DESC:
TECHNICAL FIELD
[0001] The present invention relates generally to the field of electro-mechanodiagnostics. More specifically, the present invention relates to a system for estimation of deformability of a cell using electrical impedance data.

BACKGROUND
[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] Study of mechanical properties of a cell can provide several important insights. Many techniques have been proposed and developed so far to study the mechanical properties of cells. Among these techniques, microfluidics has dominated in the last decade owing to low sample volume and high-throughput measurement in cell deformability. Microfluidic cell-transit analyzers study the mechanical properties of cells by measurements of Deformability Index and transit time of cells. However, such systems and methods necessitate utilization of high-speed camera(s) for measurement of the deformability index, which is/are expensive, bulky and time-consuming and hence, limit(s) the portability and ease of operation. Accordingly, none of these system and/or methods could find practical application on a commercial scale.
[0004] Electrical Impedance Spectroscopy (EIS) has been used as a tool to study properties of single cells for quite some time. Several reports indicate usage thereof for study of biological parameters. For example, Sohn et al. [Capacitance cytometry: Measuring biological cells one by one, Proc. Natl. Acad. Sci., vol. 97, no. 20, pp. 10687-10690, Sep. 2000] discloses usage thereof for quantification of DNA in single cells, Cheung et al. [Impedance spectroscopy flow cytometry: On-chip label-free cell differentiation, Cytom. Part A, vol. 65, no. 2, pp. 124-132, 2005] discloses usage thereof as a label-free technique to differentiate red blood cells with varying Glutaraldehyde concentration, Jang et al. [Microfluidic device for cell capture and impedance measurement, Biomed. Microdevices, vol. 9, no. 5, pp. 737-743, 2007] discloses usage thereof to measure electrical properties of single cells using a mechanical trap, Katsumoto et al. [Dielectric Cytometry with Three-Dimensional Cellular Modeling, Biophys. J., vol. 95, no. 6, pp. 3043-3047, 2008] discloses usage thereof to extract volumetric information of cells and differentiated between discocytes and echinocytes, Chen et al. [Classification of cell types using a microfluidic device for mechanical and electrical measurement on single cells, Lab Chip, vol. 11, no. 18, p. 3174, 2011] discloses usage of EIS for differentiating between cell lines by using transit time measurement, Zheng et al. [High-throughput biophysical measurement of human red blood cells, Lab Chip, vol. 12, no. 14, p. 2560, 2012] measured transit time to differentiate neonatal RBCs from healthy RBCs, Emaminejad et al. [Microfluidic diagnostic tool for the developing world: contactless impedance flow cytometry, Lab Chip, vol. 12, no. 21, p. 4499, 2012] discloses usage thereof for studying the electrical properties of cell population, and Adamo et al. [Microfluidics-Based Assessment of Cell Deformability, Anal. Chem., vol. 84, no. 15, pp. 6438-6443, Aug. 2012] used the transit time measurement to study deformability of HeLa cells. Katsumoto et al. [Electrical classification of single red blood cell deformability in high-shear microchannel flows, Int. J. Heat Fluid Flow, vol. 31, no. 6, pp.985-995, Dec. 2010] discloses usage of Electrical Impedance Spectroscopy (EIS) to measure the deformability of the cells in high shear flows by measuring the deformed length using the electrical measurements using FWHM technique. However, this method suffers from two major limitations viz. (i) the method does not take into account, the flow rate fluctuations, which are dominant in high shear flows, and (ii) the method does not allow measurement of deformability index in low shear flows.
[0005] Accordingly, it can be gathered that with the advent of EIS, the cell transit-time measurements have become realistic and feasible at low cost. However, measurements of transit time using the reported systems and methods have not been widely accepted for the measurement of cell deformability, owing to dynamic flow rate fluctuations of the cell(s) under analysis in microchannel.
[0006] There is, therefore, a need in the art to develop a system for estimation of deformability of a cell by measuring its deformability index and transit time using electrical impedance data. Need is also felt of a system for estimation of deformability of a cell that allows measurement of transit time, offsetting the dynamic flow rate fluctuations in microchannel.
OBJECTS OF THE INVENTION
[0007] An object of the present disclosure is to provide a system for estimation of deformability of a cell that satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.
[0008] Another object of the present disclosure is to provide a system for estimation of deformability of a cell that does not require utilization of high-speed camera(s) for measurement of the deformability index.
[0009] Another object of the present disclosure is to provide a system for estimation of deformability of a cell that allows estimation of deformability of a cell using electrical impedance data.
[0010] Another object of the present disclosure is to provide a system for estimation of deformability of a cell that allows measurement of cell’s impedance signature.
[0011] Another object of the present disclosure is to provide a system for estimation of deformability of a cell that allows monitoring and/or detection of the health or disease states of the cell.
[0012] Another object of the present disclosure is to provide a system for estimation of deformability of a cell that allows measurement of transit time thereof while offsetting the dynamic flow rate fluctuations in microchannel.

SUMMARY
[0013] The present invention relates generally to the field of electro-mechanodiagnostics. More specifically, the present invention relates to a system for estimation of deformability of a cell using electrical impedance data.
[0014] An aspect of the present disclosure provides a system for estimation of deformability of a cell, the system including: a microchannel defining a flow path for the cell, the flow path defining at least two sections, wherein a first section of said at least two sections has a diameter larger than that of a second section of said at least two sections; a first source electrode flanked by a first pair of reference electrodes, configured to measure a first electrical impedance of the cell transiting through the first section of said flow path; and a second source electrode flanked by a second pair of reference electrodes, configured to measure a second electrical impedance of the cell transiting through the second section of said flow path.
[0015] In an embodiment, the first source electrode and the first pair of reference electrodes are configured to measure the first electrical impedance of the cell in a self-referenced manner. In an embodiment, the second source electrode and the second pair of reference electrodes are configured to measure the second electrical impedance of the cell in a self-referenced manner. In an embodiment, the system affords estimation of deformability of the cell by measuring deformability index of the cell and transit time of the cell. In an embodiment, the system affords measurement of any of the size of the cell and the deformed length of the cell based on time information embedded in the first electrical impedance and the second electrical impedance, respectively. In an embodiment, the system affords impedance measurement of undeformed cell and deformed cell using the first electrical impedance and the second electrical impedance, respectively. In an embodiment, the system affords measurement of the transit time of the cell offsetting flow rate fluctuation(s) in the microchannel.
[0016] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiment/s, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS
[0017] 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.
[0018] FIG. 1 illustrates an exemplary schematic depicting the system for estimation of deformability of a cell vis-à-vis the electrical impedance measured during passage of a single cell through the section with larger diameter and the section with the narrower diameter (constricted section) along the flow path, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION
[0019] 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.
[0020] 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.
[0021] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0022] 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.
[0023] 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.
[0024] The term “cell” as used herein throughout the present disclosure denotes the biological materials including but not limited to mammalian cells, plant cells, microbes, viruses or virus like particles, organelles, beads, vesicles and like biological materials that can be subjected to the estimation of deformability, in accordance with embodiments of the present disclosure, to assess one or more properties/parameters thereof.
[0025] The present invention relates generally to the field of electro-mechanodiagnostics. More specifically, the present invention relates to a system for estimation of deformability of a cell using the electrical impedance data.
[0026] An aspect of the present disclosure provides a system for estimation of deformability of a cell, the system including: a microchannel defining a flow path for the cell, the flow path defining at least two sections, wherein a first section of said at least two sections has a diameter larger than that of a second section of said at least two sections; a first source electrode flanked by a first pair of reference electrodes, configured to measure a first electrical impedance of the cell transiting through the first section of said flow path; and a second source electrode flanked by a second pair of reference electrodes, configured to measure a second electrical impedance of the cell transiting through the second section of said flow path.
[0027] In an embodiment, the first source electrode and the first pair of reference electrodes are configured to measure the first electrical impedance of the cell in a self-referenced manner. In an embodiment, the second source electrode and the second pair of reference electrodes are configured to measure the second electrical impedance of the cell in a self-referenced manner. In an embodiment, the system affords estimation of deformability of the cell by measuring deformability index of the cell and transit time of the cell. In an embodiment, the system affords measurement of any of the size of the cell and the deformed length of the cell based on time information embedded in the first electrical impedance and the second electrical impedance, respectively. In an embodiment, the system affords impedance measurement of undeformed cell and deformed cell using the first electrical impedance and the second electrical impedance, respectively. In an embodiment, the system affords measurement of the transit time of the cell offsetting flow rate fluctuation(s) in the microchannel.
[0028] In an embodiment, the system includes PDMS (polydimethylsiloxane) microchannel defining a flow path for the cell under analysis. However, a person skilled in the pertinent art would appreciate that any other material can be utilized for fabrication of the microchannel without departing from the scope and spirit of the present invention.
[0029] In an embodiment, the flow path defines at least two contiguous sections, wherein at least one section has a width (alternatively and synonymously termed as “diameter” throughout the present disclosure) larger than that of the other section. In other words, the flow path for the cell defines at least one constricted section necessitating the deformation of the cell under analysis.
[0030] In an embodiment, the system includes electrodes i.e. two source electrodes, each flanked by a pair of reference electrodes. Preferably, the electrodes are gold electrodes. However, a person skilled in the pertinent art would appreciate that any other material can be utilized for fabrication of the electrodes without departing from the scope and spirit of the present invention.
[0031] In an exemplary embodiment, the electrodes are fabricated in a sequential manner by sputtering Cr/Au on glass slides, performing optical lithography, followed by etching Cr/Au. However, any other method, as known to or appreciated by a person skilled in the art, can be utilized to fabricate the electrodes without departing from the scope and spirit of the present disclosure.
[0032] In an embodiment, the first source electrode and each electrode of the first pair of reference electrodes are separated by a distance in order of diameter of the cell. In an embodiment, the second source electrode and each electrode of the second pair of reference electrodes are separated by a distance in order of diameter of the cell. In an exemplary embodiment, the first source electrode and each electrode of the first pair of reference electrodes are separated by 20 micron distance (i.e. diameter of blood cells). However, a person skilled in the art would appreciate that the source electrode and each electrode of the pair of reference electrodes can be separated by any other distance to serve its intended purpose as laid down in embodiments of the present disclosure, without departing from the scope and spirit of the present invention.
[0033] In an embodiment, the first source electrode and the first pair of reference electrodes are configured to measure the first electrical impedance of the cell in a self-referenced manner. In an embodiment, the second source electrode and the second pair of reference electrodes are configured to measure the second electrical impedance of the cell in a self-referenced manner. Preferably, both of the source electrodes and pairs of reference electrodes operatively coupled therewith are configured to measure the electrical impedance of the transiting cell in a self-referenced manner. This allows for offsetting the flow rate fluctuations of the cell transiting through the sections of the flow path.
[0034] In an embodiment, the electrical impedance (synonymously and interchangeably referred to as “signal”) captured from the reference electrodes were subtracted using a custom-built instrumentation amplifier and measured using a lock-in amplifier at 800 kHz (HF2LI, Zurich Instruments). Analog Devices operational amplifier (AD8066) can be used to design and build the low-noise instrumentation amplifier. This results in two peaks (+ve/-ve) in the measurement signal, corresponding to the electrode over which the cell is passing/transiting. In practice, the signal frequency can be varied from low range (10 kHz to 50 kHz) to high range (500 kHz to 4 MHz) for impedance measurements.
[0035] By placing a set of electrodes (a source electrode flanked by a pair of reference electrodes) in a constricted section of the flow path for the cell, one can measure the time difference between the +ve and the -ve peaks, amplitude of the peaks, and the width of the peaks. The gap between the signal and measurement electrode can be in the order of the cell size. However, the gap between each pair can be optimized to obtain accurate measurements of the deformed length. The amplitude of the peak provides the electrical information about the deformed cell. The amplitude width and the time difference between the peaks can be used to measure the deformed length and the velocity of the cell in the constricted section. The velocity of the cell in the constricted section for a pre-defined flow rate gives a measure of the cell-wall/channel-wall interaction. Accordingly, all the measurements provide one with an estimate of the mechanical properties of the deformed (squeezed) cell. The individual values of the peaks (-ve/+ve) measures the dielectric properties of the cell before/after constricted section and hence, allows investigation of the effect of the mechanical deformation on the cell.
[0036] FIG. 1 illustrates an exemplary schematic depicting the system for estimation of deformability of a cell vis-à-vis the electrical impedance (impedance signal) measured during passage of a single cell through the pathway defining at least one constricted section, in accordance with an embodiment of the present disclosure. The time of transit of positive and negative peaks is a measure of the size of the cell. In an exemplary embodiment, the source electrode provides an input signal of 100mV @ 1MHz.
[0037] As illustrated in FIG. 1, the system (100) includes a microchannel (102) defining a flow path (104) for the cell (106), the flow path defining at least two sections, wherein a first section (108) of said at least two sections has a diameter larger than that of a second section (110) of said at least two sections; a first source electrode (112) flanked by a first pair of reference electrodes (114a and 114b), configured to measure a first electrical impedance (120) of the cell transiting through the first section (108) of said flow path; and a second source electrode (116) flanked by a second pair of reference electrodes (118a and 118b), configured to measure a second electrical impedance (130) of the cell transiting through the second section (110) of said flow path.
[0038] The following distance-time relationships can be used to calculate the diameter (D) and the deformed length (L) of a single cell flowing with a velocity v1 (outside the constricted section or transiting through the section with larger diameter) and v2 (inside the constricted section) -






[0039] Accordingly, the deformability index can be calculated using the following equation –

[0040] Transit time of the cell outside the constricted section (cell transiting through the section with larger diameter) can be calculated using the following equation –

[0041] Transit time of the cell inside the constricted section (cell transiting through the section with smaller diameter) can be calculated using the following equation –

[0042] Accordingly, the system affords/enables estimation of deformability of the cell by measuring deformability index of the cell and transit time of the cell. In an embodiment, the system affords measurement of any of the deformability index of the cell and the transit time of the cell based on time information embedded in the first electrical impedance and the second electrical impedance. In an embodiment, the system affords measurement of any of the size of the cell and the deformed length of the cell based on time information embedded in the first electrical impedance and the second electrical impedance, respectively. In an embodiment, the system affords impedance measurement of undeformed cell and deformed cell using the first electrical impedance and the second electrical impedance, respectively. In an embodiment, the system affords measurement of the transit time of the cell offsetting flow rate fluctuation(s) in the microchannel.
[0043] In an embodiment, the system includes a PDMS (polydimethylsiloxane) microchannel integrated with two sets of gold electrodes. One electrode set includes a source electrode at the center flanked by two reference electrodes. The distance between the source electrode and the reference electrodes is about 20 µm (i.e. of the order of size of blood cells). The electrodes are fabricated in a sequential manner by sputtering Cr/Au on glass slides, performing optical lithography, followed by etching Cr/Au. The impedance of the cells passing over the electrodes is measured in the self-referenced manner. Signal captured from the reference electrodes were subtracted using a custom-built instrumentation amplified and measured using the lock-in amplifier at 800 kHz (HF2LI, Zurich Instruments). Analog Devices operation amplifier (AD8066) was used to design and build the low-noise instrumentation amplifier. This technique leads to two peaks (+ve/-ve) in the measurement signal, corresponding to the electrode over which the cell is passing. By placing a set of electrodes in a constricted section of the channel, the time difference between the +ve and the -ve peaks, amplitude of the peaks, and the width of the peaks can be measured. The gap between the signal and measurement electrode is again in the order of the cell size (i.e. 20 µm). The gap between each pair can be optimized to obtain accurate measurements of the deformed length. The amplitude of the peak provides the electrical information about the squeezed cell. The amplitude width and the time difference between the peaks can be used to measure the deformed length and the velocity of the cell in the constriction. The velocity of the cell in the constriction of a set flow rate gives a measure of the cell-wall/channel-wall interaction. All the measurements provide with an estimate of the mechanical properties of the squeezed cells. The individual values of the peaks (-ve/+ve) measures the dielectric properties of the cell before/after constriction and hence, allows investigation of the effect of the mechanical deformation on the cell.
[0044] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

ADVANTAGES OF THE INVENTION
[0045] The present disclosure provides a system for estimation of deformability of a cell that satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.
[0046] The present disclosure provides a system for estimation of deformability of a cell that does not require utilization of high-speed camera(s) for measurement of the deformability index.
[0047] The present disclosure provides a system for estimation of size and deformed length of a cell that allows estimation of deformability of a cell using electrical impedance data.
[0048] The present disclosure provides a system for estimation deformed and undeformed cell’s impedance signature.
[0049] The present disclosure provides a system for estimation of deformability of a cell that allows monitoring and/or detection of the health or disease states of the cell.
[0050] The present disclosure provides a system for estimation of deformability of a cell that allows measurement of transit time thereof while offsetting the dynamic flow rate fluctuations in microchannel.

,CLAIMS:
1. A system for estimation of deformability of a cell, the system comprising:
a microchannel defining a flow path for the cell, the flow path defining at least two sections, wherein a first section of said at least two sections has a diameter larger than that of a second section of said at least two sections;
a first source electrode flanked by a first pair of reference electrodes, configured to measure a first electrical impedance of the cell transiting through the first section of said flow path; and
a second source electrode flanked by a second pair of reference electrodes, configured to measure a second electrical impedance of the cell transiting through the second section of said flow path.

2. The system as claimed in claim 1, wherein the first source electrode and the first pair of reference electrodes are configured to measure the first electrical impedance of the cell in a self-referenced manner.

3. The system as claimed in claim 1, wherein the second source electrode and the second pair of reference electrodes are configured to measure the second electrical impedance of the cell in a self-referenced manner.

4. The system as claimed in claim 1, wherein the system affords estimation of deformability of the cell by measuring deformability index of the cell and transit time of the cell.

5. The system as claimed in claim 1, wherein the system affords measurement of any of the size of the cell and the deformed length of the cell based on time information embedded in the first electrical impedance and the second electrical impedance, respectively.
6. The system as claimed in claim 1, wherein the system affords impedance measurement of undeformed cell and deformed cell using the first electrical impedance and the second electrical impedance, respectively.

7. The system as claimed in claim 1, wherein the system affords measurement of the transit time of the cell offsetting flow rate fluctuation(s) in the microchannel.