 mmHg to about 760 mmHg to at least one microfluidic chip.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 608,790 filed on December 11, 2023.

[0003] Technology field

[0004] The present invention relates to an apparatus, system, and method for separating and / or analyzing extracellular matrix bodies (ECMBs) from biological fluids. Background Technology

[0005] Conventional methods for the diagnosis and prognosis of diseases involve isolating or analyzing small fractions of biological samples (e.g., biological fluids), which can detect and analyze, for example, individual cells, extracellular matrix, extracellular vesicles or soluble tissue microenvironments, proteins, and nucleic acid molecules. For example, the inventors have previously described a method for detecting and analyzing molecules using microfluidic chips and positive pressure. However, disadvantages of such positive pressure systems include low throughput, limited scalability, and reproducibility. Furthermore, existing systems tend to rely excessively on manual labor. For example, users must manually transfer each biological sample and / or set of reagents to the corresponding inlet of the microfluidic chip using a pipette or inject them with a syringe. Moreover, since positive pressure systems require a significant amount of tubing to deliver samples to the microfluidic chip, the volume of liquid for each microfluidic chip may be inconsistent. Additionally, these systems tend to have high contamination rates because they require a significant amount of time-intensive manual labor for installation and cleaning. Other existing methods for disease diagnosis and prognosis involve isolating and analyzing intact organ tissues, whole cells, and biomarkers (e.g., extracellular vesicles, such as exosomes). However, a disadvantage of these methods is that diseases cannot be detected or characterized if these isolated structures do not clearly reflect the disease state. For example, biomarkers have inherent limitations as they are often not directly related to the pathology of interest. Therefore, additional systems, devices, and methods are required to isolate and analyze biological fluids.

[0006] The present invention describes systems, apparatus, and methods useful for separating extracellular matrix (ECMB) from biological fluids, either indirectly or indirectly, from tissues or gels in an automated and high-throughput manner. In this manner, ECMB may be used, for example, for the diagnosis, prognosis prediction, and / or treatment of subjects using histochemical staining techniques, immunohistochemistry, or nucleic acid hybridization and analysis. Generally, the system described herein for separating extracellular matrix from biological fluids may comprise a holder configured to receive biological fluids, a robot configured to transfer biological fluids from the holder to a microfluidic chip, a chip connector configured to secure at least one microfluidic chip, a manifold connected to at least one microfluidic chip, and a negative pressure source connected to the manifold. The negative pressure source may be configured to apply a negative pressure of about 10 mmHg to about 760 mmHg to at least one microfluidic chip.

[0007] In some variations, the chip connector may include a base configured to contact the lower portion of at least one microfluidic chip and a cover configured to contact the upper portion of at least one microfluidic chip. In some variations, the chip connector may be configured to distribute a compressive force applied by negative pressure around the perimeter of the microfluidic chip. In some variations, the lower portion may include the perimeter of at least one microfluidic chip. In some variations, the cover may define a plurality of openings. In some variations, the base may include a first fixing device and the cover may include a second fixing device. The first fixing device and the second fixing device may be configured to align the microfluidic chip in a predetermined direction. In some variations, at least one inlet connector and at least one outlet connector may be disposed between the cover and at least one microfluidic chip.

[0008] In some variations, at least one outlet connector may include an extended body that defines a lumen and includes a plurality of steps along the length of the extended body. In some variations, at least one outlet connector may include an extended body that defines a lumen in which the inner diameter decreases in the distal direction. In some variations, one or more microfluidic chips, inlet connectors, and outlet connectors may include disposable components. In some variations, the chip connector may include durable components.

[0009] In some variations, the holder may be configured to receive one or more reagents, and the robot is configured to transfer one or more reagents from the holder to the microfluidic chip. In some variations, a sensor may be connected to at least one inlet connector. The sensor may be configured to measure one or more of flow rate and pressure. In some variations, an optical sensor may be connected to a chip connector. The optical sensor may be configured to image one or more of the microfluidic chips.

[0010] In some variations, at least one microfluidic chip may include at least one restricting channel fluidically connected between the inlet and outlet of the microfluidic chip. In some variations, at least one restricting channel may include one or more obstacles. In some variations, at least one restricting channel may have a length of about 5 mm to about 30 mm. In some variations, the cross-sectional area of ​​each channel may be about 5 μm to about 30 μm. In some variations, at least one microfluidic chip may include at least one obstacle configured to restrict fluid flow. At least one obstacle may include a pillar.

[0011] In some variations, at least one microfluidic chip may include a confining region configured to hold a first fraction of biological fluid and allow fluid flow of a second fraction of biological fluid. In some variations, the first fraction may include ECMB.

[0012] In some variations, the confinement region may include a plurality of obstacles configured to hold a first fraction. In some variations, the spacing between the plurality of obstacles within the confinement region decreases along the length of the microfluidic chip from the entrance of the confinement region to the exit of the confinement region. In some variations, the spacing between the plurality of obstacles within the confinement region is about 100 μm to about 4 μm. In some variations, each of the plurality of obstacles has a diameter of about 50 μm to about 1 mm.

[0013] In addition, the present invention describes a method for separating extracellular matrix (ECMB) from biological fluid. Generally, such a method comprises the steps of transferring biological fluid to an inlet reservoir of a microfluidic chip and applying negative pressure to the microfluidic chip. The microfluidic chip may include at least one restriction channel having an inlet and an outlet. The inlet reservoir may be fluidically connected to the inlet of at least one restriction channel and at least one column and outlet reservoir. A negative pressure of about 10 mmHg to about 760 mmHg may be applied to the outlet reservoir of the microfluidic chip, and the ECMB remains in the microfluidic chip after the biological fluid is removed from the microfluidic chip.

[0014] In some variations, the compressive force applied by negative pressure may be distributed from the outlet reservoir to the perimeter of the microfluidic chip. In some variations, at least one restricting channel may include at least one column. In some variations, at least one restricting channel may have a length of about 5 mm to about 30 mm. In some variations, at least one restricting channel may have a cross-sectional dimension of about 5 μm to about 30 μm. In some variations, at least one microfluidic chip may include at least one obstacle configured to restrict fluid flow. In some variations, at least one microfluidic chip may include a restricting region configured to hold a first fraction of biological fluid and allow fluid flow of a second fraction of biological fluid. In some variations, the restricting region may include a plurality of obstacles configured to hold the first fraction. In some variations, the spacing between the plurality of obstacles within the restricting region may decrease along the length of the microfluidic chip from the inlet of the restricting region to the outlet of the restricting region. In some variations, the spacing between the plurality of obstacles within the restricting region may be about 100 μm to about 4 μm. In some variations, each of the multiple obstacles may have a diameter of about 50 μm to about 1 mm.

[0015] Once ECMB is separated from the biological fluid, additional methods can be used to analyze the ECMB. For example, in some variations, one or more of histochemical staining (including immunohistochemical (IHC) staining and multiplex IHC staining), protein staining, nucleic acid staining, chemical fixation, and protease inhibitors can be applied to the ECMB within the microfluidic chip. In some variations, one or more biomarkers from the biological fluid and ECMB can be measured by one or more of immunoassays, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, and UV-VIS.

[0016] In some variations, biological fluids removed from the microfluidic chip may be analyzed using one or more of the following: microscopy, microfluidic devices, mass spectrometry, microarray, nucleic acid amplification, hybridization, proteomic profiling, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next-generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, fluorescence in situ hybridization (FISH), chelate complexation, quantitative HPLC, spectrophotometry, antibody array, Western blot, immunoassay, immunoprecipitation, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS / MS, RT-PCR, and quantitative PCR.

[0017] In some variations, processing the biological fluid removed from the microfluidic chip may use one or more of microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, mesh filtration, dialysis filtration, tangential flow filtration, membrane filtration, immunoaffinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC electrodynamics.

[0018] In some variations, biological fluids may include whole blood, plasma, serum, cerebrospinal fluid, intracerebrospinal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspirate, semen, amniotic fluid, vitreous fluid, aqueous humor, lymph, bile, earwax, chyme, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric fluid, mucus, pericardial fluid, pus, watery excretion (rheum), sebum, serous fluid, smegma, sputum, synovial fluid, vaginal secretions, menstrual fluid, and vomit, and one or more of fluids that have passed through one or more of tissues and gels. Brief explanation of the drawing

[0019] Figure 1 is a block diagram of an exemplary variation of the system. FIGS. 2a through 2c show perspective views of exemplary variations of the system. FIG. 2d is a side view of the system illustrated in FIG. 2c. FIG. 3a is a top view of an exemplary modification of a microfluidic chip. FIG. 3b is a cross-sectional side view of the microfluidic chip shown in FIG. 3a. Fig. 3c is a plan view of an exemplary variation of a microfluidic chip array. FIG. 4a shows a perspective view of an exemplary variation of a chip connector. FIG. 4b shows an exploded perspective view of the cover and microfluidic chip shown in FIG. 4a. FIG. 4c shows an exploded top perspective view of the base and microfluidic chip shown in FIG. 4a. FIG. 4d shows an exploded bottom perspective view of the base and microfluidic chip shown in FIG. 4a. FIGS. 5a through 5e show the top perspective, bottom perspective, side view, top view, and bottom view, respectively, of exemplary variations of an exit connector. FIGS. 6a through 6e show the top perspective, bottom perspective, top view, and bottom view, respectively, of exemplary variations of an exit connector. Figure 7 shows a flowchart of an exemplary modification for separating ECMB from biological fluid. Figures 8a and 8b are exemplary graphs of ECMB quantities based on microfluidic channel dimensions. Figures 8a through 8e are exemplary images of ECMB on microfluidic chips with different channel sizes. Figure 9 is an exemplary graph of the flow rate through a microfluidic chip with or without a chip connector. Fig. 10a is an exemplary graph of installation times for various outlet connector configurations. Fig. 10b is an exemplary graph of experimental periods for dyeing procedures in positive pressure and negative pressure systems. Fig. 10c is an exemplary graph of abnormal shapes for various outlet connector configurations. Figure 11 is an exemplary graph of the number of parallel experiments performed for positive pressure systems and negative pressure systems. FIGS. 12a and 12b are exemplary images of biological contamination in a positive pressure microfluidic system. FIGS. 12c and 12d are exemplary images of biological contamination using the negative pressure system described herein. FIG. 12e is an exemplary graph of biological contamination per area for a positive pressure system and a negative pressure system. Figures 13a and 13b are exemplary graphs of ECMB amounts based on negative pressure. Figures 13c and 13d are exemplary images of ECMB on a microfluidic chip with different negative pressures applied. FIGS. 14a, FIGS. 14c, FIGS. 14d, FIGS. 14e, and FIGS. 14i are exemplary graphs of ECMB amounts based on manifold ports. FIGS. 14b and FIGS. 14j are exemplary graphs of ECMB amounts based on pillar regions. FIGS. 14f, FIGS. 14g, and FIGS. 14h are exemplary images of ECMB on a microfluidic chip with different negative pressures applied. FIG. 15a is an exemplary graph of the amount of ECMB based on a system with or without a manifold. FIG. 15b is an exemplary graph of the amount of ECMB based on a column area and a system with or without a manifold. Figures 16a and 16b are exemplary graphs of ECMB amounts based on sound pressure. Figure 17a is an exemplary image of an ECMB on a microfluidic chip perfused with a healthy (control) human aqueous humor sample. Figure 17b is an exemplary graph of the amount of ECMB on a microfluidic chip between a control sample and a primary open-angle glaucoma (POAG) sample. FIG. 18a shows a plan view of another exemplary variation of a microfluidic chip. FIG. 18b shows a perspective view of the microfluidic chip shown in FIG. 18a. FIG. 18c shows a perspective view of the microfluidic chip shown in FIG. 18b connected to a cover. FIG. 18d shows a detailed plan view of the microfluidic chip shown in FIG. 18a. FIG. 19a is a plan view of an exemplary variation of a multi-channel microfluidic chip. FIG. 19b shows a detailed plan view of the microfluidic chip illustrated in FIG. 19a. FIG. 19c is a plan view of an exemplary variation of a multi-channel microfluidic chip array. FIG. 20a illustrates a plan view of another exemplary variation of a microfluidic chip. FIG. 20b illustrates a detailed plan view of the microfluidic chip illustrated in FIG. 20a. FIG. 20c is a plan view of an exemplary variation of a microfluidic chip array. FIG. 20d illustrates a perspective view of the microfluidic chip array illustrated in FIG. 20a connected to a cover. FIG. 20e illustrates a plan view of the cover illustrated in FIG. 20d. FIG. 20f illustrates a plan view of the microfluidic chip array and the cover illustrated in FIG. 20d. Figure 21 shows a plan view of another exemplary variation of a multi-channel microfluidic chip. FIG. 22 shows a perspective view of another exemplary variation of a chip connector. FIG. 23 shows a perspective view of an exemplary variation of a multi-chip connector. FIGS. 24a through 24d illustrate a perspective view, top view, bottom view, and side view of an exemplary variation of an inlet connector. FIGS. 25a through 25d illustrate perspective, top, bottom, and side perspective views of other exemplary variations of an inlet connector. FIGS. 26a through 26d illustrate a perspective view, top view, bottom view, bottom perspective view, and side view of other exemplary variations of an inlet connector. FIGS. 27a through 27d illustrate a perspective view, top view, bottom view, and side view of an exemplary variation of an exit connector. FIGS. 28a through 28d illustrate a perspective view, a top view, and a bottom view of other exemplary variations of an exit connector. Fig. 29a is an exemplary image of fluid flow through a single-channel microfluidic chip. Fig. 29b is an exemplary image of fluid flow through the channels of an 8-channel microfluidic chip. FIG. 30a is an exemplary graph of the non-laminar flow region based on a single-channel microfluidic chip and a multi-channel microfluidic chip. FIG. 30b is an exemplary graph of the clogging rate based on a single-channel microfluidic chip and a multi-channel microfluidic chip. FIG. 30c is an exemplary graph of the background noise based on a single-channel microfluidic chip and a multi-channel microfluidic chip. Fig. 31a is an exemplary image of a staining artifact induced by non-laminar flow through a single-channel microfluidic chip. Fig. 31b is an exemplary graph of the artifact size based on a single-channel microfluidic chip and a multi-channel microfluidic chip. Figure 32 is an exemplary graph of the amount of ECMB on a microfluidic chip based on an inlet connector containing silicon and an inlet connector without silicon. Specific details for implementing the invention

[0020] The present invention describes systems, apparatus, and methods (e.g., analysis, etc.) for separating ECMB from biological fluids. For example, the systems, apparatus, and methods described herein may be useful for diagnosing, prognosing, and / or treating a subject by: separating, concentrating, extracting, and / or immobilizing a predetermined fraction (e.g., ECMB, proteins, nucleic acids, particles, substances, molecules, extracellular vesicles or soluble tissue microenvironments, proteins) from a biological fluid into a biological fluid fraction (e.g., isolated fraction) while preserving the composition and characteristics of the fraction; improving the yield of the fraction from the biological fluid by reducing contamination to improve signal intensity for the pathology of interest; identifying one or more fractions containing disease-related biomarkers (e.g., proteins, genes, ECMB components) for disease target identification; and predicting disease risk and / or medical conditions based on the analysis of multiple biological fluid fractions.

[0021] Generally, the systems and devices described herein can separate ECMB from biological fluids in a manner that preserves the composition and / or properties of the biological fluids, thereby facilitating their use as biomarker(s) for disease diagnosis and / or monitoring of chemical or biological processes. For example, the systems and devices described herein may be additionally useful for high-throughput, scalable, and automated microfluidic processing of biological fluids for histology; reduced compression of microfluidic chips due to applied negative pressure based on distributed pressure; reduced clogging and increased laminar flow, flow consistency, and signal-to-noise ratio based on microfluidic chip channel density; reduced risk of contamination, installation time, manual processing, and cleaning; visualization of ECMB using fixation and staining (e.g., histochemical, immunohistochemical) for one or more of spatial localization and profiling (e.g., proteins, genes); and ease of identification between diseased ECMB and physiologically normal ECMB. Thus, biological fluid separation and histochemical analysis as described herein can be performed faster, more cheaply, and with higher throughput (e.g., volume) than with positive pressure microfluidic systems.

[0022] Generally, the systems and devices described herein may include a microfluidic system configured for the biological assay of ECMB in biological fluids. For example, the microfluidic system may include a plurality of disposable microfluidic chips (e.g., a microfluidic chip array) configured to receive biological fluids (e.g., samples) from a robot to facilitate automation and high-throughput processing. The system may be configured to separate ECMB from biological fluids within the microfluidic chips using a negative pressure source connected to the microfluidic chips. The separated ECMB may be exposed to one or more reagents and buffers for histochemical staining and subsequent imaging and / or analysis. Thus, a plurality of biological samples may be processed in parallel by the microfluidic system.

[0023] For example, a system for separating an extracellular matrix from a biological fluid may include a holder configured to receive the biological fluid, one or more robots configured to transfer the biological fluid from the holder to a microfluidic chip, a chip connector configured to secure at least one microfluidic chip, a manifold connected to at least one microfluidic chip, and a negative pressure source connected to the manifold. The negative pressure source may be configured to apply a negative pressure of about 10 mmHg to about 760 mmHg to at least one microfluidic chip. In some variations, the system may include a plurality of holders, a plurality of chip connectors, a plurality of manifolds, a plurality of negative pressure sources, a plurality of reservoirs, a plurality of robots, etc.

[0024] The negative pressure system of the apparatus and system described herein aids in the separation of ECMB from biological fluids with high throughput and low contamination. In this way, multiple samples can be processed (e.g., 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, by using a manifold to uniformly distribute negative pressure in parallel across multiple microfluidic chips, consistent fluid flow and reproducible results can be provided. Additionally, negative pressure applied to the outlet of a microfluidic chip allows a robot to transfer fluid to the inlet of the microfluidic chip, thereby increasing throughput and consistency and reducing manual labor. However, modifying a positive pressure microfluidic system to apply negative pressure to microfluidic chips and samples can compromise the composition and properties of the biological fluids as well as the structural integrity of the microfluidic chips, making it difficult to separate, concentrate, and / or extract biological fluid fractions. In other words, without the innovative technology described herein, applying negative pressure to positive pressure microfluidic chips and systems would produce unpredictable results.

[0025] Biological fluids may include one or more fluids that have passed through one or more of tissues and gels, among human or animal biological fluids, tissues, cells, whole blood, plasma, serum, cerebrospinal fluid, intracerebrospinal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspirate, semen, amniotic fluid, vitreous fluid, aqueous humor, lymph fluid, bile, earwax, chyme, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric fluid, mucus, pericardial fluid, pus, aqueous excretions, sebum, serous fluid, smegma, sputum, synovial fluid, vaginal secretions, menstrual fluid, vomit, tumors, carriers, reagents, solutions, conjugated moiety, etc.

[0026] Buffer solutions include MES, HCl acid buffer, acid phthalate buffer, alkaline borate buffer, acetate buffer, ammonia acetate buffer, acetone buffer, ammonia buffer, barbiturate buffer, buffered copper sulfate solution, glycerin solution, glycine buffer, palladium chloride buffer, Na2HPO4 citrate, sodium citrate buffer preparation, sodium 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, It may include one or more of TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS.

[0027] Some microfluidic chips and systems suitable for use in the systems of the present invention are described in international patent application PCT / US2021 / 023827, filed on March 24, 2021, with the title of invention "DEVICE AND METHODS FOR ISOLATING EXTRACELLULAR MATRIX BODIES" and international patent application PCT / US2019 / 052310, filed on September 21, 2019, with the title of invention "COMPOSITIONS AND METHODS FOR GLAUCOMA", each of which is incorporated herein by reference in its entirety.

[0028] I. Systems and devices

[0029] Generally, the systems and devices described herein can separate extracellular matrix (ECMB) from biological fluids at high throughput. The separated ECMB and residual biological fluids (or parts or fractions thereof) may be used to aid in diagnosis, prognosis prediction, or determining a treatment plan for a subject and determining the effectiveness of such treatment plan. A block diagram of an exemplary system (100) is shown in FIG. 1. The system (100) may include a holder (112), a reservoir (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.

[0030] In some variations, the holder (112) (e.g., material storage) may be configured to store one or more fluids (e.g., biological fluids) for transfer to the microfluidic chip (116). The holder (112) may include one or more of a tray and a container. In some variations, the storage (113) may be configured to receive a predetermined fraction of the biological fluid (e.g., waste liquid) from the microfluidic chip (116). For example, the non-ECMB fraction of the biological fluid may be transferred to the storage (113) and stored for further processing (e.g., separation, analysis) and / or disposal. Additionally or alternatively, the system (100) may include one or more pretreatment components, such as a material separation assembly, a fractionation assembly, a biological storage, etc.

[0031] In some variations, the robot (114) may be configured to transfer a fluid (e.g., biological fluid, reagent) from the holder (112) to at least one microfluidic chip (116). In some variations, the microfluidic chip (116) may be configured to receive and process the biological fluid. For example, the microfluidic chip (116) may include a channel (e.g., microfluidic channel) and at least one obstacle (e.g., pillar), and is configured to separate and retain (e.g., capture) a predetermined fraction of the biological fluid within the microfluidic chip (116), while the remaining fraction flows out of the microfluidic chip (116). In some variations, the chip connector (118) may be configured to secure at least one microfluidic chip (116). The microfluidic chip (116) may be detachably connected to the chip connector (118) to facilitate cleaning and reduce installation time. For example, the microfluidic chip (116) may be a disposable component, and the chip connector (118) may be a durable component. In some variations, the chip connector (118) may include a disposable connector (e.g., an inlet connector, an outlet connector) configured to facilitate the negative pressure applied to the microfluidic chip (116) by a negative pressure source (122) to perfuse and flow a biological fluid through the microfluidic chip (116).

[0032] In some variations, the manifold (120) may be configured to be connected to at least one microfluidic chip (116). In some variations, a negative pressure source (122) may be configured to be connected to the manifold (120). In this way, a single manifold (120) may be fluidically connected to multiple microfluidic chips (116) to facilitate applying negative pressure (e.g., vacuum, suction) to multiple microfluidic chips (116) using a single negative pressure source (122).

[0033] In some variations, the sensor (124) may include one or more sensors configured to measure one or more features (e.g., pressure, flow rate, optical image, temperature, humidity) corresponding to one or more components of the biological fluid and the system (100, 200). In some variations, the input device (126) may be configured to generate an input signal based on operator input. In some variations, the processor (128) and memory (130) may be configured to control the system (100, 200). In some variations, the communication device (132) may be configured to communicate with one or more components of the system (100, 200), as well as with a network and other computer systems. In some variations, the output device (134) may be configured to output data corresponding to the system (100, 200), such as images, flow rates, etc. of the biological fluid within the microfluidic chip (116).

[0034] In some variations, the system (100, 200) may be configured to perform a number of assays, e.g., ELISA, immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, UV-VIS, Raman, NMR, mass spectrometry, NG sequencing, protein array, ribonucleic acid array, gene array, qPCR, RT-qPCR, RT-PCR, etc. In some variations, the microfluidic chip (116) may be analyzed by the system (100, 200) and / or removed and analyzed using another device (e.g., a multi-well plate, a 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 contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, light-ray microscopy, ultrasound microscopy, positron emission tomography, computed tomography, and magnetic resonance imaging.

[0035] FIGS. 2A and 2B are perspective views of a system (200) comprising a holder (212), a storage (213), a robot (214), an end effector (215) (e.g., a pipette) connected to the robot (214), a plurality of chip connectors (218) each having a chip connector (218) holding a plurality of microfluidic chips (216), a manifold (220) including a plurality of fluid conduits (221a, 221b), and a negative pressure source (222). The system (200) may include sensors, input devices, processors, memory, communication devices, and output devices as described in relation to FIG. 1, but are not shown in FIGS. 2A through 2D for clarity. In some variations, the end effector (215) may include a plurality of pipettes configured to transfer fluid(s) stored in the holder (212) to microfluidic chips (216) fixed to each chip connector (218). In some variations, the system (200) may include a plurality of robots (214). For example, a first robot may be configured to transport biological fluid, a second robot (not shown for clarity) may be configured to transport reagent, and a third robot (not shown for clarity) may be configured to detachably connect microfluidic chips (216) to respective chip connectors (218). The third robot may be configured to assemble and disassemble disposable components, including microfluidic chips (216), from durable components of the system (200) (e.g., chip connectors (218)) to reduce manual labor and / or reduce contamination. In FIG. 2a, each microfluidic chip (216) is connected to a respective fluid conduit (221a), and each of these is connected to a manifold (220). A single fluid conduit (221b) can connect the manifold (220) to the reservoir (213) and the negative pressure source (222).

[0036] In some variations, a robot (214) may be used to load one or more fluids from a holder (212) to each inlet of a plurality of microfluidic chips (216). A negative pressure source (222) may apply suction to each of the microfluidic chips (216) through a manifold (220) and fluid conduits (221a, 221b). The fluid at the inlet (e.g., inlet reservoir) may be drawn toward each outlet of the microfluidic chip (216) while the ECMB remains within the microfluidic chip (216). The separated non-ECMB fluid is drawn through the fluid conduits (221a, 221b) and the manifold (220) and received in a reservoir (213) (e.g., wastewater treatment).

[0037] FIG. 2c is a detailed perspective view of the system (200), FIG. 2d is a corresponding detailed side view, 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). The chip connector (218) and the robot (214) are not shown in FIG. 2c and FIG. 2d for clarity.

[0038] An inlet connector (240) may be connected 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 to be transferred from the end effector (215) to the microfluidic chip (216). An outlet connector (242) may be connected between the outlet (232) of the microfluidic chip (216) and the fluid conduit (221a). In some variations, the fluid conduit (221a) may be configured to receive fluid to be transferred from the microfluidic chip (216) to the reservoir (213) (e.g., via negative pressure).

[0039] In some variations, one or more clamps (250) may be configured to detachably connect (e.g., secure, hold) the inlet connector (240) and the outlet connector (242) to the microfluidic chip (216), respectively. For example, the clamp (250) may include one or more springs (not shown) and hinges (252) configured to provide a predetermined force to hold the inlet connector (240) and the outlet connector (242) in place against the microfluidic chip (216). In some variations, a portion (254) of the clamp (250) (e.g., an actuator) is actuated (e.g., by being pressed down by a robot or operator) to release one or more of the inlet connector (240) and the outlet connector (242), thereby allowing the microfluidic chip (216) to be easily removed from the chip connector (218).

[0040] A. holder

[0041] The system described herein may include a holder (112, 212) configured to receive one or more fluids. In some variations, the holder (112, 212) may be configured to receive and store a plurality of fluids, including a biological fluid (e.g., a sample) and one or more reagents. Examples of reagents include buffers, lysis solutions, nucleic acid cleavages, cleavage inhibitors, precipitating agents, fixation reagents, carrier fluids, biological fluids, water, purified water, saline solutions, organic solvents, gelling agents, surfactants, ligands that bind to or associate with components of ECMB, combinations thereof, and reagents that interact with the biological components of the sample. In some variations, the reagents may include one or more reagents for measuring biomarker levels or amounts or comparing biomarker levels to a control group. In some variations, the holder (112, 212) may include a plurality of reservoirs that store each fluid separately without mixing. The fluid may include any suitable fluid, for example, including one or more fluids useful for sampling and / or analysis. The biological fluid for sampling may include, for example, human or animal biological fluid, tissue, cell, whole blood, plasma, serum, cerebrospinal fluid, intracerebrospinal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspirate, semen, amniotic fluid, vitreous fluid, aqueous humor, lymph fluid, bile, earwax, chyme, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric fluid, mucus, pericardial fluid, pus, aqueous excretion, semen, serous fluid, smegma, sputum, synovial fluid, vaginal secretion, menstrual fluid, vomit, tumor, carrier, reagent, solution, binding moiety, etc., among one or more fluids that have passed through one or more tissues and gels. Biological fluids useful for analysis may include carriers, reagents, binding moiety, etc. A holder (112, 212) may be placed within the system (100, 200) at a location accessible to a robot (114, 214).For example, the holder (112, 212) may be configured to receive a plurality of pipettes connected to the robot (114, 214) to transfer a plurality of fluids from the holder (112, 212) to a plurality of microfluidic chips (116, 216).

[0042] B. robot

[0043] The system described herein may include one or more robots (114, 214). Generally, the robots (114, 214) may be configured to move and transfer one or more fluids (e.g., biological fluids, reagents, antibodies, buffers) between a holder (112, 212) and at least one microfluidic chip (116, 216). For example, the 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, the robot (114, 214) may be connected to an end effector (215) comprising one or more pipettes (e.g., micropipettes, multi-channel pipettes) configured to transfer fluid (e.g., biological fluid, reagent) from a holder (112, 212) to at least one microfluidic chip (116, 216) (e.g., through the inlet connector (240) of the chip connector (118, 218). For example, biological fluid from an object may first be transferred from the holder (112, 212) to a plurality of microfluidic chips (116, 216) using the robot (114, 214). Then, using a robot (114, 214), different reagents can be transferred from a holder (112, 212) to a predetermined microfluidic chip (116, 216) for different treatments (e.g., various histochemical staining). The robot (114, 214) is connected to a processor (128) and a memory (130) to control the type and volume of fluid being transferred using one or more pipettes. In some variations, an end effector (215) may be configured to detachably connect (e.g., assemble, disassemble) a disposable component from a durable component of the system (200). Additionally, or alternatively, the system (100) may include one or more material transfer components, such as a track and a container, configured to translate along a track.

[0044] In some variations, the robot (114, 214) may be configured to detachably connect a microfluidic chip (116, 216) to one or more of a chip connector (118, 218) and a negative pressure source (122, 222). For example, the robot (114, 214) may be configured to connect the microfluidic chip (116, 216) to the chip connector (118, 218) so that the microfluidic chip (116, 216) is fluidly connected to a manifold (120, 220) and a negative pressure source (122, 222). Additionally, the robot (114, 214) may be configured to detachably connect other components (e.g., an inlet connector, an outlet connector) to the microfluidic chip (116, 216). Conversely, the robot (114, 214) may be configured to detach the microfluidic chip (116, 216) from the chip connector (118, 218) and transfer it to another device (e.g., a microscope slide, an imaging system, an analysis system). In some variations, the robot (114, 214) may be configured to remove one or more of the holder (112, 212) and reservoir (113, 213) from the system (100, 200) (e.g., to replace them with different holders (112, 212) and reservoirs (113), 213). Automated system installation, fluid transfer, and cleaning using the robot (114, 214) can reduce contamination and human error, and increase consistency and throughput, among other benefits.

[0045] In some variations, the robot (114, 214) may be, for example, a linear robot arm, an articulated robot arm, and / or a SCARA robot arm. The robot (114, 214) may include one or more segments connected 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, the robot (114, 214) may have six or more degrees of freedom. Degrees of freedom in an orthogonal coordinate system may be expressed as three translational (position) variables (e.g., surge, heave, sway) and three rotational (orientation) variables (e.g., roll, pitch, yaw). In some variations, the robot (114, 214) may have fewer than six degrees of freedom.

[0046] In some variations, the robot (114, 214) may be configured to move all areas of the system (100, 200) in up to three dimensions. The robot (114, 214) may include one or more motors that translate and / or rotate joints to move the robot (114, 214) to a desired position and orientation. In some variations, the position of the robot may be temporarily fixed when delivering fluid to a predetermined microfluidic chip or when removing one or more microfluidic chips (116, 216) from the chip connector (118, 218) (e.g., to transfer them to a microscope or other imaging system). The robot (114, 214) may be mounted on any suitable object, such as a platform (e.g., a table), a wall, or a ceiling, or may stand on its own (e.g., on the ground). Additionally, or alternatively, the robot (114, 214) may be configured to move manually.

[0047] C. microfluidic chip

[0048] The system described herein may include one or more microfluidic chips (116, 216). Generally, the microfluidic chips (116, 216) may be configured to receive 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 restricting channel, a uniform flow channel), at least one obstacle (e.g., a column) configured to restrict fluid flow, and an outlet reservoir. The restricting channel may include an inlet and an outlet. The inlet reservoir may be fluidically connected to the inlet of the restricting channel, and the outlet of the restricting channel may be fluidly connected to the outlet reservoir. The inlet reservoir may be configured to receive and store fluid delivered from the robot (114, 214). The microfluidic chips (116, 216) may be configured to process biological fluids through the channels while receiving negative pressure from the outlet of the microfluidic chips (116, 216) (e.g., an outlet reservoir).

[0049] FIGS. 3A and 3B are a plan view and a cross-sectional view of a microfluidic chip (316) comprising an inlet reservoir (310), an outlet reservoir (330), and a confining area (320) (e.g., a filter area) 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 obstacle (340) (e.g., a column). Likewise, the outlet reservoir (330) may include an outlet (332) (e.g., an opening) and at least one obstacle (340). The size, shape, and spacing of the obstacle (340) within the inlet reservoir (310) and the outlet reservoir (330) may be the same or different. In some variations, fluid may be received at the inlet (312) and flow through the confining area (320) to the outlet (332). A fluid conduit connected to a manifold and a negative pressure source (not shown in FIGS. 3a through 3c) may be connected to an outlet (332). The suction force applied by the negative pressure source through the outlet (332) can draw the fluid contained in the inlet (312) through the inlet reservoir (310), the restricted area (320), and the outlet reservoir (330).

[0050] In some variations, the confinement area (320) may be configured to confine fluid flow to separate and retain (e.g., capture) a first fraction of fluid within the confinement area (320) of the microfluidic chip (316), while the remaining second fraction of fluid may flow out of the microfluidic chip (316) through negative pressure applied to the microfluidic chip (316). In some variations, the first fraction of fluid may include ECMB. As illustrated in FIG. 3a, the confinement area (320) may include a flow barrier (322) that confines a confinement channel (324) (e.g., a uniform flow channel) and a plurality of obstacles (340) (e.g., pillars) having a plurality of gaps (326). The confinement channel (324) may be linear and may have a length equal to at least the length of the confinement area (320). In some variations, one or more of laminar flow and flow consistency may be based on the length of the confinement channel (324). For example, a restricted channel (324) that does not extend beyond one of the inlet reservoir (310) and the outlet reservoir (330) can increase one or more of laminar flow and flow consistency throughout the entire microfluidic chip (e.g., in the width and length directions of the microfluidic chip (316)).

[0051] Multiple obstacles (340) within the confining area (320) may be configured to confine (e.g., obstruct) fluid flow and retain a first fraction of the biological fluid. For example, the spacing between the multiple obstacles (340) within the confining area (320) may decrease along the length of the microfluidic chip (316) from the inlet of the confining area (320) to the outlet of the confining area (320). In some variations, the spacing between the multiple obstacles (340) within the confining area (320) may be between about 100 μm and about 4 μm. For example, the spacing between the obstacles (340) at the inlet of the confining area (320) may be about 100 μm, and the spacing between the obstacles (340) at the outlet of the confining area (320) may be about 4 μm. In some variations, the spacing may increase in steps. For example, the spacing can decrease in the order of about 100 μm, about 50 μm, about 25 μm, about 15 μm, and about 4 μm. Additionally or alternatively, the spacing between obstacles (340) can vary continuously along the length of the restricted area (320). In some variations, a greater proportion of ECMB may be captured within the portion of the restricted area (320) with smaller spacing.

[0052] The limiting channel (324) may have a length of about 5 mm to about 30 mm, about 10 mm to about 30 mm, about 15 mm to about 30 mm, about 20 mm to about 30 mm, about 5 mm to about 25 mm, about 5 mm to about 20 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, and all ranges and sub-values ​​in between. The limiting channel (324) may have a cross-sectional dimension of about 5 μm to about 30 μm, about 10 μm to about 30 μm, about 15 μm to about 30 μm, about 20 μm to about 30 μm, about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, and all ranges and sub-values ​​in between. In some variations, the limiting channel (324) may include at least one obstacle. Multiple obstacles (340) may have a diameter of about 50 μm to about 1 mm.

[0053] As illustrated in the cross-sectional side view of FIG. 1, in FIG. 3b, the microfluidic chip (316) may comprise a substrate (350) connected to a cover (360) (e.g., a top plate, a glass slide). The cover (360) placed over and above the substrate (350) facilitates the flow of fluid through the microfluidic chip (316). For example, the cover (360) may be configured to surround the substrate (350) (e.g., including an inlet reservoir (310), a confining area (320), and an outlet reservoir (330)), except for one or more inlets (312) and one or more outlets (332). In some variations, the substrate (350) may comprise a plurality of microfluidic chips (316). For example, FIG. 3b illustrates a set of eight microfluidic chips (316) arranged parallel to each other on the substrate (350). In this way, a single microfluidic chip array (370) can be used to process multiple fluid samples individually (e.g., each), thereby increasing throughput and efficiency and reducing system size. For example, a microfluidic chip array (370) having eight microfluidic chips (316) can enable independent ECMB separation for eight fluid samples by any combination of histological staining, immunohistochemical (IHC) staining, reagents, etc. In some variations, the substrate (350) may comprise a suitable material including one or more silicone-based polymers (e.g., polydimethylsiloxane (PDMS)), other polymers, thermoplastic resins, thermoplastic elastomers, hydrogels, paper, and glass.

[0054] In some variations, the restriction channel may extend beyond the length of the restriction area. For example, FIG. 18a and FIG. 18b respectively illustrate a plan view and a perspective view of a microfluidic chip (1800) comprising an inlet reservoir (1810), an outlet reservoir (1830), and a restriction area (1820) (e.g., a filter area) fluidly communicating between the inlet reservoir (1810) and the outlet reservoir (1830). The inlet reservoir (1810) may include at least one inlet obstacle (1841), and the outlet reservoir (1830) may include at least one outlet obstacle (1843). The inlet reservoir (1810) and the outlet reservoir (1830) may generally have a circular shape. FIG. 18c illustrates a microfluidic chip (1800) connected to a cover (1802). In some variations, the cover (1802) may include an inlet (1812) fluidly communicating with an inlet reservoir (1810) of the microfluidic chip (1800) and an outlet (1832) fluidly communicating with an outlet reservoir (1830). A plurality of obstacles (1840) may be disposed between the inlet reservoir (1810) and the outlet reservoir (1830). Similar to the microfluidic chip (316), the confining area (1820) may be configured to restrict fluid flow to separate and retain (e.g., capture) a first fraction of fluid within the confining area (1820) of the microfluidic chip (1800), and allow the remaining second fraction of fluid to flow out of the microfluidic chip (1800) through negative pressure applied to the microfluidic chip (1800). In some variations, the first fraction of fluid may include ECMB. As described in detail in FIG. 18d, the restricted area (1820) may include a flow barrier (1822) that limits a restricted channel (1824) (e.g., a uniform flow channel) and a plurality of obstacles (1840) (e.g., pillars) having a plurality of gaps (1826). The restricted channel (1824) may be linear and may have a length greater than the length of the restricted area (1820).A restricting channel (1824) may be configured to facilitate fluid flow through the microfluidic chip (1800) when negative pressure is applied to the outlet (1832) of the microfluidic chip (1800). For example, the restricting channel (1824) may reduce bubble formation within the microfluidic chip (1800).

[0055] A plurality of obstacles (1840) within the confining area (1820) may be configured to confine (e.g., obstruct) fluid flow and retain a first fraction of the biological fluid for analysis (e.g., image analysis). For example, FIG. 18d provides a detailed drawing of the confining area (1820) of a microfluidic chip (1800) comprising a fluid barrier (1822), a confining channel (1824), and a plurality of gaps (1821 to 1829). In some variations, the gaps between the plurality of obstacles (1840) within the confining area (1820) may decrease along the length of the microfluidic chip (1800) from the inlet (1818) of the confining area (1820) to the outlet (1819) of the confining area (1820). In some variations, the gaps between the plurality of obstacles (1840) within the confining area (1820) may be between about 100 μm and about 4 μm. For example, the first gap (1821) between the entrance (1818) of the confining area (1820) and the obstacle (1840) may be about 100 μm, and subsequently, the second gap (1823) at the exit of the confining area (1820) may be about 50 μm, the third gap (1825) about 25 μm, the fourth gap (1827) about 15 μm, and the fifth gap (1829) about 4 μm. The length of each gap, as well as the diameter of the obstacle (1840), may be the same or different. In some variations, the confining area (1820) may extend across the entire width of the microfluidic chip (1800), excluding the fluid barrier (1822) and the confining channel (1824).

[0056] In some variations, the microfluidic chip may include a plurality of restricting channels to facilitate fluid flow and reduce bubble formation and clogging within the microfluidic chip. For example, FIGS. 19a and 19b illustrate a top view of a multi-restricting channel microfluidic chip (1900) comprising an inlet reservoir (1910), an outlet reservoir (1930), and a restricting region (1920) (e.g., a filter region) fluidly communicating 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 generally have a circular shape. A plurality of obstructions (1940) may be placed between the inlet reservoir (1910) and the outlet reservoir (1930). Similar to the microfluidic chip (316, 1800), the confining region (1920) may be configured to confine fluid flow to separate and retain (e.g., capture) a first fraction of fluid within the confining region (1920) of the microfluidic chip (1900), and allow the remaining second fraction of fluid to flow out of the microfluidic chip (1900) through negative pressure applied to the microfluidic chip (1900). In some variations, the first fraction of fluid may include ECMB. FIGS. 19a through 19c illustrate a microfluidic chip (1900) having a plurality of confining channels (1924, 1926, 1928) (e.g., uniform flow channels) that may be spaced apart from each other, linear, and parallel. For example, the restricted area (1920) may include a flow barrier (1922) that limits each restricted channel (1924, 1926, 1928) as described more in detail in FIG. 19b, and a plurality of obstacles (1940) (e.g., pillars) having a plurality of gaps (1921, 1923, 1925, 1927, 1929). The restricted channels (1924, 1926, 1928) may be longer than the length of the restricted area (1920).The restricting channels (1924, 1926, 1928) may have the same or different lengths from one another. Thus, multiple restricting channels (1924, 1926, 1928) may be configured to facilitate fluid flow through the microfluidic chip (1800) while minimizing bubble formation and blockage when negative pressure is applied to the outlet reservoir (1930) of the microfluidic chip (1900). Additionally, FIG. 19c illustrates a microfluidic chip array (1902) comprising multiple multi-restricting channel microfluidic chips (1900). Since multiple fluid samples can be processed individually (e.g., each) using a single microfluidic chip array (1902), throughput and efficiency can be increased and the system size can be reduced. In some variations, the microfluidic chip array may include a plurality of microfluidic chips having different configurations (e.g., including at least one microfluidic chip (316), at least one microfluidic chip (1800), at least one microfluidic chip (1900), etc.).

[0057] A plurality of obstacles (1940) within the confining area (1920) may be configured to confine (e.g., obstruct) fluid flow and retain a first fraction of the biological fluid for analysis (e.g., image analysis). For example, FIG. 19b provides a detailed drawing of the confining area (1920) of a microfluidic chip (1900) comprising a fluid barrier (1922), a first confining channel (1924), a second confining channel (1926), a third confining channel (1928), and a plurality of gaps (1921, 1923, 1925, 1927, 1929). In some variations, the gaps between the plurality of obstacles (1940) within the confining area (1920) may decrease along the length of the microfluidic chip (1900) from the inlet (1918) of the confining area (1920) to the outlet (1919) of the confining area (1920). In some variations, the spacing between multiple obstacles (1940) within the restricted area (1920) may be between about 100 μm and about 4 μm. For example, the first spacing (1921) between the obstacles (1940) at the entrance (1918) of the restricted area (1920) may be about 100 μm, and subsequently, at the exit (1919) of the restricted area (1920), the second spacing (1923) may be about 50 μm, the third spacing (1925) about 25 μm, the fourth spacing (1927) about 15 μm, and the fifth spacing (1929) about 4 μm. The length of each spacing, as well as the diameter of the obstacles (1840), may be the same or different. In some variations, the confinement area (1920) may extend across the entire width of the microfluidic chip (1900), excluding the fluid barrier (1922) and confinement channels (1924, 1926, 1928).

[0058] FIGS. 20a and 20b illustrate a top view of a microfluidic chip (2000) comprising an inlet reservoir (2010), an outlet reservoir (2030), and a restricted area (2020) (e.g., a filter area) fluidly communicating 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 obstacle (2040). The inlet reservoir (1810) and the outlet reservoir (1830) may generally have a polygonal shape.

[0059] Multiple obstacles (2040) may be placed between the inlet reservoir (2010) and the outlet reservoir (2030). Similar to the microfluidic chip (316, 1800, 1900), the confining area (2020) may be configured to restrict fluid flow to separate and retain (e.g., capture) a first fraction of fluid within the confining area (2020) of the microfluidic chip (1800), and allow the remaining second fraction of fluid to flow out of the microfluidic chip (2000) through negative pressure applied to the microfluidic chip (2000). In some variations, the first fraction of fluid may include ECMB. The restricted area (2020) may include a flow barrier (2022) that limits a restricted channel (2024) (e.g., a uniform flow channel) as described in detail in FIG. 20b, and a plurality of obstacles (2040) (e.g., pillars) having a plurality of gaps (2021, 2023, 2025, 2027, 2029). The restricted channel (2024) may be linear and may have a length at least equal to the length of the restricted area (2020). The restricted channel (2024) may be configured to facilitate fluid flow through the microfluidic chip (2000) when negative pressure is applied to the outlet (2032) of the microfluidic chip (2000). For example, the restricted channel (2024) may reduce bubble generation within the microfluidic chip (2000).

[0060] A plurality of obstacles (2040) within the confining area (2020) may be configured to confine (e.g., obstruct) fluid flow and retain a first fraction of the biological fluid for analysis (e.g., image analysis). For example, FIG. 20b provides a detailed drawing of a confining area (2020) of a microfluidic chip (2000) comprising a fluid barrier (2022), a confining channel (2024), and a plurality of gaps (2021, 2023, 2025, 2027, 2029). In some variations, the gaps between the plurality of obstacles (2040) within the confining area (2020) may decrease along the length of the microfluidic chip (2000) from the inlet (2018) of the confining area (2020) to the outlet (2019) of the confining area (2020). In some variations, the spacing between multiple obstacles (2040) within the restricted area (2020) may be between about 100 μm and about 4 μm. For example, the first spacing (2021) between the obstacles (2040) at the entrance (2018) of the restricted area (2020) may be about 100 μm, and subsequently, at the exit of the restricted area (2020), the second spacing (2023) may be about 50 μm, the third spacing (2025) about 25 μm, the fourth spacing (2027) about 15 μm, and the fifth spacing (2029) about 4 μm. The diameter of the obstacles (2040), as well as the length of each spacing, may be the same or different. In some variations, the confinement area (2020) may extend across the entire width of the microfluidic chip (2000), excluding the fluid barrier (2022) and the confinement channel (2024).

[0061] FIG. 20c illustrates a plan view of a microfluidic chip array (2002) comprising a plurality of microfluidic chips (2000). Since a plurality of fluid samples can be processed individually (e.g., each) using a single microfluidic chip array (2002), throughput and efficiency can be increased and the system size can be reduced. FIG. 20d illustrates a microfluidic chip array (2002) connected to a cover (2004). In some variations, the cover (2004) may include an inlet (2012) fluidly communicating with an inlet reservoir (2010) of each microfluidic chip (2000) and an outlet (2032) fluidly communicating with an outlet reservoir (2030). FIG. 20e illustrates a plan view of the cover (2004) illustrated in FIG. 20d. For example, the inlets (2012) and outlets (2032) of the cover (2004) may correspond to each 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 an end effector (e.g., a pipette). For example, the distance between adjacent inlets (2012) may be about 5 mm to about 15 mm, about 7 mm to about 13 mm, about 8 mm to about 12 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, and all ranges and sub-values ​​in between. FIG. 20f shows a top view of the microfluidic chip array (2002) and the cover (2004) illustrated in FIG. 20d.

[0062] The microfluidic chip described herein may comprise a single channel (e.g., microfluidic chip (1800, 1900, 2000)) or multiple channels (e.g., microfluidic chip array 2002). In some variations, based on the channel density of the microfluidic chip (e.g., comparison of a multi-channel microfluidic chip array with a single-channel microfluidic chip), unexpected, surprising, and significant improvements may be observed in one or more of laminar flow, flow uniformity, clogging, signal-to-noise ratio, and artifacts. For example, FIG. 29a is a fluorescence image of fluid flow through a single-channel microfluidic chip (2900), and FIG. 29b is a corresponding fluorescence image of fluid flow through a channel of an 8-channel microfluidic chip array (2950). In particular, FIGS. 29a and 29b illustrate respective restricted regions (2020, 2070) comprising a plurality of obstacles (2940), restricted channels (2924, 2974), and fluid barriers (2940, 2990). For example, a secondary antibody (e.g., B100) was injected into a microfluidic chip (2900) and a microfluidic chip array (2950) at pressures of approximately 100 mmHg and approximately 75 mmHg, respectively, to achieve steady-state fluid flow. When comparing a single-channel microfluidic chip (2900) and a multi-channel microfluidic chip array (2950), the microfluidic chip array (2950) has significantly fewer blind spots (2910) than the microfluidic chip (2900). The blind spot (2910) in FIG. 29a features relatively slow / slow or turbulent (e.g., non-laminar) fluid flow, which is represented by a white line running across the length of the confining area (2020). In contrast, the fluid flow is laminar along the width and length of the channels of the microfluidic chip array (2950). The fluid flow through the obstacle (2940) and the confining channel (2974) in the microfluidic chip array (1950) is more uniform (e.g., identical) than the fluid flow passing through the obstacle (2940) and the confining channel (2924) in the microfluidic chip (2900).

[0063] The difference in laminar flow between a single-channel microfluidic chip (2900) and a multi-channel microfluidic chip array (2950) showed an unexpected and surprising difference, as shown in the graphs of FIGS. 30a to 30c. FIG. 30a is a graph (3000) of non-laminar flow regions (e.g., number of dead zones) based on a single-channel microfluidic chip and a multi-channel microfluidic chip array. For example, the number of dead zones of a single-channel microfluidic chip (e.g., microfluidic chip (2900)) was compared with the number of dead zones of a single channel of a microfluidic chip array having eight channels (e.g., microfluidic chip array (2950)). Error bars represent the standard deviation. The improved laminar flow through the multi-channel microfluidic chip array (2950) can correspond to a reduction in the frequency of dead zones. These data indicate that the disturbance to laminar flow in a single-channel microfluidic chip is significantly increased compared to the flow disturbance in a single lane of an 8-channel microfluidic chip array (bar graph, left). Surprisingly, less flow disturbance was observed in the 8-channel microfluidic chip array.

[0064] FIG. 30b is a graph (3010) of the clogging rate based on a single-channel microfluidic chip (e.g., microfluidic chip (2900)) and a multi-channel microfluidic chip (e.g., microfluidic chip (2950)). 200 μl of human plasma samples were injected into each chip (2900, 2950) and stained, and clogging was determined when there was substantially no fluid flow. FIG. 30b shows that the clogging rates of the single-channel microfluidic chip and the 8-channel microfluidic chip array are approximately 20% and approximately 2%, respectively. Enhanced laminar flow through the multi-channel microfluidic chip array (2950) can correspond to a reduction in the clogging rate. FIG. 30c is a graph (3020) of background noise (e.g., average gray value) of stained fluid samples based on a single-channel microfluidic chip (e.g., microfluidic chip (2900)) and a multi-channel microfluidic chip (e.g., microfluidic chip (2950)). Fluid flow rate: Compared to the single-channel microfluidic chip, the fluid flow rate in the multi-channel microfluidic chip array was improved, resulting in lower background noise values. For example, graph (3020) shows that the background noise in the multi-channel microfluidic chip array was reduced by approximately 15% compared to the single-channel microfluidic chip. The standard deviations for the single-channel microfluidic chip and the 8-channel microfluidic chip array are 0.83 and 0.49, respectively.

[0065] In some variations, a decrease in laminar flow in a microfluidic chip channel may correspond to an increase in artifacts and an increase in non-laminar flow. For example, FIG. 31a is an image (3100) of non-laminar flow passing through a confined area (3120) of a single-channel microfluidic chip for an eosin-stained sample (e.g., SRX 1032). In particular, the boundary (e.g., edge, perimeter) area of ​​the confined area (3120) may have a higher frequency of artifact occurrence. For example, the upper edge area (3110) of the confined area (3120) may have a higher frequency of artifacts (3130) (e.g., residual eosin stain aggregates) corresponding to flow disturbances. Artifacts and ECMBs can be distinguished based on morphological features. FIG. 31b is a graph (3140) of artifact size based on a single-channel microfluidic chip and a multi-channel microfluidic chip. The difference in artifact size between the single-channel microfluidic chip and the multi-channel microfluidic chip array was unexpected, surprising, and significant. For example, in the single-channel microfluidic chip, edge artifacts appeared at a higher frequency compared to the microfluidic chip array due to the irregular fluid flow passing through the chip. In particular, in the 8-channel microfluidic chip array, no edge artifacts were observed due to the consistent flow pattern and laminar flow passing through the array. Error bars represent the standard deviation.

[0066] FIG. 21 illustrates a top view of a multi-restricted channel microfluidic chip (2100) comprising an inlet reservoir (2110), an outlet reservoir (2130), and a restricting region (2120) (e.g., a filter region) fluidly communicating between the inlet reservoir (2110) and the outlet reservoir (2130). The inlet reservoir (2110) and the outlet reservoir (2130) may include at least one inlet obstacle (2140). The inlet reservoir (2110) and the outlet reservoir (2130) may generally have a polygonal shape. A plurality of obstacles (2140) may be placed between the inlet reservoir (2110) and the outlet reservoir (2130). Similar to the microfluidic chip (316, 1800, 1900, 2000), the confining region (2120) may be configured to confine fluid flow to separate and retain (e.g., capture) a first fraction of fluid within the confining region (2120) of the microfluidic chip (2100), and allow the remaining second fraction of fluid to flow out of the microfluidic chip (2100) through negative pressure applied to the microfluidic chip (2100). In some variations, the first fraction of fluid may include ECMB. The microfluidic chip (2100) may include a plurality of confining channels (2124, 2126, 2128) (e.g., uniform flow channels) that may be spaced apart from each other, linear, and parallel. For example, the restricted area (2120) may include a flow barrier (2122) that limits each restricted channel (2124, 2126, 2128) and a plurality of obstacles (2140) (e.g., pillars) having a plurality of gaps. The restricted channels (2124, 2126, 2128) may have a length longer than the length of the restricted area (2120). The restricted channels (2124, 2126, 2128) may have the same or different lengths from each other. Accordingly, the plurality of restricted channels (2124, 2126, 2128) may be configured to facilitate fluid flow through the microfluidic chip (1800) while minimizing bubble generation and blockage when negative pressure is applied to the outlet reservoir (2130) of the microfluidic chip (2100).In some variations, the microfluidic chip array may include a plurality of microfluidic chips (2100).

[0067] The microfluidic chip described herein may have various configurations and dimensions. For example, the obstacles described herein may have one or more of circular, spherical, triangular, square, polygonal, rhomboidal, and fin shapes. The obstacles may have the same or different shapes, sizes, and spacing. For example, the obstacles may have spacing between adjacent pillars of about 4 μm to about 100 μm, about 4 μm, about 15 μm, about 25 μm, about 50 μm, about 100 μm, and all ranges and sub-values ​​in between.

[0068] In some variations, a microfluidic chip with negative pressure applied can improve the yield (e.g., immobilization) of a predetermined fraction of a biological fluid based on the cross-sectional dimensions of the channels. For example, each channel may have cross-sectional (e.g., height) dimensions of about 5 μm to about 30 μm, about 5 μm to about 10 μm, about 5 μm to about 20 μm, about 5 μm to about 15 μm, about 10 μm to about 20 μm, about 15 μm to about 30 μm, about 8 μm, about 11 μm, about 15 μm, and all ranges and sub-values ​​in between.

[0069] The amount of ECMB captured using the negative pressure system described herein may vary depending on the contents of the fluid (e.g., healthy control sample, positive sample). FIG. 17a is an image (1700, 1702) of ECMB captured within the confined area of ​​a microfluidic chip for a glaucomatous human aqueous humor sample after eosin-Y staining. The magnified image (1702) shows the captured ECMB (a1) surrounding the pillar (a2). The high abundance of aggregated material captured in the microfluidic chip suggests that healthy aqueous humor may block the trabecular meshwork with ECMB, causing an increase in intraocular pressure. In contrast, the healthy (control) human aqueous humor sample has a lower relative content of ECMB captured in the microfluidic chip compared to the images (1700, 1702). For example, Fig. 17b is a graph (1710) of ECMB abundance (e.g., chip coverage %) as a function of sample type (healthy control sample, primary open-angle glaucoma (POAG) sample). The amount of ECMB captured in the microfluidic chip was significantly higher in the POAG sample than in the healthy control aqueous humor sample. Therefore, this data suggests that the increased ECMB in the POAG sample may cause an increase in intraocular pressure in glaucoma by blocking and obstructing the flow of ocular fluid in the trabecular meshwork.

[0070] In some variations, the amount of ECMB captured using the negative pressure system described herein may vary depending on the size of one or more microfluidic channels. For example, FIG. 8a is a graph (800) of the amount of ECMB as a function of cross-sectional channel size in a microfluidic chip. For example, graph 800 compares the total amount of ECMB captured when the cross-sectional heights of each microfluidic channel are 8 μm, 11 μm, and 15 μm. Normalized carboxyfluorocene succinimidyl ester (CFSE) signal intensity represents the total amount of protein and is correlated with ECMB abundance, and there is a linear relationship between channel size and ECMB abundance. FIG. 8b is a graph (810) of the amount of ECMB as a function of obstacle (e.g., pillar) spacing in a microfluidic chip. For example, FIG. 8b compares the total amount of ECMB across pillar regions with different height (cross-sectional) spacings. In all 15 μm (dark gray bar), 11 μm (light gray), and 8 μm (white) size channels, most of the ECMB is captured in the 100 μm, 50 μm, and 25 μm interval regions.

[0071] FIGS. 8c through 8e are exemplary images (820, 830, 840) of ECMB on a microfluidic chip having different channel cross-sectional (e.g., height) dimensions. FIG. 8c is an image (820) of an ECMB (a2) stained with CSFE with a channel dimension of 15 μm. Most of the ECMB is trapped in a column (a1) region with intervals of 100 μm, 50 μm, and 25 μm. An enlarged image (822) shows the trapped ECMB (a2) surrounding the column (a1). FIG. 8d is an image (830) of an ECMB (a2) stained with CSFE with a channel dimension of 11 μm. Most of the ECMB is trapped in a column (a1) region with intervals of 100 μm, 50 μm, and 25 μm. An enlarged image (832) shows a captured ECMB (a2) surrounding a pillar (a1). FIG. 8e is an image (840) of an ECMB (a2) stained with CSFE with a channel cross-sectional (e.g., height) dimension of 8 μm. Most of the ECMB is captured in the pillar (a1) regions at intervals of 100 μm, 50 μm, and 25 μm. An enlarged image (842) shows a captured ECMB (a2) surrounding a pillar (a1).

[0072] D. chip connector

[0073] The system described herein may include a chip connector. Generally, the chip connector (118, 218) is configured to secure one or more microfluidic chips (116, 216, 316, 1800, 1900, 2000, 2100) to improve the performance of the system (100, 200). Applying negative pressure to a microfluidic chip generates force on the structure of the microfluidic chip itself, which may reduce or hinder the processing of biological fluids using the microfluidic chip. For example, applying negative pressure to a channel of a microfluidic chip (e.g., a restriction channel) generates force at the outlet, which may compress (and / or collapse) the outlet reservoir of the microfluidic chip, which may lead to unpredictable flow rates, bubbles, and non-uniform dispersion of material within the microfluidic chip. In contrast, the chip connector described herein may be configured to secure the microfluidic chip and more uniformly distribute the negative pressure applied by a negative pressure source throughout the microfluidic chip (116, 216, 316) to maintain the structural integrity of the microfluidic chip (116, 216, 316) and provide consistent results.

[0074] FIG. 4a shows a perspective view of a chip connector (418) configured to secure a plurality of microfluidic chips (416). FIG. 4b shows an exploded perspective view of the cover (420) of the chip connector (418) and the microfluidic chip (416). As illustrated in FIG. 4a through 4d, the chip connector (418) may include a cover (420) configured to contact the upper part of the microfluidic chip (416) and a base (430) configured to contact the lower part of the microfluidic chip (416). The cover (420) may define a plurality of openings (422) and may include (e.g., overlap, cover) the perimeter of the microfluidic chip (416). The plurality of openings (422) may be configured to overlap one or more of the inlet (450) and outlet (460) of the microfluidic chip (416). Accordingly, a fluid conduit (not shown) of the manifold can be connected to a microfluidic chip (416) through an opening (422). In some variations, an inlet connector (440) can be connected to an inlet (450) and configured to receive fluid from the robot (114). Likewise, an outlet connector (442) can be connected between fluid conduits (not shown for clarity), and an outlet (460) can be configured to receive fluid transferred to a reservoir (113). The outlet connector (442) can be a connection interface between the fluid conduit and the outlet (460).

[0075] In some variations, the microfluidic chip (416) between the cover (420) and the lower part (430) can disperse the negative pressure applied to the outlet (460) of the microfluidic chip (416) to the periphery of the microfluidic chip (416) (e.g., the side of the chip without channels), thereby reducing compression at the outlet of the microfluidic chip and at the part not around the periphery of the microfluidic chip (e.g., fluid flow), and thus promote a constant flow rate and more uniform material dispersion through the microfluidic chip (416). For example, FIGS. 4c and FIGS. 4d illustrate exploded top and bottom perspective views of the base (430) of the chip connector (418) and the microfluidic chip (416), respectively, such that the periphery of the microfluidic chip (416) is in contact with the periphery of the base (430), and the lower part inside (e.g., not around the periphery) of the microfluidic chip (416) does not come into contact with the chip connector (418). This configuration allows the force applied by negative pressure at the outlet (460) (pushing the microfluid chip (416) toward the base (430)) to be distributed to the periphery of the microfluid chip (416) (parts where fluid does not flow), thereby reducing compression at the outlet (460) and parts not around the microfluid chip (416) (parts where fluid flows). In this way, the chip connector (418) can be configured to distribute the compressive force applied by negative pressure to the microfluid chip (416), thereby relieving pressure on the microfluid chip (416) and keeping the fluid channels open for fluid flow. Thus, the chip connector promotes laminar fluid flow and a constant flow rate, thereby minimizing dead zones, blockages, background noise, and artifacts.

[0076] In some variations, the chip connector (418) may include one or more fasteners configured to connect the microfluidic chip (416) to the chip connector (418). For example, as shown in FIG. 4b, the chip connector (418) may include a plurality of magnets (424) configured to securely hold the microfluidic chip (416) in place inside the chip connector (418). For example, the base (430) and the cover (420) of the chip connector (418) may each include a magnet (424) configured to align the microfluidic chip (418) to a predetermined orientation. As shown in FIG. 4b, the perimeter (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 counterforce against the negative pressure applied to the outlet (460) to further reduce compression at the outlet (460). Additionally, the fastening device may include one or more of the following: a latch, strap, clip, mount, shackle, snap, clamp, locking device, anchor, tie, adhesive, hook and loop fastener, and combinations thereof.

[0077] FIG. 22 shows an exploded perspective view of a chip connector (2200) configured to accommodate one or more microfluidic chips (2216). The chip connector (2200) may include a base (2230) configured to contact the lower portion of the microfluidic chip (e.g., the portion facing away from the cover of the microfluidic chip), an output connector (2242), and a cover (2220) configured to contact the upper portion of the microfluidic chip (2216) (e.g., the portion facing the output connector (2242)). The cover (2220) may define a plurality of openings (2222) and may include (e.g., overlap, cover) the perimeter of the microfluidic chip (2216). The plurality of openings (2222) may be configured to overlap one or more of the inlet (2250) and outlet (2260) of the microfluidic chip (2216). Accordingly, the fluid conduit of the manifold (not shown in the drawing) can be connected to the microfluidic chip (2216) through the corresponding opening (2222). In some variations, an inlet connector (not shown for clarity) may be connected to the inlet (2250) and configured to receive fluid from the robot (114). Likewise, an outlet connector (2242) may be connected between the fluid conduit (not shown for clarity) and the outlet (2260) and configured to receive fluid transferred (e.g., sucked) to the reservoir (113). In some variations, the outlet connector (2242) may be a connection interface between the fluid conduit and the outlet (2260). The chip connector (2200) may further include a base (e.g., base (430), FIG. 4c and FIG. 4d) which is not shown in FIG. 22 for clarity. The base can be configured to contact the bottom portion of the microfluidic chip (2216) in the same manner as illustrated and described in FIG. 4c and FIG. 4d.

[0078] In some variations, a microfluidic chip (2216) fixed between the cover (2220) and the base (2230) of the chip connector (2200) can reduce compression at the outlet (2260) of the microfluidic chip (2216) by dispersing the negative pressure applied to the outlet (2260) of the microfluidic chip (2216) to the periphery of the microfluidic chip (2216) (e.g., the side of the chip without channels), thereby promoting a constant flow rate and more uniform dispersion of material (e.g., the first fraction, ECMB) through the microfluidic chip (2216). This configuration can reduce compression at the outlet (2260) of the microfluidic chip (2216) by redistributing the force applied by the negative pressure at the outlet (2260) (pushing the microfluidic chip (2216) toward the base (2230)) to the periphery of the microfluidic chip (2216). In this way, the chip connector (2200) can be configured to disperse the compressive force applied to the microfluidic chip (2216) by negative pressure to relieve pressure on the microfluidic chip (2216) and to keep the fluid channel open for fluid flow. Thus, the chip connector (2200) promotes laminar fluid flow and a constant flow rate, thereby minimizing dead zones, blockages, background noise, and artifacts.

[0079] In some variations, as illustrated in FIG. 22, the chip connector (2200) may include a plurality of magnets (2224) configured to securely hold the microfluidic chip (2216) in place inside the chip connector (2200). For example, the base (2230) and the cover (2220) of the chip connector (2200) may each include at least one magnet (2224) configured to align the microfluidic chip (2200) in a predetermined orientation. As illustrated in FIG. 22, the perimeter (e.g., side) of the cover (2220) and the inner perimeter (e.g., side wall) of the base (2230) may each include one or more magnets (2224) configured to surround the microfluidic chip (2216) in the chip connector (2200).

[0080] In some variations, the chip connector (118, 218) is configured to accommodate one or more microfluidic chips (116, 216, 316, 1800, 1900, 2000, 2100) and / or a microfluidic chip array (370, 2002) to improve throughput or reduce the size of the system. Additionally, the chip connector may be configured to distribute the negative pressure applied by a negative pressure source more evenly across multiple microfluidic chips to maintain structural integrity and provide consistent results in a small space.

[0081] FIG. 23 illustrates an exploded perspective view of a chip connector (2300) configured to accommodate one or more microfluidic chip arrays (2314). For example, the chip connector (2300) may include a cover (2320) configured to contact the upper portion of the microfluidic chip array (2314) (e.g., a portion facing the inlet (2350) and outlet (2360)) and a base (2330) configured to contact the lower portion of the microfluidic chip array (2314) (e.g., a portion away from the inlet (2350) and outlet (2360). The cover (2320) may define a plurality of openings (2322) and may include (e.g., overlap, cover) the perimeter of the microfluidic chip array (2314). A plurality of openings (2322) may be configured to overlap with 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 the manifold may be connected to the microfluidic chip array (2314) through the openings (2322). In some variations, the inlet connector (2340) of the chip connector (2300) may be configured to be connected to the plurality of inlets (2350) of the microfluidic chip array (2314) to receive fluid from the robot (114). Likewise, the outlet connector (2342) of the chip connector (2300) may be configured to be connected between the fluid conduit (not shown for clarity) and the plurality of outlets (2360) of the microfluidic chip array (2314) to receive fluid transferred (e.g., sucked) to the reservoir (113). Accordingly, the outlet connector (2342) may be a connection interface between a fluid conduit and a plurality of outlets (2360). The inlet connector of the chip connector is described in relation to FIGS. 27a to 27d and FIGS. 28a to 28d, and the output connector of the chip connector is described in more detail in relation to FIGS. 24a to 24d, FIGS. 25a to 25d, and FIGS. 26a to 26d.

[0082] In some variations, a microfluidic chip array (2314) fixed between the cover (2320) and the bottom (2330) of the chip connector (2300) can reduce compression at the outlets of the microfluidic chip array (2314) by dispersing the negative pressure applied to the multiple outlets (2360) of the microfluidic chip array (2314) to the periphery of the microfluidic chip array (2314) (e.g., the side of the chip without channels), thereby promoting a constant flow rate and more uniform dispersion of material through the microfluidic chip array (2314). This configuration allows the negative pressure applied to 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), thereby reducing compression at the multiple outlets (2360) of the microfluidic chip array (2314). In this way, the chip connector (2300) can be configured to disperse the compressive force applied to the microfluidic chip array (2314) by negative pressure to relieve pressure on the microfluidic chip array (2314) and to keep the fluid channels open for fluid flow. Thus, the chip connector (2300) promotes laminar fluid flow and a constant flow rate, thereby minimizing dead zones, blockages, background noise, and artifacts.

[0083] In some variations, as illustrated in FIG. 23, the chip connector (2300) may include a plurality of magnets (2324) configured to securely hold the microfluidic chip array (2314) in place within the chip connector (2300). For example, the base (2330) and the cover (2320) of the chip connector (2300) may each include at least one magnet (2324) configured to align the microfluidic chip (2300) in a predetermined orientation. As illustrated in FIG. 23, the perimeter (e.g., side) of the cover (2320) and the inner perimeter (e.g., side wall) of the base (2330) may each include one or more magnets (2324) configured to surround the microfluidic chip array (2314) within the chip connector (2300). The chip connector (2300) may further include a base (e.g., base (430), FIG. 4c and FIG. 4d) which is not illustrated in FIG. 23 for clarity. The base may be configured to contact the bottom portion of the microfluidic chip array (2314) in the same manner as illustrated and described in FIG. 4c and FIG. 4d.

[0084] In some variations, the fluid flow rate and flow uniformity through the microfluidic chip (116, 216, 316, 416) to which negative pressure is applied can be improved by securing the microfluidic chip (116, 216, 316, 416) to the chip connector (118, 218, 418, 2200, 2300). FIG. 9 illustrates the improvement in the flow rate and flow uniformity between a microfluidic chip connected to the chip connector and a microfluidic chip without the chip connector as described herein. In particular, FIG. 9 is a graph (900) of the flow rates for three fluid flow processes (e.g., PBS priming, BVH stained with CFSE, PBS washing) under negative pressure conditions of about 100 mmHg in System 910 (e.g., System 100, dark gray bar) using a microfluidic chip and a chip connector and System 920 (light gray bar) using a microfluidic chip without a chip connector. In particular, the flow rate of PBS priming was 10.4 ± 3.27 μl / min when there was no chip connector and 11.4 ± 0.36 μl / min when there was a chip connector. The signal-to-noise ratio (SNR) (e.g., mean to standard deviation of flow rate) increased 9.8-fold from 3.2 when there was no chip connector to 31.3 when there was a chip connector. For BVH stained with CFSE, the flow rate was 3.9 ± 1.42 μl / min without a chip connector and 4.8 ± 0.10 μl / min with a chip connector. The signal-to-noise ratio increased 17.9-fold, from 2.8 without a chip connector to 50.0 with a chip connector. The flow rate of the washing reagent PBS was 4.9 ± 0.79 μl / min without a chip connector and 6.7 ± 0.22 μl / min with a chip connector. The signal-to-noise ratio also increased 4.8-fold, from 6.2 without a chip connector to 30.0 with a chip connector. The standard deviation was consistently larger in the microfluidic chip without a chip connector.Pressure redistribution across the entire microfluidic chip by the chip connector described herein leads to low standard deviation, high SNR, and predictable fluid flow, which further enables the automation of the system described herein.

[0085] In some variations, the chip connector (118, 218, 418) may include at least one inlet connector (440) and at least one outlet connector (442) disposed between the cover (420) and the microfluidic chip (416). In some variations, applying negative pressure to the microfluidic chip (416) may generate a force at the connection interface between the microfluidic chip (416) and the fluid conduit, which may reduce or hinder the processing of biological fluids using the microfluidic chip (416). For example, the outlet (460) of the microfluidic chip (416) may be connected to a fluid conduit (e.g., a vacuum tube) and a negative pressure source (122, 222) (not shown) of the manifold (120, 220). Negative pressure applied through the fluid conduit and at the outlet (460) of the microfluidic chip (416) can otherwise separate the fluid connection between the microfluidic chip (416) and the manifold (120, 220), causing leakage. In some variations, one or more of the connectors (440, 442) described herein are connected 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 manifold (120, 220), thereby providing a consistent flow rate and uniform material distribution (e.g., by preventing leakage). In some variations, one or more of the inlet connector (440) and the outlet connector (442) can be made disposable to reduce contamination and installation time.

[0086] FIGS. 5a through 5e illustrate, respectively, a top perspective view, a bottom perspective view, a side view, a top view, and a bottom view of an outlet connector (500). In some variations, the outlet connector (500) (e.g., gasket) comprises an extended body defining a lumen (510) and may comprise a plurality of steps (520) along the length of the extended body. For example, the outlet connector (500) may have four steps (e.g., steps) of different diameters, but the outlet connector described herein may have two, three, four, five, or more steps. The steps of the outlet connector (500) increase the contact area between the fluid conduit and the microfluidic chip, thereby strengthening the connection interface.

[0087] FIGS. 6a through 6e illustrate the top perspective, bottom perspective, top view, and bottom view, respectively, of the outlet connector (600). In some variations, the outlet connector (600) may include an extended body defining a lumen (610) in which the inner diameter decreases in the distal direction. For example, the lumen (610) may be inclined 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 be conical, and the extended body may be cylindrical. The lumen (610) with varying inner diameters can increase the tolerance when connecting a fluid conduit to the outlet connector (600), thereby shortening installation time and reducing leakage due to misalignment.

[0088] In some variations, a single inlet connector may be configured to deliver (e.g., receive) fluid to multiple microfluidic chips, thereby reducing the complexity of the chip connector and shortening installation and cleaning times. FIGS. 24a through 24d illustrate perspective, top, bottom, and side perspective views of an inlet connector (2400). FIGS. 25a through 25d illustrate perspective, top, bottom, and side perspective views of another inlet connector (2500). FIGS. 26a through 26d illustrate perspective, top, bottom, and side perspective views of another inlet connector (2600). In some variations, the inlet connector (2400, 2500, 2600) (e.g., gasket) may include a body (2420, 2520, 2620) that defines a plurality of lumens (2410, 2510, 2610). The plurality of lumens (2410, 2510, 2610) may be configured to fluidly communicate with a corresponding plurality of inlet reservoirs of a microfluidic chip array (e.g., a plurality of individual microfluidic chips). The body (2420, 2520, 2620) may include one or more collars (2424, 2524, 2624) configured to extend the length of the lumens (2410, 2510, 2610) and to strengthen the connection interface by facilitating safe fluid communication between the end effector (215) (e.g., pipette) and each microfluidic chip.

[0089] In some variations, the inlet connector (2400, 2500, 2600) may include a body comprising a plurality of lumens 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, 2624)). Each lumen (2410, 2510, 2610) may have an increased inner diameter in one or more of the distal or proximal directions. For example, as illustrated in FIGS. 24a through 24d, the lumen (2410) may be inclined so that the inner diameter increases in both the distal and proximal directions. In particular, the lumen (2410) may have an hourglass shape inside the body (2420) of the inlet connector (2400) and a cylindrical shape in the portion passing through the collar (2424). In some variations, as illustrated in FIGS. 26a through 26d, the lumen (2610) may be inclined such that the inner diameter increases from the distal end to the proximal end. For example, the lumen (2610) may have a conical shape inside the body (2620) of the inlet connector (2600) and a cylindrical shape in the portion passing through the collar (2624). In some variations, the inlet connector described herein may surprisingly, unexpectedly, and significantly increase the amount of ECMB captured within the microfluidic chip. For example, FIG. 32 is a graph (3200) of the amount of ECMB on a microfluidic chip (e.g., within a limited area) based on an inlet connector containing silicon (e.g., inlet connector (2400, 2500, 2600)) and an inlet connector without silicon (e.g., inlet connector (2400, 2500, 2600)). In particular, homogenized bovine vitreous humor was injected into a microfluidic chip through each of the 8-channel inlet connectors (e.g., inlet connectors (2400, 2500, 2600)) and then stained with eosin. Inlet connectors containing silicon (e.g., silicon surface) can reduce protein attachment to the inlet connector surface, thereby increasing the amount of ECMB captured in the microfluidic chip.Error bars represent the standard deviation.

[0090] In some variations, a single outlet connector may be configured to transfer (e.g., draw) fluid from multiple microfluidic chips, thereby reducing the complexity of the chip connector as well as shortening installation and cleaning times. For example, the single outlet connector has a lower standard deviation of flow rate compared to conventional connectors. FIGS. 27a through 27d illustrate perspective, top, bottom, and side perspective views of an outlet connector (2700) (e.g., gasket). FIGS. 28a through 28d illustrate perspective, top, bottom, and side perspective views of other outlet connectors (2800). In some variations, the outlet connector (2700, 2800) may include a body (2730, 2830) containing one or more lumens (2740, 2840). Lumens (2740, 2840) may be configured to receive fluid from multiple outlets of the microfluidic chip to be transported through a single outlet of the outlet connector. Additionally, the body (2730, 2830) and one or more lumens (2740, 2840) may be configured to redistribute negative pressure applied to the microfluidic chip to reduce compression at each outlet of the multiple microfluidic chips, thereby improving the uniformity of the flow rate.

[0091] In some variations, the lumen (2740, 2840) may define a distal portion (e.g., the portion facing the exit of the microfluidic chip), a proximal portion (e.g., the portion approaching the collar 2834), and an inner diameter. As illustrated in FIGS. 27c and 28c, the lumen (2740, 2840) may have a defined inner diameter at the proximal portion and be sloped so that the inner diameter increases toward the distal end at the proximal portion. For example, the lumen (2740, 2840) may have a cylindrical shape at the proximal portion and a groove shape at the distal portion. In some variations, as illustrated in FIGS. 28a through 28d, the body (2830) of the outlet connector may include one or more collars (2834) 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.

[0092] In some variations, the inlet and outlet connectors may comprise one or more suitable materials, including silicone-based polymers, other polymers, thermoplastic resins, thermoplastic elastomers, etc. For example, one or more inlet and outlet connectors may comprise silicone-coated plastic. Inlet and outlet connectors containing silicone can further improve flow consistency. The silicone may include liquid silicone rubber, high-viscosity silicone, solid silicone rubber, room temperature curing (RTV) silicone, fluorosilicone, combinations thereof, etc.

[0093] In some variations, the shape of the disposable inlet and outlet connectors described herein can reduce the setup time of the system by improving operational aspects such as assembly and cleaning of the negative pressure system. For example, FIG. 10a is a graph (1000) of the setup time of a 16 microfluidic chip system using a conventional (e.g., inclined) outlet connector and the outlet connector described herein. Disposable outlet connectors with improved shapes (e.g., tiered, conical) can reduce the setup time by improving the fluid seal between the chip connector and the fluid conduit of the manifold compared to the inclined outlet connector and by being easy to operate.

[0094] In some variations, the total experimental time for an exemplary staining procedure can be shortened by using a negative pressure system compared to a positive pressure system. For example, FIG. 10b is a graph (1010) of the total time (e.g., setup, experiment, and washing) for immunofluorescence (IF) and CSFE staining procedures for a positive pressure system and a negative pressure system. In particular, the average time for an IF experiment is shortened by about 12 hours when using a negative pressure system compared to a positive pressure system. The average time for a CSFE stain is shortened by about 8.25 hours when using a negative pressure system compared to a positive pressure system. This increase in efficiency may be partly due to disposable outlet connectors and negative pressure generators that eliminate the time-consuming flushing process for system decontamination (e.g., taking up to about 8 hours). For example, disposable outlet connectors can be replaced between experiments instead of being washed, thereby shortening washing time and the total experimental time, and reducing variability between connectors due to washing deviations.

[0095] In some variations, the improved fluid seal of the disposable inlet and outlet connectors described herein can reduce contamination of the microfluidic chip corresponding to abnormal shapes compared to reusable connectors used in positive pressure systems. FIG. 10c is a graph (1020) of the average frequency of abnormal BVH shapes when using a slanted outlet connector and the outlet connector described herein. For example, incomplete sealing (e.g., air leakage) at the connection interface between the fluid conduit, the outlet connector, and the microfluidic chip may be associated with an increase in abnormal shapes. Incomplete sealing may occur in part due to misalignment of the outlet connector with respect to one or more of the fluid conduit and the microfluidic chip. The improved shape of the outlet connector described herein reduces installation time and the frequency of abnormal shapes.

[0096] E. manifold

[0097] The system described herein may include a manifold (120, 220). Generally, the manifold (120, 220) may be configured to be connected to a plurality of microfluidic chips (116, 216, 316) and a negative pressure source (122, 222). For example, the manifold (120, 220) may be fluidly connected to each microfluidic chip (116, 216, 316) fixed by a single negative pressure source (122, 222) and a chip connector (118, 218), so that the negative pressure generated by the negative pressure source (122, 222) can be applied to each microfluidic chip (116, 216, 316) connected to the manifold (120, 220). In some variations, the manifold (120, 220) may include a plurality of fluid conduits (e.g., vacuum tubes, fluid lines) configured to be detachably connected to each microfluidic chip (116, 216, 316) and a negative pressure source (122, 222). Thus, the manifold can reduce the size of the system (100) because each microfluidic chip does not require its own negative pressure source.

[0098] In some variations, manifolds and negative pressure sources significantly improve throughput through scaling up and increasing efficiency without consistency or a reduction in flow rate. FIG. 11 is a graph (1100) of the number of parallel (e.g., simultaneous) experiments performed for the positive pressure system and negative pressure system described herein. For example, a manifold connected to eight microfluidic chips, each having eight channels, enables 64 simultaneous experiments to be performed, significantly improving throughput compared to a positive pressure system that does not use negative pressure and a manifold. On the other hand, the positive pressure system relies on pumps and sample injection mechanisms with limited connectivity.

[0099] The microfluidic system comprising the manifold and negative pressure source described herein not only improves efficiency and throughput but also enables this in a relatively uniform manner that could not be achieved by conventional methods. For example, FIG. 14a is a graph (1400) of the amount of ECMB based on a 3-port manifold at a negative pressure of 100 mmHg. As can be seen, there is no statistically significant difference in the amount of ECMB recovered from each port (Kruskal-Wallis, p=0.863). However, even at higher negative pressures (e.g., 400 mmHg), there is no statistically significant difference in the amount of ECMB captured. For example, FIG. 14i is a graph (1480) of the amount of ECMB based on a 3-port manifold at a negative pressure of 400 mmHg. There is no statistically significant difference in the amount of ECMB from each port (Fischer's, p=0.481).

[0100] FIG. 14b is a graph (1410) of the amount of ECMB across pillar regions with different spacing for different ports of the manifold at 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 is captured in the 100 μm, 50 μm, and 25 μm spacing regions, respectively. As shown, there is no statistically significant difference between the amounts of ECMB in each pillar region between the different ports (100 μm region - Fisher's, p=0.744; 50 μm region - Kruskal-Wallis, p=0.882; 25 μm region - Fischer's, p=0.912; 15 μm region - Kruskal-Wallis, p=0.490; 4 μm region - Kruskal-Wallis, p=0.230). FIG. 14c is a graph (1420) of ECMB amounts based on an 8-port manifold. There is no statistically significant difference in the amount of ECMB at each port (p=0.078). Thus, this manifold distributes the negative vacuum pressure (e.g., 100 mmHg) to each port and each microfluidic chip in a substantially uniform manner. However, even at higher negative pressures (e.g., 400 mmHg), there is no statistically significant difference in the amount of ECMB captured. For example, FIG. 14j is a graph (1490) of the amount of ECMB across bar regions with different spacing for different ports of the manifold at 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 is captured in the 100 μm, 50 μm, and 25 μm spacing regions, respectively. As shown, there is no statistically significant difference in ECMB amounts between the respective pillar regions of different ports (100 μm region - Fischer's, p=0.160, 50 μm region - Fischer's, p=0.745, 25 μm region - Kruskal-Wallis, p=0.475, 15 μm region - Kruskal-Wallis, p= 0.393, 4 μm region - Kruskal-Wallis, p=0.114).

[0101] Similar to Fig. 14a, there is no statistical difference in the amount of ECMB recovered from each port of the 8-port manifold. Fig. 14d is a graph (1430) of the amount of ECMB across pillar regions with different spacing in the 8-port manifold. The microfluidic chip has pillar spacing regions of 100 μm (dark gray bar), 50 μm (dark gray bar), 25 μm (medium gray bar), 15 μm (light gray bar), and 4 μm (white bar), and most of the ECMB is captured in the 100 μm, 50 μm, and 25 μm spacing regions. As shown, there is no statistically significant difference in ECMB amounts between each pillar spacing region between 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).

[0102] The fluid flow rate passing through a microfluidic chip to which negative pressure is applied through an 8-port manifold can be relatively uniform. For example, FIG. 14e is a graph (1440) of the flow rates for three fluid flow processes (e.g., PBS priming, BVH stained with CFSE, PBS washing) using an 8-port manifold at a negative pressure of about 100 mmHg. There is no statistically significant difference in the flow rates of PBS priming (dark gray bars), BVH and CFSE (medium gray bars), and PBS washing (light gray bars) between the respective ports (PBS priming - 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), there is no statistically significant difference in ECMB capture.

[0103] FIGS. 14f through 14i are images showing ECMBs captured in a microfluidic chip connected to a manifold of a negative pressure system. For example, FIG. 14f is an image (1450) of an ECMB (a2) stained with CSFE in a microfluidic chip connected to a 3-port manifold at a negative pressure of 100 mmHg. Most of the ECMB is captured in a column (a1) region with intervals of 100 μm, 50 μm, and 25 μm. An enlarged image (1452) shows the captured ECMB (a2) surrounding the column (a1). FIG. 14b is an image (1460) of an ECMB (a2) stained with CSFE in a microfluidic chip connected to an 8-port manifold at a negative pressure of 100 mmHg. Most of the ECMB is captured in a column (a1) region with intervals of 100 μm, 50 μm, and 25 μm. An enlarged image (1462) shows the captured ECMB (a2) surrounding the pillar (a1). FIG. 14h is an image (1470) of the ECMB (a2) stained with CSFE in a microfluidic chip connected to a 3-port manifold at a negative pressure of 400 mmHg. Most of the ECMB is captured in the pillar (a1) regions at intervals of 100 μm, 50 μm, and 25 μm. An enlarged image (1472) shows the captured ECMB (a2) surrounding the pillar (a1).

[0104] FIG. 15a is a graph (1500) of the amount of ECMB using a system with a manifold and a system without a manifold at a negative pressure of 400 mmHg. There is no statistically significant difference between the amount of ECMB of the microfluidic chip connected to the manifold and the microfluidic chip not connected to the manifold (Welch's t, p=0.400).

[0105] Fig. 15b is a graph (1510) of the amount of ECMB across different spacing pillar regions for the case with and without a manifold at a negative pressure of 400 mmHg. For the case with a manifold (dark gray bars) and for a single microfluidic chip without a manifold (light gray bars), most of the ECMB is trapped in the 100 μm, 50 μm, and 25 μm spacing regions. There is no statistically significant difference in the amount of ECMB in each pillar region for the case 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).

[0106] F. negative pressure source

[0107] The system described herein may include a negative pressure source (122, 222). Generally, the negative pressure source (122, 222) may be configured to be connected to a manifold (120, 220) and at least one microfluidic chip (116, 216, 316). Applying negative pressure can help reduce fouling 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) connected to the negative pressure source (122, 222) may be configured to control fluid flow and negative pressure. In some variations, the negative pressure source (122, 222) may be configured to apply a negative pressure of about 10 mmHg to about 760 mmHg to each microfluidic chip (116, 216, 316) of the system (100, 200).

[0108] FIGS. 12a and 12b are images (1200, 1200) showing biological contamination in a positive pressure microfluidic system after immunofluorescence staining using bovine vitreous humor stained for fibronectin, taken using a fluorescence microscope. Arrows indicate biological contaminants. A positive pressure system with a reusable inclined outlet connector was cleaned according to standard operating procedures, a new microfluidic chip was placed in the positive pressure system, and a buffer flush was provided. FIGS. 12c and 12d are images (1230, 1240) showing biological contamination using the system described herein, the system was cleaned according to standard operating procedures, a new microfluidic chip was placed in the chip connector, a buffer flush was performed, and a new outlet connector was provided. FIG. 12e is a graph (1250) of the biological contamination per unit area of ​​a positive pressure system and a negative pressure system. A system using the negative pressure and outlet connector described herein significantly reduces contamination compared to a positive pressure system, thereby improving the precision and reliability of the system described herein.

[0109] In some variations, the amount of ECMB trapped in the microfluidic chip may not increase linearly in proportion to the amount of applied negative pressure. For example, FIG. 13a is a graph (1300) of the amount of ECMB for negative pressures of 100 mmHg and 400 mmHg. The amount of ECMB for each negative pressure is statistically not different (Student's t, p=0.988). Similarly, FIG. 13b is a graph (1310) of the amount of ECMB across barbed regions with different spacing for negative pressures of 100 mmHg and 400 mmHg. At a negative pressure of 100 mmHg (dark gray bar) and 400 mmHg (light gray bar), most of the ECMB is trapped in the 100 μm, 50 μm, and 25 μm spacing regions. There is no statistically significant difference in the amount of ECMB in each pillar region under each negative pressure condition (100 μm region - Welch's t, p=0.211; 50 μm region - Student's t, p=0.738; 25 μm region - Student's t, p=0.335; 15 μm region - Mann-Whitney U, p=0.238; 4 μm region - Mann-Whitney U, p=0.657).

[0110] FIGS. 13c and FIGS. 13d are images showing ECMBs trapped in a microfluidic chip connected to a negative pressure system under different negative pressure conditions. For example, FIG. 13c is an image (1320) of an ECMB (a2) stained with CSFE under a negative pressure condition of 100 mmHg. Most of the ECMB is trapped in a column (a1) region with intervals of 100 μm, 50 μm, and 25 μm. An enlarged image (1322) shows the trapped ECMB (a2) surrounding the column (a1). FIG. 13d is an image (1330) of an ECMB (a2) stained with CSFE under a negative pressure condition of 400 mmHg. Most of the ECMB is trapped in a column (a1) region with intervals of 100 μm, 50 μm, and 25 μm. The enlarged image (1332) shows the captured ECMB (a2) surrounding the column (a1).

[0111] In some variations, applying a negative pressure of about 10 mmHg to about 760 mmHg to the microfluidic chip can effectively separate ECMB from the biological fluid. For example, FIGS. 16a and FIGS. 16b are graphs (1600, 1610) of the amount of ECMB captured in the microfluidic channels of the microfluidic chip using negative pressures of 10 mmHg to 760 mmHg and conical outlet connectors (e.g., FIGS. 6a to 6d) and stepped outlet connectors (e.g., FIGS. 5a to 5e), respectively. In FIG. 16a, the amount of ECMB captured at a negative pressure of 5 mmHg is statistically significantly less than the amount captured at negative pressures between 10 mmHg and 760 mmHg, according to a t-test (Welch's, p = 0.001). Likewise, in Fig. 16b, the amount of ECMB captured at a negative pressure of 5 mmHg is statistically significantly less than the amount captured at negative pressures between 10 mmHg and 760 mmHg according to a t-test (Student's, p < 0.001).

[0112] G. sensor

[0113] The system described herein may include one or more sensors (124). Generally, the sensor (124) may be configured to measure one or more features (e.g., pressure, flow rate, optical image, temperature, humidity) corresponding to one or more of a biological fluid, a holder (112, 212), a reservoir (113), 213, a robot (114, 214), a microfluidic chip (116, 216, 316), a chip connector (118, 218), a manifold (120, 220), and a negative pressure source (122, 222). In this way, the system and the fluid may be monitored during use. In some variations, the sensor may be connected to or integrated with any component of the system (100), such as an inlet connector, a chip connector, etc.

[0114] In some variations, the sensor may be an optical sensor connected to a component of the system (100, 200), e.g., a chip connector (118, 218). The optical sensor may be configured to image one or more channels of the microfluidic chip (116, 216, 316) for histochemical and morphological studies. The optical sensor may be used to receive a light signal (e.g., a light beam) reflected by the fluid within the microfluidic chip (116, 216, 316). The received light may be processed in a processor (128) and memory (130) to generate signal data that produces sample data. The optical sensor may be configured to image one or more identifiers (e.g., a label, a barcode) and identifiers of the microfluidic chip (116, 216, 316). In some variations, the optical sensor may include one or more of a lens, a camera, and a measuring optical device. For example, the optical sensor may include a charged coupled device (CCD) or a complementary metal-oxide semiconductor (CMOS) optical sensor and may be configured to generate an image signal transmitted to an output device (134) (e.g., a display). For example, the optical sensor may include a camera having an image sensor (e.g., a CMOS or CCD array with or without a color filter array and associated processing circuitry). Additionally, or alternatively, in some variations, the sensor may be an ultrasonic sensor configured to generate an ultrasonic signal used to determine a fluid flow rate.

[0115] In some variations, the system (100, 200) may include a radiation source (e.g., UV light) configured to emit a light signal (e.g., illumination) to a microfluidic chip (116, 216, 316) for visualization, imaging, and / or cleaning. In some variations, the radiation source may include one or more of a light-emitting diode, a laser, a microscope, an optical sensor, a lens, and a flash lamp. For example, the radiation source may be configured to generate light that can be transmitted by a fiber optic cable, or one or more LEDs may be configured to provide illumination. In another example, a fiberscope comprising a flexible fiber bundle may be configured to receive and transmit light from an external light source.

[0116] H. Input device

[0117] Generally, an input device (126) of a system (100, 200) can serve as a communication interface between an operator and the system (100, 200). The input device (126) may be configured to receive input data and output data to one or more of a robot (114), a sensor (124), and an output device (134). For example, the operator's control of the input device (126) (e.g., foot controller, joystick, keyboard, touchscreen) may be processed by a processor (128) and a memory (130) to cause the input device (126) to output a control signal to one or more of a robot (114, 214), a sound pressure source, and a sensor (124). As another example, an image generated by a sensor (124) may be processed by a processor (128) and a memory (130) and displayed on an output device (134) (e.g., a display). Sensor data obtained from one or more sensors (124) can be output as visual, auditory, and / or tactile feedback through one or more output devices (134).

[0118] Some variations of the input device may include one or more switches configured to generate control signals. The input device may be connected to or disconnected from other components of the system (100, 200). For example, the input device (126) may be placed in a different room from the robot (114, 214) and the microfluidic chip (116, 216, 316) to reduce potential contamination. The control signals may include, for example, robot signals, sound 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 the control signals to a wired and / or wireless receiver of the controller. The robot signals (e.g., for movement, position, and orientation control) may control robot joint movements of at least four degrees of freedom and may include yaw and / or pitch rotation. For example, an input device (126) including a touch surface may be configured to detect contact and movement on the touch surface using various touch sensing technologies including capacitive, resistive, infrared, optical imaging, distributed signal, acoustic pulse recognition, and surface acoustic wave technology.

[0119] In a variation of an input device (126) comprising at least one switch, the switch may include, for example, a button (e.g., hard key, soft key), a touch surface, a keyboard, an analog stick (e.g., joystick), a directional pad, a mouse, a trackball, a jog dial, a step switch, a rocker switch, a pointer device (e.g., stylus), a motion sensor, an image sensor, and a microphone. The motion sensor may receive movement data of the operator from an optical sensor and classify the operator's gestures as control signals. The microphone may receive audio and recognize the operator's voice as a control signal. In a variation of a system comprising multiple input devices, different input devices may generate different types of signals. For example, some input devices (e.g., a button, an analog stick, a directional pad, and a keyboard) may be configured to generate robot signals, and other input devices (e.g., a step switch, a rocker switch) may be configured to control sound pressure sources (122, 222) and sensors (124).

[0120] I. processor

[0121] The system (100, 200) illustrated in FIG. 1 may include a robot (114, 214), a negative pressure source (122, 222), a sensor (124), a processor (128) communicating with the sensor (124), and a machine-readable memory (130) (e.g., a controller). The processor (128) may be connected to the system (100, 200) via a wired or wireless communication channel. The processor (128) may be located in the same or a different room as the microfluidic chip (116, 216, 316). The processor (128) may be configured to control one or more components of the system (100, 200), for example, a robot (114, 214) configured to transfer fluid to the microfluidic chip, or an optical sensor configured to visualize the separated ECMB using the microfluidic chip (116, 216, 316).

[0122] The processor (128) may be implemented to fit various general-purpose or special-purpose computing systems or configurations. Various exemplary computing systems, environments and / or configurations that may be suitable for use with the systems and devices disclosed herein may include, but are not limited to, personal computing devices, network devices, servers or server computing devices (e.g., routing / connection components), portable (e.g., handheld) or laptop devices, multiprocessor systems, microprocessor-based systems, and software or other components located within or implemented in a distributed computing network.

[0123] Examples of portable computing devices include smartphones, personal digital assistants (PDAs), mobile phones, tablet PCs, phablets (personal computing devices larger than smartphones but smaller than tablets), wearable computers in the form of smartwatches, and portable music devices, as well as portable or wearable augmented reality devices that interact with the worker's environment through sensors and can use a head-mounted display for visualization, eye tracking, and user input.

[0124] The processor (128) can control one or more robots (114, 214) and sound pressure sources (122, 222) by integrating data received from memory (130) and worker input. Memory (130) can also store instructions that cause the processor (128) to execute modules, processes, and / or functions related to the system (100, 200). The processor (128) may be a suitable processing unit configured to execute instructions or a set of codes and may include one or more data processors, image processors, graphics processing units, physics processing units, digital signal processors, and / or central processing units. For example, the processor (128) may be a general-purpose processor, a Field Programmable Gate Array (FPGA), or an Application Specific Integrated Circuit (ASIC) configured to execute application processes and / or other modules, processes, and / or functions related to the system and / or related network. Basic device technology can be provided in 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 technology (e.g., silicon junction polymer and metal junction polymer-metal structures), mixed analog and digital technology, and combinations thereof.

[0125] J. memory

[0126] Some variations of the memory (130) described herein relate to a computer storage product having a non-volatile computer-readable medium (also referred to as a non-volatile processor-readable medium) comprising instructions or computer code for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-volatile in that it does not itself contain volatile radio signals (e.g., radio electromagnetic waves that transmit information in a transmission medium such as air or a cable). The medium and the computer code (also referred to as code or algorithm) may be designed and manufactured for a specific purpose. Examples of non-volatile computer-readable media include magnetic storage media, e.g., hard disks, floppy disks, and magnetic tapes; optical storage media, e.g., compact discs / digital video discs (CD / DVD), compact disc read-only memory (CD-ROM), and holographic devices; magneto-optical storage media, e.g., optical discs; solid-state storage devices, e.g., solid-state drives (SSDs) and solid-state hybrid drives (SSHDs); and carrier wave signal processing modules. 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), are described herein. Other variations described herein relate to computer program products and may include, for example, instructions and / or computer code disclosed herein.

[0127] The systems, devices, and / or methods described herein may be implemented by software (executed on hardware), hardware, or a combination thereof. Software modules (executed on hardware) may 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 used to generate web services, and files containing high-level instructions executed by a computer using an interpreter. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

[0128] K. communication device

[0129] In some variations, the system (100, 200) described herein may communicate with network and computer systems through a communication device (128). Additionally, the system (100, 200) may communicate with other devices through one or more wired and / or wireless networks. A wireless network may refer to any type of digital network that is not connected by any kind of cable. Examples of wireless communication in a wireless network include, but are not limited to, cellular, radio, satellite, and microwave communication. A wireless network may be connected to a wired network to connect to the Internet, voice and data networks of other telecommunications carriers, business networks, and private networks. Wired networks are generally transmitted via copper twisted pair, coaxial cable, and / or fiber optic cable. Wired networks include a number of different types, including wide area networks (WAN), metropolitan area networks (MAN), local area networks (LAN), internet area networks (IAN), campus area networks (CAN), global area networks (GAN), the Internet, and virtual private networks (VPN). Hereinafter, the term "network" generally refers to any combination of wireless, wired, public, and private data networks that are interconnected via the Internet to provide an integrated networking and information access system.

[0130] Cellular communication may include 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 of multiple cellular networks or use a mix of cellular, Wi-Fi, and satellite communication. In some variations, the communication device (132) may include a radio frequency receiver, a transmitter, and / or an optical (e.g., infrared) receiver and transmitter. The communication device (132) may communicate with a robot (114, 214), a sound pressure source (122, 222), a sensor (124), an input device (126), an output device (134), a network, a database, a server, a combination thereof, etc. via wired and / or wireless means.

[0131] L. Output device

[0132] The output device (134) of the system (100, 200) may be configured to output data corresponding to the system and may include one or more display devices, audio devices, and haptic devices. For example, through the display device, an operator may 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 a light-emitting diode (LED), a liquid crystal display (LCD), an electroluminescent display (ELD), a plasma display panel (PDP), a thin film transistor (TFT), an organic light-emitting diode (OLED), an electronic paper / e-ink display, a laser display, and / or a holographic display.

[0133] The audio device may output fluid data, sensor data, system data, alarms, and / or warnings as audible sounds. For example, the audio device may output a warning sound when sensor data (e.g., pressure, flow rate) exceeds a predetermined range or when a malfunction is detected in the robot. As another example, audio may be output when the system ignores operator input to prevent potential hazards (e.g., robot collision, excessive sound pressure) that may occur to the operator and / or the system. In some variations, the audio device may include at least one of a speaker, a piezoelectric audio device, a magnetostrictive speaker, and / or a digital speaker. In some variations, the operator may communicate with other users using the audio device and a communication channel. For example, the operator may establish an audio communication channel (e.g., VoIP call) with a remote operator and / or observer.

[0134] A haptic device may be integrated into one or more input and output devices to provide the operator with additional sensory output (e.g., force feedback). For example, the haptic device may generate a tactile response (e.g., vibration) to confirm the operator's input to an input device (e.g., a touch surface). Additionally, or alternatively, haptic feedback may indicate that the operator's input has been ignored by the system to prevent potential hazards to the operator and / or the system (e.g., robot collision, excessive sound pressure).

[0135] II. method

[0136] Additionally, the present invention describes a method for separating ECMB from biological fluids. The method described herein may be useful for separating ECMB and biological fluids at high throughput, thereby aiding in further analysis, e.g., by analyzing ECMB or biological fluids (or parts or fractions thereof), to diagnose subjects and / or establish treatment plans for subjects and monitor the effectiveness of treatment plans. For example, analysis of a predetermined fraction of biological fluid or ECMB may determine the amount of disease-associated biomarkers, which may be useful for diagnosis and target identification. Biomarkers may be used to support disease prognosis as well as disease screening and prediction, to facilitate treatment selection, to further classify patients for clinical trials, and / or to monitor both the safety and therapeutic signal of subjects after the initiation of treatment and during the course of treatment. In some variations, pharmacokinetic biomarkers may function as endpoints or as surrogate endpoints for clinical trials.

[0137] FIG. 7 is a flowchart generally illustrating a method for separating ECMB from a biological fluid (700) using any of the systems and devices described herein. The method (700) may include the step of transferring a biological fluid (e.g., about 400 μl sample) to an inlet reservoir of a microfluidic chip (702). For example, the microfluidic chip may include at least one restricting channel (e.g., a uniform flow channel) having an inlet and an outlet. The inlet reservoir may be fluidly connected to the inlet of at least one restricting channel, at least one obstacle (e.g., a column), and an outlet reservoir. Optionally, the microfluidic chip may be primed with a buffer before receiving the biological fluid. A vacuum seal may be established and maintained within the system to minimize air bubbles within the microfluidic chip.

[0138] In some variations, a negative pressure of about 10 mmHg to about 760 mmHg may be applied to the outlet reservoir of the microfluidic chip (704). The applied negative pressure may remove the non-ECMB portion of the biological fluid from the microfluidic chip while allowing the ECMB to remain within the microfluidic chip. For example, when the fluid flows through the microfluidic chip, the shape of the ECMB within the fluid is deformed by contact (e.g., bending) with an obstacle (e.g., a pillar) placed within the microfluidic chip, thereby fixing the ECMB within the microfluidic chip (e.g., the ECMB is attached to the pillar).

[0139] In some variations, ECMB within the microfluidic chip can be processed (706). For example, ECMB within the microfluidic chip can be applied to one or more of 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 in any of the analytical methods described above.

[0140] In some variations, one or more pre-determined antibodies (e.g., extracellular matrix substances, extracellular vesicle markers), washing solutions, and reagents can be applied to the microfluidic chip to enable IHC staining. For example, various antibodies (e.g., extracellular matrix, cancer-related markers, extracellular vesicle markers) can be applied to the microfluidic chip IHC for multispectral imaging.

[0141] In some variations, isolated fractions on the microfluidic chip may be sequentially stained to aid in the removal of material within the microfluidic chip. For example, staining a sample with an antibody may create a first spatial arrangement on the microfluidic chip. Subsequent staining of the microfluidic chip (e.g., with a different set of reagents) may create a second spatial arrangement different from the first spatial arrangement as the sample moves within the microfluidic chip. Consequently, it may be difficult to usefully compare the first and second spatial arrangements. Accordingly, chemical fixation (e.g., crosslinking) may be applied to fix biological fluids (e.g., ECMB) on the microfluidic chip. For example, the microfluidic chip may accommodate carbodiimide fixatives and aldehyde fixatives configured to crosslink and fix biological fluids onto the microfluidic chip. In some variations, the inlet and outlet of the microfluidic chip may be sealed.

[0142] In some variations, one or more biomarkers among biological fluids and ECMB can be measured by one or more of immunoassays, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared, and UV-VIS.

[0143] In some variations, the biological fluid may be analyzed as a whole biological fluid fraction before transferring the biological fluid (708). In some variations, the ECMB remaining in the microfluidic chip may be analyzed as an isolated fraction after applying negative pressure (710). In some variations, the biological fluid removed from the microfluidic chip may be analyzed as an eluent fraction after applying negative pressure (712). In some variations, the biological fluid removed from the microfluidic chip may be processed using one or more of microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, mesh filtration, ultrafiltration, dialysis filtration, tangential flow filtration, membrane filtration, immunoaffinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC electrodynamics.

[0144] In some variations, biological fluids removed from the microfluidic chip (e.g., eluent fraction, total fluid, captured material) may be analyzed using one or more of the following: microscopy, microfluidic devices, mass spectrometry, microarray, nucleic acid amplification, hybridization, proteomic profiling, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next-generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, in situ fluorescence hybridization (FISH), chelation complexation, quantitative HPLC, spectrophotometry, colorimetric assay, chemiluminescence assay, immunofluorescence assay, light scattering, antibody array, Western blot, immunoassay, immunoprecipitation method, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS / MS, RT-PCR, and quantitative PCR.

[0145] Although the above variations have been described in detail by way of examples and embodiments for clarity and understanding, specific changes and variations may be made, and it will be obvious that such are intended to be included within the scope of the appended claims. Additionally, it should be understood that the components and features of the systems and devices described herein may be used in any combination. Descriptions of specific elements or features in specific drawings are not intended to be limiting and should not be interpreted 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 do not need to be performed sequentially. Some steps are optional, so not all steps of the method need to be performed.

Claims

Claim 1 A system for separating extracellular matrix (ECMB) from a biological fluid, comprising: a holder configured to receive the biological fluid; a robot configured to transfer the biological fluid from the holder to a microfluidic chip; a chip connector configured to secure at least one microfluidic chip; a manifold connected to at least one microfluidic chip; and a negative pressure source connected to the manifold configured to apply a negative pressure of about 10 mmHg to about 760 mmHg to at least one microfluidic chip. Claim 2 A system according to claim 1, wherein the chip connector comprises a base configured to contact the lower part of at least one microfluidic chip and a cover configured to contact the upper part of at least one microfluidic chip. Claim 3 A system according to claim 1 or 2, wherein the chip connector is configured to distribute the compressive force applied by negative pressure around the microfluidic chip. Claim 4 A system according to any one of claims 1 to 3, wherein the lower portion comprises the circumference of at least one microfluidic chip. Claim 5 A system according to any one of paragraphs 1 to 4, wherein the cover defines a plurality of openings. Claim 6 A system according to any one of claims 1 to 5, wherein the base comprises a first fixing device and the cover comprises a first fixing device, and the first fixing device and the second fixing device are configured to align the microfluidic chip in a predetermined orientation. Claim 7 A 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 a cover and at least one microfluidic chip. Claim 8 A system according to any one of claims 1 to 7, wherein at least one outlet connector comprises an extended body that defines a lumen and includes a plurality of steps along the length of the extended body. Claim 9 A system according to any one of claims 1 to 8, wherein at least one outlet connector comprises an extended body that defines a lumen in which the inner diameter decreases in the distal direction. Claim 10 A system according to any one of claims 1 to 9, wherein one or more microfluidic chips, an inlet connector, and an outlet connector comprise disposable components. Claim 11 A system according to any one of claims 1 to 10, wherein the chip connector comprises a durable component. Claim 12 A system according to any one of claims 1 to 11, wherein the holder is configured to receive one or more reagents and the robot is configured to transfer one or more reagents from the holder to a microfluidic chip. Claim 13 A system according to any one of claims 1 to 12, further comprising a sensor connected to at least one inlet connector, wherein the sensor is configured to measure one or more of flow rate and pressure. Claim 14 A system according to any one of claims 1 to 13, further comprising an optical sensor connected to a chip connector, wherein the optical sensor is configured to image one or more of the microfluidic chips. Claim 15 A system according to any one of claims 1 to 14, wherein at least one microfluidic chip comprises at least one limiting channel fluidically connected between the inlet and outlet of the microfluidic chip. Claim 16 A system according to any one of claims 1 to 15, wherein at least one restriction channel comprises one or more obstacles. Claim 17 A system according to any one of claims 1 to 16, wherein at least one limiting channel has a length of about 5 mm to about 30 mm. Claim 18 A system according to any one of claims 1 to 17, wherein at least one limiting channel has a cross-sectional dimension of about 5 μm to about 30 μm. Claim 19 A system according to any one of claims 1 to 18, wherein at least one microfluidic chip comprises at least one obstacle configured to restrict fluid flow. Claim 20 A system according to any one of paragraphs 1 through 19, wherein at least one obstacle comprises a column. Claim 21 A system according to any one of claims 1 to 20, wherein at least one microfluidic chip comprises a restricted area configured to hold a first fraction of biological fluid and allow fluid flow of a second fraction of biological fluid. Claim 22 A system in any one of paragraphs 1 through 21, wherein the first fraction comprises ECMB. Claim 23 A system according to any one of paragraphs 1 through 22, wherein the restricted area comprises a plurality of obstacles configured to have a first fraction. Claim 24 A system according to any one of claims 1 to 23, wherein the spacing between a plurality of obstacles within a restricted area decreases along the length of the microfluidic chip from the entrance of the restricted area to the exit of the restricted area. Claim 25 A system according to any one of claims 1 to 24, wherein the spacing between a plurality of obstacles within a restricted area is about 100 μm to about 4 μm. Claim 26 A system according to any one of claims 1 to 25, wherein each of the plurality of obstacles has a diameter of about 50 μm to about 1 mm. Claim 27 A method for separating extracellular matrix (ECMB) from a biological fluid, comprising the step of transferring the biological fluid to an inlet reservoir of a microfluidic chip, wherein the microfluidic chip comprises: at least one restriction channel having an inlet and an outlet fluidically connected to the inlet of at least one restriction channel; at least one column; and an outlet reservoir; and the step of applying a negative pressure of about 10 mmHg to about 760 mmHg to the outlet reservoir of the microfluidic chip, wherein the ECMB remains in the microfluidic chip after the biological fluid is removed from the microfluidic chip. Claim 28 A method according to claim 27, further comprising the step of dispersing the compressive force applied by negative pressure from the outlet reservoir to the circumference of the microfluidic chip. Claim 29 A method according to claim 27 or 28, wherein at least one limiting channel comprises at least one column. Claim 30 A method according to any one of claims 27 to 29, wherein at least one limiting channel has a length of about 5 mm to about 30 mm. Claim 31 A method according to any one of claims 27 to 30, wherein at least one limiting channel has a cross-sectional dimension of about 5 μm to about 30 μm. Claim 32 A method according to any one of claims 27 to 31, wherein at least one microfluidic chip comprises at least one obstacle configured to restrict fluid flow. Claim 33 A method according to any one of claims 27 to 32, wherein at least one microfluidic chip comprises a restricted area configured to hold a first fraction of biological fluid and allow fluid flow of a second fraction of biological fluid. Claim 34 A method according to any one of paragraphs 27 through 33, wherein the restricted area comprises a plurality of obstacles configured to have a first fraction. Claim 35 A method according to any one of claims 27 to 34, wherein the spacing between a plurality of obstacles within a restricted area decreases along the length of the microfluidic chip from the entrance of the restricted area to the exit of the restricted area. Claim 36 A method according to any one of claims 27 to 35, wherein the spacing between a plurality of obstacles within a restricted area is about 100 μm to about 4 μm. Claim 37 A method according to any one of claims 27 to 36, wherein each of the plurality of obstacles has a diameter of about 50 μm to about 1 mm. Claim 38 A method according to any one of claims 27 to 37, further comprising the step of applying one or more of histochemical staining, immunohistochemical (IHC) staining, multiplex IHC staining, multispectral imaging, protein staining, nucleic acid staining, chemical fixation, and protease inhibitor to an ECMB within a microfluidic chip. Claim 39 A method comprising, in any one of claims 27 to 38, further a step of measuring one or more biomarkers among biological fluid and ECMB by one or more of immunoassay, microscopy, immunohistochemistry, fluorescence in situ hybridization, immunofluorescence, infrared and UV-VIS. Claim 40 A method according to any one of claims 27 to 39, further comprising the step of analyzing a biological fluid removed from a microfluidic chip using one or more of a microscope, a microfluidic device, mass spectrometry, a microarray, nucleic acid amplification, hybridization, proteomic profiling, fluorescence hybridization, immunohistochemistry, nucleic acid analysis or sequencing, next-generation sequencing, flow cytometry, chromatography, electrophoresis, immunostaining, fluorescence assay, fluorescence in situ hybridization (FISH), chelation complexation, quantitative HPLC, spectrophotometry, antibody array, Western blot, immunoassay, immunoprecipitation, ELISA, LC-MS, LC-MRM, radioimmunoassay, 2D gel mass spectrometry, LC-MS / MS, RT-PCR, and quantitative PCR. Claim 41 A method according to any one of claims 27 to 40, further comprising the step of treating the biological fluid removed from the microfluidic chip using one or more of microfluidic separation, affinity chromatography, centrifugation, differential centrifugation, density gradient centrifugation, mesh filtration, dialysis filtration, tangential flow filtration, membrane filtration, immunoaffinity capture, magnetic bead capture, size exclusion chromatography, electrophoresis, and AC electrodynamics. Claim 42 A method according to any one of claims 27 to 41, wherein the biological fluid comprises one or more of whole blood, plasma, serum, cerebrospinal fluid, intracerebrospinal fluid, urine, saliva, sweat, tears, synovial fluid, pleural fluid, gastric fluid, peritoneal fluid, breast milk, nipple aspirate, semen, amniotic fluid, vitreous fluid, aqueous humor, lymph fluid, bile, earwax, chyme, chyme, endolymph, perilymph, exudate, feces, ejaculate, gastric acid, gastric fluid, mucus, pericardial fluid, pus, aqueous excretion, sebum, serous fluid, smegma, sputum, synovial fluid, vaginal secretion, menstrual fluid, vomit, and fluids that have passed through one or more of tissues and gels.

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