Claims:1. A microfluidic system for determining mechanical properties of biological cells, comprising:
a first microfluidic channel comprising an inlet and an outlet, the inlet being configured to receive a fluidic medium containing cells into the first microfluidic channel, and the outlet being configured to dispense the fluidic medium from the first microfluidic channel; and
a second microfluidic channel comprising a first air inlet and a second opposite air inlet;
wherein the first and second microfluidic channels intersect perpendicularly to each other to form an intersection region;
wherein each of the first and second air inlets receives pressurized air that flows towards the intersection region, and wherein the air flow creates, in the intersection region, an air/liquid interface which squeezes the cells as they pass through the air/liquid interface.
2. The microfluidic system as claimed in claim 1, wherein the air/liquid interface creates a constriction in the intersection region which squeezes the cells as they pass through the air/liquid interface.
3. The microfluidic system as claimed in claim 1, wherein the constriction having a width smaller than a width of the first microfluidic channel.
4. The microfluidic system as claimed in claim 3, wherein the constriction having a width as small as 2 µm.
5. The microfluidic system as claimed in claim 1, further comprising an imaging unit configured to capture a plurality of images of the cells passing through the air/liquid interface.
6. The microfluidic system as claimed in claim 1, further comprising a data processing unit configured to calculate one or more mechanical properties of the cells.
7. The microfluidic system as claimed in claim 1, wherein the mechanical properties of biological cells comprising cell deformability, cell shape, cell stiffness, and intracellular structure.
8. A method for determining mechanical properties of biological cells, comprising the steps of:
(a) providing a microfluidic system comprising:
a first microfluidic channel comprising an inlet and an outlet, the inlet being configured to receive a fluidic medium containing cells into the first microfluidic channel, and the outlet being configured to dispense the fluidic medium from the first microfluidic channel;
a second microfluidic channel comprising a first air inlet and a second opposite air inlet;
wherein the first and second microfluidic channels intersect perpendicularly to each other to form an intersection region;
(b) letting a fluidic medium containing cells pass through the first microfluidic channel so as to produce a flow;
(c) flowing pressurized air into the second microfluidic channel through the first and second air inlets, wherein the pressurized air flows towards the intersection region, and wherein the air flow creates, in the intersection region, an air/liquid interface which squeezes the cells as they pass through the air/liquid interface;
(d) obtaining a plurality of images of the cells as they pass through the first microfluidic channel; and
(e) calculating one or more mechanical properties of the cells from the obtained plurality of images.
9. The method as claimed in claim 9, wherein the one or more mechanical properties of the cells comprising cell deformability, cell shape, cell stiffness, and intracellular structure.
, Description:
FIELD OF THE INVENTION
[0001] The present disclosure pertains to technical field of microfluidic devices. In particular, the present disclosure pertains to air-liquid or liquid-liquid interface based microfluidic cell squeezer for determining mechanical properties of a biological system such as single cell.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Cells are active materials that respond to changes in their environment by changing their mechanical properties. With the advent of advanced characterization techniques [“Cell mechanics and the cytoskeleton” Fletcher, D. A., & Mullins, R. D. (2010), Nature, 463(7280), 485-492] combined with cell biology, the mechanical responses can be detected at single cell level, which helps in investigating fundamental questions in cell biology. Towards this, researchers have developed techniques such as 1. Optical Tweezers [“Assessment of red blood cell deformability in type 2 diabetes mellitus and diabetic retinopathy by dual optical tweezers stretching technique” R. Agrawal et al. (2016), Scientific Reports 6, Article number 15873]; 2. Magnetic Probes [“Magnetic nanoparticle-mediated massively-parallel mechanical modulation of single-cell behavior” Tseng, P., Judy, J. W., & Di Carlo, D. (2012), Nature methods, 9(11), 1113-1119]; 3. MEMS Platforms [“Microengineered Platforms for Cell Mechanobiology” Kim et al. (2009), Annual review of biomedical engineering, 11, 203-233]; 4. Atomic Force Microscopy [“Investigating cell mechanics with atomic force microscopy” Haase, K., & Pelling, A. E. (2015), Journal of The Royal Society Interface, 12(104), 20140970]; and 5. Microfluidic Cell Transit Analyzers [“Hydrodynamic stretching of single cells for large population mechanical phenotyping” Gossett et al (2012). Proceedings of the National Academy of Sciences, 109(20), 7630-7635].
[0004] However, most of the aforementioned prior art techniques possess drawbacks and limitations which seriously limit their effectiveness and/or flexibility. For example, the prior art cell deformability measurement techniques are slow and low-throughput, and some lead to localized heating resulting in potential cell damage. Amongst the prior art techniques, the microfluidic cell transit analyzers are attractive as they allow high throughput analysis of cell population. Conventional cell transit analyzers use a mechanical (solid) constriction (smaller than size of cell) to slow the cell. The slowing down has been shown to depend on stiffness of cells, and estimations of mechanical properties of cell can be made by measuring the transit time of cell passage. However, the presence of physical walls naturally leads to the problem of cell-substrate interactions. Cell-substrate interaction due to cell adhesion interferes with the measurement of mechanical properties of cells. Also, the conventional microfluidic analyzers have fixed channel width, which cannot be altered dynamically for different cell sizes and are prone to channel clogging owing to their small dimensions.
[0005] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.
[0006] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0007] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability.

OBJECTS OF THE INVENTION
[0008] It is an object of the present disclosure to provide a new and improved microfluidic system that overcomes one or more disadvantages associated with previously known devices and techniques that investigate cell biomechanics.
[0009] It is a further object of the present disclosure to provide a new and improved microfluidic system for investigating mechanical properties of a biological system such as single cell.
[0010] It is another object of the present disclosure to provide a microfluidic system that enables measurement of cell deformability at high throughputs.
[0011] It is another object of the present disclosure to provide a microfluidic system that can be used for different cells with comparable sizes, thereby offering flexibility and scalability.
[0012] It is another object of the present disclosure to provide a microfluidic system that solves the channel clogging problem inherent in conventional bio-microfluidic devices.
[0013] It is another object of the present disclosure to provide a microfluidic system which eliminates cell-substrate interactions and provides more accurate estimate of mechanical properties of biological cells.
[0014] It is another object of the present disclosure to provide a microfluidic system that allows dynamic adjustment of microfluidic channel width to achieve desirable channel width.
[0015] It is another object of the present disclosure to provide a microfluidic system that enables real time adjustment of microfluidic channel dimension.
[0016] It is yet another object of the present disclosure to provide a quick, cost-effective and scalable method for investigating mechanical properties of a biological system such as single cell.

SUMMARY
[0017] Aspects of the present disclosure relate to a new and improved microfluidic system which can precisely measure mechanical properties of a biological system such as single cell. The microfluidic system disclosed herein can enable measurement of cell mechanics at high throughputs, and can solve the channel clogging problem inherent in conventional bio-microfluidic devices. Further, the disclosed microfluidic system can eliminate cell-substrate interactions and provide more accurate estimate of mechanical properties of biological cells.
[0018] In an aspect, the present disclosure provides a microfluidic system for determining mechanical properties of biological cells through single cell deformation, wherein the system can include:
a first microfluidic channel including an inlet and an outlet, the inlet being configured to receive a fluidic medium containing cells into the first microfluidic channel, and the outlet being configured to dispense the fluidic medium from the first microfluidic channel; and
a second microfluidic channel including a first air inlet and a second opposite air inlet;
wherein the first and second microfluidic channels intersect perpendicularly to each other to form an intersection region;
wherein each of the first and second air inlets receives pressurized air that flows towards the intersection region, and wherein the air flow creates, in the intersection region, an air/liquid interface which squeezes the cells as they pass through the air/liquid interface.
[0019] According to embodiments of the present disclosure, the air/liquid interface creates a constriction in the intersection region which squeezes cells as they pass through the air/liquid interface. The constriction can have a width which is smaller than a width of the first microfluidic channel. Further, the constriction width can be adjusted to any desired working width by controlling the pressure of air supplied via the first and second air inlets to the second microfluidic channel. Thus, the disclosed microfluidic system can replace the conventional solid mechanical constriction with a tunable air/liquid interface.
[0020] In one preferred embodiment, air can be flowed into the second microfluidic channel through the first and second air inlets to create virtual constriction having a width in the range of 1 µm to 7 µm.
[0021] In an embodiment, the microfluidic system disclosed herein can further include an imaging unit / sensor array configured to image deformation of each cell passing through the air/liquid interface. In another embodiment, the microfluidic system can include a data processing unit configured to use data from the imaging unit / sensor array to calculate one or more mechanical properties of the cells.
[0022] According to embodiments of the present disclosure, the disclosed microfluidic system can be used to measure single cell mechanical properties including, but not limited to, cell deformability, cell shape, cell stiffness, and intracellular structure.
[0023] In another aspect, the present disclosure provides a method for determining mechanical properties of biological cells using the microfluidic system disclosed herein.
[0024] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0026] FIG. 1 illustrates a schematic view of a microfluidic system for determining mechanical properties of cells in accordance with one embodiment of the present disclosure.
[0027] FIG. 2 schematically shows an air/liquid interface as a cell squeezer in a high throughput microfluidic channel, in accordance with embodiments of the present disclosure.
[0028] FIG. 3 illustrates changing the microfluidic channel width from 7 µm to 2 µm by controlling the pressure of air supplied via the first and second air inlets to the second microfluidic channel, in accordance with embodiments of the present disclosure.
[0029] FIG. 4 is a graph showing average transit time of Red Blood Cells (RBCs) passing through different constriction widths, in accordance with embodiments of the present disclosure.
[0030] FIG. 5 shows superimposed time lapse images of RBCs at the Inlet, Centre and Outlet of the junction for different virtual constrictions, in accordance with embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0031] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0032] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[0033] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0034] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0035] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
[0036] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0037] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0038] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0039] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0040] Reference will now be made in detail to the exemplary embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.
[0041] Embodiments of the present disclosure provide a technique for measuring mechanical properties of individual cells through single cell compression. Specifically, embodiments of the present disclosure use a microfluidic system which is configured to produce virtual constrictions in microfluidic channel to cause deformation of the cells. The cell deformation behavior can then be obtained to extract the mechanical properties of each cell passing through the constriction. The microfluidic system disclosed herein can enable measurement of cell mechanics at high throughputs, and can solve the channel clogging problem inherent in conventional bio-microfluidic devices. Further, the disclosed microfluidic system can eliminate cell-substrate interactions and provide more accurate estimate of mechanical properties of biological system.
[0042] In an aspect, the present disclosure provides a microfluidic system for determining mechanical properties of biological cells through single cell deformation, wherein the system can include:
a first microfluidic channel including an inlet and an outlet, the inlet being configured to receive a fluidic medium containing cells into the first microfluidic channel, and the outlet being configured to dispense the fluidic medium from the first microfluidic channel; and
a second microfluidic channel including a first air inlet and a second opposite air inlet;
wherein the first and second microfluidic channels intersect perpendicularly to each other to form an intersection region;
wherein each of the first and second air inlets receives pressurized air that flows towards the intersection region, and wherein the air flow creates, in the intersection region, an air/liquid interface which squeezes the cells as they pass through the air/liquid interface.
[0043] The disclosed microfluidic system can be made of any material or combination of materials suitable for use in providing microfluidic channels that can allow biological cells to pass through the microfluidic channels. Preferably, the microfluidic system can be made of a polymeric material. More preferably, the microfluidic system can be made of polydimethylsiloxane (PDMS). The first and second microfluidic channels can have a height in the range of 2 µm to 10 µm, preferably 5 µm, and a width in the range of 10 µm to 20 µm, preferably 10 µm. The microfluidic system can be fabricated using micro-fabrication techniques such as, but not limited to, soft lithography. The fabricated microfluidic system may then be bonded onto a rigid supporting substrate like glass.
[0044] According to embodiments of the present disclosure, the air/liquid interface creates a constriction in the intersection region which squeezes cells as they pass through the air/liquid interface. The constriction can have a width which is smaller than a width of the first microfluidic channel through which fluidic medium and cells traverse. Further, the width of the constriction can be adjusted to any desired working width by controlling the pressure of air supplied via the first and second air inlets to the second microfluidic channel. Thus, the disclosed microfluidic system can replace the conventional solid mechanical constriction with a tunable air/liquid interface.
[0045] In one preferred embodiment, air can be flowed into the second microfluidic channel through the first and second air inlets to create virtual constriction as small as 1 µm. Although the preferred embodiments use air to create virtual constrictions in the microfluidic channel, other gases could be used without deviating from the scope of the present disclosure.
[0046] In some embodiments, a fluidic medium containing cells can pass through the first microfluidic channel system at low flow rates. Pressure or force can be applied to the fluidic medium in order to drive a cell through the virtual constriction.
[0047] In an embodiment, the microfluidic system disclosed herein can further include an imaging unit configured to image deformation of each cell passing through the air/liquid interface. The imaging unit may be any system capable of providing one or more images of each moving cell, and capturing deformation of each cell as it travels through the first microfluidic channel. In one preferred embodiment, the imaging unit can be a digital high speed camera capable of capturing several thousand frames per second.
[0048] In another embodiment, the microfluidic system can include a data processing unit configured to use data from the imaging unit to calculate mechanical properties of the cells.
[0049] According to embodiments of the present disclosure, the disclosed microfluidic system can be used to study mechanical properties of cells such as, but not limited to, cell deformability, cell shape, cell stiffness, and intracellular structure. In some embodiments, stiffness of a cell can be measured by measuring the transit time of cell passing through the constriction. However, other possibilities to extract mechanical properties can be also applied.
[0050] FIG. 1 illustrates an exemplary configuration of cell mechanics measuring system 100 constructed in accordance with embodiments of the present disclosure. The microfluidic system 100 can consist of a plus-shaped polydimethylsiloxane (PDMS) channel-network fabricated using soft lithography. The microfluidic system 100 can include a first microfluidic channel 102, a second microfluidic channel 108 and an intersection region 112. The first and second microfluidic channels 102 and 108 can have a height of 5 µm, and a width of 10 µm. As shown in FIG. 1, the first and second microfluidic channels can intersect perpendicularly to each other to form the intersection region 112. The first microfluidic channel 102 can include an inlet 104 through which a fluidic medium containing cells can be flowed. The opposing outlet end 106 can be used to dispense of the fluidic medium flowing from the first microfluidic channel. Cells suspended in a buffer or solution can be caused to flow from the inlet end 104 tothe outlet end 106, and a flow speed of the buffer or solution can be adjusted as required. The second microfluidic channel 108 can include a first air inlet 110-a and a second opposite air inlet 110-b through which a pressurized gas (e.g. air) can be introduced into the second microfluidic channel 108.
[0051] As shown in FIG. 2, when pressurized air is caused to flow from the air inlet ends 110-a and 110-b toward the intersection region 112, the flow of air creates an air/liquid interface 116 at the intersection region. The air/liquid interface 116 thus produced creates a virtual constriction in the intersection region which squeezes cells as they pass through the air/liquid interface 116.The constriction can be sized such that only a single cell can pass through the constriction at one time. As the cell 114 approaches and passes through the constriction, the constriction applies pressure (e.g. mechanical compression) to the cell 114, and squeezes the cell 114 (shown as cell 114-a). In an exemplary embodiment, the microfluidic system 100 can create a stiffer air/liquid interface (e.g. air/water interface, s ~ 70 mN) which can be used to deform a softer biological material (e.g. Red Blood Cell, s ~ 20 mN/m).
[0052] A pressure control device can be used to control the pressure of air supplied through the inlets 110-a and 110-b in order to achieve different virtual channel widths. FIG. 3 illustrates changing the width of the microfluidic channel 102 from 7 µm to 2 µm by controlling air pressure at both the air inlet ends 110-a and 110-b. Thus, the microfluidic system 100 can replace the conventional solid mechanical constriction with a tunable air/liquid interface. Further, the microfluidic system 100 can eliminate the problem of clogging of the microfluidic channel, and cell-substrate interactions which often hide the true deformability of cells.
[0053] In exemplary testing, Red Blood Cell (RBC) buffer was caused to flow from the inlet end 104 to the outlet end 106 at low flow rates, and air pressure was maintained at the first and second inlets 110-a and 110-b. The air/liquid interfaces produced were found to be intact for long durations (> 5 h). In order to validate the effect of interfaces squeezing the cells, high speed imaging of the RBCs flowing in the first microfluidic channel was done. The transit time, which is a direct estimate of the stiffness of the cells, was analyzed using image processing. It was found that the transit time of the RBCs increased as the constriction size decreased as shown in FIGs. 4 and 5, thereby confirming the effectiveness of the disclosed microfluidic system 100 in mechanobiological applications. It was further observed that the air/liquid interfaces did not deform as cells passed between them and this confirmed that the constriction of liquid-air interface works in exactly the same way as a mechanical (solid) constriction.
[0054] In another aspect, the present disclosure provides a method for determining mechanical properties of biological cells, wherein the method can include the steps of:
(a) providing a microfluidic system comprising:
a first microfluidic channel comprising an inlet and an outlet, the inlet being configured to receive a fluidic medium containing cells into the first microfluidic channel, and the outlet being configured to dispense the fluidic medium from the first microfluidic channel;
a second microfluidic channel comprising a first air inlet and a second opposite air inlet;
wherein the first and second microfluidic channels intersect perpendicularly to each other to form an intersection region;
(b) letting a fluidic medium containing cells pass through the first microfluidic channel so as to produce a flow;
(c) flowing pressurized air into the second microfluidic channel through the first and second air inlets, wherein the pressurized air flows towards the intersection region, and wherein the air flow creates, in the intersection region, an air/liquid interface which squeezes the cells as they pass through the air/liquid interface;
(d) obtaining a plurality of images of the cells as they passthrough the first microfluidic channel; and
(e) calculating one or more mechanical properties of the cells from the obtained plurality of images.
[0055] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the appended claims.

ADVANTAGES OF THE PRESENT INVENTION
[0056] The present disclosure provides a new and improved microfluidic system that provides precise measurement of biomechanics of individual cells.
[0057] The present disclosure provides an improved microfluidic system that enables measurement of cell deformability at high throughputs.
[0058] The present disclosure provides a microfluidic system that solves the channel clogging problem inherent in conventional bio-microfluidic devices.
[0059] The present disclosure provides a microfluidic system which eliminates the problem of cell-substrate interaction which is a major problem in the existing techniques.
[0060] The present disclosure provides a microfluidic system that replaces the conventional solid mechanical walls with a tunable air/liquid or liquid/liquid interface.
[0061] The present disclosure provides a microfluidic system that can tune the microfluidic channel width dynamically and can work for variety of cells with comparable sizes.
[0062] The present disclosure provides a microfluidic system that allows dynamic adjustment of microfluidic channel width using tunable air/liquid or liquid/liquid interface, thereby achieving stable channel widths as small as 2 µm.
[0063] The present disclosure provides a microfluidic system that enables real time adjustment of microfluidic channel dimension.
[0064] The present disclosure provides a quick, cost-effective and scalable method for investigating mechanical properties of a biological system such as single cell.