This application claims the benefit of priority of Singapore Patent Application No. 10201909776U, filed 21 Oct. 2019, the content of it being hereby incorporated by reference in its entirety for all purposes.
The present disclosure relates to a method of isolating exosomes from blood.
The present disclosure also relates to a microfluidic device operable to carry out the method, and a method of identifying diabetes mellitus based on the microfluidic device and method.
Extracellular vesicles (EVs), including exosomes (˜50-200 nm) and microvesicles (˜100 nm-1 μm), are produced by cells upon physiological or pathological cues and serve as mediators for intercellular communications. While circulating EVs in blood are promising diagnostics biomarkers in cancer and diabetes, isolation of blood-borne exosomes involves laborious ultracentrifugation or commercial precipitation kits with high protein contamination.
The complexity of blood with the high cellular components (˜50% v/v), and the similar size range between EVs and platelets (˜2-3 μm) present a huge technical challenge for EVs isolation. A current standard for EVs isolation may involve multi-step differential and ultracentrifugation (typically ˜1,000×g for 10 minutes to remove cellular components; ˜2,000×g for 20 minutes to obtain platelet-free plasma; ˜20,000×g for 60 minutes to pellet microvesicles; ˜175,000×g for 70 minutes to pellet exosomes). Such a standard may be commonly used to purify EVs, but the process is laborious and the EVs yield and purity may be highly dependent on user operation and blood collection method.
In one example, immunomagnetic bead-based capture of exosomes appears more effective, but may lead to biased analysis depending on the binding targets.
Commercial products based on filtration and/or precipitation are also available for isolating EVs from blood sera. However, despite their user-friendliness, purities are lower than the conventional/standard methods and there tends to be a risk of losing EVs functionalities after elution.
Several other exosomes isolation and detection microfluidics platforms have been developed, with the most common being affinity capture using well-established exosomal surface markers (CD81 or CD63) on microchannel surfaces or microbeads. These technologies were demonstrated with plasma or serum samples, which require additional sample processing (centrifugation) steps to deplete the blood cells. Throughput and flow rate tend to be low in these devices (˜4-20 μL min−1) as they need to facilitate exosomes binding within channels or mixing with capture beads. This happens to limit direct whole blood processing as the large RBCs generates a background interference that significantly hinders binding of EVs to antibody-functionalized surfaces.
Another strategy was developed via size-based exclusion to isolate exosomes by membrane crossflow filtration, or microporous ciliated micropillars using silicon nanowires. These label-free approaches tend to be non-selective in trapping EVs which result in higher yield and unbiased analysis. Throughput may be scaled up easily with larger filtration footprint, but device operations are largely limited by clogging issues and low EVs recovery.
In another example, a microfluidic technology termed “High-resolution Dean Flow Fractionation (HiDFF)” for sub-micron binary particle sorting was developed. HiDFF exploits a non-equilibrium differential Dean migration of particles across a channel to achieve continuous size-based separation of small microparticles and nanoparticles (˜50 nm-1 μm). This provides for fractionation of small particles with high separation resolution at high throughput (˜70-100 μL min−1) and purified particles may be continuously collected off chip for downstream analysis. However, such technology may be specific and does not sufficiently provide the resolution needed for exosome isolation.
There is thus a need to provide for a solution that addresses one or more of the limitations mentioned above. The solution should at least provide for a method of isolating EVs such as exosomes, and/or a multiplexed EVs fractionation tool with sub-300 nm separation resolution. The solution should also provide a capability of direct isolation of circulating exosomes from whole blood.
In a first aspect, there is provided for a method of isolating exosomes from blood, the method includes:
providing a microfluidic device having a spiral-shaped channel in fluid communication with (i) two inlet ports and (ii) at least two outlet ports, wherein one of the two inlet ports is proximal to an inner wall of the spiral-shaped channel and the other inlet port is proximal to an outer wall of the spiral-shaped channel, wherein at least one of the outlet ports is in fluid communication with a container configured to store isolated exosomes;
introducing a blood sample into the inlet port proximal to the outer wall and introducing a sheath fluid into the inlet port proximal to the inner wall to form a diluted sample in the spiral-shaped channel;
driving the diluted sample through the spiral-shaped channel; and
recovering the exosomes in the container,
wherein the at least two outlet ports include a first outlet port which is in fluid communication with the container configured to store the isolated exosomes,
wherein the spiral-shaped channel in fluid communication with the first outlet port includes a first outlet channel which connects the spiral-shaped channel to the first outlet port and is longer than other outlet channels respectively connecting the spiral-shaped channel to the other outlet ports.
In another aspect, there is provided for a microfluidic device operable to isolate exosomes from blood, the microfluidic device includes:
a spiral-shaped channel in fluid communication with (i) two inlet ports and (ii) at least two outlet ports, wherein one of the two inlet ports is proximal to an inner wall of the spiral-shaped channel and the other inlet port is proximal to an outer wall of the spiral-shaped channel; and
a container in fluid communication with at least one of the outlet ports, wherein the container is configured to store isolated exosomes,
wherein the at least two outlet ports include a first outlet port which is in fluid communication with the container configured to store the isolated exosomes,
wherein the spiral-shaped channel in fluid communication with the first outlet port comprises a first outlet channel which connects the spiral-shaped channel to the first outlet port and is longer than other outlet channels respectively connecting the spiral-shaped channel to the other outlet ports.
In another aspect, there is provided a method of identifying diabetes mellitus, the method includes:
providing a blood sample and introducing the blood sample into the microfluidic device described in various embodiments of the aspect mentioned above;
operating the microfluidic device; and
isolating exosomes according to the method described in various embodiments of the first aspect mentioned above to identify diabetes mellitus.
The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the present disclosure. In the following description, various embodiments of the present disclosure are described with reference to the following drawings, in which:
The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the present disclosure may be practiced.
Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.
The present disclosure introduces a strategy for rapid and continuous isolation of extracellular vesicles, such as exosomes, from whole blood directly, which facilitates and improves the influence of centrifugal-induced Dean migration in, for example, a spiral microfluidic device. The strategy allows scalable, single-step size-based purification of exosomes without the need for ultracentrifugation or additional labelling or processing steps.
The strategy includes a method and a device which provide improved separation or fractionation resolution to isolate extracellular vesicles, such as exosomes, in a sample. The sample may be a blood sample. The present method and microfluidic device provide several advantages over existing methods as it is label-free, able to process whole blood directly at high throughput, and workable with micro-sized and nano-sized features without clogging. The terms “separation” and “fractionation” herein may be used interchangeably.
Details of various embodiments of the present method and microfluidic device, and advantages associated with the various embodiments are now described below. The various embodiments and advantages are also demonstrated through the examples provided further below herein.
In the present disclosure, there is provided a method of isolating exosomes from blood. The method includes providing a microfluidic device having a spiral-shaped channel in fluid communication with (i) two inlet ports and (ii) at least two outlet ports, wherein one of the two inlet ports is proximal to an inner wall of the spiral-shaped channel and the other inlet port is proximal to an outer wall of the spiral-shaped channel. At least one of the outlet ports may be in fluid communication with a container configured to store isolated exosomes.
The present method may include introducing a blood sample into the inlet port proximal to the outer wall and introducing a sheath fluid into the inlet port proximal to the inner wall to form a diluted sample in the spiral-shaped channel. The blood sample and sheath fluid can be introduced by means of a syringe, by dipping the device into a sample, or other means.
With one of the two inlet ports proximal to an inner wall of the spiral-shaped channel and the other inlet port proximal to an outer wall of the spiral-shaped channel, this facilitates and improves the influences of inertial focusing separation in a curved channel (i.e. the spiral-shaped channel). Such inertial focusing separation in a curved channel may be termed herein “Dean flow separation”. Said differently, the described arrangement of the two inlets port helps to set up a flow condition in a spiral-shaped channel such that the forces acting on the exosomes advantageously renders the exosomes in a sample to be completely isolated.
Moreover, with one of the two inlet ports proximal to an inner wall of the spiral-shaped channel and the other inlet port proximal to an outer wall of the spiral-shaped channel, the introduction of the blood sample and sheath fluid in the manner as described herein also assists for the exosomes to be significantly influenced by Dean flow, such that the exosomes get channeled to the inner wall of the spiral-shaped channel before exiting the spiral-shaped channel. In other words, the exosomes experience a centripetal force that drives the exosomes toward a point as the “center” of a curvature of the spiral-shaped channel as the exosomes flow in the spiral-shaped channel. Following this flow configuration, it can be easily understood that the inner wall of the spiral-shaped channel is the wall proximal to the “center” and the outer wall of the spiral-shaped channel is the wall that is away from the “center”. The term “proximal” herein includes within its meaning “near”, “at”, or “in vicinity of”.
The term “sheath fluid” herein refers to a variety of fluids, including aqueous or nonaqueous fluids and/or fluids that may include additional material components, e.g., soluble chemical components or suspensions or emulsions of at least partially insoluble components. As a non-limiting example, the sheath fluid can be a buffer that is compatible with blood cells, such as phosphate-buffered saline (abbreviated PBS). The term “buffer” herein means any compound or combination of compounds that control the pH of the environment in which they are dissolved or dispersed. Concerning the pH value, buffers diminish the effect of acids or basis added to the buffer solution. Buffers generally can be broken into two categories based upon their solubility. Both categories of buffer separately, or in combination, can be employed. “Water-soluble buffers” typically have a solubility in water of at least 1 gm in 100 ml, at least 1 gm in 75 ml, or at least 1 gm in 30 ml, etc. Examples of water-soluble buffers include, but are not limited to PBS, meglumine, sodium bicarbonate, sodium carbonate, sodium citrate, calcium gluconate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, sodium tartarate, sodium acetate, calcium glycerophosphate, tromethamine, magnesium oxide or any combination of the foregoing. “Water-insoluble buffers” typically have a solubility in water less than 1 gm in 1,000 ml, less than 1 gm in 5,000 ml, or less than 1 gm in 10,000 ml, etc. Examples of water-insoluble buffers include, but are not limited to magnesium hydroxide, aluminum hydroxide, dihydroxy aluminum sodium carbonate, calcium carbonate, aluminum phosphate, aluminum carbonate, dihydroxy aluminum amino acetate, magnesium oxide, magnesium trisilicate, magnesium carbonate, and combinations of the foregoing. Buffer can also be supplemented with supporting agents, such as salts, detergents, BSA (bovine serum albumin), etc.
The present method may include driving the diluted sample through the spiral-shaped channel, and recovering the exosomes in the container, wherein the at least two outlet ports comprise a first outlet port which is in fluid communication with the container configured to store the isolated exosomes. The diluted sample can be driven by a force of capillary attraction. Alternatively, the diluted sample can be driven any pump, by electrical forces, or by other means for driving samples through the inlets, spiral-shaped channel, and out of the outlets.
In the present method, the spiral-shaped channel in fluid communication with the first outlet port includes a first outlet channel which connects the spiral-shaped channel to the first outlet port and is longer than other outlet channels respectively connecting the spiral-shaped channel to the other outlet ports. An “outlet channel” herein refers to a channel that connects the spiral-shaped channel to an outlet port. For example, the first outlet channel is the channel that connects the spiral-shaped channel to the first outlet port from which the exosomes exit. The first outlet channel is termed herein as such, as it is the channel which connects to and is in fluid communication with the first outlet port. Accordingly, a second/third/fourth outlet channel refers to respective channel that connects to and is in fluid communication with the second/third/fourth outlet port, respectively. The first outlet channel is demonstrated in the form of a serpertine channel only by way of a non-limiting example (see examples section herein, also see, e.g.
In various embodiments, the first outlet channel may have a length ranging from 0.5 cm to 1.5 cm, 1 cm to 1.5 cm, etc. This can confer an advantage of collecting a small fraction (e.g. —0.5-4%) of the entire volume output and collecting the fluid exiting from the inner wall region.
Further advantageously, the present method avoids the need for a centrifugation step (i.e. use of a centrifugation machine). Said differently, the method does not include a centrifugation step.
In various embodiments, introducing the blood sample and the sheath fluid may include introducing the sheath fluid at a higher flow rate compared to a flow rate for introducing the blood sample. For example, introducing the blood sample and the sheath fluid may include introducing the blood sample into the inlet port proximal to the outer wall and introducing the sheath fluid into the inlet port proximal to the inner wall at a flow rate ratio of 1:5 to 1:50, 1:10 to 1:50, 1:20 to 1:50, 1:30 to 1:50, 1:40 to 1:50, etc. These flow rate ratios help to initially confine the blood sample at the outer channel wall so as to have a controlled and tighter Dean-induced lateral migration of exosomes towards the inner wall for efficient exosomes separation.
In various embodiments, the spiral-shaped channel may be defined as having a width ranging from 150 μm to 500 μm, 200 μm to 500 μm, 250 μm to 500 μm, 300 μm to 500 μm, 350 μm to 500 μm, 400 μm to 500 μm, 450 μm to 500 μm, etc. These channel width dimensions are suitable to process high cell concentration samples (e.g. whole blood) with minimal channel clogging issues.
In various embodiments, the spiral-shaped channel may be defined as having a height ranging from 30 μm to 100 μm, 40 μm to 100 μm, 50 μm to 100 μm, 60 μm to 100 μm, 70 μm to 100 μm, 80 μm to 100 μm, 90 μm to 100 μm, etc. These channel height dimensions help to prevent the larger cells (˜5 to 20 um) from flowing close to the inner wall where the exosomes migrate to or are located.
In various embodiments, the spiral-shaped channel may be defined as having a length ranging from 3 cm to 10 cm, 4 cm to 10 cm, 5 cm to 10 cm, 6 cm to 10 cm, 7 cm to 10 cm, 8 cm to 10 cm, 9 cm to 10 cm, etc. These channel length dimensions render formation of the secondary Dean vortices in the spiral-shaped channel that are sufficiently stable to induce exosome migration to inner wall of the spiral-shaped channel.
In various embodiments, the spiral-shaped channel may be defined as having a width to height aspect ratio ranging from 3 to 7, 4 to 7, 5 to 7, 6 to 7, etc. These aspect ratios help to prevent the larger cells (e.g. —5 to 20 urn) from flowing close to the inner wall where the exosomes migrate to or are located.
In various embodiments, the spiral-shaped channel may be defined as having a radius curvature ranging from 0.3 cm to 1 cm, 0.4 cm to 1 cm, 0.5 cm to 1 cm, 0.6 cm to 1 cm, 0.7 cm to 1 cm, 0.8 cm to 1 cm, 0.9 cm to 1 cm, etc. These radii of curvature render formation of secondary Dean vortices in the spiral-shaped channels. The radius of curvature of the spiral-shaped channel is the distance measured from a center of the spiral-shaped channel's cross-section to the “centripetal” center, such that the radius measured is orthogonal to the motion of fluid flowing in the spiral-shaped channel. In other words, the centripetal center is a fixed point of the center of curvature of the path.
The spiral-shaped channel may be termed herein a “spiral-shaped microchannel” due to one or more dimensions being in the micron-sized range. The spiral-shaped channel may have one or more of the dimensions and/or ratios mentioned above. In certain embodiments, the spiral-shaped channel may be a semi-spiral-shaped channel. This means the spiral-shaped channel is a channel forming a semi-circle (see e.g.
In various embodiments, the two inlet ports may be arranged in a manner where the spiral-shaped channel horizontally spirals around the inlet ports and the at least two outlet ports may be arranged away from the spiral-shaped channel (see e.g.
In various embodiments, driving the diluted sample may include driving the diluted sample to flow in the spiral-shaped channel to have a Reynolds number ranging from 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, etc., and/or a Dean number ranging from 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, etc. As used herein, Reynolds number means ρυL/μ, wherein p represents density of a liquid, υ represents velocity of the liquid, represents characteristic length of a flow channel, and μ represents viscosity of the liquid. As used herein, Dean number refers to a product of the Reynolds number (based on axial flow υ through a channel of diameter D) and the square root of the curvature ratio, i.e. Re√[D/(2Rc)], wherein Rc the radius of curvature of the path of the channel.
In certain embodiments, the at least two outlet ports may include four outlet ports. The number of outlet ports may depend on the components, other than exosomes, to be isolated from the sample.
In various embodiments, the spiral-shaped channel may gradually expand or furcate to a width of 500 μm to 3000 μm. In other words, the end of the spiral-shaped channel that is connected to the one or more outlet ports may furcate into the outlet channels that connect to their respective outlet ports. In certain embodiments, the first outlet port and/or first outlet channel may have a width ranging from 20 μm to 100 μm and the other outlet ports and/or other outlet channels then have a width that adds up to 500 μm to 3000 μm. As one example, the spiral-shaped channel may gradually expand or furcate to a width of 1000 μm, wherein the width of the first port outlet port and/or first outlet channel may be 50 μm, the width of the second outlet port and/or second outlet channel may be 50 μm, the width of the third outlet port and/or third outlet channel may be 50 μm, the width of the fourth outlet port and/or fourth outlet channel may be 4th outlet may be 800 μm.
In various embodiments, the first outlet port may have a width ranging from 20 μm to 100 μm, 30 μm to 100 μm, 40 μm to 100 μm, 50 μm to 100 μm, 60 μm to 100 μm, 70 μm to 100 μm, 80 μm to 100 μm, 90 μm to 100 μm, etc. Such widths assist in size-based separation of exosomes, increasing the yield of exosomes isolated.
The present disclosure also provides for a microfluidic device operable to isolate exosomes from blood. Embodiments and advantages described in various embodiments for the method of the first aspect can be analogously valid for the present microfluidic device subsequently described herein, and vice versa. As the various embodiments and advantages have already been described above and examples demonstrated herein, they shall not be iterated for brevity.
The microfluidic device includes a spiral-shaped channel in fluid communication with (i) two inlet ports and (ii) at least two outlet ports, wherein one of the two inlet ports is proximal to an inner wall of the spiral-shaped channel and the other inlet port is proximal to an outer wall of the spiral-shaped channel, and a container in fluid communication with at least one of the outlet ports, wherein the container is configured to store isolated exosomes.
In various embodiments, the at least two outlet ports may include a first outlet port which is in fluid communication with the container configured to store the isolated exosomes, wherein the spiral-shaped channel in fluid communication with the first outlet port includes a first outlet channel which connects the spiral-shaped channel to the first outlet port and is longer than other outlet channels respectively connecting the spiral-shaped channel to the other outlet ports.
In various embodiments, the first outlet channel may have a length ranging from 0.5 cm to 1.5 cm. Other lengths of the first outlet channel are already described above in embodiments relating to the method of the first aspect.
In various embodiments, the inlet port proximal to the inner wall of the spiral-shaped channel is operable to introduce the sheath fluid at a higher flow rate than the inlet port proximal to the outer wall of the spiral-shaped channel.
In various embodiments, the inlet port proximal to the outlet wall of the spiral-shaped channel and the inlet port proximal to the inner wall of the spiral-shaped channel are operable to introduce the blood sample and the sheath fluid at a flow rate ratio of 1:5 to 1:50. Other flow rate ratios are already described above in embodiments relating to the method of the first aspect.
The spiral-shaped channel may be defined as having a width ranging from 150 μm to 500 μm, a height ranging from 30 μm to 100 μm, a length ranging from 3 cm to 10 cm, a width to height aspect ratio ranging from 3 to 7, and/or a radius curvature ranging from 0.3 cm to 1 cm. Other dimensions and ratios are already described above in embodiments relating to the method of the first aspect.
The spiral-shaped channel may be a semi-spiral-shaped channel. The semi-spiral-shaped channel may have a length as described in various embodiments of the method of the first aspect.
In various embodiments, the two inlet ports may be arranged in a manner where the spiral-shaped channel horizontally spirals around the inlet ports and the at least two outlet ports may be arranged away from the spiral-shaped channel. In certain embodiments, the two inlet ports may be arranged away from the spiral-shaped channel and the at least two outlet ports may be arranged in a manner where the spiral-shaped channel horizontally spirals around the at least two outlet ports.
The at least two outlet ports may include four outlet ports.
The spiral-shaped channel may gradually expand or furcate to a width of 500 μm to 3000 μm.
The first outlet port may have a width ranging from 20 μm to 100 μm.
The present microfluidic device may be used in ex vivo and/or in vitro identification of diabetes, such as diabetes mellitus. As described above and in the examples section herein, a sample such as blood sample may be drawn from a subject. Then, without the need for the subject to be present, the blood sample may be, injected as an example, into the present microfluidic device.
The present disclosure also provides for a method of identifying diabetes, such as diabetes mellitus, the method may include providing a blood sample and introducing the blood sample into the microfluidic device described according to various embodiments above, operating the microfluidic device, and isolating exosomes according to the method described in various embodiments of the first aspect to identify diabetes mellitus.
Embodiments and advantages described for the present method of the first aspect and for the microfluidic device can be analogously valid for the present method of identifying diabetes mellitus described herein, and vice versa. As the various embodiments and advantages have already been described above and examples demonstrated herein, they shall not be iterated for brevity. Demonstration/Application of the present method and microfluidic device to identify type 2 diabetes are described in the examples section below.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the present disclosure.
In the context of various embodiments, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements.
In the context of various embodiments, the punctuation “—”, the term “about” or “approximately” as applied to a numeric value encompasses the exact value and a reasonable variance.
As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
Unless specified otherwise, the terms “comprising” and “comprise”, and grammatical variants thereof, are intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, unrecited elements.
The present disclosure relates to a method and a microfluidic device operable to isolate extracellular vesicles, particularly or specifically exosomes, from blood.
The present method and microfluidic device may involve, for example, the use of a 2-inlet and at least a 2-outlet (e.g. 4-outlet) system spiral microchannel that can demonstrate multiplexed size-based fractionation of EVs (exosomes (˜50-200 nm) and microvesicles (˜100 nm-1 μm), platelets (˜2 μm) and blood cells (>8 μm) into the different outlets. The present method and microfluidic device provide for a label-free, low-cost EVs purification approach with desirably high throughput (˜20 μL undiluted whole blood (WB)/min), are scalable by device stacking, and offer higher exosomes yield as compared to ultracentrifugation. WB denotes whole blood. The spiral-shaped channel is termed herein a “spiral-shaped microchannel” due to its micron-sized dimension. The terms “spiral” and “spiral-shaped” herein are used exchangeably to describe the structure of the channel.
The present method and microfluidic device involve a spiral-shaped or curvilinear channel in which a sample suspected to contain exosomes can flow. The spiral-shaped or curvilinear channel may have at least a first end and a second end, wherein the spiral-shaped channel can have at least two inlet ports proximal to (i.e. at or near) the first end and at least two outlet ports proximal to the second end, wherein the at least two outlet ports contain a first outlet port (O1) that can be in fluid communication with an additional channel length, which provides for more fluidic resistance, to render sub-300 nm separation resolution for the separation of exosomes. This additional channel length may be termed herein a “first connecting channel” or a “first outlet channel” as described above.
In certain embodiments, the additional channel length connected to a first outlet port (O1) can be, for example, 0.5 cm to 1.5 cm. The additional channel length connected to the first outlet port (O1) can be, for example, 0.5 cm to 1.5 cm, and have a flow rate of 1 to 15 uL/min for example. The first outlet port (O1) is proximal to or located at the inner wall region. In other words, the first outlet port is positioned to receive exosomes that flows proximal to the inner wall region of the spiral-shaped microchannel The outlet port (O1) will collect a fraction (˜0.5 to 4%) of the total volume output.
In various embodiments, the microfluidic device may have more than one flow channel to allow multiplexing.
Advantageously, the present microfluidic device allows for sub-300 nm separation resolution. Advantageously, the proposed microfluidic device is able to completely isolate exosomes from whole blood.
The present method and microfluidic device may be referred to herein as “HiDFF” technology and/or “ExoDFF” technology. The present method and microfluidic device are described in further details, by way of non-limiting examples, as set forth below.
The 2-inlet, 4-outlet spiral microdevice (300 μm (w)×50 μm (h)) was fabricated in polydimethylsiloxane (PDMS) using standard soft lithography techniques and has a radius of 0.5-0.6 cm and a total length of ˜6.5 cm (
During device operation, diluted WB (1:1) is perfused through the outer inlet while sheath buffer (1× phosphate buffer saline (PBS)) is perfused through the inner inlet at a higher flow rate (1:10) to confine the sample stream near the outer wall. As smaller blood components (platelets and EVs, particle size (ap)/hydraulic diameter (Dh) <0.07) traverse through the channel, they experience lateral drag forces (FD) and migrate towards the inner wall due to influence of Dean vortices (
Computational fluid dynamics (CFD) modelling using ANSYS FLUENT was conducted to study the outlet design. Exosomes were assumed to behave like fluid particles due to its tiny size (<200 nm) and their streamlines were tracked starting from sample inlet and throughout the channel. The channel resistance of outlet 1 was first investigated by varying its channel length (denoted as 0x and 2.5x resistance). The flow trajectories of 50 nm fluorescent beads obtained experimentally, and streamlines profile predicted using CFD were then compared between different channel length of outlet 1 (
To characterize the present device and to study the flow rate conditions, fluorescent polystyrene microbeads (ap/h<0.07) of defined sizes (e.g. 50 nm, 1 μm, 2 μm, 3 μm) were used to visualize streamline positions and successful multiplexed bead separation (
As proof-of-concept for whole blood processing, whole blood was diluted in a ratio of 1:1 with PBS and perfused into the HiDFF device. High-speed imaging clearly indicated efficient removal of platelets (˜2-3 μm) and larger blood cells (˜5-15 μm) via outlets 3 and 4, respectively (
Non-limiting examples of certain parameters that may be used for the present device are set out below. The parameters serve to structurally describe the present device and is not intended to limit the device to such parameters.
Inlet Configuration:
Sample inlet is configured at the outer side of the spiral channel.
Sheath inlet is configured at the inner side of the spiral channel.
Sample to sheath flow rate ratio can be 1:5 to 1:50.
Channel Configuration:
Channel length: 3-10 cm
Channel width: 150-500 μm
Channel height: 30-100 μm
Channel aspect ratio (width/height):3-7
Radius of curvature: 0.3-1 cm
Operable Reynolds number range: 20-100
Operable Dean number range: 2-10
Sample can migrate along the top and bottom channel wall from the outer wall towards the inner wall region.
Outlet Configuration:
Exosome outlet can be at the inner wall of the spiral channel.
Outlet design can have more than 1 outlet and up to 10 outlets.
Exosome outlet can collect a fraction (˜0.5 to 4%) of the total volume output.
Exosome outlet width can be from 20-100 μm.
Extracellular vesicles (EV) are mediators of intracellular communication in health and diseases. Despite significant interest in EV-based biomarkers in liquid biopsy, clinical utilities remain limited due to difficulties in isolating EV from whole blood with high yield and reproducibility. Inertial microfluidics is widely used for cell separation (˜10 to 20 μm in diameter) but remains challenging for smaller nanoparticles (<1 μm) due to negligible inertial forces for particle equilibrium focusing. The present device is a unique microfluidic separation technology for direct isolation of circulating EV from whole blood using an inertia-based method. This label-free approach enables simultaneous fractionation of nanoscale EV (exosomes, 50 to 200 nm in diameter) and medium-sized EV (microvesicles (MV), 100 nm to 1 μm in diameter) from whole blood based on differential wall-induced lift forces in spiral microchannels. Besides achieving a three-fold increase in EV yield and complete depletion of cellular constituents, the gentle sorting method also leads to a significant (ten-fold) reduction of platelet-derived MV as compared to ultracentrifugation (UC) due to minimal shear-induced platelet lysis. In a pilot clinical study of healthy (n=9) and type 2 diabetes mellitus (T2DM) (n=12) subjects, higher EV levels were detected in T2DM patients (P<0.05) using the present method, and identified a subset of “high-risk” T2DM subjects with abnormally high (˜10 to 50-fold) amount of platelet (CD41a+) or leukocyte-derived (CD45+) MV by immunophenotyping the sorted EV. In vitro endothelial cell assay further revealed that the “high-risk” T2DM EV induced significantly higher vascular inflammation (ICAM-1 expression) (P<0.05) as compared to healthy and T2DM EV, thus reflecting a pro-inflammatory phenotype. Overall, the method presented here is a scalable and versatile EV research tool which reduces manual labour, cost and processing time. This facilitates the further development of EV-based diagnostics using liquid biopsy, and a combinatory EV immuno- and functional phenotyping strategy can potentially be used for rapid vascular risk stratification in T2DM.
The present method and microfluidic device are versatile to cater for another example of the multiplexing strategy disclosed herein, wherein the position of outlets and inlets were switched to create another second generation HiDFF (hereafter termed as 2nd Gen ExoDFF) so that the inlets of each subunit spiral can be connected easily without modifying the outlet design, wherein the abbreviation “ExoDFF” denotes for Exosomes Dean Flow Fractionation. In other words, for the present ExoDFF device, the inlets are positioned away from the spiral channel and the outlets are positioned in a manner where the channel spirals around the outlets (e.g. surround the outlets—see center and rightmost images of
As proof-of-concept, 4 subunits of spiral channel were designed and fabricated to become high throughput ExoDFF (ExoDFFHT) (
Separation performance of the present ExoDFFHT was further quantified using 200 nm beads. Nanoparticle tracking analaysis (NTA) results showed that highest separation efficiency of 2nd Gen ExoDFF and ExoDFFHT were at 20:400 μL/min at each spiral (
Blood borne EVs collected from 1st Gen HiDFF, ExoDFFHT and ultracentrifugation (UC) were next compared using NTA and Western blot. NTA showed that ExoDFFHT and UC small EVs exhibited similar particle size distribution below 200 nm corresponding to small EVs (
As mentioned above, the present method and microfluidic device is versatile. In another strategy of the present disclosure for high throughput sample processing, the ExoDFF spiral length was shortened by 4-5 times to a “half-loop” channel design (i.e. semi-spiral channel). The idea was to increase the flow rate to achieve similar EV sorting performance. This was demonstrated using beads (50 nm, 500 nm, 1 um) in 2 half loop designs of 300 μm width (same as ExoDFF) (
Isolation of circulating extracellular vesicles (EV) from whole blood using a 4-outlet HiDFF (hereinafter the chip may also be referred to as ExoDFF only for simplicity and to signify both the present HiDFF and ExoDFF configurations are operable for this example) was demonstrated, wherein the present 4-outlet HiDFF device has the longer first outlet channel and the inlet ports are arranged in a manner where the spiral-shaped channel spirals around the inlet ports and the outlet ports are arranged away from the spiral-shaped channel. Diluted whole blood (1:1 PBS) and sheath fluid were perfused into the ExoDFF device at the optimized flow rates (Re˜40, 40 μLmin−1 (sample) and 400 μLmin−1 (sheath)). Re denotes Reynolds number. As shown in high speed images, larger blood cells (˜6-15 μm) remained close to the channel outer wall prior outlets and were efficiently removed via O4 (
A low-cost and label-free microfluidic strategy is herein developed for direct and scalable isolation of circulating exosomes from whole blood. The present methods and microfluidic device are straightfoward to use, requires minimal user operation, and can be readily translated to clinical settings to accelerate exosome biology research, and development of point-of-care exosome diagnostic tools.
The development of a single-step, high-throughput and label-free exosome sorting method has great commercial interest ranging from genomics and proteomics studies, to large-scale exosomes biomanufacturing and point-of-care clinical diagnostics. With the present technology, it can advance the development of a “sample-in-answer-out” liquid biopsy-based testing system (
The advantages of the present technology include the passive separation principle, user-friendliness (only syringe pumps needed) and low-cost operation (no need for labelling or processing). Of note, the microdevice is also portable and easily integrated to other platforms for downstream detection or analysis.
While the present disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as defined by the appended claims. The scope of the present disclosure is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.
Number | Date | Country | Kind |
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10201909776U | Oct 2019 | SG | national |
Filing Document | Filing Date | Country | Kind |
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PCT/SG2020/050601 | 10/21/2020 | WO |