Method and device for electroseparation of charged particles

Abstract

An electroseparator is provided comprising an injection portion having a first inlet configured to receive a sample solution at a sample solution flow rate, the sample solution comprising at least one target charged particle; a separation portion fluidly connected to the injection portion; a collection portion fluidly connected to the separation portion the collection portion comprising a first outlet configured to dispense the at least one target particle and a second outlet configured to dispense a carrier solution; an anode end; a cathode end opposite the anode end; the cathode end electrically coupled to the anode end for delivering a constant electric field across the separation portion. Also provided is a method of separating charged particles comprising providing a sample solution having a flow rate, comprising at least one target charged particle and at least one non-target charged particle, and applying a constant electric field across the sample solution.

Description

Method and Device for Electroseoaration of Charged ParticlesFIELD

[0001] The present disclosure relates generally to electroseparation.BACKGROUND

[0002] Current approaches to electroseparation typically comprise the superimposition of an electrical field onto a conventional dead-end filtration setup. In particular, a filtration chamber is provided and an electrical field is then applied for inducing electrophoretic flux of charged particles towards an oppositely charged electrode. Such systems typically apply a constant applied voltage.SUMMARY

[0003] The present disclosure integrates a uniform electrical field within a bifurcating microchannel that may separate vesicles from unwanted particles, such as proteins, across a laminar interface in a pellet-free manner by targeting the electrical properties of particles. This approach may have higher recovery yield and may be faster and more automated than ultracentrifugation.

[0004] Currently available electrical field-based platforms used for isolation of charged particles, for example extracellular vesicles, utilize constant applied voltage (Vc), that may experience voltage drops (VDroP) of up to 95% depending on the duration of the isolation period. Voltages below certain thresholds may reduce the ability to fine-tune parameters, or tunability, for particular isolation targets. This may, in turn, negativity impact isolation efficiencies. To provide voltages that remain sufficiently high, even with a voltage drop, most current electrical field-based isolation platforms work under high applied voltages (for example, 100 V to 500 V). However, applying a high voltage can create issues that hinder device performance. For example, it can result in bubble generation which negatively impacts isolation efficiency in micron-scale fluidic channels. Using high voltage can also add significant complexity when attempting to tune device operation for different types of samples.

[0005] The method and device disclosed herein may avoid the requirement to apply constant applied voltage, instead utilizing constant electrical current (lc) to provide a stable andefficient working condition within the device to separate target charged particles, such as populations of extracellular vesicles, including exosomes.

[0006] In one aspect is provided an electroseparator comprising: an injection portion having a first inlet configured to receive a sample solution at a sample solution flow rate, the sample solution comprising at least one target charged particle; a separation portion fluidly connected to the injection portion; a collection portion fluidly connected to the separation portion the collection portion comprising a first outlet configured to dispense the at least one target particle and a second outlet configured to dispense a carrier solution; an anode end;

[0007] a cathode end opposite the anode end; the cathode end electrically coupled to the anode end for delivering a constant electric field across the separation portion.

[0008] In one embodiment, the constant electric field is between about 6 V / cm and about 9 V / cm.

[0009] In another embodiment, the electroseparator further comprises a second inlet configured to receive a buffer solution at a buffer solution flow rate.

[0010] In another embodiment, the buffer solution flow rate is about two or more times that of the sample solution flow rate.

[0011] In another embodiment, the first inlet and the second inlet converge to form a channel.

[0012] In another embodiment, the first inlet and the second inlet are each configured for producing laminar flow in the channel.

[0013] In another embodiment, the channel splits to form the first outlet and the second outlet.

[0014] In another embodiment, the first outlet and the second outlet are each configured for producing laminar flow for reducing convective mixing of the target charged particle and the carrier solution.

[0015] In another embodiment, the channel is about 1.2 mm in width, about 0.15 mm in height, and about 35 mm in length.

[0016] In another embodiment, the electroseparator further comprises a filter positioned between the injection portion and the separation portion for filtering the sample solution and the buffer solution.

[0017] In another aspect is provided a method for separating charged particles from a sample solution comprising: providing a sample solution having a flow rate, the sample solution comprising at least one target charged particle and at least one non-target charged particle, the at least one non-target charged particle having a different charge to the at least one target charged particle; applying a constant electric field across the sample solution for separating the at least one target charged particle from the at least one non-target charged particle.

[0018] In one embodiment, the method further comprises providing a buffer solution having a flow rate, the buffer solution flowing lateral to the sample solution for establishing laminar flow.

[0019] In another embodiment, the electric field is from about 6 V / mm to about 9 V / mm.

[0020] In another embodiment, the method further comprises selecting the electric field according to one or more properties of the sample solution or according to one or more electrical properties of the at least one target charged particle.

[0021] In another embodiment, the one or more properties of the sample solution is selected from the group consisting of: pH, ionic strength, electrical conductivity, surface charge density of the at least one target charged particle, zeta potential of the at least one target charged particle, and electrical conductivity of the at least one target charged particle.

[0022] In another embodiment, the at least one target charged particle is from about 4 nm to about 200 pm in diameter.

[0023] In another embodiment, the at least one target charged particle is from about 20 nm to about 5 pm in diameter.

[0024] In another embodiment, the target charged particle is selected from the group consisting of: extracellular vesicles, proteins, DNA, and RNA.

[0025] In another embodiment, the target charged particle is: a natural macromolecule, such as a protein, a lipid, a nucleic acid, or other biomolecule or bioactive molecule; a synthetic macromolecule, such as a nanoplastic, a microplastic, graphene, or a carbon nanotube; or substances and structures composed of macromolecular units, including extracellular vesicles, viruses, vaccine / drug delivery systems, and both eukaryotic and prokaryotic cells.

[0026] In another embodiment, the target charged particle is an exosome.

[0027] In another embodiment, the method further comprises removing particles greater than about 450 nm in diameter from the electrolyte solution prior to contacting the electrolyte solution to the feed side of the filter.

[0028] In another embodiment, the buffer solution flow rate is at least about twice that of the sample solution flow rate.

[0029] In another aspect is provided an electroseparator comprising: an anode end; a cathode end opposite the anode end for establishing an electric field; at least one filter disposed between the anode and the cathode end, the at least one filter defining a plurality of parallel channels; the anode and the cathode end electrically coupled to deliver a constant electric current across the at least one filter.

[0030] In another embodiment, each of the at least one filter comprises a porous membrane.

[0031] In another embodiment, each of the porous membranes comprises pores sized about 30 nm to about 5 pm.

[0032] In another embodiment, each of the at least one filters comprises pores sized about 30 nm to about 40 nm.

[0033] In another embodiment, the plurality of parallel channels are each configured to receive fluid.

[0034] In another embodiment, the plurality of parallel channels each terminate in an outlet for draining fluid.

[0035] In another embodiment, the plurality of channels comprises at least a first channel for loading a sample, and a second channel for receiving a filtrate via the at least one filter.

[0036] In another aspect is provided a method for separating charged particles from an electrolyte solution comprising: providing an electrolyte solution comprising at least one target charged particle; providing a filter sized to separate the at least one target charged particle from the electrolyte solution, the filter having a feed side and a filtrate side opposite the feed side; applying an electric field across the filter; contacting the electrolyte solution to the feed side of the filter; and applying a constant electrical current across the filter for separating the at least one target charged particle from the electrolyte solution across the filter.

[0037] In an embodiment, the method, further comprises contacting a buffer solution to the filtrate side of the filter for receiving filtrate.

[0038] In another embodiment, the filtrate comprises the at least one target charged particle.

[0039] In another embodiment, the electric field is from about 1 V / mm to about 20 V / mm.

[0040] In another embodiment, the method further comprises selecting the constant electrical current according to one or more properties of the electrolyte solution or according to one or more electrical properties of the at least one target charged particle.

[0041] In another embodiment, the one or more properties of the electrolyte solution is selected from the group consisting of: pH, ionic strength, electrical conductivity, surface charge density of the at least one target charged particle, zeta potential of the at least one target charged particle, and electrical conductivity of the at least one target charged particle.

[0042] In another embodiment, the at least one target charged particle is from about 4 nm to about 200 pm in diameter.

[0043] In another embodiment, the at least one target charged particle is from about 20 nm to about 5 pm in diameter.

[0044] In another embodiment, the at least one target charged particle is from about 30 nm to about 200 nm in diameter.

[0045] In another embodiment, the target charged particle is: a natural macromolecule, such as a protein, a lipid, a nucleic acid, or other biomolecule or bioactive molecule; a synthetic macromolecule, such as a nanoplastic, a microplastic, graphene, or a carbon nanotube; or substances and structures composed of macromolecular units, including extracellular vesicles, viruses, vaccine / drug delivery systems, and both eukaryotic and prokaryotic cells.

[0046] In another embodiment, the target charged particle is selected from the group consisting of: extracellular vesicles, proteins, DNA, and RNA.

[0047] In another embodiment, the target charged particle is an exosome.

[0048] In another embodiment, the method further comprises removing particles greater than about 450 nm in diameter from the electrolyte solution prior to contacting the electrolyte solution to the feed side of the filter.

[0049] In another embodiment, the electrolyte solution and the buffer solution have a flow rate of about 10 pL / min to about 30 mL / min.

[0050] In another embodiment, the electrolyte solution and the buffer solution have an ionic strength of about 0.001 mS / cm to about 25 mS / cm.BRIEF DESCRIPTION OF THE FIGURES

[0051] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0052] FIG.1 depicts a schematic of an electrofiltration platform according to this disclosure;

[0053] FIG. 2 depicts resistor-capacitor modeling of the electrofiltration platform of FIG.1 accommodating an electrolyte solution between two electrodes under the effect of an applied electric field;

[0054] FIG. 3 depicts resistor-capacitor modeling of the electrofiltration platform of the prior art with constant applied voltage;

[0055] FIG. 4 depicts resistor-capacitor modeling of the electrofiltration platform of FIG.1 with constant applied current;

[0056] FIG. 5 depicts an example of a fabricated platform of FIG.1 ;

[0057] FIG. 6 depicts a schematic of an electrode-electrolyte interface; and

[0058] FIG. 7 depicts a simulated electrical potential distribution in an electrolyte between two electrodes.

[0059] FIG. 8 depicts the performance of an electroseparator according to the present disclosure operating under (a) constant applied voltage and (b) constant applied current. Protein concentration at the outlets of the device (Ch#2: outlet channel 2, Ch#3: outlet channel 3) was measured over time.

[0060] FIG. 9 depicts that electrical conductivity of biological solutions, such as culture supernatant containing extracellular vesicles, may be dependent on the volume fractions of charged particles.

[0061] FIG. 10 depicts that exosomes collected after isolation using an electrofiltration technique may be completely recovered from the device.

[0062] FIG. 11 shows Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) analyses of the isolated particle fractions obtained from the electroseparator to determine the concentration of particles expressing exosome-specific markers CD63, CD81 , and CD9. (a) Particle fractions recovered from outlet channel 2 (Ch2) and channel 3 (Ch3), as depicted schematically in FIG. 1 , indicate that exosomes may migrate from channel 2 to channel 3 after the application of an electrical field, (b) A scatter plot of fluorescence intensity of particles collected from channel 3 suggests that the average size of the captured particles bound to CD63 antibodies (normalized to non-specific binding of proteins bound to mouse IgG, MlgG) was less than 60 nm, which may suggest the existence of exosomes. (c) The measurement of exosomal content in the samples collected from channel 2 (Ch2) and the culture supernatant injected into the device indicate that exosomes remained within channel 2 when no electrical force was applied to push particles to translocate towards channel 3.

[0063] FIG. 12 shows the characterization of isolated exosome fractions: Dynamic light scattering of isolated exosome fraction (a representative sample with three consecutive runs) using (a) ultracentrifugation and (b) electro- filtration technique. Ultracentrifugation is a widely used method for isolation of exosomes, and dynamic light scattering size characterization of isolated exosomes displayed an average diameter of 230 nm (Polydispersity Index = 0.3) with two picks at 60 nm and 260 nm. This indicated that ultracentrifuged isolated exosomes were co-purified with some portion of particles sized out of the range of exosomes suggesting the presence of impurities such as protein aggregates and cluster of larger vesicles. In contrast, size distribution of particles isolated by electro-filtration technique (b) represented a clean peak size at 160 nm, strongly resembling size characteristics for exosomes with minimal copurification of impurities, (c) Transmission electron microscopy of the exosome fraction displayed nanostructures with a cup-shaped morphology. The sample purified using the electrofiltration technique is relatively clean compared to ultracentrifugation, as it may effectively separate and remove unwanted materials based on their size and charge.

[0064] FIG. 13 shows cell culture and SP-IRIS analyses of culture supernatant. Phase contrast microscopy image of (a) mesenchymal stem cells and (b) adenocarcinomic human alveolar basal epithelial cells (A549) on day 3, at 10x magnification (scale bar: 200 pm), along with colocalization charts for particles within culture supernatant bound to CD63 antibodies on ExoView chips, normalized to non-specific binding of proteins bound to mouse IgG.

[0065] FIG. 14 shows isolation and characterization of small EV fractions from the culture supernatant of mesenchymal stem cells (MSCs) and adenocarcinomic human alveolar basal epithelial cells (A549). (a) Schematic representation of the procedure utilized for obtaining small EV fractions from culture supernatant using ultracentrifugation. Obtained pellet from 20 mL culture supernatant of two experimental groups: (b) MSC culture and (c) A549 culture. Colocalization chart for each experimental group represents the isolated particle fractions bound to CD63 antibodies on ExoView chips. Inserts (i) for each experimental group represents transmission electron microscopy of isolated small EV. Inserts (ii) for each experimental group represents the scatter plot of fluorescence intensity of particles bound to CD63 antibodies on ExoView chips, normalized to non-specific binding of proteins bound to mouse IgG (MlgG). (d) depicts a graph of intensity by diameter of MSC-derived small EVs resuspended in PBS on the left and a graph of intensity by diameter of A549-derived small EVs resuspended in PBS on the right.

[0066] FIG. 15 shows pellet-free and continuous separation of small EVs in a microchannel under the effect of an externally applied electrical force, (a) Schematic of the proposed technique for separation of small EVs (orange points) from proteins (green points), and (b) fabricated platform (total length of 35 mm for main channel, height of 0.15 mm, and width of 1.2 mm). Sample solution (mixture of proteins and small EVs resuspended in buffer saline) is injected into the microchannel (Inlet II) and continuously flowing while being confined by a buffer flow injected into the microchannel (Inlet I). This platform works in conjunction with a uniform electrical field to accelerate the translocation of small EVs (orange points) toward Outlet I (positive electrode side). Unwanted particles (green points) are collected from Outlet II. Insert (i) shows a brightfield microscopy image of the microchannel and insert (ii) represents a scanning electron microscopy of the cross-section of the microchannel.

[0067] FIG. 16 shows the effect of buffer flow force on particle confinement during translocation in the absence of an electrical force. Simulated translocation of particles (a) when no buffer was injected into the microchannel, and (b) when the buffer flow rate was twice that of the sample flow rate, (c) The protein content of samples collected from two outlets under different buffer flow forces: no buffer (Buffer Flow = 0), equal buffer and sample flow rates (Buffer Flow = X), and buffer flow rate is twice that of sample flow rate (Buffer Flow = 2X). Sample solution injected into the Inlet II is phosphate buffer saline containing human albuminproteins (1 mg / mL). (d) Collected samples from two outlets under different buffer flow forces, stained with fluorescent CD63 antibodies. Red dots represent particles bound to CD63 antibodies run on the ExoView. Sample solution injected into the Inlet II is phosphate buffer saline containing small EVs derived from mesenchymal stem cells (small EVs obtained from 5 mL of culture supernatant).

[0068] FIG. 17 shows microfluidic electrophoretic platform for separation of small EVs from proteins and characterization of the particle fractions obtained from the fabricated platform (a) The schematic of the sample preparation for processing using the fabricated platform and ultracentrifugation. Compared to proteins, small EVs with a greater surface charge experience a greater electrical force and migrate faster, thereby being collected from Outlet I in the fabricated platform. Main portion of proteins along with neutral and positively charged impurities are collected from Outlet II (b) Measured electrophoretic mobility of HSA proteins and small EVs derived from mesenchymal stem cells (MSC_sEV) and adenocarcinomic human alveolar basal epithelial cells (A549_sEV), dispersed in diluted phosphate buffer saline (PBS) with an ionic strength of 17 mM, and pH of 7 (*P-value < 0.05). (c) Protein content of the samples collected from Outlet I, where two experimental groups of sample solutions are introduced into the device through Inlet I. The solutions contain HSA proteins at a concentration of 5 mg / mL mixed with small EV fractions obtained from culture supernatant of mesenchymal stem cells (MSC_sEV) and adenocarcinomic human alveolar basal epithelial cells (A549_sEV). (d) Intensity-based size distribution (measured using dynamic light scattering) of the collected samples from Outlet I when two experimental groups of sample solutions, including MSC_sEV (blue and black lines) and A549_sEV (red and green lines), are introduced into the device and being processed under different applied electrical forces, (e) Quantified particles expressing exosome-specific markers, collected from Outlet I under different applied electrical forces. Colocalization pie charts represents particles bound to CD63 run on the ExoView, normalized to non-specific binding of proteins bound to mouse IgG (MlgG).

[0069] FIG. 18 shows an evaluation of microfluidic device performance for separation of small EVs from protein impurities, (a) Yield of recovery and (b) purity for separated small EV fractions using ultracentrifugation (UC) and the proposed microfluidic device (MD) under different applied electrical force, for two experimental groups: small EVs derived frommesenchymal stem cells (MSC_sEV) and adenocarcinomic human alveolar basal epithelial cells (A549_sEV). (c) Quantification of total recovered particles expressing exosome-specific markers of CD9, CD63, and CD8, normalized to non-specific binding of proteins bound to mouse IgG (MlgG).

[0070] FIG. 19 shows a colocalization pie chart for fresh culture medium and culture supernatant collected from mesenchymal stem cells (MSCs) and adenocarcinomic human alveolar basal epithelial cells (A549), presenting particle fractions bound to CD63, CD81 , and CD9 antibodies on ExoView chips.

[0071] FIG. 20 shows a colocalization pie chart for isolated small extracellular vesicles (EVs) from culture supernatant collected from mesenchymal stem cells (MSCs) and adenocarcinomic human alveolar basal epithelial cells (A549), presenting particle fractions bound to CD81 , and CD9 antibodies on ExoView chips.

[0072] FIG. 21 shows the effect of buffer flow force on particle confinement during translocation in the absence of an electrical force where protein solution is injected into the device (a) with buffer flow 1x and (b) with buffer flow 2x.

[0073] FIG. 22 shows the effect of buffer flow force on particle confinement during translocation in the absence of an electrical force.

[0074] FIG. 23 shows protein content of the samples collected from Outlet II, where two experimental groups of sample solutions are introduced into the device through Inlet I.

[0075] FIG. 24 shows quantified particles expressing exosome-specific markers, collected from Outlet I under different applied electrical forces. Colocalization pie charts represents particles bound to CD81 and CD9 run on the ExoView, normalized to non-specific binding of proteins bound to mouse IgG (MlgG).

[0076] FIG. 25 shows a schematic of the proposed platform which is composed of one adhesive layer (patterned with a microchannel with two bifurcations at both ends of the main channel) and two polydimethylsiloxane (PDMS) layers.

[0077] FIG. 26 shows water contact angle measurement, (a) Contact angle of a water drop on a solid surface (0) is defined using the Young’s equation (ys= ysi+ yi cos 0), where yi, Ysi, and Ys are liquid surface tension, solid-liquid interfacial tension, and solid surface tension, respectively. The measured water contact angle determines whether the surface is hydrophilicor hydrophobic, (b) Measured contact angles of polymethylmethacrylate (PMMA) and Polydimethylsiloxane (PDMS) substrates with and without oxygen plasma treatment.DETAILED DESCRIPTION

[0078] Generally, the present disclosure provides an electroseparation device and method that avoids the requirement to apply constant applied voltage and avoids the requirement of the application of tags or labels on isolation targets of interest. Isolation targets of interest may be, for example, biologically derived or not biologically derived.

[0079] Various compositions, systems and methods will be described herein to provide an example of at least one embodiment of the claimed subject matter. No embodiment described herein limits any claimed subject matter and any claimed subject matter may cover compositions, systems, devices and methods that differ from those described below. The claimed subject matter is not limited to compositions, systems, devices and methods having all of the features of any one composition, system, device or method described below or to features common to multiple or all of the compositions, systems, devices and methods described below. It is possible that any composition, system, device or method described below is not an embodiment of any claimed subject matter. Any subject matter that is disclosed in a composition, system, device or method described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicants, inventors or owners do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.

[0080] Furthermore, it will be appreciated that for simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein may be practiced without these specific details. In other instances, well- known methods, procedures and components have not been described in detail so as not to obscure the embodiments described herein. Also, the description is not to be considered as limiting the scope of the embodiments described herein.

[0081] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0082] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0083] As used herein, the wording “and / or” is intended to represent an inclusive-or. That is, “X and / or Y” is intended to mean X or Y or both, for example. As a further example, “X, Y, and / or Z” is intended to mean X or Y or Z or any combination thereof.

[0084] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “formed from”, “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0085] Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively.

[0086] The following examples are intended to illustrate certain exemplary embodiments of the present disclosure. However, the scope of the present disclosure is not limited to the following examples.

[0087] EXAMPLES

[0088] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.

[0089] EXAMPLE 1. Electroseparation of Charged Particles

[0090] FIG. 1 , shows the device may comprise multiple parallel channels, and may be configured to stack filters of the same or varying sizes to separate particles from a fluid such as an electrolyte solution received by the channels.

[0091] FIG. 2 shows an electric circuit model composed of resistors and capacitors may be used when the describing a device accommodating a fluid such as an electrolyte solution under the effect of an applied electric field. FIG. 2 shows resistor-capacitor modeling of the electrofiltration platform of FIG.1 accommodating an electrolyte solution between two electrodes under the effect of an applied electric field.

[0092] As shown in FIG. 3, current electrical-based systems may utilize constant applied voltage. The effective voltage (VEff) within the isolation platform may be lower than the applied voltage (VEff < Vc) due to the naturally occurring voltage-drop that may result from electrochemical reactions at the electrode-electrolyte interface and formation of an electric double layer (EDL) at the surface of electrodes (FIG. 6 and FIG. 7). The effective voltage, and accordingly the effective electric field, drops to 5% or less of its initial value (FIG. 4). By contrast, constant current power sources (lc= V / R) may be able to adjust effective voltage (VEff) in response to the resistance (REiectroiyte) of a fluid such as a biological sample or electrolyte solution injected into the device to ensure that the applied electrical field (Ec) remains constant during the isolation procedure (FIG. 2). This may be especially important, as the resistance of a sample changes with time when exposed to an electric field. Constant voltage devices may be unable to respond similarly to the dynamic nature of biological sample resistance.

[0093] The method and electroseparator disclosed herein may allow for the continuous and label-free isolation of charged particles, and particularly extracellular vesicles (30 nm < diameter < 5 pm) and more specifically exosomes (30 nm < diameter < 200 nm). The device may be an electrical field-based microfluidic system to separate charged particles from electrolyte solutions, including target population of extracellular vesicles (for example, exosomes), based on their size and electrical properties by combining filtration and electrical force in an integrated microfluidic platform (FIG. 1 and FIG. 2).

[0094] The technology may be label-free as it may not require the presence of foreign molecules (for example, antibodies) to capture a target population of suspended extracellular vesicles, thereby eliminating the complicated, time-consuming, and expensive procedures used in label-based techniques. The electrical properties of the target particles and the electrolyte solutions in which they are suspended, may be parameters that directly impact the efficiency of isolation procedures in any electrical field-based platforms.

[0095] In electrical-based systems that utilize constant applied voltage, the effective voltage within the isolation platform may be lower than the applied voltage (VEff « Vc) due to the naturally occurring voltage-drop that results from electrochemical reactions at the electrode-electrolyte interface and formation of an electric double layer (EDL) at the surface of electrodes (FIG. 4 and FIG. 5).

[0096] The effective voltage, and accordingly the effective electric field, may drop to 5% or less of its initial value (FIG. 4 and FIG. 5). By contrast, constant current power sources (lc= V / R) may be able to adjust effective voltage (VEff) in response to the resistance ( Eiectroiyte) of biological samples injected into the device to ensure that the applied electrical field (Ec) remains constant during the isolation procedure (FIG. 2). This may be especially important, as the resistance of a sample may change with time when exposed to an electric field. Constant voltage devices are unable to respond similarly to the dynamic nature of biological sample resistance.

[0097] Shown in FIG. 8 is the performance of an electroseparator according to the present disclosure operating under (a) constant applied voltage and (b) constant applied current. Protein concentration at the outlets of the device (Ch#2: outlet channel 2, Ch#3: outlet channel 3) was measured over time. Results indicate that the electroseparator’s performance remained stable in constant current mode compared to the constant voltage mode, which showed fluctuations.

[0098] The electrical conductivity of biological solutions containing charged particles may be a parameter that may determine the required electrical force for operation of the electroseparator. Results indicate that electrical conductivity of biological solutions, such as culture supernatant containing extracellular vesicles, may be dependent on the volume fractions of charged particles as shown in FIG. 9.

[0099] The assessment of the device's performance indicates that exosomes collected after isolation using an electroseparation technique may be completely recovered from the device, as experiments showed no particles remained attached to the membrane after washing (FIG 10).

[0100] FIG. 11 shows Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) analyses of the isolated particle fractions obtained from the electroseparator to determine the concentration of particles expressing exosome-specific markers CD63, CD81 , and CD9. (a) Particle fractions recovered from outlet channel 2 (Ch2) and channel 3 (Ch3), as depicted schematically in FIG. 1 , indicate that exosomes may migrate from channel 2 to channel 3 after the application of an electrical field, (b) A scatter plot of fluorescence intensity of particles collected from channel 3 suggests that the average size of the captured particles bound to CD63 antibodies (normalized to non-specific binding of proteins bound to mouse IgG,MlgG) was less than 60 nm, which may suggest the existence of exosomes. (c) The measurement of exosomal content in the samples collected from channel 2 (Ch2) and the culture supernatant injected into the device indicate that exosomes remained within channel 2 when no electrical force was applied to push particles to translocate towards channel 3.

[0101] FIG. 12 shows the characterization of isolated exosome fractions: Dynamic light scattering of isolated exosome fraction (a representative sample with three consecutive runs) using (a) ultracentrifugation and (b) electro- filtration technique. Ultracentrifugation is a widely used method for isolation of exosomes, and dynamic light scattering size characterization of isolated exosomes displayed an average diameter of 230 nm (Polydispersity Index = 0.3) with two picks at 60 nm and 260 nm. This indicated that ultracentrifuged isolated exosomes were co-purified with some portion of particles sized out of the range of exosomes suggesting the presence of impurities such as protein aggregates and cluster of larger vesicles. In contrast, size distribution of particles isolated by electro-filtration technique (b) represented a clean peak size at 160 nm, strongly resembling size characteristics for exosomes with minimal copurification of impurities, (c) Transmission electron microscopy of the exosome fraction displayed nanostructures with a cup-shaped morphology. The sample purified using the electrofiltration technique is relatively clean compared to ultracentrifugation, as it may effectively separate and remove unwanted materials based on their size and charge.

[0102] Based on an analysis of the collected computational and experimental data, considerations have been identified that may provide a tunable platform capable of isolating target populations of charged particles in the presence of a tightly controlled electrical field force. These considerations may increase isolation efficiency in terms of sample processing time, target population purity and target population recovery yield.

[0103] EXAMPLE 2. Pellet-Free Separation of Extracellular Vesicles Using Electrophoretic Force

[0104] Small extracellular vesicles (EVs), commonly known as exosomes, are small membrane-enclosed structures secreted by cells and can be found in biological fluids. They contain biomolecules and bioactive molecules that may have potential as diagnostic and therapeutic tools in clinical applications. However, many studies on clinical applications of these vesicles fail to fully utilize their potential due to difficulties in separating them from unwanted biological components present in bodily fluids. Although ultracentrifugation is themost prevalent method for isolation of these vesicles from biological fluids, it is a timeconsuming pellet-based technique that results in low yield and vesicle damage, which presents significant challenges to the clinical utility of small extracellular vesicles.

[0105] Extracellular vesicles (EVs) are heterogeneous populations of nano to microsized (30 nm -2 pm in diameter) membrane-enclosed structures which are secreted by cells and can be found in various biological fluids including cell culture supernatant and bodily fluids such as blood and urine. Among the different types of EVs, exosomes, which are small EVs ranging from 30 nm to 150 nm in diameter, have garnered significant attention for their role in various physiological and pathological processes within living systems. These vesicles are structures with a lipid bilayer membrane, secreted by almost every known type of cell, that encapsulates a bioactive cargo in the form of proteins, lipids, and genetic information such as nucleic acids. Initially believed to only serve as a mechanism for cells to eliminate waste materials, accumulating evidence suggests that small EVs are part of a complex system of intercellular communication. Significant strides have been made in this field, acknowledging the potential of small EVs as diagnostic and therapeutic tools in clinical applications. However, the field continues to face a significant hurdle in the development and translation of EV-based technologies for clinical applications, mainly due to an inability to efficiently isolate small EVs from biological fluids.

[0106] Ultracentrifugation (UC) is the most prevalent method utilized for isolation of small EVs. However, it is a time-consuming process and demands highly skilled technicians, as well as large sample volumes that exceed the typical liquid biopsies used in clinical applications. In addition, UC requires the high spin speeds that can potentially damage small EVs and decrease the viability of their content for downstream analysis. Another significant constraint that undermines downstream analyses is the low yield of small EVs isolated using UC. This method causes small EVs to sediment at the bottom wall of the centrifuge tube and form a pellet. The resulting pellet is typically non-compact which may slide down the tube wall and mix with the supernatant again, causing a significant loss of the small EV fraction. Additionally, harvesting the pelleted particles from the tube wall typically leads to further loss of the small EVs as well as damaging their integrity, significantly reducing the final yield of isolated population of small EVs. These shortcomings render UC unsuitable for clinical sampleprocessing and highlight the importance of developing pellet-free methods that can offer higher yield, faster processing time, and better preservation of EV integrity.

[0107] The majority of current microfluidic platforms for the isolation of small EVs are label-based techniques utilizing foreign molecules (e.g., antibodies) as a label to capture these vesicles. These capture molecules may have detrimental effects on the native characteristics of the small EVs, thereby being incompatible with the processing of clinical samples. In addition, label-based methods are generally complicated, costly due to expensive labelling procedures, and time-consuming.

[0108] Existing platforms are plagued by several limitations that hinder their capability to isolate small EVs effectively. In particular, most current label-free methods are not designed specifically for manipulation of nanoscale particles such as small EVs but rather are often modified versions of cell-oriented sorting systems.

[0109] Extensive research on label-free isolation of nanoparticles within microfluidic systems, including sieving, deterministic lateral displacement, field flow fractionation, viscoelastic, and acoustic techniques, has led to a broad recognition that isolating small EVs is considerably more challenging than larger particles such as cells. This is primarily due to the dominant effects of diffusion and Brownian motion at the nanoscale. For example, for target particles at the microscale, such as cells, fluid flow in a microchannel exerts a strong convective force that dominates over diffusion effects. However, for nanoscale particles like small EVs, diffusion has a considerable impact on particle displacement within the microchannel.

[0110] To distinguish small EVs from unwanted particles that partially overlap in size and density, such as naturally occurring proteins in biological fluids, isolation parameters may be combined with other physical characteristic of nanoparticles to minimize diffusion effects during the separation procedure within a microchannel. One of these physical characteristics may be the electrical surface charge which has rarely been studied but can distinguish small EVs from impurities such as proteins.

[0111] Current electrical-based microfluidic platforms suffer from several significant limitations. One of the major drawbacks is the requirement for high applied voltages (typically ranging from 100-500 volts) which can cause denaturation of biological components and bubble generation that impede device performance in small-scale fluidic channels likemicrochannels. Consequently, the accuracy and reliability of results can be compromised, and isolation efficiency can be hindered.

[0112] The present disclosure relates to a strategy that utilizes a bifurcating microchannel integrated with an electrical force, under the effect of an applied voltage of less than 2 volts, to separate small EVs from impurities like proteins across a laminar interface in a label-free and pellet-free manner. This technique may target the electrical properties of particles to discriminate between small EVs and proteins without using any labels. Small EVs derived from mesenchymal stem cells (MSCs) and adenocarcinomic human alveolar basal epithelial cells (A549) were the target particles in this study, suspended in phosphate-buffered saline (PBS) solution containing human insulin albumin (HSA) proteins. In this study, HSA protein was used as a candidate reference for the impurity because it is the most prevalent protein found in biological fluids including cell culture supernatant. The separated small EV fractions using the proposed method in this study were characterized based on their purity and recovery yield, and device performance was compared to UC.

[0113] Operation of Pellet-Free Separation Device

[0114] A unique microfluidic platform, designed with a bifurcating microchannel and integrated with an applied electrical field (E-field), is disclosed herein to demonstrate a continuous and pellet-free separation of small EVs from impurities such as proteins across a laminar interface. FIG. 15 shows the fabricated platform and a representative schematic.

[0115] The disclosed platform included three different regions: (i) injection region, (ii) separation region, and (iii) collection region. The platform accepted two different laminar streams of buffer solution and sample solution (including small EVs and proteins). Flow may remain laminar in the separation region (Reynolds number < 0.05) due, for example, to the small dimensions of main channel (total length of 35 mm, width of 1.2 mm, and height of 0.15 mm), thereby the two flows do not mix.

[0116] In addition, the inlet and outlet were considered with a design to avoid convective mixing at the contact point of two streams in the initiation and termination of the main channel, for example, a curved design. FIG. 15A demonstrates that a mixture of small EVs and proteins was injected at Inlet I, while a buffer solution was introduced into the system through Inlet II to confine all particles to one lateral side of the main channel. This step was necessary to prevent particle translocation from the lower stream to the upper stream owingto convection and diffusion effects. To direct small EVs towards Outlet I, a uniform E-field was applied to the separation region through two electrodes placed on the two lateral sides of the main channel.

[0117] Under the effect of a uniform E-field, when the field (E) is constant in magnitude and direction between two oppositely charged plates, the electrical force (F) on a charged particle (q) is given by Equation 1 so that uncharged particles are not influenced by the applied E-field. Given that the target particles in this study are small EVs with negative surface charge, they repel the negative electrode and move towards the positive electrode, thereby being collected from Outlet I (FIG. 15A).

[0118] By contrast, positively charged impurities are practically kept away from the small EVs as they travel through the channel. Although this assists in reducing the amount of impurity that would be copurified with small EVs, it should be considered that contaminants like proteins, possessing negative surface charge, migrate along with small EVs and contaminate purified EVs. Since the migration velocity (v) of a charged particle depends on both the strength of the applied E-field (E) and the particle's electrophoretic mobility (p), as formulated in Equation 2, separation efficiency can be improved by controlling particle migration through the magnitude of the applied E-field. Therefore, the electrophoretic mobility of a charged particle can serve as a deterministic factor in determining the required magnitude of the applied E-field for particle translocation within the fabricated platform.

[0119] F=qE (Equation 1)

[0120] v=pE (Equation 2)

[0121] To determine the minimum required buffer fluid flow to thoroughly confine all particles towards the sidewall of the microchannel, translocation of particles in the absence of the electrical force was simulated using COMSOL Multiphysics. The result showed that, in the absence of buffer fluid flow, particles passed through the separation region and divided into two streams just before the Y-shaped junction at the termination of the main channel (FIG. 16A). As shown in FIG 16B, it was demonstrated that the minimum buffer fluid flow rate (injected via Inlet I) required to drive particles towards Outlet II would be twice that of the sample fluid flow rate (injected via Inlet II) in the absence of an electrical force. This finding was experimentally validated when a blue dye solution was introduced into the device under similar simulated conditions (FIG. 16A and FIG. 16B, right).

[0122] Device operation was further examined by introducing protein solutions with various concentration into Inlet I (FIG. 21). FIG. 16C shows the measured protein content of the samples collected from Outlet I and Outlet II under different buffer flow conditions with no applied electrical forces. The protein solution introduced into Inlet I had a concentration of 1 mg / mL. The results confirmed that all protein particles were released from Outlet II when sample and buffer flow rates were 10 pL / min and 20 pL / min, respectively. Under similar experimental conditions, small EV sample solutions were injected into the device, and the obtained particle fractions from Outlet I and Outlet II were stained with fluorescent antibodies to visualize small EVs bound to exosome-specific markers. As shown in FIG. 15D, there were no EVs in Outlet I to be captured on ExoView chips through antibodies targeting exosome CD63 marker when buffer flow (Inlet I) was 20 pL / min (small EV sample flow from Inlet II was 10 pL / min). Representative plots depicting particles bound to CD81 and CD9 antibodies on ExoView chips are provided in Example 3 and FIG. 22.

[0123] All these findings indicated that buffer fluid flow may be at least two times of sample fluid flow to facilitate all particles being perfectly confined to the sidewall in the absence of an electrical force. The experiment was repeated at various buffer and sample flow rates, and the results consistently showed that a minimum ratio of two between buffer and sample streams was necessary for efficient particle confinement. To minimize the negative impact of applied voltage, such as bubble generation, without compromising processing time, the experiments were continued with an applied sample flow rate of 10 pL / min. It should be highlighted that the speed of particle translocation across the channel is dependent on the strength of E-field strength (corresponding to applied voltage), which is in turn determined by the sample flow rate. Indeed, particles move faster across the channel as the strength of the E-field increases. However, the time required for a particle to cross the channel must be shorter than the time required for the sample to flow through the channel. Therefore, a higher voltage is necessary as sample flow rate increases, resulting in an increased risk of electrolysis and associated side effects. The experiments showed that a flow rate of 10 pL / min was the maximum flow rate that could ensure the required voltage with minimal side effects of electrolysis and bubble formation within the fabricated platform. While lower flow rates result in lower voltage requirements, they increase processing time.

[0124] A various range of applied electrical forces (6-15 V / cm) was tested in this study for translocation of small EVs. The results showed that the microchannel was clogged when the applied E-field was greater than 15 V / cm due to the excessive generation of bubbles within the microchannel. Therefore, device performance for lateral translocation of small EVs was examined under the effect of an applied E-field < 15 V / cm to ensure a relatively consistent fluid flow through the microchannel enabling the collection of samples from Outlet I for further analysis. It should be noted that preventing bubble generation is not realistic when applying a constant voltage, thereby bubble generation still occurred to some degree at the applied E- field of 13 and 15 V / cm. It should be highlighted that all applied E-fields in this study were less than the critical limit of 104 V / cm to avoid thermal denaturation of biological particles under the impact of the produced Joule heating. The critical limit of E-field (Ec) was defined using Equation 3, in which Vtmand R, are cell transmembrane voltage and cell radius, respectively.

[0125] |Vtm|=1.5R|EC| (Equation s)

[0126] Continuous Separation of Small EVs

[0127] To investigate device performance for a continuous and pellet-free separation of small EVs from protein impurities, a mixture of small EVs (already isolated from culture supernatant using UC) and HSA proteins (5 mg / mL, corresponding to the protein concentration of culture supernatant) was suspended in diluted PBS (17 mM). As shown in FIG. 17A, the resulting sample solution was divided into two individual aliquots for being proceeded by the fabricated device and UC to separate small EV fractions and evaluate device performance. In this configuration, a laminar fluid flowing the microchannel propelled a mixture of particles (proteins and small EVs) through the separation region within the microchannel while sample and buffer streams were injected at the flow rates of 10 pL / min and 20 pL / min, respectively. When there was no electrical force across the microchannel, all particles were confined to one sidewall (adjacent to the negative electrode) of the microchannel and collected from Outlet II. In the presence of an applied E-field, driving a lateral translocation of negatively charged particles across the microchannel, small EVs migrated toward positive electrode and accumulated at Outlet I. Given that proteins also move along small EVs, recovered particle fractions obtained from Outlet I were analysed using SP-IRIS and BOA assay to determine concentration of impurities and quantity of particles expressing exosome-specific markers CD63, CD81 and CD9.

[0128] To experimentally investigate the required electrical force for lateral migration of small EVs across the microchannel, while minimizing translocation of protein, the electrophoretic mobility of HSA proteins and small EVs (derived from MSCs and A549 cells) was measured under similar conditions (ionic strength of 17 mM and pH of 7.5). As shown in FIG. 17B, the distribution of electrophoretic mobility of small EVs collected from culture supernatant confirmed the existence of negatively charged groups on the surface of EVs and agreed reasonably well with the published data. The results showed that small EVs, in both experimental groups (MSCs and A549 cells), had a greater magnitude of electrophoretic mobility compared to the proteins. As shown in FIG. 17B, electrophoretic mobility measurements indicated a mean value of -0.76 pm.cmA / .s for HSA proteins, while small EVs derived from MSCs and A549 cells exhibited a mean value of -1.43 pm.cm / V.s and -1.66 pm.cmA / .s, respectively. Therefore, it was confirmed that small EVs would undergo greater lateral migration than proteins under the same applied electrical force, allowing them to move faster towards Outlet I.

[0129] As shown in FIG. 17C and 17E, all experimental groups of proteins and small EVs experienced a greater lateral translocation at higher levels of E-field. FIG. 17C shows the protein content of each sample collected from Outlet I, indicating that protein concentration increases with E-field strength up to 9 V / cm. Example 3 includes representative plots for the samples collected from Outlet II (FIG. 23). The results showed that the protein concentration of samples collected from Outlet I sharply decreased when the E-field was higher than 9 V / cm because the negatively charged proteins became stuck to the surface of the positive electrode, thereby preventing their recovery from Outlet I (FIG. 17C). This issue becomes more critical when it comes to the recovery of small EVs, as they have higher electrophoretic mobility and are more likely to become stuck at the surface of electrodes when the E-field is higher than 9 V / cm. According to the DLS analysis presented in FIG. 17D, the particles collected from Outlet I under the influence of E-fields of 6 V / cm and 9 V / cm showed a relatively homogeneous size distribution with an average diameter of less than 200 nm (polydispersity index between 0.3 to 0.4), which falls within the standard range for small EVs. However, no significant particles were collected from Outlet I at E-fields below 6 V / cm or above 9 V / cm. This finding was further confirmed by examining the quantity and size distribution of particles expressing exosome surface markers (CD9, CD63, CD81) using SP-IRIS technology. FIG. 17E displays thecolocalization pie charts for each sample collected for Outlet I, indicating the portion of colocalized particles expressing two or more exosome-specific markers. Additionally, it shows quantity of particles expressing exosome-specific markers bound to CD63 antibodies on ExoView chips. Example 3 includes representative plots for the particles bound to CD81 and CD9 antibodies on ExoView chips. (FIG. 24). As depicted in FIG. 17E, the portion of captured particles bound to exosome-specific markers was significantly increased in Outlet I when there was an applied electric field of 6 V / cm and 9 V / cm. This supports the migration of small EVs from the sidewall near the negative electrode, where particles were already confined by the buffer flow force, towards the positive electrode adjacent to Outlet I. As depicted from the colocalization pie charts in FIG. 17E, when the applied E-field was above 9 V / cm, there was no significant particles recovered from Outlet I to express two or more exosome-specific markers.

[0130] These findings confirmed that almost all small EVs were stuck at the surface of the positive electrode when E-field was higher than 9 V / cm, thereby significantly lowering EV recovery at Outlet I (FIG. 17E and FIG. 18A). Indeed, for such a continuous separation technique, the recovery efficiency of the target particles would be limited at higher levels of E- field, where the applied electrical force contributed to sticking the charged particles at the surface of the electrode. As shown in FIG. 18A, recovered particle fractions from Outlet I, in the presence of the applied E-field of 6 V / cm, demonstrated greater quantities of total particles expressing one of the three exosome-specific markers compared to the E-field of 9 V / cm. It was also demonstrated that lower amounts of proteins were copurified with small EVs at Outlet I when the applied E-field was 6 V / cm (FIG. 18B). Although particles could be recovered in greater quantities with higher purity when the applied E-field was 6 V / cm, it does not mean that the recovered particles are all small EVs. As shown in FIG. 18C, relatively greater quantities of recovered particles expressed exosome-specific markers (CD9, CD81 , and CD 63) when the applied E-field was 9 V / cm in both experimental groups of MSC-derived small EVs and A549-derived small EVs. The findings indicate that, when subjected to an applied E-field of 9 V / cm, 33% of the total recovered particles in the A549 experimental group and 27% of the total recovered particles in the MSC experimental group expressed all three markers. In contrast, a lower percentage of recovered particles in the MSC and A549 experimental groups, specifically 19% and 22% respectively, showed expression of all three markers (FIG. 18C).

[0131] As shown in FIG. 18, the proposed technique enabled the separation of small EVs from proteins with higher than 85% purity. Although small EV fractions obtained from UC copurified about 2% of the total proteins that exist in the culture supernatant, it should be highlighted that the EV recovery efficiency using the proposed technique in this study was significantly higher than UC. For instance, in MSC experimental group, the results showed that approximately 84% of small EVs injected into the fabricated platform could be recovered at Outlet I after application of an E-field of 6 V / cm, while UC recovered less than 30% of total particles that exist within culture supernatant (FIG. 18C).

[0132] These findings suggest keeping the applied E-field as low as 6 V / cm to lower copurification of impurities while collecting a significant quantity of small EVs in a continuous and pellet-free manner. However, increasing the magnitude of the applied E-field may provide sufficient electrical force for the translocation of small EVs, as indicated by the increased expression of exosome-specific markers in recovered particles under the effect of an E-field of 9 V / cm. Notably, an in-depth investigation is necessary to determine the potential parameters that impact the required electrical force for translocation of small EVs. Nevertheless, most electrical-based manipulation techniques face limitations due to the lack of validated methods that define the driving forces of processes where the electrostatic contribution of nanosized particles like small EVs is determinative.3 The results of this study highlight the fact that impurities with a negative surface charge like proteins translocate along with small EVs under the effect of an E-filed, thereby finding an optimized working condition in such a microfluidic platform still requires more investigation on the parameters influencing electrophoretic behaviour of both protein and small EVs. To improve the separation efficiency in this technique, it is essential to have knowledge about the electrical properties of small EVs, including net surface charge. Most published studies to date that assessed surface charge of EVs relied on zeta potential which alone does not reflect electrical properties of EVs accurately due to the neglect of ion effects tightly bound to the EV surface. Besides surface charge, considering the electrical conductivity of the sample solution and particle concentration in the sample solution would be crucial for the development of an efficient electrical-based separation techniques.

[0133] Without wishing to be bound by theory, the inventors outline the below general principals believed to contribute to the functionality and advantages of the device of this embodiment.

[0134] Characterization of EVs collected from culture supernatant

[0135] Human adipose-derived MSCs and A549 cells were statically cultured, and FIG. 13A and FIG.13 B 1 show a phase contrast microscopy image of the cells on day 3 at the time of collecting culture supernatant. The collected supernatant was analysed using the SP-IRIS technique to confirm the existence of small EVs within the supernatant. As depicted in the colocalization pie chart (FIG. 13), culture supernatant showed a large degree of colocalized particles expressing two or more exosome-specific markers (CD63, CD81 and CD9) which are endosome-specific tetraspanins. These markers are exosome-specific transmembrane proteins enriched in exosomal membranes, thereby identifying small EVs from other compartments which are non-exosomal. The results indicated a large population of small EVs present within the culture supernatant collected from both experimental groups of cells. All representative plots for the particles within culture supernatant bound to CD63, CD81 and CD9 antibodies on ExoView chips have been reported in Example 3, FIG. 19.

[0136] Using UC (FIG 14A), small EV fractions were isolated from culture supernatant, and subsequently examined by SP-IRIS and DLS. ExoView analysis for exosome-related surface markers (CD81 , CD63 and CD9) confirmed the presence of small EVs in the UC- isolated particle fractions with a large degree of colocalized particles expressing two or more exosome-specific markers (FIG. 14B and FIG 14C). The scatter plot of fluorescence intensity of particles bound to CD63 antibodies on ExoView chips, for both experimental groups of small EVs derived from MSCs and A549 cells, indicated an average diameter smaller than 60 nm for the captured particles which confirmed the existence of small EVs.

[0137] Representative plots for the UC-isolated particle fractions bound to CD81 , and CD9 antibodies on ExoView chips are shown at FIG. 20.

[0138] Analysis of the size distribution of UC-isolated particles from both MSC and A549 culture supernatant using DLS (FIG 14D) revealed the co-isolation of protein aggregates and large compartments with small-EVs. Additionally, the pellet-based nature of UC may result in the formation of significant small EV clusters. For instance, as seen in the representative graph for small EVs derived from A549 cells (FIG. 14), there were three peaks at average diameters of 12 nm (corresponding to protein aggregates co-isolated with small EVs), 60 nm (corresponding to small EV fractions), and 243 nm (corresponding to clusters of small EVs or large compartments co-isolated with small EVs).

[0139] Material

[0140] Polydimethylsiloxane (PDMS) base polymer and curing agent were obtained from Dow Corning Corp (Sylgard 184). Tygon Microbore tubing (outside diameter of 1.52 mm, inside diameter of 0.51 mm) and pressure sensitive adhesive (AR 90880) were purchased from Cole-Parmer (Canada) and Research Adhesive (USA), respectively. Dulbecco's phosphate- buffered saline (10X PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and human serum albumin (HSA, 100 pg / pL) were purchased from Thermo Fisher (Thermo Fisher Scientific, Canada) and Invitrocare (Invitrocare Inc., United States), respectively. Radioimmunoprecipitation assay (RIPA) lysis buffer and protease inhibitors cocktail (Set III, EDTA-Free) were purchased from Sigma-Aldrich (Millipore Sigma, Canada).

[0141] Method

[0142] Electroseparation Device Fabrication. A PDMS-based microfluidic platform was used to fabricate an electro separation device. The PDMS-based microfluidic platform was composed of three layers (FIG. 25); the top and bottom layers were flat PDMS layers, and the middle layer was a double-sided adhesive tape patterned with a bifurcating microchannel (height of 0.15 mm, width of 1.2 mm, and a total length of 35 mm in main channel).

[0143] The device was fabricated using an adhesive-based technique previously developed by the authors in their earlier research.

[0144] FIG. 15 depicts the resulting fabricated device. Aside from its biocompatibility and low fabrication costs, there was a substantial interest in using PDMS to fabricate the device instead of materials like polymethylmethacrylate (PMMA). This interest stemmed from the electrostatic interactions between microchannel surface and biological particles within the sample solutions. While the poor wettability of PDMS surfaces presents a significant drawback that can lead to nonspecific adsorption on the device substrate and cause sample loss, this issue can be readily addressed through oxygen plasma treatment. Additionally, storing PDMS substrates in water after plasma treatment can help delay hydrophobic recovery which beneficial for using the microfluidic device for weeks. FIG. 26 shows water contact angle measurements for PDMS and PMMA substrates before and after plasma treatment. The results indicate that, following plasma treatment, PDMS surfaces become more hydrophilic compared to PMMA. This indicates that after undergoing oxygen plasma treatment, PDMS- based microchannels can reduce the adsorption of charged particles like small EVs andproteins, ultimately minimizing sample loss of EVs that come into contact with the microchannel walls.

[0145] Bifurcation nodes were designed with a curved structure to avoid the turbulence of fluid streams. Microchannel was designed using AutoCAD software (Autodesk, USA) and a digital craft cutting machine (Cricut Maker 3, Cricut, CA) was utilized to cut the adhesive sheets into the pieces with the desired channel pattern. Both sidewalls of the main channel were covered with an electrically conductive copper tape to apply a constant voltage across the microchannel. The PDMS base polymer and curing agent were mixed at a ratio of 10:1 , and the resulting mixture was poured onto a glass Petri dish and then cured in an oven at 120 °C for 20 minutes. The generated PDMS sheet was subsequently peeled off from the Petri dish and cut into two flat PDMS layers of appropriate dimensions. Four holes were punched into one of the PDMS layers (the device top layer) to create the inlet and outlet ports of the microchannel. Both the PSA and PDMS surfaces treated with oxygen plasma PE-25, Plasma- Etch, Inc.) for 1 minute at 15 W, with an oxygen flow rate of 25 mL / min and a chamber pressure of 700 mTorr, to increase their surface energy and promote bonding between the materials. After treatment, the microchannel-patterned PSA layer was positioned between the two PDMS layers, and the entire assembly was slightly compressed for 15 seconds. To determine if the fabricated platform would be prone to leakage, a blue dye solution was introduced into the inlets of the microchannel at the flow rates of 1-5 mL / min. The results showed that microchannels successfully tolerated the high influx (5 mL / min) of dye solution without destroying the bond. In addition to oxygen plasma treatment utilized during the fabrication procedure, the fully bonded PDMS device subsequently underwent a second round of oxygen plasma treatment and the microchannel was immediately filled with de-ionized water after the treatment.

[0146] Buffer and sample solutions were injected into the fabricated platform using a syringe pump (Harvard Apparatus, PHD 2000 Infusion). Due to the interaction between the electrode and biological samples, side effects of electrolysis and bubble formation are unavoidable that may disturb the fluid flow within the microchannel. Therefore, buffer solution and carrier fluid for the particles were considered to be diluted PBS with low ionic strength (17 mM), and device operating conditions was set for the applied E-field of 6-15 V / cm where there was no significant bubble generation to disturb fluid flowing within the system.

[0147] Cell Culture. Human adipose-derived MSCs and A549 cells were obtained under ethical approval from the University of Calgary Health Research Ethics Board. MSCs were statically cultured in PPRF-msc6 medium according to the protocols developed in the Pharmaceutical Production Research Facility (PPRF).A549 cells were statically cultured in DMEM supplemented with 10% FBS. To deplete FBS-related EVs before A549 cell culture, the complete culture medium was ultracentrifuged at 100,000 g and 4°C for 18.5 hours (Beckman Coulter Optima L-100K, 70 Ti rotor, 38,000 rpm, k factor = 148) and followed by vacuum filtration through a 0.22 pm sterile filter 26. Both experimental groups of cells were seeded (MSCs inoculated at 5,000 cells / cm2 and A549 cells inoculated at 10,000 cells / cm2) in tissue culture flasks with cell growth areas of 75 cm2 (T-75) and fed with fresh medium every 3 days, while incubating in a humidified atmosphere containing 5% carbon dioxide at 37°C. Based on the PPRF protocols, all tissue culture flasks utilized for MSCs expansion were coated with 0.1% gelatine. The spent culture medium (culture supernatant) was collected every 3 days to isolate small EVs, while the cells were being passaged. Cell morphology was assessed during the cell culture process using an Axiovert 200M phase-contrast inverted microscope (Carl Zeiss Canada Ltd., Toronto, Canada).

[0148] Preparation of Small EV Sample Solution. To investigate device performance for separation of small EVs from protein impurities, a mixture of small EVs (obtained from culture supernatant collected on day 3) and HSA proteins (5 mg / mL) was suspended in diluted PBS (ionic strength of 17 mM). Since PBS (1X) with molar concentration of approximately 170 mM possesses high electrical conductivity (>16 mS / cm), all particle samples (proteins and small EVs) were dispersed in diluted PBS (17 mM) to maintain the electrodes of the device in an unsaturated state and to minimize bubble generation during separation procedure. The sample solution was divided into two aliquots, with each aliquot being processed separately using the fabricated device and UC to separate small EVs from proteins. FIG. 17A illustrates the sample preparation procedure used to evaluate the performance of the device in this study.

[0149] The protein concentration of the culture supernatant was monitored for three days and ranged between 4 mg / mL and 5 mg / mL. On day 3, the average concentration was approximately 5 mg / mL, which was selected for sample preparation. Culture supernatant was collected on day 3 and centrifuged at 300 g and 4°C for 5 min (Thermo Scientific Sorvall Legend XT Centrifuge, TX-750 rotor) to remove dead cells, and then at 2,000 g and 4°C for 10min (Thermo Scientific Sorvall Legend XT Centrifuge, TX-750 rotor) to remove very large particles including cell debris and apoptotic bodies. The supernatant was collected and centrifuged at 10,000 g and 4°C for 30 min (Thermo Scientific Sorvall RC-5C Plus Centrifuge, HS-4 rotor), and then at 100,000 g and 4°C for 2 hours (Beckman Coulter Optima L-100K Ultracentrifuge, 70 Ti rotor, k factor = 148) to obtain the small EV pellet. The supernatant was discarded, and the small EV pellet was resuspended in PBS for sample preparation and further analysis.

[0150] Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS). An ExoView R100 instrument (NanoView Biosciences, Boston, MA, USA) was used to identify and characterize separated particle fractions based on the expression level of exosome- specific markers CD81 , CD63, and CD9, using the SP-IRIS technology. The standard manufacturer protocol was followed, wherein 50 pL of each sample was diluted with 1 mL of incubation buffer (supplied by NanoView Biosciences) and incubated on an antibody-coated microarray chip (supplied by NanoView Biosciences) for 18 hours at 23°C. The chips were then washed and stained with corresponding fluorescently conjugated antibodies (supplied by NanoView Biosciences), followed by imaging of individual captured EVs.

[0151] Bicinchoninic Acid (BCA) Assay. To estimate the purity of the separated small EVs, the protein content of each experimental group (culture supernatant, UC-isolated EV fractions, and small EV fractions obtained from the fabricated platform) was measured using a BCA protein assay (Pierce BCA Protein Assay kit, Thermo Fisher Scientific, CA). According to the standard manufacturer protocols, each sample (25 pL) was mixed with a working reagent (200 pL, 50:1 ratio of assay reagents A to B) within a 96 well plate. Then, the plate was incubated for 30 min at 37 °C, followed by measuring each sample’s absorbance at 562 nm using a spectrophotometer (BioTek Synergy HTX Multi-Mode Microplate Reader). Sample absorbance was converted to the protein content (mg / mL) via a standard curve generated for bovine serum albumin solutions with various protein concentrations according to the standard manufacturer protocols.

[0152] Dynamic Light Scattering (DLS). A Zetasizer Nano ZS (Malvern Instruments Ltd., Cambridge, UK) was utilized to measure electrophoretic mobility and size distribution of isolated EV fractions using DLS technique, and the recorded data were analyzed using Zetasizer Software 7.12 (Malvern Instruments Ltd). All measurements were conducted underpersistent experimental conditions of temperature (25.00 ± 0.1 °C), electrolyte viscosity (0.887 cP), ionic strength (17 mM), and pH (neutral pH of 7.5).

[0153] Computational Simulation. The proposed design for the microfluidic channel was modeled using COMSOL Multiphysics (version 5.5, www.comsol.com) which utilizes the Electrophoretic T ransport and Laminar Flow interfaces to simulate the effect of buffer fluid flow on confining particles towards the sidewall of the microchannel. In this computational study, two streams of fluid flow solutions were utilized, which included a buffer solution and a sample solution containing small EVs and proteins. In this model, small EVs and proteins were treated as rigid charged nanoparticles, while the bulk electrolyte solution in each stream was assumed to be an electroneutral solution with equal concentrations of cations and anions. The fluid flow was set up with the Laminar Flow interface (Navier-Stokes equations) and the Electrophoretic Transport interface supported particle transport by diffusion, convection and migration in electric fields.

[0154] Statistical Analysis. Where applicable, three sets of measurements were performed for each experimental group and the values were presented as the mean ± standard deviation (SD). Two-tailed unpaired t-tests were used to compare between two experimental groups and a p-value less than 0.05 was indicative of a statistically significant difference.

[0155] Non-Binding Conclusions

[0156] The proposed platform in this disclosure may enable the separation of small EVs from proteins (main impurities that exist in biological fluids) while avoiding the possible detrimental effects of label-based methods. The disclosed technique was a pellet-free separation technique utilizing an E-filed to facilitate the translocation of small EVs through a unique microchannel with two bifurcations at both ends of the main channel.

[0157] The results verified that the proposed technique was more efficient and faster than UC which is currently the common technique for the isolation of small EVs.

[0158] The proposed platform discriminated small EVs from proteins and provided a pellet-free population of small EVs in minutes with a higher recovery rate compared to UC. Considering that bubble generation at the interface of electrodes and biological solutions would be the common limiting factor in electrical-based microfluidic devices, as it disturbs the laminar flow of the fluid and lowers the efficiency of separation, this technique demands an in-depthinvestigation of parameters influencing electrophoretic behaviour of particles within a microchannel.

[0159] To perform a separation of small EVs, specifically from raw biological samples (e.g., culture supernatant and bodily fluids), the proposed platform may be performed with optimization under the lower strength of an applied E-field to isolate EVs with a higher purity and recovery yield. Given the results of this study along with advantages in terms of being label-free and pellet-free, the proposed isolation platform represents a great potential for making a significant contribution to enabling the clinical application of microfluidic devices for the development of the EV-based diagnostic and therapeutic tools.

[0160] EXAMPLE 3 - Supplemental Information for Pellet-Free Separation of Extracellular Vesicles Using Electrophoretic Force

[0161] FIG. 19 shows a colocalization pie chart for fresh culture medium and culture supernatant collected from mesenchymal stem cells (MSCs) and adenocarcinomic human alveolar basal epithelial cells (A549), presenting particle fractions bound to CD63, CD81 , and CD9 antibodies on ExoView chips. It shows that there is no colocalized particles in fresh culture medium that contain no cells and no extracellular vesicles (EVs) to express two or more EV-specific markers. In contrast, a significant proportion of collected particles on Exoview chips are found in culture supernatant that express two or more EV-specific markers.

[0162] FIG. 20 shows a colocalization pie chart for isolated small extracellular vesicles (EVs) from culture supernatant collected from mesenchymal stem cells (MSCs) and adenocarcinomic human alveolar basal epithelial cells (A549), presenting particle fractions bound to CD81 , and CD9 antibodies on ExoView chips. It shows that a significant proportion of collected particles in small EV pellets expresses two or more EV-specific markers.

[0163] FIG. 21 shows the effect of buffer flow force on particle confinement during translocation in the absence of an electrical force. The protein content of samples collected from two outlets under different buffer flow forces: (a) equal buffer and sample flow rates (Buffer Flow = X), and (b) buffer flow rate is twice that of sample flow rate (Buffer Flow = 2X). Sample solution injected into the Inlet II is phosphate buffer saline containing human albumin proteins with different concentration (Solution 1 : 3 mg / mL, Solution 2: 2 mg / mL, Solution 3: 1 mg / mL).

[0164] FIG. 22 shows the effect of buffer flow force on particle confinement during translocation in the absence of an electrical force. Collected samples from two outlets when buffer flow rate is twice that of sample flow rate (Buffer Flow = 2X), stained with fluorescent CD81 and CD9 antibodies. Grean dots represent particles bound to CD81 antibodies and blue dots represent particles bound to CD9 antibodies run on the ExoView. Sample solution injected into the device is phosphate buffer saline containing small extracellular vesicles (EVs) derived from mesenchymal stem cells (small EVs obtained from 5 mL of culture supernatant).

[0165] FIG. 23 shows protein content of the samples collected from Outlet II, where two experimental groups of sample solutions are introduced into the device through Inlet I. The solutions contain human serum albumin (HSA) proteins at a concentration of 5 mg / mL mixed with small extracellular vesicles (EVs) fractions obtained from culture supernatant of mesenchymal stem cells (MSC_sEV) and adenocarcinomic human alveolar basal epithelial cells (A549_sEV).

[0166] FIG. 26A-B show water contact angle measurement, (a) Contact angle of a water drop on a solid surface (0) is defined using the Young’s equation (ys= ysi+ yi cos 0), where yi, Ysi, and Ys are liquid surface tension, solid-liquid interfacial tension, and solid surface tension, respectively. The measured water contact angle determines whether the surface is hydrophilic or hydrophobic, (b) Measured contact angles of polymethylmethacrylate (PMMA) and Polydimethylsiloxane (PDMS) substrates with and without oxygen plasma treatment.

[0167] FIG. 24 shows quantified particles expressing exosome-specific markers, collected from Outlet I under different applied electrical forces. Colocalization pie charts represents particles bound to CD81 and CD9 run on the ExoView, normalized to non-specific binding of proteins bound to mouse IgG (MlgG).

[0168] FIG. 25 shows a schematic of the proposed platform which is composed of one adhesive layer (patterned with a microchannel with two bifurcations at both ends of the main channel) and two polydimethylsiloxane (PDMS) layers.

[0169] EXAMPLE 4

[0170] It will be appreciated that, in some examples, charged particles may include a diverse group of particles, including: (i) natural macromolecules (e.g., proteins, lipids, a nucleic acid, and other biomolecules or bioactive molecules); (ii) synthetic macromolecules, such as nano / microplastics, graphene, and carbon nanotubes; and (iii) substances and structurescomposed of macromolecular units, including extracellular vesicles, viruses, vaccine / drug delivery systems, and both eukaryotic and prokaryotic cells.

[0171] In some examples, the charged particles may range from about 4 nm to about 200 pm.

[0172] Additional Embodiments

[0173] In some aspects, the disclosure relates to electrofiltration using constant current and related methods according to the following Embodiments, among others:

[0174] Embodiment 1. An electroseparator comprising: an injection portion having a first inlet configured to receive a sample solution at a sample solution flow rate, the sample solution comprising at least one target charge particle; a separation portion fluidly connected to the injection portion; a collection portion fluidly connected to the separation portion the collection portion comprising a first outlet configured to dispense the at least one target particle and a second outlet configured to dispense a carrier solution; an anode end; a cathode end opposite the anode end; the cathode end electrically coupled to the anode end for delivering a constant electric field across the separation portion.

[0175] Embodiment 2. The electroseparator of Embodiment 1 , wherein the constant electric field is between about 6 V / cm and about 9 V / cm.

[0176] Embodiment 3. The electroseparator of Embodiment 1 or Embodiment2, further comprising a second inlet configured to receive a buffer solution at a buffer solution flow rate.

[0177] Embodiment 4. The electroseparator of Embodiment 3, wherein the buffer solution flow rate is about two or more times that of the sample solution flow rate.

[0178] Embodiment 5. The electroseparator of Embodiment 3, wherein the first inlet and the second inlet converge to form a channel.

[0179] Embodiment 6. The electroseparator of any one of Embodiments 3 to 5, wherein the first inlet and the second inlet are each configured for producing laminar flow in the channel.

[0180] Embodiment 7. The electroseparator of Embodiment 5 or 6, wherein the channel splits to form the first outlet and the second outlet.

[0181] Embodiment 8. The electroseparator of Embodiment 2, wherein the first outlet and the second outlet are each configured for producing laminar flow for reducing convective mixing of the target charged particle and the carrier solution.

[0182] Embodiment 9. The electroseparator according to of any one of Embodiments 5 to 8, wherein the channel is about 1.2 mm in width, about 0.15 mm in height, and about 35 mm in length.

[0183] Embodiment 10. The electroseparator according to any one of Embodiments 1 to 9, further comprising a filter positioned between the injection portion and the separation portion for filtering the sample solution and the buffer solution.

[0184] Embodiment 11. A method of separating charged particles from a sample solution comprising: a. providing a sample solution having a flow rate, the sample solution comprising at least one target charged particle and at least one non-target charged particle, the at least one non-target charged particle having a different charge to the at least one target charged particle; b. applying a constant electric field across the sample solution for separating the at least one target charged particle from the at least one non-target charged particle.

[0185] Embodiment 12. The method according to Embodiment 11 , further comprising providing a buffer solution having a flow rate, the buffer solution flowing lateral to the sample solution for establishing laminar flow.

[0186] Embodiment 13. The method according to Embodiment 11 or 12, wherein the electric field is from about 6 V / mm to about 9 V / mm.

[0187] Embodiment 14. The method according to any one of Embodiments 11 to 33, further comprising selecting the electric field according to one or more properties of the sample solution or according to one or more electrical properties of the at least one target charged particle.

[0188] Embodiment 15. The method of Embodiment 14, wherein the one or more properties of the sample solution is selected from the group consisting of: pH, ionic strength, electrical conductivity, surface charge density of the at least one target charged particle, zeta potential of the at least one target charged particle, and electrical conductivity of the at least one target charged particle.

[0189] Embodiment 16. The method according to any one of Embodiments 11 to 15, wherein the at least one target charged particle is from about 4 nm to about 200 pm in diameter.

[0190] Embodiment 17. The method according to any one of Embodiments 11 to 16, wherein the at least one target charged particle is from about 20 nm to about 5 pm in diameter.

[0191] Embodiment 18. The method according to any one of Embodiments 11 to 17, wherein the target charged particle is selected from the group consisting of: extracellular vesicles, proteins, DNA, and RNA.

[0192] Embodiment 19. The method according to any one of Embodiments 11 to 17, wherein the target charged particle is: a natural macromolecule, such as a protein, a lipid, a nucleic acid, or other biomolecule or bioactive molecule; a synthetic macromolecule, such as a nanoplastic, a microplastic, graphene, or a carbon nanotube; or substances and structures composed of macromolecular units, including extracellular vesicles, viruses, vaccine / drug delivery systems, and both eukaryotic and prokaryotic cells.

[0193] Embodiment 20. The method according to any one of Embodiments 11 to 19, wherein the target charged particle is an exosome.

[0194] Embodiment 21. The method according to any one ofEmbodiments 11 to 20, further comprising removing particles greater than about 450 nm in diameter from the electrolyte solution prior to contacting the electrolyte solution to the feed side of the filter.

[0195] Embodiment 22. The method according to any one of Embodiments 11 to 21 , wherein the buffer solution flow rate is at least about twice that of the sample solution flow rate.

[0196] Embodiment 23. An electroseparator comprising: an anode end; a cathode end opposite the anode end for establishing an electric field; at least one filter disposed between the anode and the cathode end, the at least one filter defining a plurality of parallel channels; the anode and the cathode end electrically coupled to deliver a constant electric current across the at least one filter.

[0197] Embodiment 24. The electroseparator of Embodiment 23, wherein each of the at least one filter comprises a porous membrane.

[0198] Embodiment 25. The electroseparator of Embodiment 24, wherein each of the porous membranes comprises pores sized about 30 nm to about 5 pm.

[0199] Embodiment 26. The electroseparator of Embodiment 25, wherein each of the at least one filters comprises pores sized about 30 nm to about 40 nm.

[0200] Embodiment 27. The electroseparator of Embodiment 23, wherein the plurality of parallel channels are each configured to receive fluid.

[0201] Embodiment 28. The electroseparator of Embodiment 23, wherein the plurality of parallel channels each terminate in an outlet for draining the fluid.

[0202] Embodiment 29. The electroseparator of Embodiment 23, wherein the plurality of channels comprises at least a first channel for loading a sample, and a second channel for receiving a filtrate via the at least one filter.

[0203] Embodiment 30. A method of separating charged particles from an electrolyte solution comprising: providing an electrolyte solution comprising at least one target charged particle; providing a filter sized to separate the at least one target charged particle from the electrolyte solution, the filter having a feed side and a filtrate side opposite the feed side; applying an electric field across the filter; contacting the electrolyte solution to the feed side of the filter; and applying a constant electrical current across the filter for separating the at least one target charged particle from the electrolyte solution across the filter.

[0204] Embodiment 31. The method of Embodiment 30, further comprising contacting a buffer solution to the filtrate side of the filter for receiving filtrate.

[0205] Embodiment 32. The method of Embodiment 31 , wherein the filtrate comprises the at least one target charged particle.

[0206] Embodiment 33. The method according to any one of Embodiments 30 to32, wherein the electric field is from about 1 V / mm to about 20 V / mm.

[0207] Embodiment 34. The method according to any one of Embodiments 30 to33, further comprising selecting the constant electrical current according to one or more properties of the electrolyte solution or according to one or more electrical properties of the at least one target charged particle.

[0208] Embodiment 35. The method of Embodiment 34, wherein the one or more properties of the electrolyte solution is selected from the group consisting of: pH, ionic strength, electrical conductivity, surface charge density of the at least one target charged particle, zetapotential of the at least one target charged particle, and electrical conductivity of the at least one target charged particle.

[0209] Embodiment 36 The method according to any one of Embodiments 30 to 35, wherein the at least one target charged particle is from about 4 nm to about 200 pm in diameter.

[0210] Embodiment 37. The method according to any one of Embodiments 30 to 36, wherein the at least one target charged particle is from about 20 nm to about 5 pm in diameter.

[0211] Embodiment 38. The method according to any one of Embodiments 30 to 37, wherein the at least one target charged particle is from about 30 nm to about 200 nm in diameter.

[0212] Embodiment 39. The method according to any one of Embodiments 30 to 38, wherein the target charged particle is: a natural macromolecule, such as a protein, a lipid, a nucleic acid, or other biomolecule or bioactive molecule; a synthetic macromolecule, such as a nanoplastic, a microplastic, graphene, or a carbon nanotube; or substances and structures composed of macromolecular units, including extracellular vesicles, viruses, vaccine / drug delivery systems, and both eukaryotic and prokaryotic cells.

[0213] . Embodiment 40. The method according to any one of Embodiments 30 to 39, wherein the target charged particle is selected from the group consisting of: extracellular vesicles, proteins, DNA, and RNA.

[0214] Embodiment 41. The method according to any one of Embodiments 30 to 40, wherein the target charged particle is an exosome.

[0215] Embodiment 42. The method according to any one of Embodiments 30 to 41 , further comprising removing particles greater than about 450 nm in diameter from the electrolyte solution prior to contacting the electrolyte solution to the feed side of the filter.

[0216] Embodiment 43. The method according to any one of Embodiments 31 to 42, wherein the electrolyte solution and the buffer solution have a flow rate of about 10 pL / min to about 30 mL / min.

[0217] Embodiment 44. The method according to any one of Embodiments 30 to 43, wherein the electrolyte solution and the buffer solution have an ionic strength of about 0.001 mS / cm to about 25 mS / cm.

[0218] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[0219] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0220] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[0221] REFERENCES

[0222] (1) Van Niel, G.; d'Angelo, G.; Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nature reviews Molecular cell biology 2018, 19 (4), 213-228.

[0223] (2) Kalluri, R.; LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science 2020, 367 (6478), eaau6977.

[0224] (3) Hassanpour Tamrin, S.; Sanati Nezhad, A.; Sen, A. Label-Free Isolation ofExosomes Using Microfluidic Technologies. ACS Nano 2021. DOI:10.1021 / acsnano.1 C03469.

[0225] (4) Coughlan, C.; Bruce, K. D.; Burgy, O.; Boyd, T. D.; Michel, C. R.; Garcia-Perez, J. E.; Adame, V.; Anton, P.; Bettcher, B. M.; Chial, H. J. Exosome isolation by ultracentrifugation and precipitation and techniques for downstream analyses. Current Protocols in Cell Biology 2020, 88 (1), e110.

[0226] (5) Le, M.-C. N.; Fan, Z. H. Exosome isolation using nanostructures and microfluidic devices. Biomedical Materials 2021, 16 (2), 022005.

[0227] (6) Abreu, C. M.; Costa-Silva, B.; Reis, R. L.; Kundu, S. C.; Caballero, D.Microfluidic platforms for extracellular vesicle isolation, analysis and therapy in cancer. Lab on a Chip 2022, 22 (6), 1093-1125.

[0228] (7) Mishra, V.; Heath, R. J. Structural and biochemical features of human serum albumin essential for eukaryotic cell culture. International Journal of Molecular Sciences 2021 , 22 (16), 8411.

[0229] (8) Park, Y.-M.; Hong, S. O.; Lee, P. C.; Kim, J. M. Vortex dynamics at the junction of Y-shaped microchannels in dilute polymer solutions. Korea-Australia Rheology Journal 2019, 31 (4), 189-194.

[0230] (9) Yap, Y.; Chai, J.; Wong, T.; Nguyen, N.; Toh, K.; Zheng, H. Particle transport in microchannels. In ASME International Mechanical Engineering Congress and Exposition, 2006; Vol. 47845, pp 53-60.

[0231] (10) Hymel, S. J.; Lan, H.; Fujioka, H.; Khismatullin, D. B. Cell trapping in Y- junction microchannels: A numerical study of the bifurcation angle effect in inertial microfluidics. Physics of Fluids 2019, 31 (8), 082003.

[0232] (11) Meyari, M.; Salehi, Z.; Zarghami, R.; Saeedipour, M. Numerical investigation of particle separation in Y-shaped bifurcating microchannels. Particuology 2021 , 56, 142-151.

[0233] (12) Humphries, S. Principles of charged particle acceleration; CourierCorporation, 2013.

[0234] (13) Barany, S. Electrophoresis in strong electric fields. Advances in colloid and interface science 2009, 147, 36-43.

[0235] (14) Davies, R. T.; Kim, J.; Jang, S. C.; Choi, E.-J.; Gho, Y. S.; Park, J.Microfluidic filtration system to isolate extracellular vesicles from blood. Lab on a Chip 2012, 12 (24), 5202-5210.

[0236] (15) Jachimska, B.; Wasilewska, M.; Adamczyk, Z. Characterization of globular protein solutions by dynamic light scattering, electrophoretic mobility, and viscosity measurements. Langmuir 2008, 24 (13), 6866-6872.

[0237] (16) Russell, B.; Jachimska, B.; Kralka, I.; Mulheran, P.; Chen, Y. Human serum albumin encapsulated gold nanoclusters: effects of cluster synthesis on natural protein characteristics. Journal of Materials Chemistry B 2016, 4 (42), 6876-6882.

[0238] (17) Midekessa, G.; Godakumara, K.; Ord, J.; Viil, J.; Lattekivi, F.;Dissanayake, K.; Kopanchuk, S.; Rinken, A.; Andronowska, A.; Bhattacharjee, S. Zetapotential of extracellular vesicles: toward understanding the attributes that determine colloidal stability. Acs Omega 2020, 5 (27), 16701-16710.

[0239] (18) Zhang, Y.; Liu, Y.; Liu, H.; Tang, W. H. Exosomes: biogenesis, biologic function and clinical potential. Cell & bioscience 2019, 9 (1), 1-18.

[0240] (19) Cocucci, E.; Meldolesi, J. Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends in cell biology 2015, 25 (6), 364-372.

[0241] (20) Thery, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.;Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G. K. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of extracellular vesicles 2018, 7 (1), 1535750.

[0242] (21) Hirama, H.; Otahara, R.; Kano, S.; Hayase, M.; Mekaru, H.Characterization of Nanoparticle Adsorption on Polydimethylsiloxane-Based Microchannels. Sensors 2021 , 21 (6), 1978.

[0243] (22) Tan, S. H.; Nguyen, N.-T.; Chua, Y. C.; Kang, T. G. Oxygen plasma treatment for reducing hydrophobicity of a sealed polydimethylsiloxane microchannel. Biomicrofluidics 2010, 4 (3), 032204.

[0244] (23) Duangkanya, K.; Kopwitthaya, A.; Chanhorm, S.; Infahsaeng, Y. Oxygen plasma treatment time induced hydrophilicity of polydimethylsiloxane (PDMS) thin films for liquid lenses application. Materials Today: Proceedings 2022, 65, 2442-2445.

[0245] (24) Zhao, L. H.; Lee, J.; Sen, P. N. Long-term retention of hydrophilic behavior of plasma treated polydimethylsiloxane (PDMS) surfaces stored under water and Luria-Bertani broth. Sensors and Actuators A: Physical 2012, 181 , 33-42.

[0246] (25) Jung, S.; Sen, A.; Rosenberg, L.; Behie, L. A. Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells. Cytotherapy 2010, 12 (5), 637-657. DOI: 10.3109 / 14653249.2010.495113.

[0247] (26) Shelke, G. V.; Lasser, C.; Gho, Y. S.; Lotvall, J. Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. Journal of extracellular vesicles 2014, 3 (1), 24783.

[0248] (27) Yuan, Y.; Lee, T. R. Contact angle and wetting properties. In Surface science techniques, Springer, 2013; pp 3-34.

Claims

WHAT IS CLAIMED IS:

1. An electroseparator comprising: an injection portion having a first inlet configured to receive a sample solution at a sample solution flow rate, the sample solution comprising at least one target charged particle; a separation portion fluidly connected to the injection portion; a collection portion fluidly connected to the separation portion the collection portion comprising a first outlet configured to dispense the at least one target particle and a second outlet configured to dispense a carrier solution; an anode end; a cathode end opposite the anode end; the cathode end electrically coupled to the anode end for delivering a constant electric field across the separation portion.

2. The electroseparator of claim 1 , wherein the constant electric field is between about 6 V / cm and about 9 V / cm.

3. The electroseparator of claim 1 or claim 2, further comprising a second inlet configured to receive a buffer solution at a buffer solution flow rate.

4. The electroseparator of claim 3, wherein the buffer solution flow rate is about two or more times that of the sample solution flow rate.

5. The electroseparator of claim 3, wherein the first inlet and the second inlet converge to form a channel.

6. The electroseparator of any one of claims 3 to 5, wherein the first inlet and the second inlet are each configured for producing laminar flow in the channel.

7. The electroseparator of claim 5 or 6, wherein the channel splits to form the first outlet and the second outlet.

8. The electroseparator of claim 5, wherein the first outlet and the second outlet are each configured for producing laminar flow for reducing convective mixing of the target charged particle and the carrier solution.

9. The electroseparator according to of any one of claims 5 to 8, wherein the channel is about 1.2 mm in width, about 0.15 mm in height, and about 35 mm in length.

10. The electroseparator according to any one of claims 1 to 9, further comprising a filter positioned between the injection portion and the separation portion for filtering the sample solution and the buffer solution.

11. A method for separating charged particles from a sample solution comprising: a. providing a sample solution having a flow rate, the sample solution comprising at least one target charged particle and at least one non-target charged particle, the at least one non-target charged particle having a different charge to the at least one target charged particle; b. applying a constant electric field across the sample solution for separating the at least one target charged particle from the at least one non-target charged particle.

12. The method according to claim 11, further comprising providing a buffer solution having a flow rate, the buffer solution flowing lateral to the sample solution for establishing laminar flow.

13. The method according to claim 11 or 12, wherein the electric field is from about 6 V / mm to about 9 V / mm.

14. The method according to any one of claims 11 to 13, further comprising selecting the electric field according to one or more properties of the sample solution or according to one or more electrical properties of the at least one target charged particle.

15. The method of claim 14, wherein the one or more properties of the sample solution is selected from the group consisting of: pH, ionic strength, electrical conductivity, surface charge density of the at least one target charged particle, zeta potential of the at least one target charged particle, and electrical conductivity of the at least one target charged particle.

16. The method according to any one of claims 11 to 15, wherein the at least one target charged particle is from about 4 nm to about 200 pm in diameter.

17. The method according to any one of claims 11 to 16, wherein the at least one target charged particle is from about 20 nm to about 5 pm in diameter.

18. The method according to any one of claims 11 to 17, wherein the target charged particle is selected from the group consisting of: extracellular vesicles, proteins, DNA, and RNA.

19. The method according to any one of claims 11 to 17, wherein the target charged particle is: a natural macromolecule, such as a protein, a lipid, a nucleic acid, or other biomolecule or bioactive molecule; a synthetic macromolecule, such as a nanoplastic, a microplastic, graphene, or a carbon nanotube; or substances and structures composed of macromolecular units, including extracellular vesicles, viruses, vaccine / drug delivery systems, and both eukaryotic and prokaryotic cells.

20. The method according to any one of claims 11 to 19, wherein the target charged particle is an exosome.

21. The method according to any one of claims 11 to 20, further comprising removing particles greater than about 450 nm in diameter from the electrolyte solution prior to contacting the electrolyte solution to the feed side of the filter.

22. The method according to any one of claims 11 to 21 , wherein the buffer solution flow rate is at least about twice that of the sample solution flow rate.

23. An electroseparator comprising: an anode end; a cathode end opposite the anode end for establishing an electric field; at least one filter disposed between the anode and the cathode end, the at least one filter defining a plurality of parallel channels; the anode and the cathode end electrically coupled to deliver a constant electric current across the at least one filter.

24. The electroseparator of claim 23, wherein each of the at least one filter comprises a porous membrane.

25. The electroseparator of claim 24, wherein each of the porous membranes comprises pores sized about 30 nm to about 5 pm.

26. The electroseparator of claim 25, wherein each of the at least one filters comprises pores sized about 30 nm to about 40 nm.

27. The electroseparator of claim 23, wherein the plurality of parallel channels are each configured to receive fluid.

28. The electroseparator of claim 23, wherein the plurality of parallel channels each terminate in an outlet for draining fluid.

29. The electroseparator of claim 23, wherein the plurality of channels comprises at least a first channel for loading a sample, and a second channel for receiving a filtrate via the at least one filter.

30. A method for separating charged particles from an electrolyte solution comprising: a. providing an electrolyte solution comprising at least one target charged particle; b. providing a filter sized to separate the at least one target charged particle from the electrolyte solution, the filter having a feed side and a filtrate side opposite the feed side; c. applying an electric field across the filter; d. contacting the electrolyte solution to the feed side of the filter; and e. applying a constant electrical current across the filter for separating the at least one target charged particle from the electrolyte solution across the filter.

31. The method of claim 30, further comprising contacting a buffer solution to the filtrate side of the filter for receiving filtrate.

32. The method of claim 31 , wherein the filtrate comprises the at least one target charged particle.

33. The method according to any one of claims 30 to 32, wherein the electric field is from about 1 V / mm to about 20 V / mm.

34. The method according to any one of claims 30 to 33, further comprising selecting the constant electrical current according to one or more properties of the electrolyte solution or according to one or more electrical properties of the at least one target charged particle.

35. The method of claim 34, wherein the one or more properties of the electrolyte solution is selected from the group consisting of: pH, ionic strength, electrical conductivity, surfacecharge density of the at least one target charged particle, zeta potential of the at least one target charged particle, and electrical conductivity of the at least one target charged particle.

36. The method according to any one of claims 30 to 35, wherein the at least one target charged particle is from about 4 nm to about 200 pm in diameter.

37. The method according to any one of claims 30 to 36, wherein the at least one target charged particle is from about 20 nm to about 5 pm in diameter.

38. The method according to any one of claims of claim 30 to 37, wherein the at least one target charged particle is from about 30 nm to about 200 nm in diameter.

39. The method according to any one of claims 30 to 38, wherein the target charged particle is: a natural macromolecule, such as a protein, a lipid, a nucleic acid, or other biomolecule or bioactive molecule; a synthetic macromolecule, such as a nanoplastic, a microplastic, graphene, or a carbon nanotube; or substances and structures composed of macromolecular units, including extracellular vesicles, viruses, vaccine / drug delivery systems, and both eukaryotic and prokaryotic cells.

40. The method according to any one of claims 30 to 39, wherein the target charged particle is selected from the group consisting of: extracellular vesicles, proteins, DNA, and RNA.

41. The method according to any one of claims 30 to 40, wherein the target charged particle is an exosome.

42. The method according to any one of claims 30 to 41 , further comprising removing particles greater than about 450 nm in diameter from the electrolyte solution prior to contacting the electrolyte solution to the feed side of the filter.

43. The method according to any one of claims 31 to 42, wherein the electrolyte solution and the buffer solution have a flow rate of about 10 pL / min to about 30 mL / min.

44. The method according to any one of claims 31 to 43, wherein the electrolyte solution and the buffer solution have an ionic strength of about 0.001 mS / cm to about 25 mS / cm.

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