Device and method for isolating extracellular matrix bodies

Abstract

Provided are devices, methods and systems for separating extracellular matrix bodies. More specifically, the invention discloses devices, methods and systems for separating extracellular matrix bodies from biological samples for diagnosis and prognosis of subjects. Devices can have restricted passages for separating extracellular matrix bodies and uniform flow channels for accurate pressure measurement.

Description

Technical Field

[0001] The present invention relates to devices, methods and systems for isolating extracellular matrix bodies. More specifically, the present invention discloses devices, methods and systems for isolating extracellular matrix bodies from biological samples for diagnosis and prognosis of subjects. Background Art

[0002] Conventional methods for disease diagnosis and prognosis may require analysis or separation of biological samples of very small fractions. The corresponding analysis of components from very small fractions can be used for diagnosis and prognosis of diseases. For example, individual cells or even individual nucleic acid molecules can be detected and analyzed. However, a shortcoming of this type of conventional method is to rely on very small amounts of biological materials and corresponding small signals to make decisions. Useful signals may be lost in other unmeasured biological materials.

[0003] Conventional methods for disease diagnosis and prognosis often require the acquisition of a biological sample to be obtained and a well-known structure to be isolated from the sample for further analysis. For example, intact organ tissues, whole cells, exosomes, and other well-known structures can be isolated and characterized. Disadvantages of such methods include the inability to detect or characterize disease when the well-known structure does not readily reflect the disease state.

[0004] Other drawbacks of conventional methods for disease diagnosis and prognosis include the use of biomarkers that are only associated with specific well-known structural elements of the subject's biological sample, such as exosomes. In these approaches, biomarkers are often inherently limited by not being directly associated with the pathology of interest. Conventional methods often attempt to consider a number of biomarkers that may have a remote or partial association with the disease, hoping that statistics will provide a diagnostic answer. Often a combination of biomarkers is required, and the results are highly unpredictable.

[0005] Other drawbacks of conventional devices include the inability to accurately measure flow and pressure in complex fluids containing biological components, which can result in changes in flow and pressure due to the interaction of the biological components with the device performing the measurement.

[0006] There is a need for devices and systems for separating particles from biological samples for diagnostic purposes that can provide increased sample isolates of specific biological relevance. More specifically, there is a need for devices, methods and systems for separating important components from biological samples for diagnosis and prognosis of disease.

[0007] There is a pressing need for methods and apparatus for separating particles from biological samples that can isolate relevant fractions of biological material that more closely correspond to a disease state and measure differential pressure and flow in the fluid of interest. Summary of the invention

[0008] The present invention provides devices and systems for separating particles from biological samples that are applicable to a variety of diseases and conditions. Methods and systems for separating particles from biological samples are provided, which can increase the biologically relevant sample isolates. The devices and methods of the present invention can be used to separate important components from biological samples for diagnosis and prognosis of diseases.

[0009] In some embodiments, the methods and devices of the present invention for separating particles from biological samples can be used for various biological samples, including body fluids, tissues, and cells. In certain embodiments, the methods of the present disclosure can separate relevant fractions of biological materials that closely correspond to disease states.

[0010] The disclosed method and apparatus can be used for separating the important fraction of biological samples, analyzing the components relevant to the diagnosis and prognosis of the disease. In some embodiments, a significant amount of biological material and corresponding improved signal levels can be obtained for decision making. Aspects of the present invention include preserving the compositions and properties of extracellular matrix bodies (EMB) as disease indicators.

[0011] In some aspects, the present invention provides an enhanced ability to detect or characterize disease using extracellular matrix bodies, which can more easily reflect disease states.

[0012] In further aspects, the devices and methods of the present disclosure can provide increased signals for relevant biomarkers that correlate specific biofractions with disease states. The biomarkers of the present disclosure can be correlated with disease and provide a diagnostic tool.

[0013] The device of the present disclosure can provide accurate measurement of the flow rate and pressure of the fluid containing biological components by maintaining continuous and large amounts of flow in the device, so that the sensor can accurately measure the differential pressure and flow rate. The device and system disclosed herein can provide improved measurement of the flow rate and pressure of the fluid containing biological components by arranging channels that maintain continuous and large amounts of flow. In some embodiments, the device of the present disclosure may have one or more channels that do not significantly restrict the flow of the fluid, thereby maintaining continuous and large amounts of flow in the system.

[0014] The devices and systems of the present disclosure can separate unique subpopulations or subset fractions of biological samples. In some embodiments, the unique subset fractions of biological samples can be associated with a disease. In certain embodiments, the unique subset fractions of biological samples can be essentially composed of extracellular matrix bodies.

[0015] In a further aspect, the present disclosure provides devices and methods for separating, detecting and / or analyzing ultrastructural components of a fluid containing biological material or molecules.In certain embodiments, the ultrastructural components may be associated with a disease.

[0016] Embodiments of the present invention can be used to isolate, extract, and utilize extracellular matrix bodies (EMBs) as a source of a variety of specific disease biomarkers.

[0017] The present invention includes devices for separation, detection and analysis of extracellular matrix bodies, biological particles and complexes for a variety of uses.

[0018] In certain aspects, the extracellular matrix bodies can serve as biomarkers by virtue of their morphological characteristics. In further aspects, the extracellular matrix bodies can function by containing isolated biochemical markers that may be present in a disease pathway.

[0019] Aspects of the invention may further provide diagnostic systems comprising means for detecting and measuring biomarkers via disease-associated extracellular matrix bodies (EMBs) and / or bioparticles or complexes.

[0020] In further embodiments, the present disclosure describes methods and apparatus for preparing and analyzing samples of biological material.

[0021] Biological material samples can include body fluids, tissues, and cells.

[0022] Embodiments of the present invention include the following:

[0023] A device for separating biological sample fractions, comprising:

[0024] one or more restricted passages having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the passage;

[0025] a plurality of spaced-apart obstacles positioned in the restricted passage to provide flow resistance, wherein the spacing between the obstacles decreases in a direction from the inlet end to the outlet end; and

[0026] an inlet reservoir for containing a fluid, wherein the inlet fluid reservoir is in fluid communication with the inlet end of the restrictive passage;

[0027] One or more uniform flow channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel, wherein the inlet end is in fluid communication with the inlet reservoir.

[0028] The above device also includes a pressure source for applying pressure to the fluid in the inlet reservoir; and / or a flow sensor in fluid communication with the inlet reservoir, which is used to measure the flow rate and pressure of the fluid at the inlet reservoir.

[0029] The device may also include an outlet reservoir in fluid communication with the outlet ends of the restricting channel and the uniform flow channel.

[0030] The above device, wherein the restricted channel includes a barrier band having a window of at least about 1 micron, or at least about 2 microns, or at least about 4 microns, or at least about 10 microns, or at least about 25 microns, or at least about 50 microns, or at least about 100 microns, or at least about 200 microns, or at least about 500 microns.

[0031] The above device, wherein the restricted channel includes a window of about 1-4 microns, or about 1-15 microns, or about 4-35 microns, or about 4-100 microns, or about 4-200 microns.

[0032] The above device, wherein 1-90% of the flow in the device is in the uniform flow channel, or 1-75% of the flow in the device is in the uniform flow channel, or 1-50% of the flow in the device is in the uniform flow channel, or 1-25% of the flow in the device is in the uniform flow channel.

[0033] The restriction channel and the uniform flow channel may be integrated with the same chip or substrate. The restriction channel and the uniform flow channel may be located in different chips or substrates. The restriction channel may be a microfluidic channel.

[0034] The above device also includes means for analyzing the biological sample in the channel.

[0035] The above-mentioned device also includes a device for analyzing the proteomic composition, lipidomic composition, transcriptomic composition or carbohydrate composition of the biological sample in the channel.

[0036] The above device also includes means for measuring the level of the separated fraction of the sample in the channel.

[0037] The above device also includes means for measuring the level of the biomarker in the separated fractions of the sample in the channel.

[0038] The above device, wherein the plurality of obstacles include posts integrated with the channel.

[0039] The above device, wherein the multiple obstacles include one or more of the following: a portion of the uveal meshwork of a human or animal, a portion of the corneoscleral meshwork of a human or animal, or a portion of the juxtacanalicular meshwork of a human or animal.

[0040] The plurality of obstacles may include glass beads, magnetic beads, gel particles, dextran particles, or polymer particles.

[0041] Biological samples can be composed of human or animal body fluids, blood, tissues or cells. Biological samples contain a carrier fluid.

[0042] The above device, wherein the biological sample contains one or more reagents.

[0043] In the above device, the limiting channel also includes a binding portion for binding biomarkers or biomolecules of the sample.

[0044] The biological sample may be from a subject undergoing diagnosis or prognosis.

[0045] The above device also includes a tortuous fluid mixing region in the confining channel. The above device, wherein the confining channel or the continuous flow channel has a fluorinated coating.

[0046] The present invention also relates to a method for extracting extracellular matrix bodies from a biological sample by allowing the biological sample to flow from the inlet end to the outlet end of the device of claim 1; and reversing the direction of the fluid flow toward the inlet end of the device.

[0047] A microfluidic system for separating fractions of a biological sample, the system comprising:

[0048] A microfluidic device comprising

[0049] one or more restricted passages having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the passage;

[0050] a plurality of spaced-apart obstacles positioned in the restricted passage to provide flow resistance, wherein the spacing between the obstacles decreases in a direction from the inlet end to the outlet end; and

[0051] an inlet reservoir for containing a fluid, wherein the fluid reservoir is in fluid communication with the inlet end of the restrictive passage; and

[0052] one or more uniform flow channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel, wherein the inlet end is in fluid communication with an inlet reservoir;

[0053] A drive unit including a pressure source;

[0054] a source unit comprising a fluid source, wherein a pressure source is in fluid communication with the fluid source and an inlet reservoir of the microfluidic device;

[0055] a sensor unit including a sensor in fluid communication with the inlet reservoir for measuring a flow rate and a pressure of the fluid at the inlet reservoir and sending the flow and pressure data to the processor; and

[0056] an on-chip analyzer unit comprising one or more instruments for analyzing the separated fractions in the microfluidic device and sending the analysis data to the processor; and

[0057] Processor for receiving and displaying flow rate, pressure and analysis.

[0058] A composition comprising a fraction of a biological sample extracted from a device of the present disclosure. The composition can be used to treat a human or animal body. The composition can be used for diagnosis or prognosis of a subject.

[0059] A method for preparing a biological sample, the method comprising separating an extracellular matrix body from a biological sample. The size of the extracellular matrix body can be 0.5 to 5,000 microns, or 1 to 1,000 microns, or 1 to 200 microns, or 4 to 100 microns. Separation of the extracellular matrix body can be carried out by ultrafiltration or centrifugation. Separation of the extracellular matrix body can be carried out by a device disclosed herein.

[0060] The above method further comprises fixing the extracellular matrix bodies on the glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking.

[0061] A method for preparing a biological sample of extracellular matrix by fixing the extracellular matrix on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking.

[0062] BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The top view of the microfluidic chip embodiment of the present invention is shown. In this form, a silicon wafer master 101 is printed with three microfluidic channel chip patterns 103. Silicon wafer 101 can be used as a substrate. Photoresist can be poured on the substrate and exposed to ultraviolet light, which forms the pattern of the microfluidic chip 103. Wafer and photoresist form a mold together, and PDMS can be poured on the mold. Once solidified, PDMS can be peeled off from the mold, and each wafer can give a casting of three microfluidic chips. These castings can be adhered to a slide to form a final microfluidic chip.

[0064] Figure 2 A top view of a microfluidic chip insert in one embodiment of the device of the present invention is shown. The chip has two restricted channels 203, each of which is 2500um wide and 25,000um long in this example. Restricted channels 203 include columns of different diameters and intervals, as shown in circles. The chip has a third uniform flow channel 205, which has columns of various sizes and intervals that do not significantly restrict flow. The chip has an inlet reservoir 201 and an outlet reservoir 207, which also include larger columns. The dotted arrow shows the flow direction from the inlet reservoir toward the outlet reservoir.

[0065] Figure 3 Displays the corresponding Figure 2 Top view of the . Figure 3 PDMS polymer pillars 301 of various sizes, represented by circles, are shown. Dashed arrows show the flow of biological fluids through the three channels.

[0066] Figure 4 Display corresponds to Figure 2 Top view of the inlet reservoir. Figure 4 Posts 401 are shown as circles. Dashed arrows show the flow of biological fluid through the three channels.

[0067] Figure 5 Displays the corresponding Figure 2 Top view of the inlet reservoir area. Figure 5 Posts 501 are shown as circles. Dashed arrows show the flow of biological fluid through the three channels.

[0068] Figure 6 Display corresponds to Figure 2 Top view of the channel area. Figure 6 Posts 601 are shown, represented by circles. Dashed arrows show the flow of biological fluid through the channel. The microfluidic channel devices of the present invention may have areas of posts or obstacles of varying sizes and / or spacing for creating turbulence or restricting flow.

[0069] Figure 7 Displays the corresponding Figure 2 Magnified top view of the channel area. Figure 7 The pillars 701 are shown as circles. The dashed arrows show the flow of the biological fluid through the channel. This view shows the transition from the 50um gap between the pillars to the 25um gap in the confining channel.

[0070] Figure 8 Displays the corresponding Figure 2 Magnified top view of the channel area. Figure 8 Posts 801 are shown as circles. Dashed arrows show the flow of biological fluid through the channel. This view shows the transition from larger gaps to smaller gaps between posts in the confining channel.

[0071] Fig. 9 Displays the corresponding Figure 2 Magnified top view of the channel area. Fig. 9 Posts 901 are shown as circles. Dashed arrows show the flow of biological fluid through the channel.

[0072] Fig.10 Displays the corresponding Figure 2 Magnified top view of the channel area. Fig.10The pillars 1001 are represented by circles. The dashed arrows show the flow of the biological fluid through the channel. This view shows the channel in an area with blunt pillar obstacles 1001, which can create turbulent flow.

[0073] Fig.11 Displays the corresponding Figure 2 An enlarged top view of the outlet reservoir 1107. Fig.11 Posts 1101, 1103 and 1105 of various sizes are shown. The dashed arrows show the flow of biological fluid through the channel. In this embodiment, each outer limiting channel comprises a barrier 1102 formed by very small and closely spaced posts.

[0074] Fig.12 Displays the corresponding Figure 2 An enlarged top view of the inlet reservoir 1201. Fig.12 Various sizes of pillars 1203 are shown. The outer confinement channel 1207 includes pillars of different sizes and spacing. The uniform flow channel 1205 includes pillars of uniform size and spacing. The dashed arrows show the direction of flow of the biological fluid through the outer channel.

[0075] Fig.13 A top view of a microfluidic chip in one embodiment of the device of the present invention is shown. Three microfluidic inserts are shown. The dashed arrows show the flow direction of the biological fluid.

[0076] Fig.14 A perspective view of one embodiment of a microfluidic channel device of the present invention is shown having blunt post obstacles 1401 to restrict flow. Fig.14 yes Fig.15 The dashed arrows show the flow direction of the biofluid.

[0077] Fig.15 A perspective view showing one embodiment of the microfluidic channel device of the present invention. Fig.15 Displays the corresponding Figure 2 View of the channel area. Fig.15 Blunt post obstacles 1501 of varying spacing in a restricting channel are shown. In this embodiment, a restricting channel may have post obstacles 1501 organized into bands of varying spacing between posts. The dashed arrows show the direction of flow of the biological fluid.

[0078] Fig.16An elevational side view of an embodiment of a microfluidic chip of the present invention is shown. An inlet reservoir 1605 is in fluid communication with a fluid line 1601 for introducing biological fluid and / or other fluids into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and a hole 1604 defined in a glass cover slip. The biological fluid passes through the inlet reservoir 1605 to a microfluidic channel 1606. The dashed arrows show the direction of flow of the biological fluid.

[0079] Fig.17 Displays the corresponding Figure 2 The entrance area and Fig.16 1602. The dotted arrows show the direction of flow of the biological fluid.

[0080] Fig.18 An elevational side view of an embodiment of a microfluidic chip 1614 of the present invention is shown. An inlet reservoir is in fluid communication with a fluid line 1601 for introducing a biological fluid into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and an aperture 1604 defined in a glass cover slip 1613. The biological fluid passes through the inlet reservoir to a microfluidic channel 1606 and flows to an outlet reservoir 1607. A probe adjuster 1612 may be provided to adjust the height of the probe 1602 to form a good seal with the probe adapter 1603 and the aperture 1604. The dashed arrows show the direction of flow of the biological fluid.

[0081] Fig.19 Displays the corresponding Figure 2 Magnified top view of the channel area. Fig.19 Posts 1701 are shown as circles. For this embodiment, some representative lengths of the post band region in the outer channel are shown in microns.

[0082] Fig. 20 Displays the corresponding Figure 2 Micrograph of a magnified top view of the channel area. Fig. 20 The pillars are shown as dots. For this embodiment, some representative lengths of the pillar strip area in the outer channel are shown in microns. The dashed arrows show the flow direction of the biological fluid.

[0083] Fig.21 Displays the corresponding Figure 2 A top view of one embodiment of a microfluidic device of FIG. 2200 . A biological fluid can be introduced into an inlet region reservoir 2202 using a delivery probe 2201. The dashed arrow shows the direction of the biological fluid flow toward the outlet reservoir region 2203. A zoomed in view of this embodiment shows some representative lengths of the pillar band region in the outer channel in microns. For this embodiment, the dotted lines in the zoomed in view show the possible tortuous paths of the biological fluid between obstacles.

[0084] Fig. 22 One embodiment of the microfluidic system of the present invention is shown having a processor, a fluid drive unit, a fluid source unit, a sensor unit, an on-chip unit, and an off-chip unit.

[0085] Fig.23 showed that aqueous humor from primary open-angle glaucoma patients increased the pressure in the microfluidic device. Fig.23 The relative pressure (mm Hg) change in the artificial trabecular meshwork formed by the pillars in the microfluidic channel is shown when human aqueous humor obtained from a patient with severe primary open-angle glaucoma is injected. The fluid flow rate is kept constant at 2 μl per minute, and the baseline system pressure is measured using an external pressure sensor. Human aqueous humor samples are injected at the time points indicated by the arrows and the letter "a." The pressure rises steadily to a maximum of about 41 mm Hg at 27 minutes. Fig.23 showed that aqueous humor from patients diagnosed with POAG glaucoma increased the pressure in the device.

[0086] Fig.24 (Top) shows a confocal micrograph of the microfluidic chip after capturing EMBs in human aqueous humor from a patient with primary open-angle glaucoma. The proteins of the EMBs were labeled with the fluorescent marker carboxyfluorescein succinimidyl ester (CFSE, indicated by arrows). The circles are pillars in the confining channel. Fig.24 (Bottom) shows EMBs isolated in a microfluidic channel around a pillar.

[0087] Fig.25 Illustrated is the use of size exclusion filters to separate extracellular matrix bodies from biological fluids. Bovine vitreous humor was first filtered with a 5 μm cellulose acetate syringe filter, then with a 1 μm syringe-tip filter, then with a 0.45 μm syringe-tip filter, and then with a 0.22 μm filter. Each fraction was characterized using wide-field microscopy. Fig.25 Unenriched bovine vitreous humor 4301 is shown being drawn into a 1 mL syringe 4305 with a 22 g needle and extruded through a 5 μm needle tip filter 4309. The filtrate was collected and filtered through a 1 μm needle tip filter 4311. The filtrate was collected and filtered through a 0.45 μm needle tip filter 4313. The filtrate was collected and extruded through a 0.22 μm needle tip filter 4315. Biofluid fractions were collected after each filtration step for optical microscopy. The microscopy images show that as the filtrate passes through successively smaller sized filters, the larger bodies are reduced.

[0088] Fig.26 Representative transmission electron microscopy (TEM) images of EMBs separated by size exclusion filters are shown. Fig.26 a and Fig.26 b shows the presence of extracellular matrix bodies present in the natural biological fluid of bovine vitreous humor. To separate and recover extracellular matrix bodies from complex biological fluids, a series of syringe-based filtrations were performed using cellulose filters with pore sizes ranging from 5 μm to 0.22 μm. The extracellular matrix bodies were stained with Alcian blue stain. Fig.26 c shows the bovine vitreous fraction separated by filtration through a 5 μm needle tip filter train. Fig.26 d shows the bovine vitreous fraction separated by filtration through a 1 μm needle tip filter train. Fig.26 e shows the bovine vitreous fraction separated by filtration through a 0.45 μm needle tip filter train. Fig.26 f shows bovine vitreous fractions separated by filtration through a series of 0.22 μm needle tip filters. The image shows a relative reduction in larger ECM bodies as the filtrate series is passed through smaller filter sizes.

[0089] Fig. 27 Illustrated is the use of centrifugation to separate extracellular matrix bodies from biological fluids. Four precipitates 9705, 9713, 9721 and 9729 are obtained by sequential centrifugation. Bovine vitreous body is resuspended 9701 and placed in a 1ml tube and centrifuged (Sorvall Legend RT) at 350g for 10 minutes at 4°C to form precipitate 1 9705. The supernatant of a 50 μl aliquot is stored for analysis and is marked as supernatant 1 9709, and the remaining supernatant is transferred to a new tube and formed precipitate 2 9713 at 4°C with 2000g centrifugation (Eppendorf, 5417R series, F45-30-11Eppendorf rotor) for 10 minutes. The supernatant of a 50 μl aliquot is stored for analysis and is marked as supernatant 2 9717, and the remaining supernatant is transferred to a new tube. The supernatant was then centrifuged at 10,000 g for 10 minutes at 4°C to form precipitate 3 9721. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 3 9725, and the remaining supernatant was transferred to a new tube. The supernatant was then centrifuged at 20,000 g for 10 minutes at 4°C to give precipitate 4 9729. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 4, and the remaining supernatant was transferred to a new tube.

[0090] Fig.28 Representative transmission electron microscopy (TEM) images of EMBs isolated by serial centrifugation are shown. Fig.28 a shows the extracellular matrix present in bovine vitreous humor. Fig.28 In b, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 450 g shows that extracellular matrix bodies are present in the pellet fraction. Fig.28 In c, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 2,000 g shows that extracellular matrix bodies are present in the pellet fraction. Fig.28 In d, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 10,000 g shows that extracellular matrix bodies are present in the pellet fraction. Fig.28 In e, representative TEM micrographs of samples collected from the pellet after centrifugation at 20,000 g show that extracellular matrix bodies are present in the pellet fraction.

[0091] Fig.29 The dose-response behavior of the compound bivalirudin TFA on intraocular pressure (IOP) in bovine vitreous humor is shown. The differential pressure is measured using the microfluidic device of the present invention.

[0092] Fig.30 The dose-response behavior of the compound colistin sulfate on intraocular pressure (IOP) in bovine vitreous humor is shown. The differential pressure is measured using the microfluidic device of the present invention.

[0093] Fig.31 The dose-response behavior of the compound polymyxin B sulfate on intraocular pressure (IOP) in a bovine vitreous humor glaucoma model is shown. The differential pressure is measured using the microfluidic device of the present invention.

[0094] Fig.32 The separation and extraction of extracellular matrix bodies in confined channels of a microfluidic device of the present disclosure is shown. Fig.32 a and Fig.32 a Representative micrograph showing a microfluidic chip perfused with a biofluid of bovine vitreous humor. The letter "p" marks a post in the channel. After perfusion, extracellular matrix bodies are separated between and around the posts. Fig.32 c and Fig.32 d shows a representative micrograph of the channel after extraction of the ECM bodies. The ECM bodies were removed from the chip, which showed significantly fewer ECM bodies after extraction.

[0095] Fig.33 Shown is the off-chip proteomic analysis of isolated and extracted bovine extracellular matrix bodies by LC / MS. Biomarkers specific to extracellular matrix bodies were detected.

[0096] Fig.34 The separation of extracellular matrix bodies in a confining channel of a microfluidic device of the present disclosure and their subsequent extraction are shown. The image scale bar is 50 μm. Fig.34a shows a representative wide-field micrograph of a microfluidic device perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, and hyaluronic acid counterstained with Alcian blue (grey signal, bright field). Fig.34 A shows the signal from the extracellular matrix body (arrow) between the pillars (p) captured in the device. The chip was perfused with biofluid for at least 60 minutes. After perfusion, aggregation was separated between the pillars and was observed with mass-like formation. The material smaller than the extracellular matrix body material has been discharged through the outlet port. Fig.34 b shows the extraction of extracellular matrix bodies from a confining channel of a microfluidic device. The device was perfused with a mild detergent, 0.1% sodium dodecyl sulfate (SDS), and the flow direction was reversed from the outlet to the inlet. Fig.34 b shows that significantly fewer ECM bodies are present in the channel after elution and indicates that the ECM bodies have been extracted. Fig.34 c shows a higher magnification image of the extracellular matrix bodies (arrows) trapped between the pillars (p) after perfusion. Fig.34 d shows extraction with detergent and reverse flow, again showing significantly fewer ECM bodies in the channel after extraction.

[0097] Fig.35 Show the on-chip immunohistochemical staining of the extracellular matrix body in the device channel of the present disclosure.The microfluidic device is injected with the fluid containing the homogenized bovine vitreous body suspended in the biological fluid.After the fluid is perfused into the device, the fluid flows through the inlet and flows out via the outlet.The larger extracellular matrix body (arrow) is captured between the pillars (labeled as Lp).The chip is perfused with a blocking solution to prevent the non-specific antibody binding before the antibody staining.Next, by injecting an anti-fibronectin primary antibody, the sample is incubated for 2 hours and washed to label the protein fibronectin, which is a known extracellular matrix component and integrin binding protein.Then, goat anti-rabbit FITC secondary antibodies are incubated for 1 hour and washed.Then under wide-field fluorescence and bright-field microscopy, the microfluidic chip is imaged. Fig.35 Representative wide-field fluorescence micrographs are shown. This image shows the extracellular matrix body in the microfluidic channel. The distance between the large pillars (Lp) is about 100 μm. The dot-like signals in the extracellular matrix body represent fibronectin staining (anti-fibronectin Ab, goat anti-rabbit secondary antibody with Alexa 488, FITC, white signal).

[0098] Fig.36 On-chip immunohistochemical staining showing extracellular matrix bodies in the channels of the devices of the present disclosure. Fig.36 a Representative photomicrograph bright field image showing stained extracellular matrix bodies (arrows) in the channel. Image scale bar is 20 μm. The control image has no fluorescent signal, which indicates that Fig.36The signal in a is specific for fibronectin. Fig.36 b shows the extracellular matrix bodies stained in the channel again (arrows). Image scale bar is 50 μm. Again, the control image has no fluorescent signal, which indicates that Fig.36 The signal in b is specific for fibronectin.

[0099] Fig.37 On-chip immunohistochemical staining showing extracellular matrix bodies in the channels of the devices of the present disclosure. Fig.37 Shown in a biofluid containing an extracellular matrix body, a representative microphotograph of on-chip immunohistochemical staining of beadlecan protein, a component of the extracellular matrix of cartilage, is a known cancer biomarker. The microfluidic chip is injected with a fluid containing homogenized bovine vitreous suspended in a biofluid. After the biofluid is perfused into the device, the sample flows through the inlet and flows out through the outlet. Larger extracellular matrix bodies (arrows) are captured between the columns (marked as "p"). The chip is perfused with a blocking solution to prevent nonspecific antibody binding before antibody staining. Beadlecan is labeled by injecting an anti-beadlecan primary antibody, incubating the sample for 2 hours and washing. Then, goat anti-rabbit TRITC secondary antibodies are incubated for 1 hour and washed. The microfluidic chip is then imaged under wide-field fluorescence and bright-field microscopy. Fig.37 The extracellular matrix bodies in the microfluidic channel between the pillars (p) are shown. The punctate signals represent beaded proteoglycan staining (white signal). The control image has no fluorescent signal, which indicates that Fig.37 The signal in is specific for globulin.

[0100] Fig.38 On-chip immunohistochemical staining showing extracellular matrix bodies in the channels of the device of the present disclosure. Fig.38 a Representative photomicrograph bright field image showing on-chip immunohistochemical staining of beaded proteoglycan. The control image showed no fluorescent signal, indicating Fig.38 The signal in a is specific for globulin. Image scale bar is 10 μm. Fig.38 b Representative photomicrographs showing bright field images of on-chip immunohistochemical staining of beadlecan. The control images showed no fluorescent signal, indicating that Fig.38 The signal in b is specific for globulin. Image scale bar is 10 μm. Fig.38 It has also been shown that Alcian blue, a marker for hyaluronic acid, can be used as a stain for EMB.

[0101] Fig.39 Extracellular matrix bodies on glass surfaces were visualized using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking and Sirius red dye staining for collagen. Fig.39Signals for the ECM bodies were shown (dark staining), indicating that EDC cross-linking retained the ECM bodies on the surface.

[0102] Fig.40 Extracellular matrix bodies on glass surfaces were visualized using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking and Sirius red dye staining for collagen. Fig.40 Signals for collagen chains within the ECM bodies (dark staining) were shown, indicating that EDC cross-linking retained the ECM bodies on the surface. Fig.40 It has also been shown that Sirius red stain can be used to stain EMBs.

[0103] Fig.41 Shown is the visualization of off-chip analysis of extracellular matrix bodies on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking and DNA staining using Hoechst dye. Fig.41 The DNA signal in the extracellular matrix bodies is shown (Hoechst dye, DAPI filter, white signal, DNA).

[0104] Fig.42 On-chip separation and detection of extracellular matrix bodies are shown. Fig.42 Shown are representative micrographs of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, and counterstained with Alcian blue for hyaluronic acid, dark stain, bright field. Fig.42 Signals from extracellular matrix bodies trapped near the large pillars (circles) of the device are shown. The chip was perfused with biofluid for at least 60 minutes. Clustered formations of extracellular matrix bodies were observed. Image scale bar is 50 μm.

[0105] Fig.43 Shown are on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Fig.43 The representative low-power fluorescence micrograph of the channel after perfusion of bovine vitreous humor extracellular matrix body is shown, and the bovine vitreous humor extracellular matrix body is counterstained with carboxyfluorescein succinimidyl ester (CFSE) to protein. The microfluidic chip is injected with the fluid containing homogenized bovine vitreous. After the fluid is perfused into the device, the fluid flows through the inlet and flows out via the outlet. Larger extracellular matrix body is captured near the column (marked as "p"). The image scale is 25 μm.

[0106] Fig.44 Shown are on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Fig.44Shown are representative micrographs of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, and counterstained with Sirius red for collagen, dark stain, bright field. Fig.44 a shows the signal from the extracellular matrix bodies between the posts of the device (labeled "p").The chip was perfused with biological fluid for at least 60 minutes. Fig.44 b shows the same image with a fluorescence filter and demonstrates the detection of collagen with Sirius Red (light grey signal). Image scale bar 50 μm. Fig.44 c and Fig.44 d shows a similar image at a higher magnification. The image scale bar is 10 μm.

[0107] Fig.45 Show the frequency size distribution of human extracellular matrix bodies present in the human aqueous humor biofluid from health and pre-disease state, suspected glaucoma and pre-glaucoma.Aqueous humor is obtained from 8 patients, healthy samples or pre-glaucoma diagnosis, and the intraocular pressure range is 9 to 25mmHg.Human samples are not processed by centrifugation or other means.The size of the extracellular matrix body is determined by using carbodiimide EDC fixative that the sample is cross-linked to a slide, dyed with uranyl acetate and imaged with wide-field microscopy.Automation program (ImageJ) is used to quantify size in all eight samples.The size (area) range of the extracellular matrix body is about 1.67 μm 2 About 67×10 3 μm 2 . Fig.45 Shows 0-200μm 2 Counting of extracellular matrix bodies within the range.

[0108] Fig.46 The frequency size distribution of human extracellular matrix bodies present in the human aqueous humor biofluid from health and pre-disease state, suspected glaucoma and early glaucoma is shown. Aqueous humor is obtained from 8 patients, healthy samples or early glaucoma diagnosis, and the intraocular pressure range is 9 to 25mmHg. Human samples are not processed by centrifugation or other means. The size of the extracellular matrix body is determined by using carbodiimide EDC fixative that the sample is cross-linked to a slide, dyed with uranyl acetate and imaged with wide-field microscopy. In all eight samples, an automated program (ImageJ) is used to quantify the size. The size (area) range of the extracellular matrix body is about 1.67 μm 2 About 67×10 3 μm 2 . Fig.46 Shown 201-1000μm 2 Counting of extracellular matrix bodies within the range.

[0109] Fig.47Show the frequency size distribution of human extracellular matrix bodies present in the human aqueous humor biofluid from health and pre-disease state, suspected glaucoma and pre-glaucoma.Aqueous humor is obtained from 8 patients, healthy samples or pre-glaucoma diagnosis, and the intraocular pressure range is 9 to 25mmHg.Human samples are not processed by centrifugation or other means.The size of the extracellular matrix body is determined by using carbodiimide EDC fixative that the sample is cross-linked to a slide, dyed with uranyl acetate and imaged with wide-field microscopy.Automation program (ImageJ) is used to quantify size in all eight samples.The size (area) range of the extracellular matrix body is about 1.67 μm 2 About 67×10 3 μm 2 . Fig.47 Shown 1001-67,000μm 2 Counting of extracellular matrix bodies within the range.

[0110] Fig.48 The size distribution of bovine vitreous extracellular matrix bodies separated and extracted from the microfluidic device of the present invention is shown. Extracellular matrix bodies in bovine vitreous fluid biofluid after separation and extraction from the microfluidic device of the present invention. The chip was perfused with biofluid for at least 60 minutes. After 60 minutes of perfusion, the ECM bodies were separated near the limiting channel pillars. Next, the chip was treated with a detergent (0.1% sodium dodecyl sulfate, SDS) and samples were extracted from the chip by reverse flow, which caused the extracellular matrix bodies to flow out of the inlet port. Fractions of the eluate were collected at intervals of 10 minutes for a total of 80 minutes. The samples were fixed on a slide and stained with Alcian blue and imaged with wide-field microscopy. The size was quantified using an automated program (ImageJ). The size (area) of the extracellular matrix bodies can reach about 16×10 3 μm 2 . Fig.48 The counts of extracellular matrix bodies in each eluate fraction are shown, which increase over time. This experiment demonstrates that the microfluidic device of the present invention can be used to separate and extract extracellular matrix bodies of various sizes.

[0111] Fig.49 Off-chip analysis showing extracellular matrix bodies can be accomplished by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for retention. Fig.49 a shows a representative TEM image of extracellular matrix bodies from native bovine vitreous humor obtained by cross-linking with EDC. Extracellular matrix bodies were observed by fixation with EDC. Fig.49 b shows a similar image taken without EDC cross-linking. Without EDC fixation, no extracellular matrix bodies were observed.

[0112] Fig.50Shown are extracellular matrix bodies isolated from human plasma samples of patients with early-stage pancreatic ductal adenocarcinoma (PDAC) compared to healthy controls. Fig.50 a Shows a representative wide-field fluorescence micrograph image of a PDAC sample. Fig.50 a shows the extracellular matrix bodies retained in the microfluidic channel. Fig.50 a further shows that larger extracellular matrix bodies (arrows) stay between the pillars (marked as "p"). The width between the pillars is about 100 μm. The punctate signals from the staying extracellular matrix bodies represent fibronectin staining (anti-fibronectin Ab, goat anti-rabbit secondary Ab with Alexa 488, FITC, white signal). Staining shows abundant signals and punctate staining within EMBs ( Fig.50 a, arrow). Fig.50 b shows a similarly obtained fluorescence micrograph of an age-matched healthy control human plasma sample. Fig.50 b shows that the amount of fibronectin signal is significantly reduced ( Fig.50 b, gray signal, arrow). When treated under the same conditions, the healthy control signal is much smaller than the disease signal. The image scale bar is 20 μm. DETAILED DESCRIPTION OF THE INVENTION

[0114] The present invention discloses devices and systems for separating particles from biological samples suitable for various diseases and conditions by separating and using organisms, materials and / or molecules. The devices and systems for separating particles from biological samples can be used to increase the level of sample separations related to the biology of interest. The devices and methods of the present invention can be used to separate important components from biological samples for diagnosis and prognosis of diseases.

[0115] The device for separating particles from biological samples of the present invention can be used for various biological materials and samples, including body fluids, blood, tissues, cells and tumors. In certain embodiments, the methods of the present disclosure can separate relevant fractions of biological materials corresponding to disease states.

[0116] The present disclosure provides devices that can be used to separate important fractions of biological samples and analyze their components related to the diagnosis and prognosis of diseases. In some embodiments, significant amounts of biological material and correspondingly increased signal levels can be obtained.

[0117] Aspects of the present invention include separating and preserving the composition and property of extracellular matrix bodies (EMB) from biological fluids or materials. By preserving the composition and property of the extracellular matrix bodies (EMB) separated or extracted from biological samples, fluids or materials, EMB can be used for diagnosis or indication of disease, or for monitoring the change of chemical or biological processes or sample materials.

[0118] In some aspects, the present invention provides an enhanced ability to detect or characterize extracellular matrix bodies, which may more readily reflect disease states.

[0119] In further aspects, the devices and methods of the present disclosure can provide increased signals for relevant biomarkers that correlate specific biofractions with disease states. The biomarkers of the present disclosure can be correlated with disease and provide a diagnostic tool.

[0120] The device of the present disclosure can provide improved measurement of flow and / or pressure in a fluid containing a biological component by maintaining continuous and large amounts of flow in the device. In some embodiments, the sensor can accurately measure differential pressure and flow when maintaining a large amount of flow. The device and system disclosed herein can provide improved flow and pressure measurement of a fluid containing a biological component by maintaining a continuous and large amount of flow channel arrangement. In some embodiments, the device of the present disclosure may have one or more channels that do not substantially restrict the flow of the fluid, thereby maintaining a continuous and large amount of flow in the system.

[0121] In addition to restricted channels, the devices disclosed herein can also provide improved flow and pressure measurements of fluids containing biological components by using one or more uniform flow channels that maintain continuous and bulk flow.

[0122] In certain embodiments, the devices of the invention will have 1-90% of the flow from uniform flow channels, or 1-75% of the flow from uniform flow channels, or 1-50% of the flow from uniform flow channels, or 1-25% of the flow from uniform flow channels.

[0123] In certain embodiments, the devices of the invention will have at least 25% of the flow from uniform flow channels, or at least 50% of the flow from uniform flow channels, or at least 75% of the flow from uniform flow channels, or at least 90% of the flow from uniform flow channels.

[0124] In some embodiments, extracellular matrix bodies having a diameter size of about 0.5 to about 5,000 microns or more may become lodged in a microfluidic channel or may cause a blockage in the channel, thereby increasing pressure.

[0125] In other embodiments, extracellular matrix bodies having a diameter size of about 1 to about 200 microns or more may become lodged in a microfluidic channel, or may cause blockages in the channel, thereby increasing pressure.

[0126] In a further aspect, the present disclosure provides devices and methods for separating, detecting and / or analyzing ultrastructural components of a fluid containing biological material or molecules.In certain embodiments, the ultrastructural components may be associated with a disease.

[0127] Embodiments of the present invention can be used to isolate, extract, and utilize extracellular matrix bodies (EMBs), which are a source of a variety of specific biomarkers.

[0128] The present invention includes devices for separating, detecting and analyzing the composition of extracellular matrix bodies, biological particles and complexes for various applications.

[0129] In certain aspects, the extracellular matrix bodies can serve as biomarkers by virtue of their morphological characteristics. In further aspects, the extracellular matrix bodies can function by containing isolated biochemical markers that may be present in a disease pathway.

[0130] Aspects of the invention may further provide diagnostic systems comprising means for detecting and measuring biomarkers via disease-associated extracellular matrix bodies (EMBs) and / or bioparticles or complexes.

[0131] In further embodiments, the present disclosure describes methods and apparatus for preparing and analyzing samples of biological material.

[0132] Extracellular matrix bodies (EMBs) can be complex and can be composed of proteins, lipids, carbohydrates, nucleic acid molecules, biological particles, small vesicles such as extracellular vesicles or exosomes, and combinations thereof.

[0133] Samples of biological material may include body fluids, tissues, and cells.

[0134] Examples of body fluid samples include any body fluid, including whole blood, plasma, blood components, CSF, urine, semen, synovial fluid, pleural fluid, vaginal fluid, gastric fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, ear fluid, breast milk, and any other body fluid, and combinations thereof.

[0135] In a further aspect, the microfluidic devices and systems of the present invention can be used to separate and extract biological particles from a sample.

[0136] The microfluidic devices and systems of the present invention can be used to purify or isolate extracellular matrix bodies or complexes having a diameter greater than about 0.5 microns up to particles having a diameter of about 5,000 microns, or extracellular matrix bodies or complexes having a diameter greater than about 2 microns up to particles having a diameter of about 700 microns.

[0137] In a further aspect, the microfluidic devices and systems of the present invention can be used to measure relative viscosity and flow properties of biological and clinical fluids.

[0138] In certain aspects, the microfluidic devices and systems of the present invention can be used to separate and extract disease-associated biological particles from a sample.

[0139] In some aspects, the microfluidic devices and systems of the present invention can be used to measure intraocular pressure in ocular fluid.

[0140] Devices and systems

[0141] The present invention provides microfluidic devices and systems for measuring pressure and / or flow in a fluid.

[0142] The present invention improves the measurement of flow and pressure of a fluid by providing a continuous and large flow rate and flow volume, thereby accurately measuring differential pressure and / or flow.

[0143] In a further aspect, the microfluidic devices and systems of the present invention can be used to detect, separate and extract biological particles or other components of biological materials or fluids.

[0144] The microfluidic devices and systems of the present invention can be used to measure relative viscosity and flow properties of biological and clinical fluids.

[0145] The microfluidic devices and systems of the present invention may include a microfluidic chip that may be held in a substrate.

[0146] In some aspects, the microfluidic devices of the present invention can have channels with obstacles. The obstacles can affect and / or restrict the flow of fluid in the channels.

[0147] In some aspects, the microfluidic device of the present invention can have a channel with an obstacle strip. The obstacle strip can span the width of the channel so that the obstacle strip will affect the flow and flux of the fluid along the channel.

[0148] In some embodiments, the spacing between obstacles can be constant in a strip.The spacing between obstacles in a channel can be used to control the size of the openings in the channel for fluid flow.

[0149] In other embodiments, the microfluidic devices of the present invention can have a channel with multiple continuous obstacle strips. In certain embodiments, the spacing between obstacles in the strips can decrease in one direction along the length of the channel in multiple strips. Thus, the spacing between obstacles in the strips can increase in the opposite direction along the length of the channel in multiple strips.

[0150] For example, a microfluidic device of the present invention can have a restricted channel in which a plurality of bands arranged sequentially in one direction have a spacing of 1,000 microns between obstacles, which is adjacent to a band with a spacing of 500 microns, which is adjacent to a band with a spacing of 200 microns, which is adjacent to a band with a spacing of 200 microns, which is adjacent to a band with a spacing of 100 microns, which is adjacent to a band with a spacing of 100 microns, which is adjacent to a band with a spacing of 50 microns, which is adjacent to a band with a spacing of 50 microns, which is adjacent to a band with a spacing of 25 microns, which is adjacent to a band with a spacing of 10 microns, which is adjacent to a band with a spacing of 10 microns, and which is adjacent to a band with a spacing of 4 microns.

[0151] In a further aspect, the microfluidic device of the present invention can have a channel with strips of obstacles, wherein the spacing between obstacles in one strip is minimal. The strip with the smallest spacing between obstacles can be a barrier strip.

[0152] In additional aspects, the microfluidic devices of the invention can have a restricted channel wherein the barrier band has a spacing between obstacles of at least 1 micron, or at least 2, or at least 4, or at least 5, or at least 10, or at least 50, or at least 100, or at least 200 microns.

[0153] In certain aspects, the microfluidic device of the present invention can be a chip having a height of 7 to 25 microns.

[0154] In operation, the microfluidic devices and systems of the present invention can be used to separate, extract and / or purify biological particles. In certain embodiments, the restricted channel may have a band with a gradually decreasing interval between obstacles, one of which is a barrier band. The method of the present invention can use such a band arrangement to separate larger particles from smaller particles and fluids by causing a fluid containing particles to flow from an inlet to an outlet of the channel in a direction that gradually decreases in interval. In these methods, larger particles can be separated and retained along the channel, while smaller particles and fluids leave the channel at the outlet. By reversing the flow direction of the extraction fluid to extract larger particles from the inlet, larger particles can be extracted from the channel at the inlet.

[0155] In operation, the microfluidic devices and systems of the present invention can be used to separate, extract and / or purify biological particles extracted from the channel at the outlet.

[0156] In some embodiments, larger particles can be extracted from the channel at the outlet by adding a detergent to break up the larger particles.

[0157] Figure 1A top view showing a microfluidic chip embodiment of the present invention is shown. In this form, a silicon wafer master 101 is printed with three microfluidic channel chip patterns 103. Silicon wafer 101 can be used as a substrate. A photoresist can be poured onto the substrate and exposed to UV light, which forms the pattern of the microfluidic chip 103. The wafer and the photoresist together form a mold, and PDMS can be poured onto the mold. Once solidified, PDMS can be peeled off from the mold, and each wafer can give castings of three microfluidic chips. These castings can be adhered to a glass slide to form a final microfluidic chip.

[0158] Microfluidic chip of the present invention can have the passage for limiting fluid flow, and the inlet and outlet for fluid flow.Pump can be used for applying the head pressure (head pressure) of fluid at the inlet.In some embodiments, can use decompression or vacuum pressure to regulate flow at the outlet.

[0159] Figure 2 A top view of a microfluidic chip insert in one embodiment of the device of the present invention is shown. The chip has two restricted channels 203, each of which is 2,500um wide and 25,000um long in this example. The restricted channels 203 include posts of different diameters and spacings, as shown in circles. The chip has a third uniform flow channel 205, which has posts of uniform size and spacing that do not significantly restrict flow. The chip has an inlet reservoir 201 and an outlet reservoir 207, which also include larger posts. The dotted arrow shows the direction of flow from the inlet reservoir to the outlet reservoir. The restricted channel can have a point of maximum restriction of flow, which is a barrier 202 that restricts flow. The barrier 202 can restrict flow and change the pressure in the channel and system so that the differential pressure and / or flow rate can be related to the composition of the fluid.

[0160] Microfluidic chip of the present invention can have one or more passages for limiting fluid flow, and one or more uniform or continuous flow channels. In some embodiments, uniform flow channel does not limit the fluid flow in the channel. Uniform continuous flow channel can include blunt obstacles for generating turbulent flow and / or for making the tortuous path of fluid flow.

[0161] Figure 3 Displays the corresponding Figure 2 Top view of the . Figure 3 PDMS polymer pillars 301 of various sizes, represented by circles, are shown. Dashed arrows show the flow of biological fluids through the three channels.

[0162] In certain embodiments, blunt or non-blunt obstacles may be provided in a confining fluid channel to produce a tortuous or eddy pattern of flow in certain areas. Obstacles in the channel may be formed as circular or other shaped pillars.

[0163] In certain embodiments, the obstacles restricting the passageway can provide a Reynolds number greater than 500, or greater than 1000, or greater than 10,000, or more.

[0164] In other embodiments, continuous uniform flow channels may be located between various restrictive channels. In certain embodiments, the uniform flow channels and restrictive channels may be arranged in any order and used in any number.

[0165] Figure 4 Display corresponds to Figure 2 Top view of the inlet reservoir. Figure 4 Posts 401 are shown as circles. Dashed arrows show the flow of biological fluid through the three channels.

[0166] Figure 5 Display corresponds to Figure 2 Top view of the inlet reservoir area. Figure 5 Posts 501 are shown as circles. Dashed arrows show the flow of biological fluid through the three channels.

[0167] Figure 6 Display corresponds to Figure 2 Top view of the channel area. Figure 6 Posts 601 are shown represented by circles. Dashed arrows show the flow of biological fluid through the channel. The microfluidic channel devices of the present invention have regions of posts or obstacles of varying spacing and / or size to create turbulent or restricted flow.

[0168] In certain embodiments, the microfluidic channel devices of the present disclosure may have a region that mimics the trabecular meshwork.

[0169] The device of the present invention may include a network structure composition containing an extracellular matrix body or complex. The extracellular matrix body or complex used in the network structure composition can be extracted or purified from glaucoma eye fluid. The eye fluid can be from an animal or clinical source.

[0170] In further embodiments, the microfluidic chip of the present invention may have one or more channels for restricting fluid flow and one or more uniform flow channels. The uniform flow channels may include blunt obstacles for generating turbulence and / or tortuous paths for causing fluid flow.

[0171] In further embodiments, the microfluidic chip of the present invention may have 1-20 channels for restricting fluid flow and 1-10 uniform flow channels, which are arranged in any order on the substrate. The uniform flow channels may be distributed in any manner relative to the restricting flow channels.

[0172] In certain embodiments, the uniform flow channel can alternate in a co-linear or parallel position relative to the restrictive flow channel. In other embodiments, the uniform flow channel can be above or below the restrictive flow channel. In some embodiments, the uniform flow channel can be arranged in a substrate separate from the chip containing the restrictive flow channel.

[0173] In further embodiments, the uniform flow channel may provide fluid communication from the inlet reservoir to the outlet reservoir. In certain embodiments, the uniform flow channel may provide fluid communication from the outlet reservoir to a source of fluid entering the inlet reservoir.

[0174] In certain embodiments, the total cross-sectional area of ​​the uniform flow channel may be greater than or less than the total cross-sectional area of ​​the restrictive flow channel in the microfluidic device of the present invention. In various embodiments, the uniform flow channel may not include obstacles and may not have tortuous fluid flow. In such embodiments, the uniform flow channel may have laminar or turbulent fluid flow.

[0175] The microfluidic chip of the present invention can have one or more restriction channels for limiting the flow of fluid. The restriction flow can be attributed to various arrangements of blunt or non-blunt obstacles or pillars in the channel. In some embodiments, the pillars can present the shape of the flowing fluid, such as circular, spherical, triangular, square, polygonal, rhombus, fin-shaped and combinations thereof.

[0176] Figure 7 Displays the corresponding Figure 2 Magnified top view of the channel area. Figure 7 The pillars 701 are shown as circles. The dashed arrows show the flow of the biological fluid through the channel. This view shows the transition from the 50um gap between the pillars to the 25um gap in the confining channel.

[0177] Figure 8 Displays the corresponding Figure 2 Magnified top view of the channel area. Figure 8 Posts 801 are shown as circles. Dashed arrows show the flow of biological fluid through the channel. This view shows the transition from larger gaps to smaller gaps between posts in the confining channel.

[0178] Fig. 9 Displays the corresponding Figure 2 Magnified top view of the channel area. Fig. 9 Posts 901 are shown as circles. Dashed arrows show the flow of biological fluid through the channels.

[0179] In further embodiments, restricted flow in a channel may be due to various arrangements of blunt or non-blunt obstacles or posts in the channel, where the size and spacing of the obstacles vary with distance along the channel.

[0180] In certain embodiments, the size and / or spacing of blunt or non-blunt obstacles or posts in a restricted passage may vary with the distance along the passage. The size and / or spacing of blunt or non-blunt obstacles may decrease with the distance along the passage. At certain locations in a restricted passage, the size and / or spacing of blunt or non-blunt obstacles may be reduced to a level that provides maximum flow restriction or barrier.

[0181] Fig.10 Displays the corresponding Figure 2 Magnified top view of the channel area. Fig.10 Posts 1001 are shown as circles. Dashed arrows show the flow of a biological fluid through the channel. This view shows a channel with a region of blunt post obstacles 1001, which can create turbulent flow.

[0182] Fig.11 Displays the corresponding Figure 2 An enlarged top view of the outlet reservoir 1107. Fig.11 Posts 1101, 1103 and 1105 of various sizes are shown. Dashed arrows show the flow of biological fluid through the channel. In this embodiment, each outer limiting channel includes a barrier 1102 formed by very small and closely spaced posts.

[0183] In further embodiments, various arrangements of blunt or non-blunt obstacles or posts in the restricted passage can be used to restrict flow to any level. A wide range of blunt and / or non-blunt obstacle spacing and / or patterns can be used in the restricted passage. The fluid can have a tortuous path in the restricted flow passage. The spacing of obstacles in the restricted passage and / or the tortuosity of the fluid path can increase in the flow direction along the distance of the passage.

[0184] Fluid outflow from the channels of the microfluidic chip of the present invention can be collected in an outlet reservoir at the outlet end of the channel. Fluid inflow or insertion into the channels of the microfluidic chip of the present invention can be achieved through a reservoir at the inlet end of the channel.

[0185] Fig.12 Displays the corresponding Figure 2 An enlarged top view of the inlet reservoir 1201. Fig.12 Various sizes of posts 1203 are shown. The outer confinement channel 1207 includes posts of different sizes and spacing. The uniform flow channel 1205 includes posts of uniform size and spacing. The dashed arrows show the direction of biological fluid flow through the outer channel.

[0186] Fig.13 A top view of a microfluidic chip in an embodiment of the device of the present invention is shown. Three microfluidic inserts are shown. The dashed arrows show the flow direction of the biological fluid.

[0187] Fig.14 A perspective view of one embodiment of a microfluidic channel device of the present invention is shown having blunt post obstacles 1401 that restrict flow. Fig.14 yes Fig.15 The dashed arrows show the flow direction of the biofluid.

[0188] Fig.15 A perspective view showing one embodiment of the microfluidic channel device of the present invention. Fig.15 Displays the corresponding Figure 2 View of the channel area. Fig.15 The pillar obstacles 1501 of different spacing in the restricting channel are shown. In this embodiment, the restricting channel can have pillar obstacles 1501, which are organized into bands of different spacing between pillars. The dotted arrows show the flow direction of the biological fluid. The continuous uniform flow channel 1517 can be arranged separately from the restricting channel 1515.

[0189] Fig.16 An elevational side view of an embodiment of a microfluidic chip of the present invention is shown. An inlet reservoir 1605 is in fluid communication with a fluid line 1601 for introducing biological fluid and / or other fluids into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and a hole 1604 defined in a glass cover slip. The biological fluid passes through the inlet reservoir 1605 to a microfluidic channel 1606. The dashed arrows show the direction of flow of the biological fluid.

[0190] Fig.17 Displays the corresponding Figure 2 The entrance area and Fig.16 1602. The dotted arrows show the direction of flow of the biological fluid.

[0191] Fig.18 An elevational side view of an embodiment of a microfluidic chip 1614 of the present invention is shown. An inlet reservoir is in fluid communication with a fluid line 1601 for introducing a biological fluid into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and an aperture 1604 defined in a glass cover slip 1613. The biological fluid passes through the inlet reservoir to a microfluidic channel 1606 and flows to an outlet reservoir 1607. A probe adjuster 1612 may be provided to adjust the height of the probe 1602 to form a good seal with the probe adapter 1603 and the aperture 1604. The dashed arrows show the direction of flow of the biological fluid.

[0192] Fig.19 Displays the corresponding Figure 2 Magnified top view of the channel area. Fig.19Pillars 1701 are shown as circles. For this embodiment, some representative lengths of the pillar band region in the channel are shown in microns.

[0193] Fig. 20 Displays the corresponding Figure 2 Micrograph of a magnified top view of the channel area. Fig. 20 The pillars are shown as dots. For this embodiment, some representative spacings of the pillars in the bands in the channels are shown in microns. The dashed arrows show the direction of flow of the biological fluid.

[0194] Fig.21 Displays the corresponding Figure 2 A top view of one embodiment of a microfluidic device. Fig.21 It is shown that a biological fluid can be introduced into the inlet region reservoir 2202 using a delivery probe 2201. The dashed arrow shows the direction of the biological fluid flowing to the outlet reservoir region 2203. The enlarged view of this embodiment shows some representative spacing of the center posts in the channel in microns. For this embodiment, the dotted lines in the enlarged view show the possible tortuous path of the biological fluid between obstacles.

[0195] Fig. 22 An embodiment of the microfluidic system of the present invention is shown. Processor 102 can send control signals and / or receive signals from fluid drive unit 101, and fluid drive unit 101 provides driving fluid, such as compressed gas, to fluid source unit 103. Fluid source unit 103 can contain fluid of interest, biological fluid, carrier and / or reagent. Fluid of interest, biological fluid, carrier and / or reagent can flow to sensor unit 105, which can monitor the flow rate and / or pressure of fluid. Fluid of interest, biological fluid, carrier and / or reagent can flow to on-chip unit 107, which can include microfluidic device of the present invention. Fluid of interest, biological fluid, carrier and / or reagent can enter the inlet reservoir of the microfluidic chip of the present invention in on-chip unit 107. Fluid of interest, biological fluid, carrier and / or reagent can arrive at the outlet reservoir of the microfluidic chip of the present invention in on-chip unit 107 and flow to off-chip unit 109. Processor 102 can receive data from sensor unit 105 and record flow and / or pressure. On-chip unit 107 may include analytical tools for spectrometry, such as irradiation and photodetectors. Off-chip unit 109 may include various analytical tools, such as microscopy tools, imagers and analyzers, chromatographs, mass spectrometers, and / or magnetic resonance analyzers. Processor 102 may send control signals and / or receive data from on-chip unit 107 and off-chip unit 109.

[0196] In some aspects, the fluid composition in the system or device of the present invention can be analyzed by various techniques. For example, the fluid composition can be analyzed by imaging technology.

[0197] Examples of imaging techniques include electron microscopy, stereomicroscopy, widefield microscopy, polarizing microscopy, phase contrast microscopy, multiphoton microscopy, differential interference contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, x-ray microscopy, and ultrasound microscopy.

[0198] Examples of imaging techniques include positron emission tomography, computed tomography, and magnetic resonance imaging.

[0199] Examples of assay techniques include colorimetry, chemiluminescence, spectrophotometry, immunofluorescence, and light scattering.

[0200] In some embodiments, the present invention can provide a device for measuring the pressure and flow rate of a fluid composition. In certain embodiments, the device can have a mesh structure composition that stays in the channel to provide flow resistance. The mesh structure composition can have any one or more of the uveal mesh structure, the corneal scleral mesh structure, and the tubular parametrial mesh structure. For example, these mesh structures can be simulated with obstacles in the restricted channel, or provided from the extraction of ocular fluids, body fluids, or clinical samples.

[0201] The extracellular matrix bodies or complexes used in the mesh structure composition can be composed of various biomolecules or complex particles and can range in diameter from about 0.5 to about 5,000, or 0.5 to 1,000, or 1 to 200, or 1 to 100, or 1 to 50, or 1 to 25, or 1 to 10, or 1 to 5 microns.

[0202] Extracellular matrix bodies or complexes that can be separated in the devices of the invention have diameters ranging from about 0.5 to about 5,000, or 0.5 to 1,000, or 2 to 700, or 1 to 200, or 1 to 100, or 1 to 50, or 1 to 25, or 1 to 10, or 1 to 5 microns.

[0203] In some embodiments, the channel may include obstacles such as glass beads, microbeads, magnetic beads, gel particles, dextran particles, or polymer particles. The obstacles may also be composed of glass fibers, polymer fibers, inorganic fibers, organic fibers, or metal fibers.

[0204] In other embodiments, the device or channel of the present invention may include a binding agent, an affinity detector or an immunogenic agent attached to a device element that is fluidically connected to the sample fluid. The device may have reagents for internal capture and / or detection of biomolecules from the sample. In certain embodiments, the device may have reagents for internal capture and / or detection of biomarkers of the sample fluid.

[0205] In certain embodiments, the uveal meshwork or restrictive channel may have fenestrations of about 25 microns. The corneoscleral meshwork or restrictive channel may have fenestrations of about 2-15 microns. The paratubular meshwork or restrictive channel may have fenestrations of about 1 to 4 microns or less.

[0206] The device may further include a fluid reservoir for containing a fluid composition, such that the fluid reservoir is in fluid communication with the inlet of the channel to introduce the fluid composition into the inlet of the channel.

[0207] The device of the present disclosure may have a drive source or pressure source for applying pressure to the drive fluid composition.The drive fluid may enter the fluid reservoir to drive the fluid composition into the inlet of the microfluidic channel.

[0208] The device of the present invention may have a sensor unit in fluid communication with the fluid composition for measuring the flow rate and pressure of the fluid composition at the inlet of the channel and transmitting the flow rate and pressure to the processor.

[0209] Signals and data from system device units can be received by the processor. The processor can display flow rate and pressure. The memory or medium can store instructions or files, such as machine-readable storage media. The machine-readable storage medium can be non-transitory.

[0210] The processor of the present disclosure may be a general-purpose or special-purpose computer. The processor may execute instructions stored in a machine-readable storage device or medium. The processor may include an integrated circuit chip, a microprocessor, a controller, a digital signal processor, any of which may be used to receive and / or transmit data and execute stored instructions. The processor may also perform calculations and conversions of data, and / or store data in a memory, medium, or file. The processor may receive and execute instructions, which may include one or more steps of executing the method of the present invention. The apparatus of the present invention may include one or more non-transient machine-readable storage media, one or more processors, one or more memory devices, and / or one or more user interfaces. The processor may have an integrated display for displaying data or converting data.

[0211] In some aspects, the disclosed systems can have a device with microfluidic channels. One or more channels can be arranged in a microfluidic chip.

[0212] The system of the present disclosure may include an on-chip unit having one or more detectors for analyzing the composition of the fluid within the channel or at the entrance to the channel or exiting the exit. The detector may also be arranged to detect the composition of the fluid within the channel.

[0213] The systems of the present disclosure may include an off-chip unit having one or more detectors for analyzing a fluid composition extracted from a microfluidic channel.

[0214] In certain embodiments, the extracellular matrix bodies or complexes of the network structures compositions used in the systems or devices of the present disclosure may include fixatives, stabilizing components, or cross-linking components that can transform the structure into a stable, homogeneous composition.

[0215] Examples of stabilizing components include fixatives described herein, cross-linking compounds described herein, organic solvents, polypeptides, and pharmaceutically acceptable organic salts.

[0216] The cross-linked extracellular matrix bodies or complexes may be reversibly cross-linked or irreversibly cross-linked.

[0217] In some embodiments, the devices of the present invention may include an extracellular matrix body or complex as a network composition that can be used to identify or screen for active agents. The network composition may include a drug delivery excipient.

[0218] In other embodiments, device of the present invention can be used for measuring the quantity or level of extracellular matrix body or complex in the test sample.Measurement of the quantity or level of extracellular matrix body or complex in the test sample can provide diagnostic marker level for the test sample.Device of the present invention can be used for identifying glaucoma or early glaucoma of experimenter.

[0219] In further embodiments, the device of the present invention can be used to measure pressure related to the amount or level of extracellular matrix bodies or complexes in a test sample.The pressure value in the channel can be directly related to the amount or level of extracellular matrix bodies or complexes in a test sample.

[0220] In certain embodiments, the devices of the invention can be used to measure a measure that can be related to the amount or level of an extracellular matrix body or complex in a test sample.The measure of the composition in a channel can be directly related to the amount or level of an extracellular matrix body or complex in a test sample.

[0221] Examples of assays include colorimetric assays, chemiluminescent assays, spectrophotometric assays, immunoassays, or light scattering assays.

[0222] Apparatus for analyzing samples in a microfluidic device includes analytical tools such as radiation sources and photodetectors for spectrometry and spectroscopy, as well as immunolabeling and detection, as further shown in the Examples herein.

[0223] Apparatus for analyzing samples in a microfluidic device includes imaging tools, such as an irradiation source and microscopy, as further shown in the Examples herein.

[0224] Compositions and methods of extraction

[0225] In some embodiments, a composition may include a fraction of a biological sample extracted from a microfluidic device.

[0226] In certain embodiments, compositions extracted from a microfluidic device may be used to treat the human or animal body.

[0227] In additional embodiments, the composition extracted from the microfluidic device can be used for diagnosis or prognosis of a subject.

[0228] The composition of isolated and / or extracted extracellular matrix bodies can be combined with a pharmaceutical carrier and one or more pharmaceutical excipients.

[0229] The morphology of the isolated and / or extracted extracellular matrix bodies may be modified by the isolation and / or extraction process.

[0230] The morphology of the isolated and / or extracted extracellular matrix bodies may be chemically modified.

[0231] In some embodiments, the composition of the extracellular matrix can be isolated and / or extracted for use in treating the human or animal body.

[0232] In further embodiments, compositions can comprise sample, has removed the extracellular matrix body therefrom by separation and / or extraction process, for the treatment of human body or animal body.In certain embodiments, at least 25% or at least 50% or at least 75% or at least 90% extracellular matrix body of sample has been removed by separation and / or extraction process, for the treatment of human body or animal body.

[0233] In some aspects, extracting a composition from a microfluidic device can be a method for preparing a biological sample for diagnosis or prognosis of a subject.

[0234] In certain embodiments, the method of isolating extracellular matrix bodies can be performed by ultrafiltration or centrifugation or by a microfluidic device of the present disclosure.

[0235] Embodiments of the present invention further include immobilizing the extracellular matrix bodies on the glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking.

[0236] All publications cited in this specification, including patents, patent application publications, and non-patent publications, are each expressly incorporated herein by reference in their entirety for all purposes.

[0237] Although the foregoing disclosure has been described in detail by way of example for the purpose of clear understanding, it is apparent to those skilled in the art that certain changes and modifications are encompassed within the present disclosure and may be implemented without undue experimentation within the scope of the appended claims, which are presented by way of illustration and not limitation. The present invention includes all such additional embodiments, equivalents, and modifications. The present invention includes any combination or mixture of features, materials, elements, or limitations of the various exemplary components, embodiments, and claimed embodiments.

[0238] The terms “a”, “an”, “the” and similar terms in describing the present invention and in the claims should be construed to include both the singular and the plural. Example

[0239] Example 1. Pressure and flow measurements in a microfluidic device using disease-related biological fluids. Isolation of extracellular matrix bodies in a microfluidic device. Fig.23 showed that aqueous humor from primary open-angle glaucoma patients increased the pressure in the microfluidic device. Fig.23 Figure 2 shows the change in relative pressure (mm Hg) within the microfluidic model trabecular meshwork when injected with human aqueous humor obtained from a patient with severe primary open-angle glaucoma. The microfluidic channel flow rate was kept constant at 2 μl per minute, and the baseline system pressure was measured using an external pressure sensor. Human aqueous humor samples were injected at the time points indicated by the arrows and the letter "a". The pressure rose steadily to a maximum of approximately 41 mm Hg at 27 minutes. Fig.23 showed that aqueous humor from patients diagnosed with POAG glaucoma increased the pressure in the device.

[0240] Example 2. Isolation of disease-associated extracellular matrix bodies in a microfluidic device. Fig.24 (Figure above) shows the Fig.23 Confocal micrograph of the microfluidic chip after isolation of EMBs from human aqueous humor of a primary open-angle glaucoma patient at the end of the experiment shown. Fig.24 (Top) Shows protein content in aqueous humor labeled with the fluorescent marker carboxyfluorescein succinimidyl ester (arrows). Circles are pillars in the confinement channel. Fig.24 (Bottom) shows isolated EMBs trapped between pillars in a microfluidic channel (arrows).

[0241] Example 3. Separation of extracellular matrix bodies from biological fluids using a size exclusion filter. This example demonstrates that EMBs can be separated by size exclusion. EMBs can be separated by size exclusion and distinguished from smaller particles (e.g., free extracellular vesicles or other small vesicles).

[0242] Fig.25Illustration of the separation of extracellular matrix bodies from biological fluids using size exclusion filters. Bovine vitreous humor was first filtered through a 5 μm cellulose acetate syringe filter, then a 1 μm needle tip filter, then a 0.45 um needle tip filter, and then a 0.22 μm filter. Each fraction was characterized using wide field microscopy.

[0243] Fig.26 Representative wide-field microscopy images showing separation of EMBs by size exclusion filters. Fig.26 a and Fig.26 b shows the presence of extracellular matrix bodies in natural biofluids of bovine vitreous humor. To separate and recover extracellular matrix bodies from complex biofluids, a series of syringe-based filtrations were performed using cellulose filters with pore sizes ranging from 5 μm to 0.22 μm. The extracellular matrix bodies were fixed on a slide with EDC and stained with Alcian blue stain. Fig.26 c shows the bovine vitreous fraction separated by filtration through a 5 μm needle tip filter train. Fig.26 d shows the bovine vitreous fraction separated by filtration through a 1 μm needle tip filter train. Fig.26 e shows the bovine vitreous fraction separated by filtration through a 0.45 μm needle tip filter train. Fig.26 f shows bovine vitreous fractions separated by filtration through a series of 0.22 μm needle tip filters. The image shows a relative reduction in larger ECM bodies as the filtrate series is passed through smaller filter sizes.

[0244] This example shows that EMB can be isolated by size exclusion. Analysis of the isolated EMB shows that it is composed of DNA, RNA and proteins as well as hyaluronic acid or collagen, which are components of the extracellular matrix.

[0245] The vitreous humor is a highly hydrated tissue, with a water content between 98-99.7%, and is primarily composed of an extracellular matrix. The major component of the extracellular matrix is ​​the protein collagen. Collagen is modified with carbohydrates and, once released from the cell, assembles into collagen fibrils. Extracellular matrix bodies can be attached to the fibrils, as well as to other locations in the extracellular matrix.

[0246] Bovine eyes were dissected to remove orbital fat and extraocular muscles attached to the eyeball. The eyeball was rinsed with 5 ml of ice-cold Tris-buffered saline (TBS) (pH 8.0) containing 50 mM Tris-HCl, 150 mM NaCl at 4°C for 1 minute. The vitreous was separated by making a sclerotomy incision 4 mm or 8 mm posterior to the limbus using a 16 g needle and then making a circumferential sagittal incision with scissors to separate the eyeball into anterior and posterior cups. Scissors were used to cut and remove the formed vitreous and to sever adhesions between the vitreous and ocular structures. Tissue samples were rinsed with TBS (pH 8.0) at 4°C for 1 minute. Vitreous specimens were collected in 15 mL centrifuge tubes and homogenized using an immersion blender. Aliquots of the homogenized bovine vitreous humor (BVH) were transferred to 1 mL centrifuge tubes. Bovine vitreous was resuspended in TBS buffer for further studies and frozen at -80°C until use.

[0247] An aliquot of homogenized bovine vitreous humor diluted to 1 mL with buffered saline was loaded into a 1 mL syringe using a 22 gauge needle. The needle was replaced with a 5 μm cellulose acetate syringe filter and the bovine vitreous humor was squeezed through the filter by applying uniform downward pressure. The filtrate was collected into a new 1 mL tube and an aliquot of 80 μL of the filtrate was saved for imaging. Then, following the same steps above, the filtrate collected from the 5 μm filtration was loaded into a 1 μm needle tip filter and an aliquot was saved for imaging. Next, following the same procedure, the filtrate from the 1 μm filter was squeezed through a 0.45um needle tip filter and an aliquot was saved for imaging. Finally, the filtrate from the 0.45 μm filter was squeezed through a 0.22 μm filter. The filtrate was recovered from each filtration step.

[0248] Use wide field microscope to visualize the components of each fraction.By placing biological fluid on a slide, cross-linking sample with EDC, and dyeing the material containing hyaluronic acid with Alcian blue, each sample is imaged.For the dyeing of sample, each filtrate is incubated with 1% Alcian blue (Sigma 1% Alcian blue in 3% acetic acid pH2.5 B8483) at room temperature with a ratio of 1: 1v / v for 30 minutes.After incubation, 40 μL dyeing filtrate is placed on a slide and covered with a cover glass, and imaged using bright field microscopy.Color bright field images are captured on an inverted phase contrast microscope (ZiessAxiovert 200) equipped with an Axiocam105 color camera (Zeiss), and images are processed using Zen software (Zeiss, version 4.3).

[0249] Embodiment 4. uses sequence centrifugation to separate extracellular matrix body from biological fluid.This embodiment shows that EMB can be separated by centrifugation.EMB can be separated by centrifugation and distinguished from smaller particles (such as free exosomes or other small vesicles).In order to obtain cell-free vitreous sample, first clarify vitreous by a series of low-speed centrifugation.

[0250] Fig. 27 The method of using centrifugation to separate extracellular matrix bodies from biological fluids is illustrated. Four sediments 9705, 9713, 9721 and 9729 are obtained by sequential centrifugation. Bovine vitreous body is resuspended 9701 and placed in 1ml tube and centrifuged (Sorvall Legend RT) at 350g for 10 minutes at 4°C to form sediment 1 9705. The supernatant of 50 μl aliquots is preserved for analysis and is marked as supernatant 1 9709, and the remaining supernatant is transferred to a new tube and formed sediment 2 9713 at 4°C with 2000g centrifugation (Eppendorf, 5417R series, F45-30-11Eppendorf rotor) for 10 minutes. The supernatant of 50 μl aliquots is preserved for analysis and is marked as supernatant 2 9717, and the remaining supernatant is transferred to a new tube. The supernatant was then centrifuged at 10,000 g for 10 minutes at 4°C to form precipitate 3 9721. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 3 9725, and the remaining supernatant was transferred to a new tube. The supernatant was then centrifuged at 20,000 g for 10 minutes at 4°C to give precipitate 4 9729. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 4, and the remaining supernatant was transferred to a new tube.

[0251] Fig.28 Representative transmission electron microscopy (TEM) images of EMBs isolated by serial centrifugation are shown. Fig.28 a shows the extracellular matrix present in bovine vitreous humor. Fig.28 In b, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 450 g shows that extracellular matrix bodies are present in the pellet fraction. Fig.28 In c, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 2,000 g shows that extracellular matrix bodies are present in the pellet fraction. Fig.28 In d, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 10,000 g shows that extracellular matrix bodies are present in the pellet fraction. Fig. 27 In e, representative TEM micrographs of samples collected from the pellet after centrifugation at 20,000 g show that extracellular matrix bodies are present in the pellet fraction.

[0252] Example 5. Dose-response in a microfluidic device for testing the intraocular pressure-lowering activity of agents in a glaucoma model. In order to use bivalirudin TFA as an active agent for treating glaucoma, the dose-response behavior of bivalirudin TFA on intraocular pressure (IOP) was determined. Bivalirudin TFA exhibited an EC50 of 1.2 nM for treating bovine vitreous humor.

[0253] The compound Bivalirudin TFA was tested in bovine vitreous humor (BVH) in a microfluidic chip device. A 25% solution of homogenized BVH in PBS buffer was prepared and diluted with an equal amount of compound solution to a total BVH concentration of 12.5%. The samples were vortexed and incubated at 37°C for 1 hour. PBS buffer or PBS containing 10% ethanol or DMSO was used as a control and incubated with BVH under the same conditions.

[0254] The test compound-BVH solution was introduced into the reservoir of the microfluidic chip device and the flow rate and pressure change were recorded. The effect of various concentrations of the compound on the treatment of bovine vitreous humor was tested. 7ul of each test solution was injected into the microfluidic chip through the injector. After the sample was injected, the flow rate and pressure change were continued to be recorded for 50 minutes. The relative change of chip pressure during the whole experiment was obtained.

[0255] Fig.29 Dose-dependent response curves for treating bovine vitreous humor with the compound bivalirudin TFA are shown. The EC50 value is taken as the point at which the logarithmic function of the micromolar concentration of the compound on the x-axis produces a half-maximal response. The logarithmic function of the micromolar concentration of the drug is plotted on the x-axis, and the percentage of the maximum response is plotted on the y-axis. The maximum response is obtained by taking the value of the response to the highest drug concentration. The response is calculated by taking the absolute difference between the control value and the test value for each concentration.

[0256] Example 6. Dose-response in a microfluidic device for testing the intraocular pressure-lowering activity of an agent in a glaucoma model. In order to use colistin sulfate as an active agent for treating glaucoma, the dose-response behavior of colistin sulfate on intraocular pressure (IOP) was determined. Colistin sulfate exhibited an EC50 of 0.36 nM for treating bovine vitreous humor.

[0257] The compound colistin sulfate was tested in bovine vitreous humor (BVH) in a microfluidic chip device. A 25% solution of homogenized BVH in PBS buffer was prepared and diluted with an equal amount of compound solution to a total BVH concentration of 12.5%. The samples were vortexed and incubated at 37°C for 1 hour. PBS buffer or PBS containing 10% ethanol or DMSO was used as a control and incubated with BVH under the same conditions.

[0258] The test compound-BVH solution was introduced into the reservoir of the microfluidic chip device and the flow rate and pressure change were recorded. The effect of various concentrations of the compound on the treatment of bovine vitreous humor was tested. 7ul of each test solution was injected into the microfluidic chip through the injector. After the sample was injected, the flow rate and pressure change were recorded for another 50 minutes. The relative change of chip pressure during the entire experiment was obtained.

[0259] Fig.30 Dose-dependent response curves for the treatment of a bovine vitreous glaucoma model with the compound colistin sulfate are shown. The EC50 value is taken as the point on the x-axis where the logarithmic function of the micromolar concentration of the compound produces a half-maximal response. The logarithmic function of the micromolar concentration of the drug is plotted on the x-axis and the percentage of the maximum response is plotted on the y-axis. The maximum response is obtained by taking the value of the response to the highest drug concentration. The response is calculated by taking the absolute difference between the control value and the test value for each concentration.

[0260] Example 7. Dose-response in a microfluidic device for testing the activity of an agent in reducing intraocular pressure in a glaucoma model. In order to use polymyxin B sulfate as an active agent for treating glaucoma, the dose-response behavior of polymyxin B sulfate on intraocular pressure (IOP) was determined. Polymyxin B sulfate showed an EC50 of 4.3 nM for treating a bovine vitreous humor glaucoma model.

[0261] The compound polymyxin B sulfate was tested in a bovine vitreous humor (BVH) glaucoma model in a microfluidic chip device. A 25% solution of homogenized BVH in PBS buffer was prepared and diluted with an equal amount of compound solution to a total BVH concentration of 12.5%. The samples were vortexed and incubated at 37°C for 1 hour. PBS buffer or PBS containing 10% ethanol or DMSO was used as a control and incubated with BVH under the same conditions.

[0262] The test compound-BVH solution was introduced into the reservoir of the microfluidic chip device and the flow rate and pressure change were recorded. The effect of various concentrations of the compound on the treatment of bovine vitreous humor was tested. 7ul of each test solution was injected into the microfluidic chip through the injector. After the sample was injected, the flow rate and pressure change were recorded for another 50 minutes. The relative change of chip pressure during the entire experiment was obtained.

[0263] Fig.31 Dose-dependent response curves for the treatment of a bovine vitreous glaucoma model with the compound polymyxin B sulfate are shown. The EC50 value is taken as the point on the x-axis where the logarithmic function of the micromolar concentration of the compound produces a half-maximal response. The logarithmic function of the micromolar concentration of the drug is plotted on the x-axis and the percentage of the maximum response is plotted on the y-axis. The maximum response is obtained by taking the response to the highest drug concentration. The response is calculated by taking the absolute difference between the control value and the test value for each concentration.

[0264] Example 8. Isolation and extraction of extracellular matrix bodies in a microfluidic device. A microfluidic device was used to separate bovine vitreous extracellular matrix bodies. After separation, the extracellular matrix bodies were extracted.

[0265] Fig.32 The separation of extracellular matrix bodies in a confining channel of a microfluidic device of the present disclosure is shown. Fig.32 a and Fig.32 a shows a representative micrograph of a microfluidic chip perfused with bovine vitreous humor. The letter "p" marks a post in the channel. After perfusion, extracellular matrix bodies were isolated between and around the posts. Fig.32 c and Fig.32 D shows the representative microphotograph of the channel after the extraction of the extracellular matrix body. The extracellular matrix body is extracted from the chip using a gentle detergent 1% sodium dodecyl sulfate (SDS) and a reverse flow outward from the inlet port. The extracellular matrix body is removed from the chip, and it shows significantly less extracellular matrix body after extraction.

[0266] Example 9. Detection of biomarkers for extracellular matrix bodies by proteomic profiling. Bovine vitreous extracellular matrix bodies were isolated and their proteomic profiles were analyzed off-chip using LC / MS.

[0267] Fig.33 Shown is an on-chip proteomic analysis of bovine extracellular matrix bodies by LC / MS. Biomarkers specific to extracellular matrix bodies were detected.

[0268] The concentrated bovine vitreous extracellular matrix aggregates were resuspended in 50 μl 1% sodium dodecyl sulfate (SDS, Sigma), and were centrifuged at room temperature for 10 minutes at 25 Kg and precipitated again. The precipitate was dissolved in 20 μl of 2X SDS, 50 mM dithiothreitol (DTT) reducing agent, ultrasonically treated for 10 minutes and incubated at 95 ° C for 5 minutes. The precipitate and supernatant were electrophoresed to NuPAGE 10% Bis-Tris gel (1.5 mmX10 holes, Invitrogen). The gel was stained with Coomassie Brilliant Blue R250. The gel was captured and stored for further analysis.

[0269] Each gel band was reduced with 10 mM DTT at 60°C for 30 minutes and alkylated with 20 mM iodoacetamide at room temperature in the dark for 45 minutes. The samples were digested with 0.2 μg trypsin (sequencing grade, Thermo Scientific Cat#90058) and incubated at 37°C for 16 hours. The peptides were extracted twice with 5% formic acid, 60% acetonitrile and dried under vacuum.

[0270] Samples were analyzed by LC-MS using a Nano LC-MS / MS (Dionex Ultimate 3000RLS Canon system, Thermofisher) interfaced with Eclipse (ThermoFisher). 3 μl of the sample precipitate from the 12.5 μl in-gel digestion was loaded onto a fused silica trapping column (Acclaim PepMap 100, 75umx2cm, ThermoFisher). After washing with 0.1% trifluoroacetic acid (TFA) at a speed of 5 μl / min for 5 minutes, the trapping column was connected to an analytical column (NanoeaseMZ peptide BEH C18, 130A, 1.7 μm, 75 μm x 250mm, Waters) for LC-MS / MS. The peptides were fractionated at 300 nL / min using a stepwise linear gradient: 4-15% B in 30 min (where A: 0.2% formic acid, and B: 0.16% formic acid, 80% acetonitrile), 15-25% B in 40 min, 25-50% B in 44 min, and 50-90% B in 11 min. Solution B was then returned to 4% for 5 min for the next run.

[0271] The scan sequence started with the MS1 spectrum (Orbitrap analysis, resolution 120,000, scan range from m / z 375–1500, automatic gain control (AGC) target 1E6, maximum injection time 100 ms). A top S (3 s) duty cycle scheme was used to determine the number of MSMS performed per cycle. Precursor ions with charges 2–7 were selected for MSMS, and a 60 s dynamic exclusion was used to avoid duplicate sampling. Precursor ion masses were isolated in the quadrupole with an isolation window of 1.2 m / z and an automatic gain control (AGC) target of 1E5 and fragmented by high-energy collisional dissociation at 30% normalized collision energy. These fragments were scanned in the Orbitrap at a resolution of 15,000. The MSMS scan range was determined by the charge state of the precursor ion, but the lower limit was set to 110 amu.

[0272] Selected extracellular matrix-related proteins expressed in the vitreous bovine extracellular matrix body fraction were identified by proteomic profiling and are shown in Table 1 .

[0273] Table 1: Extracellular matrix proteins in the vitreous bovine extracellular matrix

[0274]

[0275]

[0276] The vitreous fraction was obtained by low speed centrifugation and separation using a microfluidic device. Higher spectral count values ​​represent greater amounts of protein.

[0277] Selected proteins known to be involved in protein aggregation found in the vitreous bovine extracellular matrix bulk fraction were identified by proteomic profiling and are shown in Table 2 .

[0278] Table 2: Protein aggregates in the bovine vitreous extracellular matrix

[0279]

[0280] Vitreous ECM aggregate fractions were obtained by low speed centrifugation and separation using a prototype microfluidic device. The proteins were classified by function, and the highlighted proteins are known to play a role in the extracellular matrix. For example, the spectral counts of complement C3 (spectral count, 408), α-enolase (spectral count, 151), and clusterin (spectral count, 74) were found to be relatively high.

[0281] Example 10. Isolation and extraction of extracellular matrix bodies in a microfluidic device. A microfluidic device was used to separate bovine vitreous extracellular matrix bodies. After separation, the extracellular matrix bodies were extracted.

[0282] Fig.34 The separation of extracellular matrix bodies and their subsequent extraction in a confining channel of a microfluidic device of the present disclosure is shown. The image scale bar is 50 μm. Fig.34 a shows a representative wide-field micrograph of a microfluidic device perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0 and counterstained with Alcian blue for hyaluronic acid (grey signal, bright field). Fig.34 A shows the signal from the extracellular matrix body (arrow) between the pillars (p) captured in the device. The chip was perfused with biofluid for at least 60 minutes. After perfusion, aggregation was separated between the pillars and was observed with mass-like formation. The material smaller than the extracellular matrix body material has been discharged through the outlet port. Fig.34 b shows the extraction of extracellular matrix bodies from a confining channel of a microfluidic device. The device was perfused with a mild detergent, 0.1% sodium dodecyl sulfate (SDS), and the flow direction was reversed from the outlet to the inlet. Fig.34 b shows that significantly fewer ECM bodies are present in the channel after elution and indicates that the ECM bodies have been extracted. Fig.34 c shows a higher magnification image of extracellular matrix bodies (arrows) trapped between pillars (p) after perfusion. Fig.34 c shows extraction with detergent and reverse flow, again showing significantly fewer ECM bodies in the channel after extraction.

[0283] Example 11. Isolation and extraction of extracellular matrix bodies in a microfluidic device This experiment demonstrated that on-chip staining can be used to detect the isolation of extracellular matrix bodies.

[0284] Fig.35 Shown in the device channel of the present disclosure on-chip immunohistochemical staining of extracellular matrix body.Microfluidic device is injected with the fluid containing the homogenized bovine vitreous body suspended in the biological fluid.After the fluid is perfused into the device, the fluid flows through the inlet and flows out via the outlet.Larger extracellular matrix body (arrow) is captured between the pillars (labeled as Lp).The chip is perfused with a blocking solution to prevent the non-specific antibody binding before the antibody staining.Next, by injecting an anti-fibronectin primary antibody, the sample is incubated for 2 hours and washed to label the protein fibronectin, which is a known extracellular matrix component and integrin binding protein.Then, goat anti-rabbit FITC secondary antibodies are incubated for 1 hour and washed.Then under wide field fluorescence and bright field microscopy, the microfluidic chip is imaged. Fig.35 Representative wide-field fluorescence micrographs are shown. This image shows the extracellular matrix body in the microfluidic channel. The distance between the large pillars (Lp) is about 100 μm. The dot-shaped signals in the extracellular matrix body represent fibronectin staining (anti-fibronectin Ab, goat anti-rabbit secondary antibodies with Alexa488, FITC, white signals).

[0285] Fig.36 On-chip immunohistochemical staining showing extracellular matrix bodies in the channels of the device of the present disclosure. Fig.36 a Representative photomicrograph bright field image showing stained extracellular matrix bodies (arrows) in the channel. Image scale bar is 20 μm. The control image has no fluorescent signal, which indicates that Fig.36 The signal in a is specific for fibronectin. Fig.36 b shows the stained extracellular matrix bodies (arrows) in the channel again. The scale bar of the image is 50 μm. Again, the control image has no fluorescent signal, which indicates that Fig.36 The signal in b is specific for fibronectin.

[0286] Example 12. Isolation and extraction of extracellular matrix bodies in a microfluidic device This experiment demonstrated that on-chip staining can be used to detect the isolation of extracellular matrix bodies.

[0287] Fig.37 On-chip immunohistochemical staining showing extracellular matrix bodies in the channels of the device of the present disclosure. Fig.37Shown in the biological fluid containing extracellular matrix body, the representative microphotograph of the on-chip immunohistochemical staining of beadlecan protein, which is a component of the extracellular matrix of cartilage.The microfluidic chip is injected with the fluid containing the homogenized bovine vitreous suspended in the biological fluid.After the biological fluid is poured into the device, the sample flows through the inlet and flows out through the outlet.The larger extracellular matrix body (arrow) is captured between the pillars (marked as "p").The chip is perfused with a blocking solution to prevent the non-specific antibody binding before the antibody staining.Beadlecan is marked by injecting an anti-beadlecan primary antibody, incubating the sample for 2 hours and washing.Then, goat anti-rabbit TRITC secondary antibodies are incubated for 1 hour and washed.Then the microfluidic chip is imaged under wide field fluorescence and bright field microscopy. Fig.37 The extracellular matrix bodies in the microfluidic channel between the pillars (p) are shown. The punctate signals represent beaded proteoglycan staining (white signal). The control image has no fluorescent signal, which indicates that Fig.37 The signal in is specific for globulin.

[0288] Fig.38 On-chip immunohistochemical staining showing extracellular matrix bodies in the channels of the device of the present disclosure. Fig.38 a Representative photomicrograph bright field image showing on-chip immunohistochemical staining of beaded proteoglycan. The control image showed no fluorescent signal, indicating Fig.38 The signal in a is specific for globulin. Image scale bar is 10 μm. Fig.38 b Representative photomicrographs showing bright field images of on-chip immunohistochemical staining of beadlecan. The control images showed no fluorescent signal, indicating that Fig.38 The signal in c is specific for globulin. Image scale bar is 10 μm.

[0289] Example 13. Ex-chip analysis of extracellular matrix bodies extracted from a microfluidic device. This experiment demonstrates ex-chip analysis of extracellular matrix bodies as can be extracted from a channel of a device of the present disclosure.

[0290] Fig.39 showed that extracellular matrix bodies could be visualized on glass surfaces using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking and staining of hyaluronic acid with Alcian blue dye. Fig.39 Signals for the ECM bodies were shown (dark staining), indicating that EDC cross-linking retained the ECM bodies on the surface.

[0291] Example 14. Ex-chip analysis of extracellular matrix bodies extracted from a microfluidic device. This experiment demonstrates the ex-chip analysis of the extracellular matrix bodies of the present disclosure.

[0292] Fig.40Shown is an off-chip analysis of the extracellular matrix that can be visualized on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking and Sirius red dye to stain collagen. Fig.40 The signal (dark staining) of collagen chains within the ECM bodies is shown, which shows that EDC cross-linking retains the ECM bodies on the surface. This also shows that EMBs can be visualized by collagen staining.

[0293] Example 15. Ex-chip analysis of extracellular matrix bodies extracted from a microfluidic device. This experiment demonstrates ex-chip analysis of extracellular matrix bodies that can be visualized on a glass surface using nucleic acid markers.

[0294] Fig.41 Shown is an off-chip analysis of extracellular matrix bodies that can be visualized on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking and DNA staining using Hoechst dye. Fig.41 Shows DNA signals in the extracellular matrix.

[0295] Example 16. Separation and detection of extracellular matrix bodies in a microfluidic device. This experiment demonstrates the separation and detection of extracellular matrix bodies in a channel of a device of the present disclosure.

[0296] Fig.42 On-chip separation and detection of extracellular matrix bodies are shown. Fig.42 Shown are representative micrographs of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, and counterstained with Alcian blue for hyaluronic acid, dark stain, bright field. Fig.42 Signals from extracellular matrix bodies trapped near the large pillars (circles) of the device are shown. The chip was perfused with biofluid for at least 60 minutes. Cluster formation of extracellular matrix bodies was observed. Image scale bar is 50 μm.

[0297] Example 17. On-chip separation, detection and analysis of extracellular matrix bodies in a microfluidic device. This experiment demonstrates on-chip separation, detection and analysis of extracellular matrix bodies in a channel of a device of the present disclosure.

[0298] Fig.43 Shown are on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Fig.43The representative low-power fluorescence micrograph of the channel after perfusion of bovine vitreous fluid extracellular matrix body is shown, and the bovine vitreous fluid extracellular matrix body is counterstained with carboxyfluorescein succinimidyl ester (CFSE) to protein. The microfluidic chip is injected with the fluid containing homogenized bovine vitreous. After the fluid is perfused into the device, the fluid flows through the inlet and flows out via the outlet. Larger extracellular matrix body is captured near the column (marked as "p"). The image scale is 25 μm.

[0299] Example 18. On-chip separation, detection and analysis of extracellular matrix bodies in a microfluidic device. This experiment demonstrates on-chip separation, detection and analysis of extracellular matrix bodies in a channel of a device of the present disclosure.

[0300] Fig.44 Shown are on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Fig.44 Shown are representative micrographs of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, and counterstained with Sirius red for collagen, dark stain, bright field. Fig.44 a shows the signal from the extracellular matrix bodies between the posts of the device (labeled "p"). The chip was perfused with biological fluid for at least 60 minutes. Fig.44 b shows the same image with a fluorescence filter and demonstrates the detection of collagen with Sirius Red (light grey signal). Image scale bar 50 μm. Fig.44 c and Fig.44 c shows a higher magnification image. The image scale bar is 10 μm.

[0301] Example 19. Isolation, detection and analysis of extracellular matrix bodies using a microfluidic device. This experiment demonstrates the isolation, detection and analysis of extracellular matrix bodies using the device of the present disclosure.

[0302] Fig.45 Show the frequency size distribution of human extracellular matrix bodies present in the human aqueous humor biofluid from health and pre-disease state, suspected glaucoma and pre-glaucoma.Aqueous humor is obtained from 8 patients, healthy samples or pre-glaucoma diagnosis, and the intraocular pressure range is 9 to 25mmHg.Human samples are not processed by centrifugation or other means.The size of the extracellular matrix body is determined by using carbodiimide EDC fixative that the sample is cross-linked to a slide, dyed with uranyl acetate and imaged with wide-field microscopy.Automation program (ImageJ) is used to quantify size in all eight samples.The size (area) range of the extracellular matrix body is about 1.67 μm 2 About 67×10 3 μm 2 . Fig.45 Display 0-200μm 2Counting of extracellular matrix bodies within the range.

[0303] Example 20. Isolation, detection and analysis of extracellular matrix bodies using a microfluidic device. This experiment demonstrates the isolation, detection and analysis of extracellular matrix bodies using the device of the present disclosure.

[0304] Fig.46 Show the frequency size distribution of human extracellular matrix bodies present in the human aqueous humor biofluid from health and pre-disease state, suspected glaucoma and pre-glaucoma.Aqueous humor is obtained from 8 patients, healthy samples or pre-glaucoma diagnosis, and the intraocular pressure range is 9 to 25mmHg.Human samples are not processed by centrifugation or other means.The size of the extracellular matrix body is determined by using carbodiimide EDC fixative that the sample is cross-linked to a slide, dyed with uranyl acetate and imaged with wide-field microscopy.Automation program (ImageJ) is used to quantify size in all eight samples.The size (area) range of the extracellular matrix body is about 1.67 μm 2 About 67×10 3 μm 2 . Fig.46 Shown 201-1000μm 2 Counting of extracellular matrix bodies within the range.

[0305] Example 21. Isolation, detection and analysis of extracellular matrix bodies using a microfluidic device. This experiment demonstrates the isolation, detection and analysis of extracellular matrix bodies using the device of the present disclosure.

[0306] Fig.47 Show the frequency size distribution of human extracellular matrix bodies present in the human aqueous humor biofluid from health and pre-disease state, suspected glaucoma and pre-glaucoma.Aqueous humor is obtained from 8 patients, healthy samples or pre-glaucoma diagnosis, and the intraocular pressure range is 9 to 25mmHg.Human samples are not processed by centrifugation or other means.The size of the extracellular matrix body is determined by using carbodiimide EDC fixative that the sample is cross-linked to a slide, dyed with uranyl acetate and imaged with wide-field microscopy.Automation program (ImageJ) is used to quantify size in all eight samples.The size (area) range of the extracellular matrix body is about 1.67 μm 2 About 67×10 3 μm 2 . Fig.47 Shows 1001-5000μm 2 Counting of extracellular matrix bodies within the range.

[0307] Example 22. Isolation, detection and analysis of extracellular matrix bodies using a microfluidic device. This experiment demonstrates the isolation, detection and analysis of extracellular matrix bodies using the device of the present disclosure.

[0308] Fig.48 The size distribution of bovine vitreous extracellular matrix bodies separated and extracted from the microfluidic device of the present invention is shown. Extracellular matrix bodies in bovine vitreous fluid biofluid after separation and extraction from the microfluidic device of the present invention. The chip was perfused with biofluid for at least 60 minutes. After 60 minutes of perfusion, the ECM bodies were separated near the limiting channel pillars. Next, the chip was treated with a detergent (0.1% sodium dodecyl sulfate, SDS) and samples were extracted from the chip by reverse flow, which caused the extracellular matrix bodies to flow out of the inlet port. Fractions of the eluate were collected at intervals of 10 minutes for a total of 80 minutes. The samples were fixed on a slide and stained with Alcian blue and imaged with wide-field microscopy. The size was quantified using an automated program (ImageJ). The size (area) of the extracellular matrix bodies can reach about 16×10 3 μm 2 . Fig.48 The counts of extracellular matrix bodies in each eluate fraction are shown, which increase over time. This experiment demonstrates that the microfluidic device of the present invention can be used to separate and extract extracellular matrix bodies of various sizes.

[0309] Example 23. On-chip separation, detection and analysis of extracellular matrix bodies in a microfluidic device. This experiment demonstrates off-chip separation, detection and analysis of extracellular matrix bodies.

[0310] Fig.49 Off-chip analysis showing extracellular matrix bodies can be accomplished by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for retention. Fig.49 a shows a representative TEM image of extracellular matrix bodies from native bovine vitreous humor obtained by cross-linking with EDC. Extracellular matrix bodies were observed by fixation with EDC. Fig.49 b shows a similar image taken without EDC cross-linking. In the absence of EDC fixation, no extracellular matrix bodies were observed.

[0311] Embodiment 24. uses microfluidic device to separate and detect extracellular matrix bodies in early pancreatic cancer with human plasma sample.This experiment shows the use of microfluidic device to separate and detect extracellular matrix bodies in early pancreatic cancer with human plasma sample.This experiment shows that the microfluidic device of the present invention can be used to separate and detect extracellular matrix bodies in early pancreatic cancer from human plasma sample.This experiment further shows that extracellular matrix bodies are useful markers for distinguishing early pancreatic ductal adenocarcinoma plasma from healthy controls.

[0312] Fig.50 Shown are extracellular matrix bodies isolated from human plasma samples of patients with early-stage pancreatic ductal adenocarcinoma (PDAC) compared to healthy controls.

[0313] In this experiment, the microfluidic chip was infused with human plasma from early-stage pancreatic ductal adenocarcinoma (PDAC). After infusion into the device, the biofluid flowed through the inlet and exited through the outlet. The results were compared with age-matched healthy controls.

[0314] The chip was perfused with a blocking solution to prevent nonspecific antibody binding before antibody staining. Next, the sample was incubated for 2 hours and washed by injecting an anti-fibronectin primary antibody, and the protein fibronectin (a known extracellular matrix component and integrin binding protein) was marked by on-chip immunohistochemical staining. Then, goat anti-rabbit FITC secondary antibody was incubated for 1 hour and washed. The microfluidic chip was then imaged under a fluorescence microscope.

[0315] Fig.50 a Shows a representative wide-field fluorescence micrograph image of a PDAC sample. Fig.50 a shows the extracellular matrix bodies retained in the microfluidic channel. Fig.50 a further shows that larger extracellular matrix bodies (arrows) stay between the pillars (marked as "p"). The width between the pillars is about 100 μm. The punctate signals from the staying extracellular matrix bodies represent fibronectin staining (anti-fibronectin Ab, goat anti-rabbit secondary Ab with Alexa488, FITC, white signal). Staining shows abundant signals and punctate staining within EMBs ( Fig.50 a, arrow).

[0316] Fig.50 b shows a similarly acquired fluorescence micrograph of an age-matched healthy control human plasma sample. Fig.50 b shows that the amount of fibronectin signal is significantly reduced ( Fig.50 b, gray signal, arrow). When treated under the same conditions, the healthy control signal is much smaller than the disease signal. The image scale bar is 20 μm.

Claims

1. A device for separating fractions of a biological sample, comprising: one or more restricted passages having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the passage; a plurality of spaced-apart obstacles positioned in the restricted passage to provide flow resistance, wherein the spacing between the obstacles decreases in a direction from the inlet end to the outlet end; as well as an inlet reservoir for containing a fluid, wherein the inlet fluid reservoir is in fluid communication with the inlet end of the restrictive passage; One or more uniform flow channels having an inlet end and an outlet end, wherein the one or more uniform flow channels are separate from and parallel to the one or more restricting channels, wherein the inlet end and the outlet end are in fluid communication through the channel, wherein the inlet end is in fluid communication with an inlet reservoir.

2. The device according to claim 1, further comprising: a pressure source for applying pressure to the fluid in the inlet reservoir; A flow sensor is in fluid communication with the inlet reservoir for measuring the flow rate and pressure of the fluid at the inlet reservoir.

3. The device of claim 1, further comprising an outlet reservoir in fluid communication with the outlet ends of the restricting channel and the uniform flow channel.

4. A device according to claim 1, wherein the limiting channel includes a barrier band having openings of at least 1 micron, or at least 2 microns, or at least 4 microns, or at least 10 microns, or at least 25 microns, or at least 50 microns, or at least 100 microns, or at least 200 microns, or at least 500 microns.

5. The device of claim 1, wherein the restricted channel comprises a window of 1-4 microns, or 1-15 microns, or 4-35 microns, or 4-100 microns, or 4-200 microns.

6. The device according to claim 1, wherein 1-90% of the flow in the device is in the uniform flow channel, or 1-75% of the flow in the device is in the uniform flow channel, or 1-50% of the flow in the device is in the uniform flow channel, or 1-25% of the flow in the device is in the uniform flow channel.

7. The device of claim 1, wherein the confinement channel and the uniform flow channel are integrated with the same chip or substrate.

8. The device of claim 1, wherein the confinement channel and the uniform flow channel are located in different chips or substrates.

9. The device of claim 1, wherein the restricting channel is a microfluidic channel.

10. The device of claim 1, further comprising means for analyzing a biological sample in the channel.

11. The device of claim 1, further comprising means for analyzing the proteomic composition, lipidomic composition, transcriptomic composition or carbohydrate composition of the biological sample in the channel.

12. The device of claim 1, further comprising means for measuring the level of the separated fractions of the sample within the channel.

13. The device of claim 1, further comprising means for measuring the level of a biomarker in a separated fraction of a sample within the channel.

14. The apparatus of claim 1, wherein the plurality of obstacles comprises posts integrated with the channel.

15. The apparatus of claim 1, wherein the plurality of obstacles comprises one or more of: A portion of the reticular formation of the uveal tract of a human or animal; Part of the corneoscleral reticular structure of a human or animal; and Part of the paratubular reticular formation in humans or animals.

16. The device of claim 1, wherein the plurality of obstacles comprises glass beads, magnetic beads, gel particles, dextran particles, or polymer particles.

17. The device according to claim 1, wherein the biological sample consists of human or animal body fluids, blood, tissues or cells.

18. The device of claim 1, wherein the biological sample comprises a carrier fluid.

19. The device of claim 1, wherein the biological sample comprises one or more reagents.

20. The device of claim 1, wherein the limiting channel further comprises a binding moiety for binding a biomarker or biomolecule of the sample.

21. The device of claim 1, wherein the biological sample is from a subject undergoing diagnosis or prognosis.

22. The device of claim 1 further comprising a tortuous fluid mixing region in the confined channel.

23. The device of claim 1, wherein the restricting channel or the continuous flow channel has a fluorinated coating.

24. The device of claim 1 having a height of 7 to 25 microns.

25. A method for extracting extracellular matrix from a biological sample, the method comprising: Allowing the biological sample to flow from the inlet end to the outlet end of the device of claim 1; and Reverse the direction of fluid flow toward the inlet end of the device.

26. A microfluidic system for separating fractions of a biological sample, the system comprising: A microfluidic device comprising first and second restricted passages each having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the respective passages; a plurality of spaced-apart obstacles positioned in the restricted passage to provide flow resistance, wherein the spacing between the obstacles decreases in a direction from the inlet end to the outlet end; as well as an inlet reservoir for containing a fluid, wherein the fluid reservoir is in fluid communication with the inlet end of the restricted passage; and a third uniform flow channel having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel, wherein the first restrictive channel, the second restrictive channel, and the third uniform flow channel are parallel to one another, wherein the inlet end is in fluid communication with the inlet reservoir; A drive unit including a pressure source; a source unit comprising a fluid source, wherein a pressure source is in fluid communication with the fluid source and an inlet reservoir of the microfluidic device; a sensor unit including a sensor in fluid communication with the inlet reservoir for measuring a flow rate and a pressure of the fluid at the inlet reservoir and sending the flow and pressure data to the processor; as well as an on-chip analyzer unit comprising one or more instruments for analyzing the separated fractions in the microfluidic device and sending the analysis data to the processor; as well as Processor for receiving and displaying flow rate, pressure and analysis.

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