Device and method for label-free isolation of extracellular vesicles

Abstract

The present invention provides device and method for label-free isolation of extracellular vesicles (EVs) from biological samples. More particularly, the present invention relates to an inertial microfluidic device which utilizes inertial lift forces to isolate small extracellular vesicles from a heterogenous sample based on their size without inducing any structural or functional damage to the extracellular vesicles. The present invention also relates to method for label-free isolation of extracellular vesicles (EVs) from biological samples using the inertial microfluidic device. The present invention offers rapid and efficient EVs isolation without mechanical damage to vesicles, thereby preserving their functionality for downstream applications in diagnostics and therapeutics. This innovative approach ensures high purity and repeatability.

Description

DEVICE AND METHOD FOR LABEL-FREE ISOLATION OF EXTRACELLULAR VESICLESFIELD OF THE INVENTION

[0001] The present invention relates to device and method for label-free isolation of sub-micron particles such as extracellular vesicles (EVs) from biological samples. More particularly, the present invention relates to an inertial microfluidic device which utilizes inertial lift forces and / or Dean forces to isolate sub-micron particles such as extracellular vesicles from a heterogenous sample based on their size without inducing any structural or functional damage to the extracellular vesicles. The present invention also relates to method for label-free isolation of sub-micron particles or extracellular vesicles (EVs) from biological samples using the inertial microfluidic device.BACKGROUND OF THE INVENTION

[0002] Extracellular vesicles (EVs) include exosomes and microvesicles, having a size of about 50 to 1000 nm, and are thus useful as markers for diagnosing disease because they retain the characteristics of original cells.

[0003] Biological fluids constitute of a variety of biological components like cells, viruses, lipoproteins, EVs, nucleic acids, etc. In this regard, sub-micron particles like EVs have immense potential as biomarkers for liquid biopsy due to the wealth of biological information (like mRNA, lipids, proteins etc.) they contain. Ultracentrifugation is the gold standard for EV isolation from any biological fluid. Ultracentrifugation has disadvantages like low yield, equipment to equipment variation in protocols, very long processing time, use of high volumes of sample, and requirement of skilled labour. The high centrifugal forces used in this method damage the EVs. There are some commercial products that do not use ultracentrifugation. Affinity-based methods use aptamers and antibodies to capture EVs. The first problem is that these reagents are expensive. The second problem is that only a few of the EV biomarkers are currently known. Therefore, this method will not capture a large subsets of EVs that do not express these markers. Label-free methods rely on physical properties of EVs like size, density, electrical and acoustic properties. There are precipitation-based label-free methods available in the market, but they require pre-processing of biologicalsamples before they can be used for isolation of EVs. At the moment, there is a gap in the market for a label-free technique / method of isolation of EVs that is one- step, fast, works with a small sample volume, and does not require manual intervention or pre-processing.

[0004] Isolation of extracellular vesicles (EVs) from biological samples like blood, urine etc. can be very useful from diagnostic point of view. The reason being that EVs have a rich source of biological information like proteins, nucleic acids, lipids etc. But isolation of EVs through conventional methods like ultracentrifugation, precipitation etc. have many shortcomings like timeconsuming protocols, lack of adequate purity, structural damages to EVs etc. So, the present invention provides a simplified method and device for isolation of EVs from biological fluids.

[0005] The present invention thus provides an inertial microfluidic device that can isolate particles from a heterogenous sample based on their size. The microfluidic device of the present invention uses a combination of inertial lift forces and secondary Dean forces to separate the EVs from other contaminating particles in the heterogenous sample. This takes approximately 15 minutes as compared to >4 hours duration via ultracentrifugation.SUMMARY OF THE INVENTION

[0006] In one aspect, the present invention provides an inertial microfluidic device which uses inertial lift forces to isolate extracellular vesicles (EVs) from a heterogenous sample. The present invention addresses limitations of conventional EVs isolation techniques, such as ultracentrifugation, which are time-consuming (>4 hours), require large sample volumes, and may damage EV structures due to high centrifugal forces. In contrast, present invention achieves rapid isolation in approximately 15 minutes using low sample volumes (around 1 mL), offering a high-throughput, scalable, and eco-friendly alternative suitable for diagnostic applications like liquid biopsy.

[0007] In another aspect, the device of the present invention needs low (approximately 1 mL) sample volume and can isolate desired particles in approximately 15 minutes without inducing structural or functional damage to the EVs.

[0008] In yet another aspect, the present invention provides a method for label-free isolation of extracellular vesicles (EVs) from biological samples using the afore-mentioned inertial microfluidic device.

[0009] In yet another aspect, the present invention discloses a device which comprises a spiral microchannel with a rectangular cross-section and a height-to-width aspect ratio between 1 and 5, designed to generate inertial lift forces and Dean flow vortices within a fluid stream. This fluid stream is formed by introducing a sample — typically a biological sample such as blood, urine, saliva, CSF, sweat, or conditioned cell culture media — and a sheath fluid (e.g., saline, HEPES Buffer, Tris Buffer, MES Buffer, filtered deionized water, or PBS) through at least one inlet. The inlet configuration is versatile, allowing the sample and sheath fluid to be introduced either separately via two inlets (first inlet for sample, second for sheath fluid) or pre-mixed through a single inlet, enhancing operational flexibility. The microchannel features at least two outlets, with a preferred embodiment including three: a first outlet collects an EV-enriched stream containing submicron particles below about 1000 nm, a second and third outlet captures larger particles above 1000 nm. This size-based separation is driven by the balance of inertial lift forces, which focus larger particles towards an equilibrium position nearer to the inner wall of the channel, and Dean drag forces, which direct smaller EVs to the outer wall of the channel, eliminating the need for labelling agents or affinity-binding techniques.

[0010] In yet another aspect, the present invention discloses a method of isolation which involves introducing the sample and sheath fluid into the microchannel at a controlled total flow rate ranging from 30 pL / min to 600 pL / min, with a preferred range of 50 pL / min to 500 pL / min, to optimize inertial focusing of EVs. In the two-inlet configuration, the sheath fluid forms a coaxial flow around the sample, hydrodynamically focusing it within the channel to enhance separation precision. The process operates continuously, enabling real-time collection of EVs, which is a significant improvement over batch-based methods. The microchannel’s interior surfaces lack affinity-based coatings, ensuring that separation relies solely on hydrodynamic forces, preserving EV integrity — a critical advantage validated by experimental data showing EV sizes from 4 nm to486 nm with peaks at 119-144 nm, and confirmed by techniques like Cryo-TEM and Western blot (markers CD63, CD81, TSGlOlj.OBJECT OF THE INVENTION

[0011] An object of the present invention is to provide a microfluidic device for the label-free isolation of extracellular vesicles (EVs) from biological samples based on their size, without requiring antibodies, chemical affinity tags, or ultracentrifugation.

[0012] An object of the present invention is to develop a microfluidic channel design, preferably spiral or curvilinear, that utilizes inertial lift forces and Dean flow effects to achieve efficient, high-throughput separation of EVs from other particles in a fluid sample.

[0013] Another object of the present invention is to offer a flexible inlet configuration, wherein the device can function with either:• A single inlet, where the sample is introduced alone, or• Dual inlets, where a sheath fluid is introduced alongside the sample to assist in flow focusing.

[0014] Another object of the present invention is to provide a microfluidic device with at least two, preferably three, outlets, allowing for the effective separation of:• EV-enriched particles (below 1000 nm]• Larger particles (above 1000 nm]

[0015] Another object of the present invention is to optimize flow rate conditions (30 pL / min to 600 pL / min, preferably 50 pL / min to 500 pL / min), ensuring controlled particle focusing and separation within the microfluidic channel.

[0016] Another object of the present invention is to design a microfluidic system that can be scaled up, incorporating multiple parallel microchannels on a single chip, thereby increasing processing throughput while maintaining efficient separation.

[0017] Another object of the present invention is to provide a continuous- flow method for EVs isolation, enabling real-time sample processing without requiring centrifugation or batch processing techniques.

[0018] Another obj ect of the present invention is to preserve the structural integrity of the EVs during separation by minimizing shear stress, allowing for the collection of intact EVs suitable for biomedical research, diagnostics, and therapeutic applications.

[0019] Another object of the present invention is to develop a cost-effective and user-friendly device that can be integrated into existing laboratory workflows, enabling rapid and reproducible EVs isolation across different sample types, including blood, plasma, urine, saliva, CSF, sweat, and conditioned cell culture media.

[0020] Another object of the present invention is to improve the purity and recovery of EVs compared to existing separation methods, ensuring a high yield of structurally intact EVs for downstream characterization and application.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing summary, as well as the following detailed description of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of assisting in the explanation of the invention, there are shown in the drawings embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentation shown therein.Figure 1: Experiments with conditioned cell culture media using one inlet device. (A) Schematic representation of the inertial microfluidic device with single inlet. Particle size distribution using DLS instrument for (B) Inlet, (C) Waste outlet and (D) Sample outlet.Figure 2: (A) Schematic representation of the device design and operation. (B) Pictorial representation of the device with red dye flowing through it.Figure 3: Simulation for fluid flowthrough high aspect ratio spiral channels. Cross sectional view of the velocity profile in the channel for radius of curvature of (A) 10 mm and (B) 5 mm. Q is in pL / min.Figure 4: Particle simulation results (A) Plot showing particle focusing distance from inner wall with respect to aspect ratio of the channel. (B) (A) Violin plot showing focusing position of particles of different sizes along the channel width. Median value is represented using a solid line. (C) Plot showing percentage ofparticles collected at inner + middle outlet (waste outlets) and outer outlet (sample outlet) (D)Plot showing the dependence of concentration of particles on particle focusing of the device for 1 pm particle. (E) Plot showing the dependence of concentration of particles on particle focusing of the device 200 nm particle. (F) Plot showing particle focusing distance from the centreline of the channel in the vertical direction.Figure 5: Experiments with polystyrene beads. (A) Normalised particle count at outlets for 7 pm, 3 pm, 1 pm particles. Particles are counted through a haemocytometer. (B) NTA plot depicting particle distribution of the sample collected from the sample outlet. (C) DLS data of the sample collected from outer outlet.Figure 6: Purity at inner, middle and outer outlets for a mixture of (A) 3 pm and 500 nm particles and (B) 1 pm and 500 nm particles. Plots show larger particles (3 pm and 1 pm) are collected at inner and middle outlet while outer outlet has high purity of 500 nm particles.Figure 7: DLS results showing (A) presence of particles more than 1 pm in the inlet (B) absence of larger particles in the outer outlet. Plot showing concentration of particles processed through (C) the microfluidic device and (D) ultracentrifugation. (E) Extracellular vesicle imaged through cryo-TEM (F) Western blot results of the sample outlet depicting bands for Anti-CD63, Anti- CD81 and TSG101.Figure 8: Experiments with Cell-Spiked Conditioned Cell Culture Media (A) Plot depicting the cell concentration in samples collected from each outlet. (B) NTA analysis showing the particle size distribution in the sample outlet. (C) Western blot analysis of collected samples, assessing the presence of calnexin (anti- Calnexin) and extracellular vesicle marker CD63 (anti-CD63).Figure 9: NTA plot showing removal of larger particles (>500 nm) from the plasma sample.DESCRIPTION OF THE INVENTION

[0022] For the purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing,for example, quantities of ingredients used in the specification are to be understood as being modified in all instances by the term "about". It is noted that, unless otherwise stated, all percentages given in present specification and appended claims refer to percentages by weight of the total composition.

[0023] Thus, before describing the present invention in detail, it is to be understood that present invention is not limited to particularly exemplified process parameters that may of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the scope of the invention in any manner.

[0024] The use of examples anywhere in present specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in present specification.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

[0026] It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0027] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances.

[0028] Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0029] As used herein, the terms “comprising” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0030] As used herein, the term “microchannel” corresponds to a small fluidic passage within the microfluidic device that facilitates fluid flow and particle separation.

[0031] As used herein, the term “microfluidic device” corresponds to a system incorporating microchannels and fluid control elements designed for manipulating small volumes of fluids, typically in the microliter to nanolitre range.

[0032] As used herein, the term “extracellular vesicles (EVs)” corresponds to Submicron-sized membrane-bound vesicles secreted by cells, typically ranging in size from 30 nm to 1000 nm, containing proteins, lipids, RNA, and other biomolecules.

[0033] As used herein, the term “sheath fluid” corresponds to a fluid introduced into the microchannel to hydrodynamically focus the sample fluid and assist in particle separation. Examples include buffer solutions such as saline, PBS, HEPES Buffer, Tris Buffer, MES Buffer, and DI water.

[0034] As used herein, the term “inertial lift forces” corresponds to hydrodynamic forces that act on particles within a fluid flow in a microchannel, causing lateral migration of particles based on their size and density.

[0035] As used herein, the term “dean flow vortices (Dean Flow)” corresponds to secondary rotational flows generated in a curved or spiral microchannel due to fluid inertia, which assists in size-based separation of particles by moving them to different equilibrium positions.

[0036] As used herein, the term “fluid stream” corresponds to the continuous flow of a liquid containing suspended particles (such as EVs) within the microchannel.

[0037] As used herein, the term “outlet” corresponds to an exit port of the microfluidic device through which the separated particles and fluid fractions are collected. In the representative embodiment,• First Outlet: Collects EVs-enriched fluid.• Second Outlet: Collects larger particles or unwanted sample components.• Third Outlet (if present): Collects intermediate-sized particles or residual sample fluid.

[0038] As used herein, the term “height-to-width aspect ratio” corresponds to the ratio of the height to the width of the microchannel's cross-section, whichaffects the efficiency of inertial focusing and Dean flow-based separation. In the representative embodiment, height-to-width aspect ratio is specifying a preferred range of 2:1 to 5:1.

[0039] As used herein, the term “total flow rate” corresponds to the combined flow rate of the sample and sheath fluid within the microchannel.

[0040] As used herein, the term “continuous flow process” corresponds to A method in which the sample continuously flows through the microchannel without stopping, allowing real-time separation of EVs.

[0041] The present invention provides an inertial microfluidic device which uses inertial lift forces to isolate small extracellular vesicles (EVs) from a heterogenous sample.

[0042] The device design of the present invention as shown in figure 2(A) is based on secondary flow due to curved surface. The dimensions of the channel is based on simulations that result in generation of secondary flows which are counter rotating vortices along the cross section of the device. The aim of the chip is to enrich EVs from a biological sample by segregating particles of size more than 1 pm. The principle of the device is based on sample introduction in the inner inlet along with sheath flow on the outer inlet. The flow rates of fluid through the two inlets will be varied for particle inertial focusing. The flow at the curved channel will induce dean flow which will make the particles separate based on the size. The outlets are designed according to the position of the different sized particles at the end of the channel. The larger particles will be concentrated at the inner wall due to dominant lift forces and will be collected in the inner wall. The particles will be focused according to their size from inner to outer wall. As a result we can enrich submicron sized extracellular vesicles (EVs) at the outer outlet.

[0043] The device is based on a High Aspect Ratio Curved (HARC) channel system. Analytical calculations of the HARC design are performed to obtain the device dimensions needed to eliminate particles bigger than 1 pm at a flow rate of 100 pL / min. The current design (figure 2B) consists of a high aspect ratio spiral channel of 2.5 loops. The width of the channel is 20 pm and height is changed for optimum aspect ratio.

[0044] The flow in the afore-mentioned device is simulated using COMSOL to determine the optimum radius of curvature and aspect ratio of the spiral channel needed to generate stable secondary flows. Simulation results show the formation of stable secondary flows at flow rates of 300 pL / min and 500 pL / min for 5 mm and 10 mm radii of curvature respectively (figure 3). Further lowering the flow rate for separation requires narrower channel widths. The device design can be manufactured outside the cleanroom using existing technology.

[0045] Particle separation experiments are performed with the design for finding optimum aspect ratio. The aspect ratio is changed from 2 to 5 to find the optimum aspect ratio for focusing of particles of size 1 pm. When the aspect ratio is kept at 5, 4 and 3 the particles are randomly scattered along the cross section. This can be observed from figure 4(A) where the focusing distance of the particles with different size is plotted with respect to aspect ratio. As the aspect ratio is changed to 2.5, the particles with size 3 pm, 1 pm and 0.8 pm are focused in the inner outlet.

[0046] This is more evident from figure 4(B), where larger particles (3 pm, 1 pm and 0.8 pm) are focused in inner outlet and middle outlet. At outer outlet, only 500 nm and 200 nm particles are collected. As the ratio is decreased to 2, same behaviour is observed.

[0047] Figure 4(C) shows the percentage of the particles collected in each outlet. The inner and middle outlet is merged and considered as waste outlet as it will contain all the larger particles as shown in previous figure. Almost 40 % of the 200 nm particles are collected in the outer outlet (sample outlet) whereas there are no larger particles collected in the sample outlet. Also sample outlet collects 20% of the 500 nm particles. So, it can be concluded that sub-micron particles are only available in the sample outlet. This device thus successfully removes micron sized particles from the sample.

[0048] Also, the concentration of the particles (1 pm and 200 nm) are varied and focusing distance is plotted in a violin plot as shown in figure 4(D) and 4(E) respectively. Concentration variation doesn’t affect the focusing efficiency.

[0049] Also figure 4(F) shows the focusing distance in the vertical direction. Similar to the horizontal direction, larger particles are focused precisely for aspect ratio of 2.5 and 2.

[0050] In an embodiment of the present invention, the biological sample or bio fluid may include, but is not particularly limited to, at least one selected from the group consisting of whole blood, serum, peritoneal fluid, breast milk, and urine.

[0051] In another aspect, the device of the present invention needs low (approximately 1 m ) sample volume and can isolate desired particles in approximately 15 minutes without inducing structural or functional damage to the EVs.

[0052] In yet another aspect, the present invention provides a method for label-free isolation of extracellular vesicles (EVs) from biological samples using the afore-mentioned inertial microfluidic device.

[0053] In preferred embodiment of the present invention, a device for isolation of particles from a sample, the device comprising: a microchannel; at least one inlet configured to introduce a sample and a sheath fluid into the microchannel to form a fluid stream; and at least two outlets for collecting separated particles from the microchannel, wherein a first outlet is configured to collect extracellular vesicles (EVs) and a second outlet is configured to collect other components of the sample, wherein the fluid stream through the microchannel generates inertial lift forces and dean flow vortices that cause sizebased separation of particles present in the sample, such that sub micron particles including extracellular vesicles (EVs) are focused into a streamline collected at least one outlet while larger particles are directed at least other outlet.

[0054] In another embodiment of the present invention, the sample and the sheath fluid are introduced either separately via a first inlet and a second inlet or pre-mixed and introduced via the at least one inlet.

[0055] In another embodiment of the present invention, the sheath fluid is buffer solution selected from the group consisting of saline, HEPES Buffer, Tris Buffer, MES Buffer, filtered deionized (DI) water and PBS (phosphate-buffered saline).

[0056] In another embodiment of the present invention, the device is microfluidic device and the microchannel is a microfluidic channel.

[0057] In another embodiment of the present invention, the sample is a biological sample.

[0058] In another embodiment of the present invention, the microchannel is spiral and has a rectangular cross-section with a height-to-width aspect ratio between 2-5.

[0059] In another embodiment of the present invention, the microchannel comprises three outlets, includes a first outlet configured to collect EV particles below about 500 nm; a second outlet configured to collect larger particles above about 500 nm; and a third outlet configured to collect a residual fluid or intermediate-sized particles.

[0060] In another embodiment of the present invention, the total flow rate through the channel is in a range of about 30 pL / min to 600 pL / min, preferably about 50 pL / min to 500 pL / min, to facilitate inertial focusing of the extracellular vesicles.

[0061] In another embodiment of the present invention, wherein a plurality of microchannels is provided in parallel on a single chip to increase the throughput of extracellular vesicles (EVs) isolation.

[0062] In preferred embodiment of the present invention, a method isolation of particles from a sample using the device as mentioned is above embodiments, the method includes introducing a sample containing extracellular vesicles (EVs) and sheath fluid into the microchannel via the at least one inlet; flowing the sample and sheath fluid through the microchannel at a controlled flow rate such that inertial lift forces and dean flow vortices induce size-based focusing of particles within the microchannel; and collecting an outlet stream enriched with extracellular vesicles (EVs) from the at least first outlets of the microchannel, while diverting larger particles to at least second outlet, wherein the isolation of the extracellular vesicles (EVs) is achieved without the any labelling agents or affinity-binding techniques.

[0063] In another embodiment of the present invention, comprising introducing a sheath fluid through a second inlet concurrently with the sample, such that the sheath fluid forms a coaxial flow around the sample to hydrodynamically focus the sample within the microchannel.

[0064] In another embodiment of the present invention, the total flow rate through the microchannel is in a range of about 30 pL / min to 600 pL / min, preferably about 50 pL / min to 500 pL / min.

[0065] In another embodiment of the present invention, the separation is carried out as a continuous flow process, enabling real-time collection of extracellular vesicles (EVs) from the sample.

[0066] In another embodiment of the present invention, the present invention provides a microfluidic device for the label-free isolation of extracellular vesicles (EVs) from a sample based on inertial lift forces and Dean flow effects. The device includes a microchannel, preferably spiral or curvilinear, designed to induce size-based particle separation; At least one inlet, wherein a single inlet may be used to introduce a sample fluid, Alternatively, two inlets may be used, where a first inlet introduces the sample and a second inlet introduces a sheath fluid to assist in flow focusing; At least two, preferably three, outlets to collect different fractions of separated particles, including a first outlet for collecting an EVs- enriched stream (submicron particles below 500 nm), a second outlet for collecting larger particles above 500 nm, a third outlet (if present) for collecting intermediate-sized particles or residual fluid, a fluid flow mechanism that generates inertial lift forces and Dean flow vortices, facilitating size-based EVs separation without requiring labelling agents or affinity-based capture.

[0067] In yet another embodiment of the present invention, the microfluidic device operates with a single inlet, where a sample fluid (e.g., blood plasma, conditioned cell culture media, urine, or serum) is introduced into the microchannel without requiring a sheath fluid. The design of the microchannel ensures that dean flow vortices are generated, causing submicron EVs to migrate toward a distinct flow path, separated from larger contaminants; at least two outlets separate the EVs-enriched stream from larger sample components; the fluid flow rate is optimized within the range of 30 pL / min to 600 pL / min, preferably 50 pL / min to 500 pL / min, ensuring efficient separation.

[0068] In yet another embodiment of the present invention, the microfluidic device includes two inlets a first inlet for introducing the sample fluid. A second inlet for introducing a sheath fluid, such as PBS, saline, DI water, or buffer solutions. The sheath fluid creates a coaxial flow, hydrodynamically focusing the sample fluid and improving the precision of EVs separation. Three outlets collect: a first outlet containing EVs (below 500 nm). a second outlet containing largerparticles (above 500 nm) and a third outlet (optional) containing intermediate particles.

[0069] In yet another embodiment of the present invention, the microchannel has a rectangular cross-section with a height-to-width aspect ratio between 2:1 and 5:1, allowing for the formation of stable Dean vortices at optimized flow rates. The Reynolds number range to be controlled between 10 and 100 for enhanced inertial focusing of EVs. Parallel microchannels on a single chip to increase throughput for high-volume EVs isolation.

[0070] In yet another embodiment of the present invention, the present invention also provides a continuous flow method for isolating EVs, comprising: introducing a sample (with or without a sheath fluid) into the microfluidic channel; flowing the sample through a spiral or curvilinear microchannel, where: inertial lift forces and Dean flow vortices induce size-based separation, sub micron EVs follow a distinct flow path separate from larger particles; collecting output streams via multiple outlets, where: One outlet collects EVs-enriched particles (<500 nm), another outlet collects larger particles (>500 nm), an optional third outlet collects intermediate-sized components. This method enables real-time EVs collection without requiring ultracentrifugation, antibody-based capture, or additional preprocessing.

[0071] In another embodiment of the present invention, a device (100) for separation of particles from a sample (102), said device (100) comprising: at least one inlet (104) configured to introduce the sample (102); microchannel (106) configured to receive said sample (102) to form a fluid stream, wherein microchannel (106) comprises: a height-to-width aspect ratio (H / W) within the range of 1 and 5, and radius of curvature (R) within the range of 1 mm-20 mm; and plurality of outlets (108) configured to receive separated sample particles from the microchannel (106), wherein total flow rate of the fluid stream, the radius of curvature (R), and height-to-width aspect ratio (H / W) are configured for size-based separation of sample particles.

[0072] In yet another embodiment of the present invention, wherein the plurality of outlets comprises at least one sample outlet (108) and at least one waste outlet (112).

[0073] In yet another embodiment of the present invention, wherein the device (100) comprises: at least one inlet (104) configured to receive the sample (102); and at least one sheath fluid inlet (110) configured to receive sheath fluid.

[0074] In yet another embodiment of the present invention, wherein the sample (102) and the sheath fluid are premixed and introduced via at least one inlet (104).

[0075] In yet another embodiment of the present invention, wherein the sample (102) comprises viscosity within the range 0.5 mPa.s to 2 mPa.s.

[0076] In yet another embodiment of the present invention, wherein the sheath fluid is a buffer solution.

[0077] In yet another embodiment of the present invention, wherein the sheath fluid is selected from the group consisting of saline, HEPES Buffer, Tris Buffer, MES Buffer, filtered deionized (DI) water and PBS (phosphate-buffered saline).

[0078] In yet another embodiment of the present invention, wherein the device (100) is microfluidic device.

[0079] In yet another embodiment of the present invention, wherein the microchannel (106) is a microfluidic channel.

[0080] In yet another embodiment of the present invention, wherein the microchannel (106) is a microfluidic channel.

[0081] In yet another embodiment of the present invention, wherein the shape of the microchannel (106) is selected from spiral or curved geometry.

[0082] In yet another embodiment of the present invention, wherein the microchannel (106) has a width within the range of 10 pm-50 pm and a height adjustable within a range of 10 pm -100 pm.

[0083] In yet another embodiment of the present invention, the device (100) as claimed in claim, comprises: at least one outlet (108) configured to collect sample particles from the fluid stream below about 1000 nm; and at least one outlet (108) configured to collect sample particles from the fluid stream above about 1000 nm.

[0084] In yet another embodiment of the present invention, wherein the total flow rate of the fluid stream through the microchannel (106) is within range of 30 |iL / min to 600 pL / min.

[0085] In yet another embodiment of the present invention, wherein the device (100) comprises plurality of microchannels (106) on a single substrate.

[0086] In another embodiment of the present invention, the present invention discloses a method for separating particles from a sample (102) based on the above-mentioned device (100) embodiments, the method comprising: introducing a sample (102) into the microchannel (106) to form a fluid stream via the at least one inlet (104); passing the fluid stream through the microchannel (106), wherein the height-to-width aspect ratio (H / W) is within a range of 1 to 5 and a radius of curvature (R) is within a range of 1 mm to 20 mm; separating the sample particles based on the size; and collecting separated particles through the plurality of outlets (108),

[0087] wherein total flow rate of the fluid stream, the radius of curvature (R), and height-to-width aspect ratio (H / W) are configured for size base separation of particles.

[0088] In yet another embodiment of the present invention, wherein the sample particles and waste particles are collected separately from at least one sample outlet (108) and at least one waste outlet (112) respectively.

[0089] In yet another embodiment of the present invention, wherein the method comprising, introducing a sheath fluid through at least one sheath fluid inlet (110) concurrently with the sample (102).

[0090] In yet another embodiment of the present invention, wherein the total flow rate through the microchannel (106) is in a range of about 30 pL / min to 600 pL / min.

[0091] In yet another embodiment of the present invention, wherein the separation is carried out as a continuous flow process.Advantages of the present invention:

[0092] The advantages of the present invention include:• Use of low sample volumes (~ 1ml)• High throughput particle isolation (~ 100 microlitres per minute)• Minimal particle damage as we do not use high centrifugal forces• Faster sample processing (~ 15 min) relative to ultracentrifugation (> 4 hours)The following examples are illustrations only and do not limit the scope of invention.EXAMPLES:Example 1

[0093] We have tested device design and operation. The microfluidic device for EVs isolation consists of a spiral channel with a width of 20 pm and a height ranging between 10 to 100 pm. The device contains two inlets and three outlets as shown in figure 2(A). Figure 2(B) shows the pictorial image of the device. The aspect ratio of the device (Height / width) ranged between 1 and 5. The device was tested with various configurations of radius of curvature ranging between 5-10 mm. The flow rates of the sample and the buffer (lx PBS) ranged between 50 pL / min and 500 pL / min. The sample is then flown through the spiral channel and is collected from the three outlets. We also tested a single-inlet device configuration, in which the sample was introduced at a flow rate of 50 pL / min.

[0094] Sample preparation and device operation for experiments with polystyrene beads: We conducted experiments with polystyrene beads suspended in filtered DI water to validate our simulation findings. A particle mixture (sizes: 7 pm, 3 pm, 1 pm, and 0.5 pm) was introduced through the inner inlet at 50 pL / min. DI water is used as a buffer and introduced into the outer inlet at 100 pL / min. Samples were collected from each outlet and particles were counted using a haemocytometer. Also, the outer outlet (sample outlet) specimen is analysed using dynamic light scattering (DLS) instrument and nanoparticle tracking analysis (NTA) instrument.

[0095] Sample preparation of conditioned cell culture media: Extracellular vesicles (EVs) are sourced from conditioned cell culture media (CCM) samples. The CCM samples are derived from cell culture of MDA-MB-231 breast cancer cells. The cells are cultured in both serum-based and serum-free media. The serum-based media consists of DMEM + 10% Fetal Bovine serum (FBS) and 1% antibiotic. The serum-free media consists of DMEM and 1% antibiotic only. The MDA-MB-231 cells are cultured in the media for 48hr until they reach 70- 80%confluency. This media is expected to have EVs, growth factors, cytokines and metabolites produced by the cells during their growth. The CCM is then later flown through the device's inner inlet (or sample inlet).

[0096] Device operation: We have done isolation using the CCM of varying volumes between 200 pl and 5 ml. The experimental setup consists of flowing the CCM sample at 50 pL / min and PBS (lx) buffer at 100 pL / min via the inner and outer inlets of the microfluidic device respectively. We also tested a single-inlet device configuration, in which the CCM sample was introduced at a flow rate of 50 pL / min. The samples coming out of the 3 outlets are collected. Each outlet sample was characterized based on particle size distribution and protein marker analysis.

[0097] Sample preparation of reconditioned blood plasma: Human blood plasma, obtained in powder form, is a cell-free and platelet-free preparation that contains extracellular vesicles (EVs) and other sub micron-sized particles. To prepare a plasma solution, the powdered plasma is reconstituted by mixing it with filtered PBS. The particle size distribution within this reconstituted plasma solution is then analyzed using Nanoparticle Tracking Analysis (NTA)The solution is passed through the spiral device to enrich EVs. Sample collected from each outlet is analysed using NTA for presence of EVs sized particles in the sample.

[0098] Sample preparation of cell spiked CCM: The CCM samples were then spiked with the MDA-MB-231 cells to check whether the device isolated micron-size cells separately from the EVs in the outlets of interest. As expected, the device removed the cells via the waste outlet, while the sample outlet with EVs was uncontaminated by cells. This was also proved with western blot characterization which showed the presence of calnexin protein (from cells) only in the waste outlet.3. Results3.1 Fluid flow simulation results

[0099] We simulated the fluid flow in the device for different flow rates and subsequently different Reynold’s number. The fluid flow within the device was simulated based on the dimensions specified in Section 3.4, with the channel width and height measuring 20 pm and 50 pm, respectively. The simulations were conducted for flow rates ranging from 100 pL / min to 500 pL / min, considering two different radii of curvature: 10 mm and 5 mm. As illustrated in figure 3(A), theonset of Dean flow was observed at a flow rate of 100 pL / min for a 10 mm radius of curvature. This effect became more pronounced at 300 pL / min, where the Dean forces exhibited increased complexity but remained unstable. At 500 pL / min, the device successfully generated stable rectangular Dean vortices for a 10 mm radius of curvature. The velocity distribution indicated that the flow was highest near the centreline of the cross-sectional surface, while the transverse flow was more prominent near the channel boundaries due to increased drag forces acting away from the vortex centre. The high aspect ratio of the microchannel facilitated the parallel streamlined formation of vortices along the channel wall, allowing sufficient time for particle alignment based on the equilibrium between Dean forces and lift forces.

[0100] With a reduction in the radius of curvature to 5 mm, the Dean number increased, promoting the formation of Dean vortices at lower flow rates, as confirmed by the cross-sectional simulation plots in figure 3(B). Stable rectangular Dean vortices were observed even at a flow rate of 100 pL / min, with progressively intensified vortex formation as the flow rate increased. At 500 pL / min, the vortices became significantly stronger, disrupting the parallel alignment of the vortices along the channel wall. Consequently, a flow rate of 100 pL / min and a radius of curvature of 5 mm were selected as the optimal operational parameters for the device.3.2 Particle simulation for high aspect ratio spiral channel

[0101] We conducted simulations to trace particle movement and assess the focusing efficiency of the device at the optimized device dimensions and flow rates. Additionally, we evaluated the optimal aspect ratio required for the effective focusing of larger particles (>1 pm). A particle suspension containing 3 pm, 1 pm, 0.8 pm, and 0.5 pm particles was introduced through the sample inlet at a flow rate of 50 pL / min, while water, serving as the buffer, was introduced through the buffer inlet at a flow rate of 100 pL / min. The trajectories of individual particles were tracked along the flow path, and their positions relative to the inner wall were determined, figure 4(A) presents the mean distance of different particle sizes from the inner wall as a function of the channel's aspect ratio.

[0102] The aspect ratio of the device was modified by adjusting the channel height while maintaining a constant width of 20 pm. The channel cross-sectionwas categorized into three regions — inner, middle, and outer outlets — corresponding to distinct outlet segments. A higher standard deviation in the mean particle position indicated reduced focusing efficiency for a given aspect ratio. At an aspect ratio of 5, particles were not effectively focused, regardless of their size, and their mean positions were randomly distributed across the inner and middle outlet regions. A similar trend was observed at an aspect ratio of 4. When the aspect ratio was reduced to 3, focusing improved for 3 pm and 1 pm particles, which were confined to the inner outlet segment, though their positions remained randomly distributed within this region.

[0103] Efficient particle focusing was observed at an aspect ratio of 2.5 for 3 pm and 1 pm particles, with standard deviations of 0.2 pm and 0.5 pm, respectively. In contrast, 0.8 pm and 0.5 pm particles were not focused ata defined position, as indicated by their high standard deviations. However, the mean particle positions exhibited spatial separation along the inner-to-outer wall axis, with larger particles (3 pm) being positioned closer to the inner wall than smaller particles (1 pm). This particle size-based focusing aligns with the intended functionality of the device. At an aspect ratio of 2, focusing efficiency was maintained, but the spatial arrangement of particle positions was disrupted. Based on these observations, an aspect ratio of 2.5 was selected for further experimentation.

[0104] To further analyze the distribution of individual particle positions, a violin plot was generated to assess the proportion of particles collected at each outlet. As depicted in figure 4(B), 3 pm particles are precisely focused and collected at the inner outlet for both aspect ratios. Similarly, all 1 pm particles are directed toward the inner outlet. For 0.8 pm particles, while the majority are concentrated in the inner outlet, a notable fraction is also collected in the middle outlet. Additionally, most 0.5 pm particles are distributed between the inner and middle outlets for both aspect ratios. However, at an aspect ratio of 2.5, a small fraction of 0.5 pm particles is also focused at the outer outlet. As expected, 0.2 pm particles predominantly concentrate in the outer outlet.

[0105] Subsequently, the percentage of particles collected in different outlets was quantified, with the inner and middle outlets designated as waste outlets, as larger particles (3 pm and 1 pm) were focused in this region, figure 4(C)illustrates that particles measuring 3 pm, 1 pm, and 0.8 pm were predominantly collected in the waste outlets, whereas 14% of 0.5 pm particles and 37% of 200 nm particles were recovered in the outer outlet (sample outlet). This finding suggests that the sample outlet of the device can be effectively utilized for the enrichment of small extracellular vesicle (EVs) -sized particles by excluding particles larger than 1 pm.

[0106] Furthermore, we simulated the effect of particle concentration on the focusing efficiency of the device. As depicted in figure 4(D), increasing particle concentration adversely affected focusing efficiency. The device maintained effective focusing of 1 pm particles at concentrations of 104and 105particles / mL. However, focusing efficiency declined at higher concentrations of 106and 107particles / mL. Despite this reduction in efficiency, all particles remained confined within the inner and middle outlet segments of the channel cross-section.3.3 Particle separation experiments

[0107] Figure 5(A) presents the percentage of particles collected in the waste outlet compared to the sample outlet. As shown in the figure 5, all 7 pm particles were focused near the inner wall, leading to their exclusive collection in the inner outlet. In the case of 3 pm particles, a broader distribution was observed between the waste outlets, indicating that their focusing position was farther from the inner wall compared to the 7 pm particles. Notably, only 2% of the 3 pm particles were collected in the sample outlet. A similar trend was observed for 1 pm particles, where an even greater displacement from the inner wall resulted in 8% of these particles being collected in the sample outlet. However, the concentration of particles larger than 1 pm in the sample outlet remained low, ensuring minimal impact on the purity of the processed sample. To further validate the particle size distribution in the sample outlet, nanoparticle tracking analysis (NTA) was performed. As depicted in figure 5(B), the dominant peak in the particle distribution was centered around 500 nm, confirming the enrichment of submicron particles and supporting our findings. This is confirmed in size distribution of particles measured through Dynamic light scattering (DLS) method where the peak is at 510 nm.

[0108] Experiments were done with a mix of particles and results are plotted in figure 6. Figure 6(A) shows the purity if particles collected in each outletfor a sample with mixture of 3 pm and 500 nm particles. It shows only 500 nm particles observed in outer outlet. This confirmed in figure 6(B) where similar observations are recorded for a combination of 1 pm and 500 nm particles.3.4 Experiments with conditioned cell culture media

[0109] We conducted experiments using conditioned cell culture media (CCM) to enrich extracellular vesicles (EVs). Additionally, ultracentrifugation (UC) was performed as a comparative EVs enrichment method. Samples collected from the waste outlets and the sample outlet of the device were analyzed for size distribution using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Protein characterization was performed via western blotting.

[0110] The inlet sample contained particles larger than 1 pm, as confirmed by the DLS plot (figure 7(A)). These larger particles were successfully removed and collected in the waste outlet, while the sample outlet contained only particles smaller than 1 pm (figure 7(B)). The NTA analysis of the sample outlet (figure 7(C)) revealed a particle size distribution ranging from 30 nm to 486 nm, with a peak at 119 nm. In contrast, the sample enriched through UC (figure 7(D)) exhibited a size distribution from 42 nm to 590 nm, with a peak at 123 nm. The mean particle size in the sample enriched using our device was determined to be 144 nm, whereas the mean size for the UC-enriched sample was 155 nm.

[0111] The concentration of EVs obtained through UC was higher than that obtained using our device, which is expected as UC allows for the collection of a pellet containing submicron-sized particles. However, the yield of EVs from our device was found to be threefold higher than that of UC. This discrepancy can be attributed to the loss of EVs at multiple stages of the UC pre-processing steps. Additionally, the final supernatant after UC was found to contain a significant number of EVs. Our device was capable of capturing approximately one-third of the EVs present in the CCM sample.

[0112] Cryogenic transmission electron microscopy (Cryo-TEM) imaging of the sample outlet confirmed the presence of EVs-like structures with the characteristic cup-shaped morphology (figure 7(E)). The vesicles observed in the images measured approximately 70 nm in diameter, which falls within the classification of small extracellular vesicles (sEVs).

[0113] Western blot analysis was conducted to assess the presence of key EVs protein markers. According to the M1SEV2023 guidelines, EVs protein markers are categorized into five groups, with specific markers varying based on factors such as cell source, sample type, and isolation methodology. In this study, we selected markers from category 1 (CD63 and CD81) and category 11 (TSG101) for analysis. The western blot results confirmed the presence of EVs in the sample outlet, as bands corresponding to the expected molecular weights of these markers were detected (figure 7 (F)). Cell lysate was used as a control for comparison. The band intensities for CD63 were comparable between the cell lysate and the sample outlet, whereas the CD81 band intensity was higher in the sample outlet compared to the cell lysate. A similar trend was observed for the TSG101 protein marker.3.5 Experiments with Cell spiked conditioned cell culture media

[0114] To validate our device, we utilized conditioned cell culture media spiked with cells. Samples were collected from each outlet and analyzed via microscopy to quantify cell concentrations. As illustrated in figure 8(A), 80% of the collected cells were recovered from the inner waste outlet, while the remaining 20% were retrieved from the middle waste outlet. Across three experimental replicates, no cells were detected in the outer sample outlet.

[0115] Following this, the particle distribution in the sample outlet was analyzed to assess the presence of larger-sized particles. The NTA analysis (figure 8(B)) revealed a particle size distribution within the extracellular vesicle (EVs) range of 30-300 nm, with a minor peak observed at 400 nm. The mean particle size in the sample outlet was determined to be 122 nm, which is consistent with the size range of small extracellular vesicles (sEVs). Notably, no peaks corresponding to particles larger than 500 nm were detected in the sample outlet, confirming the effective removal of larger contaminants.

[0116] Protein marker characterization of the waste and sample outlets was conducted using western blot analysis (figure 8(C)). CD63 was selected as an EVs-specific marker to confirm the presence of EVs in the sample, while calnexin was used to assess the presence of cancer cells in the waste and sample outlets. Cell lysate was employed as a control for protein characterization. Distinct bands corresponding to calnexin were observed in both the cell lysate and the wasteoutlet, whereas no calnexin bands were detected in the sample outlet, indicating the absence of cellular contamination. In contrast, CD63 bands were detected in both the waste and sample outlets, though additional CD63 bands were present in the waste outlet, suggesting that some EVs were also collected in the waste fraction.3.6 Experiments with stored cell free plasma

[0117] The samples collected from both the inner and outer outlets were analyzed using Nanoparticle Tracking Analysis (NTA), as illustrated in figure 9. The NTA results indicate the absence of micron-sized particles in both the inlet and outlet samples, suggesting effective filtration or exclusion of these larger particles. Notably, distinct peaks were observed in the size distribution profiles of the inlet and inner outlet samples, which were subsequently eliminated in the outer outlet samples.

[0118] This elimination is evidenced by the absence of any detectable peaks above 500 nm in the outer outlet sample, while the inner outlet sample exhibited prominent peaks at approximately 550 nm and 600 nm. To further validate these findings, a series of experiments were conducted with varying concentrations of the inlet samples. All outlet samples were systematically examined for the presence of larger-sized particles, specifically those greater than 500 nm. The analysis revealed that particles exceeding 500 nm were consistently present in both the inner and middle outlet samples. This observation confirms the efficacy of our device in selectively enriching extracellular vesicles (EVs) smaller than 500 nm from plasma samples, thereby demonstrating its utility in isolating nanoscale biological particles while excluding larger contaminants.Example 2Enrichment of EVs using inertial microfluidic device with one inlet

[0119] Extracellular vesicles (EVs) were isolated from conditioned cell culture media (CCM) using a single-inlet microfluidic device. A schematic representation of the device operation is provided in figure 1(A). CCM was introduced into the device at a controlled flow rate of 100 pL / min. The sample moved through the device and was subsequently collected at three distinct outlets. The collected fractions were analyzed for particle size distribution using dynamic light scattering (DLS). The sample collected at the inner and middle outlets areconsidered as waste sample whereas the one collected from outer outlet is considered as EVs sample. Additionally, the particle size distribution of the sample prior to device introduction was measured using DLS for comparison.

[0120] Figure 1 (B) illustrates the particle size distribution of the inlet sample, ranging from 4 nm to 6 pm, with distinct peaks observed at approximately 10 nm, 50 nm, 400 nm, and 5 pm. The presence of a substantial peak at 5 pm indicates the existence of micron-sized debris within the initial sample. As shown in figure 1(C), these larger particles were effectively collected in the waste outlet, exhibiting a particle size distribution similar to that of the inlet sample. In contrast, the sample outlet, depicted in figure 1(D), demonstrated removal of large-sized particles. The particle size distribution of the sample collected from this outlet ranged from 4 nm to 400 nm, confirming the efficient removal of micron-sized contaminants. These findings validate the functionality of the single-inlet device in effectively enriching EVs from CCM while eliminating larger micron sized contaminants.

Claims

CLAIMS1. A device (100) for separation of particles from a sample (102), said device (100) comprising: at least one inlet (104) configured to introduce the sample (102); microchannel (106) configured to receive said sample (102) to form a fluid stream, wherein microchannel (106) comprises: a height-to-width aspect ratio (H / W) within the range of 1 and 5, and radius of curvature (R) within the range of 1 mm-20 mm; and plurality of outlets (108) configured to receive separated sample particles from the microchannel (106), wherein total flow rate of the fluid stream, the radius of curvature (R), and height- to-width aspect ratio (H / W) are configured for size-based separation of sample particles.

2. The device (100) as claimed in claim 1, wherein the plurality of outlets comprises at least one sample outlet (108) and at least one waste outlet (112).

3. The device (100) as claimed in claim 1, comprises: at least one inlet (104) configured to receive the sample (102); and at least one sheath fluid inlet (110) configured to receive sheath fluid.

4. The device (100) as claimed in claim 1 or 3, wherein the sample (102) and the sheath fluid are premixed and introduced via at least one inlet (104).

5. The device (100) as claimed in claim 1, wherein the sample (102) comprises viscosity within the range 0.5 mPa.s to 2 mPa.s.

6. The device (100) as claimed in claim 3, wherein the sheath fluid is a buffer solution.

7. The sheath fluid as claimed in claim 6, wherein the sheath fluid is selected from the group consisting of saline, HEPES Buffer, Tris Buffer, MES Buffer, filtered deionized (DI) water and PBS (phosphate-buffered saline).

8. The device (100) as claimed in claim 1, wherein the device (100) is microfluidic device.

9. The device (100) as claimed in claim 1, wherein the microchannel (106) is a microfluidic channel.

10. The device (100) as claimed in claim 1, wherein the sample (102) is a fluid sample.

11. The device (100) as claimed in claim 1, wherein the shape of the microchannel (106) is selected from spiral or curved geometry.

12. The device (100) as claimed in claim 1, wherein the microchannel (106) has a width within the range of 10 pm-50 pm and a height adjustable within a range of 10 pm -100 pm.

13. The device (100) as claimed in claim 1, comprises: at least one outlet (108) configured to collect sample particles from the fluid stream below about 1000 nm; and at least one outlet (108) configured to collect sample particles from the fluid stream above about 1000 nm.

14. The device (100) as claimed in claim 1, wherein the total flow rate of the fluid stream through the microchannel (106) is within range of 30 pL / min to 600 pL / min.

15. The device (100) as claimed in claim 1, wherein the device (100) comprises plurality of microchannels (106) on a single substrate.

16. A method for separating particles from a sample (102) based on the device (100) as claimed in claim 1, the method comprising: introducing a sample (102) into the microchannel (106) to form a fluid stream via the at least one inlet (104);passing the fluid stream through the microchannel (106), wherein the height-to-width aspect ratio (H / W) is within a range of 1 to 5 and a radius of curvature (R) is within a range of 1 mm to 20 mm; separating the sample particles based on the size; and collecting separated particles through the plurality of outlets (108), wherein total flow rate of the fluid stream, the radius of curvature (R), and height- to-width aspect ratio (H / W) are configured for size base separation of particles.

17. The method as claimed in claim 16, wherein the sample particles and waste particles are collected separately from at least one sample outlet (108) and at least one waste outlet (112) respectively.

18. The method as claimed in claim 16, wherein the method comprising, introducing a sheath fluid through at least one sheath fluid inlet (110) concurrently with the sample (102).

19. The method as claimed in claim 16, wherein the total flow rate through the microchannel (106) is in a range of about 30 pL / min to 600 pL / min.

20. The method as claimed in claim 16, wherein the separation is carried out as a continuous flow process.

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