Microfluidic device for single cell processing, and method and system for single cell processing using the microfluidic device

Abstract

A microfluidic device for single cell processing is provided, comprising: a substrate; fluid channels disposed in the substrate; and a plurality of electrodes arranged adjacent to the fluid channels for determining the position of a cell in the fluid channels, the plurality of electrodes including sensing electrode pairs, each sensing electrode pair including a first sensing electrode and a second sensing electrode, wherein at least the first sensing electrode in the sensing electrode pair extends in a first direction, and the sensing electrode pair is configured to measure a differential electrical signal across a sensing region when a cell flows through a sensor portion of the fluid channel; and a bias electrode disposed between the first sensing electrode and the second sensing electrode, the bias electrode being configured to receive a bias voltage. One of the second sensing electrode and the bias electrode extends in a direction at least substantially parallel to the first sensing electrode, and the other of the second sensing electrode and the bias electrode is arranged to have an inclined orientation relative to the first sensing electrode. A method of forming the microfluidic device, and a method and system for single cell processing using the microfluidic device are also provided.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of Singapore Patent Application No. 10201908123Q, filed on September 3, 2019, the content of which is incorporated herein by reference in its entirety for all purposes. Technical field

[0003] The present invention generally relates to a microfluidic device for single - cell processing, a method of forming the microfluidic device, a method and system for single - cell processing using the microfluidic device. Background art

[0004] Due to the inherent heterogeneity of biological samples, cell separation is a necessary step in various biomedical applications. Various microfluidic techniques for cell separation and sorting have been developed, which can be classified into active methods (e.g., dielectrophoresis, acoustophoresis, magnetophoresis) and passive methods (e.g., inertial focusing, on - chip filtration, and deterministic lateral displacement). For all these cell separation techniques, the performance of the system, such as throughput, purity, and recovery rate, can be characterized. Traditionally, the performance of microfluidic cell separation and sorting can be analyzed by analyzing the collected input and output samples (e.g., by flow cytometry, hematology analyzer, imaging - based processing) or by detecting the lateral position of cells using a high - speed camera with post - image analysis, because such a position is directly related to the separation performance. The former method requires additional off - chip analysis steps or expensive equipment (e.g., flow cytometer), which is not easily accessible and not suitable for real - time analysis. The latter method requires an expensive high - speed imaging device and complex image - processing algorithms or laborious manual analysis. In addition, the high - speed camera generates a large amount of imaging data for post - analysis that requires high - end computing power, making this approach difficult to implement for real - time measurement of the single - cell lateral position for instantaneous feedback control.

[0005] A simple approach for measuring the lateral position of flowing particles (e.g., also interchangeably referred to as cells) needs to be developed. In this regard, impedance-based microfluidic devices have enabled label-free and high-throughput means for cell counting, sizing, and studying cell function and phenotype. For example, impedance-based microfluidic devices can characterize the mechanical properties of individual cells by the transit time required for the cells to pass through a constricted channel. More particularly, the transit time can be extracted from the measured electrical signal rather than using high-speed camera video recording to extract the transit time. Impedance-based microfluidic cytometers can be used to measure the position of cells in a microfluidic channel, including the lateral position (i.e., along the channel width), the cross-sectional position (i.e., along the channel width and height), and the longitudinal position (i.e., along the channel length). In H. Wang, N. Sobahi, and A. Han, Lab Chip, 2017, 17, 1264 - 1269, they proposed a microfluidic system with non-parallel electrodes to detect the lateral position of individual particles, which is indicated by the amplitude and width of the signal peak. In B. Brazey, J. Cottet, A. Bolopion, H. VanLintel, P. Renaud, and M. Gauthier, Lab Chip, 2018, 18, 818 - 831, they demonstrated a longitudinally sensitive position sensor by using a star-shaped electrode design. In M. Solsona, E. Y. Westerbeek, J. G. Bomer, W. Olthuis, and A. van den Berg, Lab Chip, 2019, 19, 1054 - 1059, they developed a microfluidic system to track the lateral position of particles by exploiting the electric field gradient induced by two opposing electrodes with increased electrodeposited area. Using this system, the lateral position of the particles is indicated by the amplitude of the peak. In R. Reale, A. De Ninno, L. Businaro, P. Bisegna, and F. Caselli, Microfluid. Nanofluid., 2018, 22, they demonstrated electrical measurements of the cross-sectional position of individual particles with two different sets of electrodes, where the lateral position of the particles can be determined by five pairs of electrodes and two resulting differential currents.

[0006] Accordingly, there is a need to provide a microfluidic device for single-cell processing, as well as a method and system for using the microfluidic device for single-cell processing, which attempt to overcome one or more of the deficiencies of conventional microfluidic devices and conventional methods and systems for single-cell processing, such as but not limited to, improving the position measurement of cells in a fluid channel (e.g., lateral position, vertical position) with improved resolution, improved flow rate, and / or improvement in the minimum measured particle size. The present invention has been developed in this context. Summary of the Invention

[0007] According to a first aspect of the present invention, there is provided a microfluidic device for single cell processing, the microfluidic device comprising:

[0008] a substrate;

[0009] a fluid channel disposed in the substrate, wherein the fluid channel is configured to form a fluid path for allowing a fluid sample including cells to flow along the channel; and

[0010] a plurality of electrodes disposed adjacent to the fluid channel for determining the position of a cell in the fluid channel, the plurality of electrodes including:

[0011] a sensing electrode pair including a first sensing electrode and a second sensing electrode, the sensing electrode pair defining a sensing region overlapping a sensor portion of the fluid channel, wherein at least the first sensing electrode in the sensing electrode pair extends in a first direction, the sensing electrode pair being configured to measure a differential electrical signal across the sensing region when a cell flows through the sensor portion of the fluid channel; and

[0012] a bias electrode disposed between the first sensing electrode and the second sensing electrode, the bias electrode being configured to receive a bias voltage,

[0013] wherein one of the second sensing electrode and the bias electrode extends in a direction at least substantially parallel to the first sensing electrode, and the other of the second sensing electrode and the bias electrode is arranged to have an inclined orientation with respect to the first sensing electrode.

[0014] According to a second aspect of the present invention, there is provided a method of forming a microfluidic device for single cell processing, the method comprising:

[0015] providing a substrate;

[0016] providing a fluid channel in the substrate, wherein the fluid channel is configured to form a fluid path for allowing a fluid sample including cells to flow along the channel; and

[0017] forming a plurality of electrodes disposed adjacent to the fluid channel for determining the position of a cell in the fluid channel, the plurality of electrodes including:

[0018] a sensing electrode pair including a first sensing electrode and a second sensing electrode, the sensing electrode pair defining a sensing region overlapping a sensor portion of the fluid channel, wherein at least the first sensing electrode in the sensing electrode pair extends in a first direction, the sensing electrode pair being configured to measure a differential electrical signal across the sensing region when a cell flows through the sensor portion of the fluid channel; and

[0019] a bias electrode disposed between the first sensing electrode and the second sensing electrode, the bias electrode being configured to receive a bias voltage,

[0020] One of the second sensing electrode and the bias electrode extends in a direction that is at least substantially parallel to the first sensing electrode, and the other of the second sensing electrode and the bias electrode is arranged to have an inclined orientation with respect to the first sensing electrode.

[0021] According to a third aspect of the present invention, there is provided a method for single cell processing using the microfluidic device for single cell processing according to the first aspect described above, the method comprising:

[0022] Applying a bias voltage to the bias electrode;

[0023] Obtaining a differential electrical signal based on the first sensing electrode and the second sensing electrode when a cell flows through a sensor portion of a fluid channel corresponding to a sensing region; and

[0024] Determining the position of the cell in the sensor portion of the fluid channel based on the differential electrical signal.

[0025] According to a fourth aspect of the present invention, there is provided a system for single cell processing, the system comprising:

[0026] The microfluidic device for single cell processing described according to the first aspect above; and

[0027] A computing system, comprising:

[0028] A memory; and

[0029] At least one processor communicatively coupled to the memory and the microfluidic device and configured to:

[0030] Apply a bias voltage to the bias electrode;

[0031] Obtain a differential electrical signal based on the first sensing electrode and the second sensing electrode when a cell flows through a sensor portion of a fluid channel corresponding to a sensing region; and

[0032] Determine the position of the cell in the sensor portion of the fluid channel based on the differential electrical signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Embodiments of the present invention will be better understood and become apparent to those of ordinary skill in the art from the following written description which is by way of example only and in conjunction with the accompanying drawings, wherein:

[0034] Figure 1A Schematic diagrams of microfluidic devices for single cell processing according to various embodiments of the present invention are depicted;

[0035] Figure 1B Another schematic diagram of a microfluidic device for single cell processing according to various embodiments of the present invention is depicted;

[0036] Figure 1C Depicts another schematic diagram of a microfluidic device for single-cell processing according to various embodiments of the present invention;

[0037] Figure 2 Depicts a schematic flow chart of a method for forming a microfluidic device for single-cell processing according to various embodiments of the present invention;

[0038] Figure 3 Depicts a schematic flow chart of a method for single-cell processing using a microfluidic device according to various embodiments of the present invention;

[0039] Figure 4 Depicts a schematic diagram of a system for single-cell processing according to various embodiments of the present invention;

[0040] Figure 5 Depicts a schematic block diagram of an exemplary computer system in which a system for single-cell processing according to various embodiments of the present invention can be implemented or practiced;

[0041] Figure 6A Shows a schematic diagram of the electrical sensing region of a microfluidic device according to various exemplary embodiments of the present invention;

[0042] Figure 6B Shows a microscopic image of the electrical sensing region;

[0043] Figure 6C Shows a schematic diagram of the sensing region and shows exemplary electro-signal curves of the measured electrical signals from a first sensing electrode and a second sensing electrode according to various exemplary embodiments of the present invention;

[0044] Figure 7A Shows a schematic diagram of another exemplary microfluidic device according to various exemplary embodiments of the present invention;

[0045] Figure 7B Shows a schematic diagram of yet another exemplary microfluidic device according to various exemplary embodiments of the present invention;

[0046] Figure 7C Shows an exemplary schematic diagram of a cross-section of a fluid channel along the channel length according to various exemplary embodiments of the present invention, and exemplary signal curves of three different particles flowing through a sensor portion of the fluid channel corresponding to the sensing region;

[0047] Figure 7D Shows an exemplary schematic diagram of a signal curve of a measured electrical signal of a particle flowing through a sensor portion of a fluid channel according to various exemplary embodiments of the present invention;

[0048] Figure 8A Depicts a graph showing the measured electrical position x of three representative beads flowing through the lower (i), middle (ii), and upper (iii) portions of a microchannel relative to the transit time t 1 / Transit time t 2 for the experimental results;

[0049] Figure 8B Shows the corresponding measured differential electrical signals according to various exemplary embodiments of the present invention Figure 8A for;

[0050] Figure 8C Shows an enlarged view of the representative electrical signals and their corresponding microscopic optical images (captured simultaneously) of three representative beads for measuring the optical position x according to various exemplary embodiments of the present invention;

[0051] Figure 9 Shows a quantitative comparison of the lateral position of 10 μm beads between the results of an electrical method and the results obtained by an optical method according to various exemplary embodiments;

[0052] Figure 10 Shows a Bland - Altman analysis comparing the lateral position x obtained by an electrical method according to various exemplary embodiments with the lateral position x obtained by an optical method;

[0053] Figures 11A to 11B Shows the analysis results of the smallest particles that can be detected by a microfluidic device with good performance according to various exemplary embodiments;

[0054] Figures 12A to 12B Shows a quantitative comparison of the lateral position of RBCs between the results obtained from a microfluidic device according to various exemplary embodiments and the results obtained by an optical method;

[0055] Figures 13A to 13D Shows the measurement of the lateral position x and electrical diameter of a mixture of 5 and 10 μm beads according to various exemplary embodiments;

[0056] Figures 14A to 14B Shows the quantitative analysis results of the lateral position of 7 μm beads between the results obtained from a microfluidic device according to various exemplary embodiments and the results obtained by an optical method;

[0057] Figure 15A Shows a schematic image for monitoring the sheath - flow - induced focusing of 7 μm beads at a certain flow rate according to various exemplary embodiments;

[0058] Figure 15B ShowsFigure 15A The pixel intensity curve graph (gray level) of the image shown in; and

[0059] Figure 15C A histogram of the electrical position x of 7μm beads sheath flow focused in different regions is shown. Detailed Description

[0060] Embodiments of the present invention provide a microfluidic device for single cell processing, a method of forming the microfluidic device, and a method and system for using the microfluidic device for single cell processing. Those skilled in the art will understand that cells can also be interchangeably referred to as particles. The microfluidic device can be used to measure positions (e.g., lateral, vertical) and determine the position of a single cell / particle in a continuous flow in a fluid channel. Analytical expressions derived from the measured electrical signals and the geometric relationship between the flowing cell, the electrodes, and the microchannel can be used to determine the position of the cell. Various embodiments of the present invention can be easily integrated with various upstream applications (e.g., cell sorting, cell focusing) to evaluate the efficiency of cell manipulation in real-time, thereby eliminating multiple steps of using high-speed cameras or off-chip analysis. For example, tracking the position of a single cell / particle plays an important role in evaluating the efficiency of microfluidic cell focusing, separation, and sorting.

[0061] Figure 1A A schematic diagram of a microfluidic device 100 for single cell processing according to various embodiments of the present invention is depicted. The microfluidic device 100 includes: a substrate 110; a fluid channel 114 disposed in the substrate 110, wherein the fluid channel is configured to form a fluid path for allowing a fluid sample including cells (or particles) to flow along the channel; and a plurality of electrodes 118 disposed adjacent to the fluid channel 114 for determining the position of the cells in the fluid channel. The plurality of electrodes 118 includes a sensing electrode pair that includes a first sensing electrode 118a and a second sensing electrode 118b, and the sensing electrode pair 118a, 118b defines a sensing region 120 that overlaps a sensor portion of the fluid channel. At least the first sensing electrode 118a in the sensing electrode pair extends in a first direction. The sensing electrode pair 118a, 118b is configured to measure a differential electrical signal on the sensing region 120 when a cell flows through the sensor portion of the fluid channel. The plurality of electrodes 118 further includes a bias electrode 118c disposed between the first sensing electrode 118a and the second sensing electrode 118b, and the bias electrode 118c is configured to receive a bias voltage. One of the second sensing electrode 118b and the bias electrode 118c extends in at least a direction substantially parallel to the first sensing electrode 118a, and the other of the second sensing electrode 118b and the bias electrode 118c is arranged to have an inclined orientation relative to the first sensing electrode 118a.

[0062] Those skilled in the art will understand that, for illustrative purposes only and not by way of limitation, Figure 1A shows an exemplary configuration (e.g., a first exemplary configuration) of the microfluidic device 100, where the second sensing electrode 118b extends in a direction that is at least substantially parallel to the first sensing electrode 118a, and the biasing electrode 118c is arranged to have an inclined orientation relative to the first sensing electrode 118a and the second sensing electrode 118b (in the sensing region). That is, the second sensing electrode 118b is one of the second sensing electrode 118b and the biasing electrode 118c that extends in a direction that is at least substantially parallel to the first sensing electrode 118a as described above, and the biasing electrode 118c is the other of the second sensing electrode 118b and the biasing electrode 118c that is arranged to have an inclined orientation relative to the first sensing electrode 118a. Those skilled in the art will understand that the microfluidic device 100 is not limited to the biasing electrode 118c being arranged to have an inclined orientation relative to the first sensing electrode 118a, and in another exemplary configuration (e.g., a second exemplary configuration), the biasing electrode 118c extends in a direction that is at least substantially parallel to the first sensing electrode 118a, and the second sensing electrode 118b is arranged to have an inclined orientation relative to the first sensing electrode 118a and the biasing electrode 118c (in the sensing region), as Figure 1B shown.

[0063] Figure 1B Depicts a schematic diagram of a microfluidic device 150 for single-cell processing according to various embodiments of the present invention, which is similar to the microfluidic device 100, except that the biasing electrode 118c extends in a direction that is at least substantially parallel to the first sensing electrode 118a, and the second sensing electrode 118b is arranged to have an inclined orientation relative to the first sensing electrode 118a and the biasing electrode 118c.

[0064] In yet another exemplary configuration (e.g., a third exemplary configuration), the plurality of electrodes further includes a pair of floating electrodes (e.g., a first floating electrode 118d, a second floating electrode 118e) that extend in a first direction, as Figure 1C shown. Figure 1C Depicts a schematic diagram of a microfluidic device 180 for single-cell processing according to various embodiments of the present invention, which is similar to the microfluidic device 100 and / or 150, except that the plurality of electrodes 118 further includes a pair of floating electrodes 118d, 118e that extend in a first direction. In other words, the pair of floating electrodes 118d, 118e can be arranged in a direction that is at least substantially parallel to the first sensing electrode 118a.

[0065] For clarity and conciseness, unless otherwise stated, the various embodiments of the present invention will be hereinafter referred to with reference to having as Figure 1AThe microfluidic device 100 of the exemplary configuration shown (i.e., the first exemplary configuration) will be described. Those skilled in the art will understand that the various features and related advantages described with reference to the first exemplary configuration can be similarly, equivalently, or correspondingly applied to the second exemplary configuration and the third exemplary configuration, and thus, for the sake of clarity and conciseness, do not need to be explicitly stated or repeated.

[0066] Compared with conventional impedance-based microfluidic devices for measuring the position of particles (e.g., lateral position, vertical position) in a fluid channel, the above configuration of the microfluidic device 100 for single-cell processing advantageously provides many advantages, such as but not limited to, improved resolution, increased flow rate, and improvement in the minimum measurable particle size (e.g., beads of about 3.6 μm). In addition, using a mixture of beads of different sizes (e.g., 5 and 10 μm beads), in addition to measuring the position (e.g., lateral position, vertical position), the characteristics (e.g., size) of a single particle / cell can be simultaneously characterized according to various embodiments of the present invention.

[0067] As described, the fluid channel is configured to form a fluid path for allowing a fluid sample including cells to flow along the channel. For example, the fluid sample can be configured to flow in the direction of the channel length (i.e., the length direction). Those skilled in the art will understand that Figure 1A the fluid channel 114 shown in Figure 1B and Figure 1C may only show a part of the fluid channel of the microfluidic device.

[0068] In various embodiments, the fluid channel can have a width (also interchangeably referred to as the channel width) and a height (also interchangeably referred to as the channel height).

[0069] In various embodiments, the first direction is along the width direction of the fluid channel. The width direction of the fluid channel is a direction that is at least substantially parallel to the channel width. For example, the width direction can be along the x-axis, as Figures 1A to 1C shown. The microfluidic device may further include a second direction. In various embodiments, the second direction can be along the height direction of the fluid channel. The height direction of the fluid channel is a direction that is at least substantially parallel to the channel height. For example, in the case where the width direction is along the x-axis, the height direction can be along the y-axis ( Figures 1A to 1C the y-axis is not shown in

[0070] In various embodiments, the first sensing electrode, the second sensing electrode, and the bias electrode are arranged to form a configuration corresponding to an N shape. For example, Figure 1A shows the first sensing electrode 118a, the second sensing electrode 118b, and the bias electrode 118c arranged in the sensor region 120 to form a configuration corresponding to an N shape.

[0071] The tilted orientation of the bias electrode relative to at least one of the first sensing electrode and the second sensing electrode can be at an angle according to the size of the fluid channel. In various embodiments, the tilted orientation of the bias electrode relative to at least one of the first sensing electrode and the second sensing electrode is at an angle within a range of about 10 degrees to about 60 degrees. In various embodiments, the tilted orientation of the bias electrode relative to at least one of the first sensing electrode and the second sensing electrode is at an angle of about 22 degrees.

[0072] In various embodiments, the position of the cell includes a lateral position in the fluid channel, which is relative to the width direction of the fluid channel and is determined based on the geometric relationship between the cell and the plurality of electrodes.

[0073] Regarding the Figure 1B described second exemplary configuration and the Figure 1C described third exemplary configuration, in various embodiments, the tilted orientation of the second sensing electrode relative to the bias electrode can be at an angle depending on the size of the fluid channel. In various embodiments, the tilted orientation of the second sensing electrode relative to the bias electrode is at an angle within a range of about 10 degrees to about 60 degrees. In various embodiments, the tilted orientation of the second sensing electrode relative to the bias electrode is at an angle of about 22 degrees.

[0074] In various embodiments, the plurality of electrodes further includes a pair of floating electrodes extending in a first direction. The pair of floating electrodes extends in a direction that is at least substantially parallel to the first sensing electrode. The pair of floating electrodes are electrodes that are not connected to any power source.

[0075] In various embodiments, the pair of floating electrodes is arranged between the pair of sensing electrodes, and the bias electrode is arranged between the pair of floating electrodes. As Figure 1C shown, the pair of floating electrodes 118d, 118e is arranged between the pair of sensing electrodes 118a, 118b, and the bias electrode 118c is arranged between the pair of floating electrodes 118d, 118e.

[0076] In various embodiments, the position of the cell includes a cross-sectional position in the fluid channel. The cross-sectional position includes a lateral position in the fluid channel, which is relative to the width direction of the fluid channel. The cross-sectional position further includes a vertical position in the fluid channel, which is relative to the height direction of the fluid channel.

[0077] In various embodiments, the bias voltage includes an alternating voltage.

[0078] In various embodiments, the differential electrical signal includes a differential current response on the sensing region.

[0079] Figure 2 Depicts a schematic flow chart of a method 200 for forming a microfluidic device for single cell processing, such as the microfluidic devices 100, 150 or 180 described herein with reference to Figure 1A 、 Figure 1B or Figure 1C The microfluidic devices 100, 150 or 180 described. Method 200 includes: providing (at 202) a substrate; providing (at 204) a fluid channel in the substrate, wherein the fluid channel is configured to form a fluid path for allowing a fluid sample including cells to flow along the channel; and forming (at 206) a plurality of electrodes disposed adjacent to the fluid channel for determining the position of cells in the fluid channel, the plurality of electrodes including a sensing electrode pair that includes a first sensing electrode and a second sensing electrode, the sensing electrode pair defining a sensing region that overlaps a sensor portion of the fluid channel, wherein at least the first sensing electrode in the sensing electrode pair extends in a first direction, the sensing electrode pair being configured to measure a differential electrical signal across the sensing region when a cell flows through the sensor portion of the fluid channel; and a bias electrode disposed between the first sensing electrode and the second sensing electrode, the bias electrode being configured to receive a bias voltage, wherein one of the second sensing electrode and the bias electrode extends in a direction that is at least substantially parallel to the first sensing electrode, and the other of the second sensing electrode and the bias electrode is disposed with an inclined orientation relative to the first sensing electrode.

[0080] In various embodiments, method 200 is used to form the microfluidic devices 100, 150 or 180 as described herein with reference to Figure 1A 、 Figure 1B or Figure 1C Thus, method 200 may also include various steps corresponding to providing or forming various configurations and / or components / elements of the microfluidic devices 100, 150 or 180 as described herein according to various embodiments, and thus these corresponding steps need not be repeated with respect to method 200 for the sake of clarity and conciseness. In other words, the various embodiments described herein in the context of the microfluidic devices 100, 150 or 180 similarly or correspondingly apply to method 200 (e.g., for manufacturing the microfluidic devices 100, 150 or 180 having various configurations and / or components / elements as described herein according to various embodiments), and vice versa.

[0081] By way of example only and not limitation, the substrate may be formed of glass (e.g., borosilicate glass), quartz or a polymer wafer. The plurality of electrodes may be formed of suitable electrode materials known in the art and thus need not be described herein. By way of example only and not limitation, each of the plurality of electrodes may be formed of an electrode material including a first layer formed of chromium (e.g., having a thickness of about 10 nm) and a second layer formed of gold on the first layer (e.g., having a thickness of about 100 nm).

[0082] Figure 3 depicts a schematic flowchart of a method 300 for single cell processing using a microfluidic device 100, 150, or 180 as described herein with reference to Figure 1A 、 Figure 1B or Figure 1C . The method 300 includes: applying (at 302) a bias voltage to a bias electrode; obtaining (at 304) a differential electrical signal based on a first sensing electrode and a second sensing electrode when a cell flows through a sensor portion of a fluid channel corresponding to a sensing region; and determining (at 306) the position of the cell in the sensor portion of the fluid channel based on the differential electrical signal.

[0083] In various embodiments, the obtained differential electrical signal includes a plurality of signal peaks corresponding to the cell flowing through the sensor portion of the fluid channel from the first sensing electrode to the second sensing electrode.

[0084] In various embodiments, the plurality of signal peaks includes a first signal peak corresponding to the cell flowing from the first sensing electrode to the bias electrode in the sensor portion of the fluid channel, and a second signal peak corresponding to the cell flowing from the bias electrode to the second sensing electrode in the sensor portion of the fluid channel. In various embodiments, the above determination of the position of the cell in the sensor portion of the fluid channel includes determining the lateral position of the cell in the fluid channel based on the width of the first signal peak and the width of the second signal peak, which is relative to the width direction of the fluid channel. In various embodiments, the width of the first signal peak corresponds to the transit time t 1 of the cell flowing from the first sensing electrode to the bias electrode in the sensor portion of the fluid channel, and the width of the second signal peak corresponds to the transit time t 2 of the cell flowing from the bias electrode to the second sensing electrode in the sensor portion of the fluid channel.

[0085] In various embodiments, the first signal peak and the second signal peak may have opposite polarities. For example, the first signal peak may be a positive peak and the second signal peak may be a negative peak. In other embodiments, the first signal peak may be a negative peak and the second signal peak may be a positive peak.

[0086] In various embodiments, the above determination of the lateral position of the cell in the fluid channel is also based on the geometric relationship between the cell and the plurality of electrodes. In other words, it is possible to determine the lateral position of the cell in the fluid channel based on the differential electrical signal (e.g., the width of the first signal peak corresponds to the transit time t 1 and the width of the second signal peak corresponds to the transit time t 2 ), as well as the geometric relationship between the flowing cell, the plurality of electrodes (e.g., the first sensing electrode, the second sensing electrode, the bias electrode) and the position of the fluid channel.

[0087] In cases where the plurality of electrodes further includes a floating electrode pair, in various embodiments, the first signal peak may include a first sub-peak. The first sub-peak may be a double peak. In other words, the first sub-peak may be a symmetric peak (e.g., a double peak) generated by three parallel electrodes (the first sensing electrode 118a, the first floating electrode 118d, and the bias electrode 118c). In various embodiments, the above determination of the position of the cell in the sensor portion of the fluid channel further includes determining the vertical position of the cell in the fluid channel based on the ratio of the amplitude of the first sub-peak to the valley value of the first sub-peak, where the vertical position is with respect to the height direction of the fluid channel.

[0088] In various embodiments, method 300 may further include determining the size (e.g., the diameter) of the cell based on the amplitude of the first signal peak and the amplitude of the second signal peak. In a non-limiting example, the size of the cell may be the diameter of the cell.

[0089] Figure 4 A schematic diagram of a system 400 for single-cell processing according to various embodiments of the present invention is depicted, such as corresponding to method 300 for single-cell processing described above with respect to Figure 3 described above. System 400 includes a microfluidic device 100, 150, or 180 for single-cell processing as described above with reference to Figure 1A , Figure 1B or Figure 1C ; and a computing system 402, the computing system 402 including: a memory 404; and at least one processor 406, which is communicatively coupled to the memory 404 and the microfluidic device 100 and is configured to: apply a bias voltage to the bias electrode; obtain a differential electrical signal based on the first sensing electrode and the second sensing electrode when a cell flows through the sensor portion of the fluid channel corresponding to the sensing region; and determine the position of the cell in the sensor portion of the fluid channel based on the differential electrical signal.

[0090] Those skilled in the art will recognize that at least one processor 406 may be configured to perform the required functions or operations through an instruction set (e.g., a software module) executable by at least one processor 406 to perform the required functions or operations. Thus, as Figure 4 shown, system 400 may include an electrical signal measurement module (or circuit) 410, which is configured to apply a bias voltage to the bias electrode; and obtain a differential electrical signal based on the first sensing electrode and the second sensing electrode when a cell flows through the sensor portion of the fluid channel corresponding to the sensing region; and a cell position determination module (or circuit) 412, which is configured to determine the position of the cell in the sensor portion of the fluid channel based on the differential electrical signal.

[0091] Those skilled in the art will appreciate that the above modules are not necessarily separate modules, and one or more modules may be implemented or implemented as a functional module (e.g., a circuit or software program) as needed or appropriate without departing from the scope of the present invention. For example, the electrical signal measurement module 410 and the cell position determination module 412 may be implemented (e.g., compiled together) as an executable software program (e.g., a software application or simply referred to as an "application"), which may be stored in the memory 404, for example, and may be executed by at least one processor 406 to perform the functions / operations described herein according to various embodiments.

[0092] In various embodiments, computing system 402 corresponds to the Figure 3 The method 300 for single cell processing described, therefore, various functions or operations configured to be performed by at least one processor 406 may correspond to various steps of the method 300 as described above according to various embodiments, and therefore, for the sake of clarity and conciseness, there is no need to repeat with respect to the system 402. In other words, various embodiments described herein in the context of the method are similarly valid for the corresponding system, and vice versa.

[0093] For example, in various embodiments, the memory 404 may store therein an electrical signal module 410 and a cell position determination module 412, which respectively correspond to various steps of the method 300 as described above according to various embodiments, and these steps can be executed by at least one processor 406 to perform the corresponding functions / operations as described herein.

[0094] According to various embodiments in the present disclosure, a computing system, a controller, a microcontroller, or any other system providing processing capabilities may be provided. Such a system may include one or more processors and one or more computer-readable storage media. For example, the computing system 402 described above may include a processor (or controller) 406 and a computer-readable storage medium (or memory) 404, which are used, for example, for various processes performed therein as described herein. The memory or computer-readable storage medium used in various embodiments may be a volatile memory, such as a DRAM (dynamic random access memory), or a non-volatile memory, such as a PROM (programmable read-only memory), an EPROM (erasable PROM), an EEPROM (electrically erasable PROM), or a flash memory, such as a floating gate memory, a charge trap memory, an MRAM (magnetoresistive random access memory), or a PCRAM (phase change random access memory).

[0095] In various embodiments, "circuitry" can be understood as any kind of logical implementation entity, which can be a dedicated circuit or a processor that executes software stored in a memory, firmware, or any combination thereof. Thus, in one embodiment, "circuitry" can be hard-wired logic circuitry or programmable logic circuitry, such as a programmable processor, e.g., a microprocessor (e.g., a complex instruction set computer (CISC) processor or a reduced instruction set computer (RISC) processor). "Circuitry" can also be a processor that executes software, such as any kind of computer program, e.g., a computer program that uses virtual machine code (e.g., Java). According to various alternative embodiments, any other kind of implementation of the various functions described in more detail below can also be understood as "circuitry". Similarly, a "module" can be part of a system according to various embodiments of the present invention, and can encompass "circuitry" as described above, or can be understood as any kind of logical implementation entity resulting therefrom.

[0096] Some portions of this disclosure are presented in the form of algorithms and functional or symbolic representations of operations on data within a computer memory, either explicitly or implicitly. These algorithmic descriptions and functional or symbolic representations are the means used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art. An algorithm is here generally considered to be a self-consistent sequence of steps leading to a desired result. These steps are those requiring physical manipulation of physical quantities, such as electrical, magnetic, or optical signals capable of being stored, transferred, combined, compared, and otherwise manipulated.

[0097] Unless otherwise specifically stated, and as will be apparent from the following, throughout this specification, discussions using terms such as "applying", "obtaining", "determining", etc. refer to actions and processes of a computer system or similar electronic device that manipulate data represented as physical quantities within the computer system and transform it into other data similarly represented as physical quantities within the computer system or other information storage, transmission, or display device.

[0098] This specification also discloses a computing system (e.g., which can also be embodied as a device or apparatus) for performing the operations / functions of the methods described herein, such as system 402. Such a system can be specifically constructed for the desired purpose, or can include a general-purpose computer or other devices selectively activated or reconfigured by a computer program stored in the computer. The algorithms presented herein are not inherently related to any particular computer or other device. According to the teachings herein, various general-purpose machines can be used with a computer program. Alternatively, it may be appropriate to construct more specialized devices to perform the required method steps.

[0099] In addition, the present specification also implicitly discloses at least a computer program or software / functional modules, because it is obvious to those skilled in the art that the various steps of the methods described herein can be implemented by computer code. The computer program is not intended to be limited to any specific programming language and its implementation. It should be understood that various programming languages and their encodings can be used to implement the teachings disclosed herein. In addition, the computer program is not intended to be limited to any specific control flow. There are many other variations of the computer program, which can use different control flows without departing from the spirit or scope of the present invention. Those skilled in the art will understand that the various modules described herein (e.g., the electrical signal measurement module 410 and / or the cell position determination module 412) can be (multiple) software modules implemented by (multiple) computer programs or (multiple) instruction sets executable by a computer processor to perform the required functions, or can be (multiple) hardware modules, which are (multiple) functional hardware units designed to perform the required functions. It should also be understood that a combination of hardware and software modules can be implemented.

[0100] In addition, the various steps of the computer programs / modules or methods described herein can be executed in parallel rather than sequentially. Such computer programs can be stored on any computer-readable medium. The computer-readable medium can include storage devices such as magnetic disks or optical disks, storage chips, or other storage devices suitable for interfacing with a general-purpose computer. When loaded and executed on such a general-purpose computer, the computer program effectively produces a device for implementing the steps of the methods described herein.

[0101] In various embodiments, there is provided a computer program product embodied in one or more computer-readable storage media (non-transitory computer-readable storage media), which includes instructions (e.g., the electrical signal measurement module 410 and / or the cell position determination module 412) executable by one or more computer processors to perform the method 300 for single-cell processing, as referred to above Figure 3 described. Thus, the various computer programs or modules described herein can be stored in a computer program product receivable by a system (such as Figure 4 the computing system 402 shown) for execution by at least one processor 406 of the computing system 402 to perform the required or desired functions.

[0102] The software or functional modules described herein may also be implemented as hardware modules. More particularly, in the hardware sense, a module is a functional hardware unit designed to be used with other components or modules. For example, a module may be implemented using discrete electronic components, or it may form part of an entire electronic circuit, such as an application specific integrated circuit (ASIC). There are many other possibilities. Those skilled in the art will appreciate that the software or (multiple) functional modules described herein may also be implemented as a combination of hardware and software modules.

[0103] In various embodiments, the computing system 402 may be implemented by any computing system (e.g., a desktop or portable computing system) including at least one processor and a memory, such as the computing system 500 schematically illustrated by way of example and not limitation Figure 5 in FIG. Various methods / steps or functional modules (e.g., the electrical signal measurement module 410 and / or the cell position determination module 412) may be implemented as software, such as a computer program executed within the computing system 500, and instruct the computing system 500 (specifically, one or more of its processors) to perform the methods / functions of the various embodiments described herein. The computing system 500 may include a computer module 502, input modules such as a keyboard 504 and a mouse 506, and a plurality of output devices such as a display 508 and a printer 510. The computer module 502 may be connected to a computer network 512 via a suitable transceiver device 514 to allow access to, for example, the Internet or other network systems, such as a local area network (LAN) or a wide area network (WAN). The computer module 502 in the example may include a processor 518 for executing various instructions, a random access memory (RAM) 520, and a read only memory (ROM) 522. The computer module 502 may also include a plurality of input / output (I / O) interfaces, such as an I / O interface 524 to the display 508 and an I / O interface 526 to the keyboard 504. The components of the computer module 502 typically communicate via an interconnect bus 528 and in a manner known to those skilled in the relevant art.

[0104] Those skilled in the art will understand that the terms used herein are for the purpose of describing various embodiments only and are not intended to limit the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof.

[0105] For ease of understanding and to put into practice, various example embodiments of the present invention will be described hereinafter by way of example only and not limitation. However, those skilled in the art will understand that the present invention can be implemented in various different forms or configurations and should not be construed as limited to the exemplary embodiments set forth hereinafter. On the contrary, these example embodiments are provided to make the present disclosure thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0106] According to various exemplary embodiments of the present invention, there is provided a microfluidic impedance flow cytometry device (also interchangeably referred to as a microfluidic device) for single cell processing, such as measuring the position of a single cell (e.g., corresponding to the microfluidic devices 100, 150, or 180 described above according to various embodiments).

[0107] In various example embodiments, the microfluidic impedance flow cytometry device can be used for lateral position measurement of single cells / particles and has an N-shaped electrode design (e.g., corresponding to a plurality of electrodes arranged adjacent to the fluid channel). A differential current (corresponding to a differential electrical signal measured across the sensing region) can be collected from the N-shaped electrodes. The differential current can encode the trajectory of the flowing single cell / particle. Figure 6A A schematic diagram of an electrical sensing region of a microfluidic device according to various exemplary embodiments of the present invention is shown. In particular, Figure 6A A schematic design of the electrical sensing region 620 in a microfluidic impedance hematocytometer is shown. The entire fluid channel (also interchangeably referred to as a microchannel) 614 can be accessed by cells introduced by a pressure-driven flow. The N-shaped electrodes according to various example embodiments can include two outer electrodes (e.g., corresponding to the first sensing electrode and the second sensing electrode 118a, 118b) and an intermediate inclined electrode (e.g., corresponding to the bias electrode 118c), which are arranged to detect the passage event of a single cell (e.g., detect the passage event of a cell individually).

[0108] In various example embodiments, the first sensing electrode, the second sensing electrode, and the bias electrode can each have a width ranging from about 10 μm to about 40 μm. In various example embodiments, in the case where the microchannel has a width of about 200 μm and a height of about 20 μm, the first sensing electrode, the second sensing electrode, and the bias electrode can each have a width of about 20 μm. In various non-limiting examples, the electrical sensing region 620 can have a length l of about 240 μm s(e.g., the space between the outer edges of two outer electrodes). In various exemplary embodiments, depending on the dimensions of the microchannel, the bias electrode 118c may be arranged in an inclined orientation at an angle in the range of about 10 degrees to about 60 degrees with respect to at least one of the first sensing electrode 118a and the second sensing electrode 118c. In various exemplary embodiments, the intermediate electrode may have an inclination angle of about 22 degrees with respect to any of the outer electrodes. Based on the measured electrical signals and the geometric relationship between the flowing particles, the electrodes, and the position of the microchannel, the lateral position of a single particle / cell flowing through the N-shaped electrode can be calculated.

[0109] Figures 6B to 6C Shows the working mechanism of a microfluidic device according to various exemplary embodiments of the present invention. Figure 6B Shows a microscopic image of the electrical sensing region or zone, which symbolically shows the electrical measurement setup. In a non-limiting example, an alternating current (AC) voltage (e.g., about 3 V at about 500 kHz) can be applied to the bias electrode 118c (e.g., the intermediate inclined electrode). A differential current response (I diff ) can be measured from the first sensing electrode and the second sensing electrodes 118a, 118b (e.g., the other two electrodes).

[0110] Figure 6C Shows a schematic diagram of the sensing region or zone and an exemplary electrical signal curve of the measured electrical signals from the first sensing electrode and the second sensing electrode. Figure 6C Shows the geometric relationship between the flowing cell, the electrodes (e.g., including the first sensing electrode, the second sensing electrode, and the bias electrode), and the position of the fluid channel. Since a single flowing cell passes through the sensor portion of the fluid channel corresponding to the sensing region, a pair of opposite signal peaks of the measured electrical signal can be generated.

[0111] According to various exemplary embodiments of the present invention, based on the relationship between the generated current and the position of the flowing particles, the electrodes, and the microchannel, a simple analytical expression for measuring the lateral position of the particles can be derived. Figure 6C Also shows an analytical expression for measuring the lateral position of the flowing particles according to various exemplary embodiments, which is derived from the measured electrical signals and the geometric relationship between the flowing particles, the electrodes, and the position of the microchannel.

[0112] The analytical expression for measuring the lateral position of the flowing particles can be derived as follows.

[0113] Equation (1) describes the relationship between the distances d 1 , d 2 and the times t 1 , t 2 as follows:

[0114]

[0115] where d 1 and d 2 are the passing distances of the flowing cells corresponding to the passing times t 1 and t 2 respectively. For example, the passing time t 1 refers to the time taken for a cell to travel from the first sensing electrode 118a to the bias electrode 118c in the sensor portion of the fluid channel, and the passing time t 2 refers to the time taken for a cell to travel from the bias electrode 118c to the second sensing electrode 118b in the sensor portion of the fluid channel. The passing distance d 1 refers to the distance that a cell travels from the first sensing electrode 118a to the bias electrode 118c in the sensor portion of the fluid channel during the passing time t 1 , and the passing distance d 2 refers to the distance that a cell travels from the bias electrode 118c to the second sensing electrode 118b in the sensor portion of the fluid channel during the passing time t 2 .

[0116] It can be assumed that the flow rate of the cells in the channel in the sensing region is constant (e.g., assuming that v 1 is equal to the flow rate v 2 along the inductive sensing region), resulting in the following equation (2):

[0117]

[0118] For example, since according to various exemplary embodiments, the inductive sensing region has a length l s of about 240 μm, it can be assumed that the flow rate of the cells in the fluid channel in the sensing region is constant. For example, in the case where the length of the sensing region is short, it can be assumed that the flow rate of the cells in the fluid channel in the sensing region is constant.

[0119] The geometric relationship between the flowing cells, the electrodes, and the position of the channel can be given by the following equation (3):

[0120]

[0121] where x is the lateral position of the cell and is defined as the distance from the lower channel wall to the center of the cell, and w is the channel width (e.g., 200 μm).

[0122] The following equation (4) can be derived by combining equations (2) and (3),

[0123]

[0124] where And M 1 、M 2 and the value of α can be known dimensions of the device. In a non - limiting example, they can be 80.3μm, 76.2μm and 22° respectively. D can be the diameter of the particle. In a non - limiting example, M 1 can be the sum of the shortest distance between the first sensing electrode 118a and the bias electrode 118c across the length l of the sensor region and 1.5 times the electrode width, while M 2 can be the sum of the shortest distance between the bias electrode 118c and the second sensing electrode 118b across the length l of the sensor region and 1.5 times the electrode width.

[0125] Since the amplitude of the peak is proportional to the volume of the particle (or cell), the following equation (5) can be used to estimate the diameter (D) of the particle (or cell).

[0126]

[0127] Where G is a calibration factor depending on the device geometry and electrical characteristics (e.g., when calibrated by 10μm beads according to various exemplary embodiments, G can be ), a represents the amplitude of the first signal peak, and b represents the amplitude of the second signal peak. According to equation (4), the lateral position x can be easily determined based on the parameters extracted from the measured electrical signal and the known dimensions of the microfluidic device.

[0128] In various exemplary embodiments, the lateral positions of the beads and human red blood cells (RBCs) measured by the devices according to various exemplary embodiments of the present invention have good correlation and consistency with those obtained by conventional microscopy imaging methods.

[0129] Figures 7A to 7B FIG. shows a schematic diagram of another exemplary microfluidic device according to various exemplary embodiments of the present invention. Compared with the microfluidic device described with reference to Figures 6A to 6C , these microfluidic devices can have different electrode designs. Referring to Figure 7A , the bias electrode 118c extends in a direction at least substantially parallel to the first sensing electrode 118a, and the second sensing electrode 118b is arranged to have an inclined orientation with respect to the first sensing electrode 118a and the bias electrode 118c. Figure 7A FIG. shows a schematic diagram of the sensing region for lateral position measurement. Similar to the working principle of the N - shaped electrodes of various exemplary embodiments, the lateral position of a single particle can be measured by an electrode design having the following equation (6).

[0130]

[0131] According to various example embodiments, the electrode design can be integrated with a pair of floating electrodes (e.g., a first floating electrode 118d and a second floating electrode 118e) extending along the channel width to measure the cross-sectional position of a single particle, and only one signal output is required. Thus, the cross-sectional position measurement of single cells or particles flowing through the microchannel can be performed with only one electrical signal output. The cross-sectional position can include the lateral position and the vertical position in the fluid channel. Figure 7B A schematic diagram of a sensing region for cross-sectional measurement of flowing particles is shown. By adding two floating electrodes 118d, 118e, the resulting signal curve encodes the height of the particle trajectory (corresponding to the vertical position in the fluid channel).

[0132] For example, when a cell passes from the first sensing electrode 118a to the bias electrode 118c, a first signal peak can be observed, and when the cell passes from the bias electrode 118c to the second sensing electrode 118b, a second signal peak can be observed. In various example embodiments, the first signal peak can be higher than the signal baseline (i.e., a positive peak), and the second signal peak can be lower than the signal baseline (i.e., a negative peak), as Figure 7B shown. Alternatively, in other embodiments, the first signal peak can be a negative peak, while the second signal peak can be a positive peak. As Figure 7B shown, the first signal peak can include a first sub-peak, and the second signal peak can include a second sub-peak. According to various example embodiments, the first sub-peak can be a double peak. In other words, the first sub-peak can be a symmetric peak (e.g., a double peak). The first sub-peak can be generated by the first three parallel electrodes (the first sensing electrode 118a, the first floating electrode 118d, and the bias electrode 118c).

[0133] Figure 7C An exemplary schematic diagram of a cross-sectional fluid channel along the channel length is shown, as well as exemplary signal curves of three different particles flowing through the sensor portion of the fluid channel corresponding to the sensing region. Referring to Figure 7C , the signal curve changes with the height of the particle trajectory in the fluid channel.

[0134] Referring to Figure 7D , the relative prominence S is related to the vertical position y of the particle. The relative prominence S is as follows:

[0135]

[0136] where Q represents the amplitude of the first sub-peak (e.g., a double peak), and Q represents the valley between the first sub-peaks.

[0137] A quadratic fit can be used to calculate the vertical position y of the cell, as shown in Equation (8):

[0138] y = h * (b0 +b 1 *S + b 2 *S 2 ) Equation (8)

[0139] where h is the channel height. The parameter b i depends on the experimental setup and can be calculated through a calibration process. Thus, it is possible to measure the cross-sectional position of a single particle (i.e., the lateral position x and the vertical position y along the cross-section) using only one electrical signal output.

[0140] An application of monitoring bead focusing is demonstrated according to an exemplary embodiment, showing good agreement with the optical quantification to be described.

[0141] Experimental Setup and Data Analysis

[0142] According to the exemplary experiments conducted, a microfluidic device with N-shaped electrodes can be fabricated. The fluid flow in the microfluidic channel can be controlled using a pressure pump (e.g., Elveflow AF1). In a non-limiting example, the entire microfluidic channel is accessed by 3.6, 5, 7, 10 μm beads and human RBCs. Driving pressures of approximately 300, 500, and 700 mbar (millibar) are applied to study the measurement accuracy of the particle lateral position at different flow rates, resulting in flow rates of approximately 25.4, 42.4, and 59.3 μl min -1 . The corresponding average particle flow rates are approximately 0.08, 0.14, and 0.21 m s -1 , which are extracted from the recorded high-speed videos. In the experiment of monitoring sheath-flow-induced particle focusing, three separate syringe pumps (KD Scientific, Holliston, Massachusetts) are used to control the fluid flow. The sample flow (e.g., 7 μm beads) is concentrated at the bottom (e.g., lower position) (15, 4, and 1 μl min -1 ), middle (e.g., middle position) (8, 4, and 8 μl min -1 ), and top (e.g., upper position) (1, 4, and 15 μl min -1 ) of the fluid channel (lateral direction x) by the sheath flow. Blue food dye is mixed in the sheath flow to optically show the particle focusing region, which is quantified by analyzing the pixel intensity curve of the microscopic image. For example, software such as ImageJ can be used to analyze grayscale images that carry only intensity information. The pixel intensity curve after subtracting the baseline is shown.

[0143] Current data was recorded using an impedance spectroscope (e.g., HF2IS, Zurich Instruments), while the trajectories of the particles were captured using a high-speed camera for comparison. Electrical data was analyzed using a customized MATLAB program (Matlab, Mathworks, USA) to provide the lateral position x (electrical position x) measured by electrical methods according to various exemplary embodiments, and the corresponding optical position x was derived from the captured high-speed video using academically published tracking software (e.g., DMV), which can provide precise multiple parameters such as particle lateral position, zone, and velocity.

[0144] Linear regression and Bland-Altman analysis were used to evaluate the correlation and consistency between the electrical method and the optical method. The root mean square deviation (RMSD) of two measurements often used in model performance studies was used as a measure of the precision of the microfluidic device for measuring the particle lateral position.

[0145] At low excitation frequencies, the electrical characteristics of cells are similar to those of insulating beads because the cell membrane acts as a capacitor to prevent electric field lines from passing through it. Since the experiment utilized a low-frequency alternating voltage of 500 kHz, measuring the particle lateral position is similar to measuring the cell lateral position. The functionality of the microfluidic impedance cytometer device according to various exemplary embodiments was verified by comparing the electrical position x of flowing 3.6, 5, 7, 10 μm beads and human red blood cells (RBCs) with their optical position x.

[0146] Figure 8A Depicts a graph showing the measured electrical position x of 10 μm beads at a flow rate of 25.4 μl min -1 with respect to t 1 / t 2 for experimental results according to various exemplary embodiments. More particularly, Figure 8A shows the lateral position of 10 μm beads obtained according to various exemplary embodiments and the lateral position obtained by the optical method, where three representative beads flow through the lower (I), middle (ii), and upper (iii) parts (or positions) of the microchannel at a flow rate of 25.4 μl min -1 The position x determined by the optical method with respect to t (determined by the electrical method) 1 / t 2 is also plotted as a reference. To compare the two methods, the t 1 / t 2 values obtained by the electrical method were used to plot the corresponding optical lateral positions. Figure 8B A graph of the corresponding measured differential electrical signal is shown in.

[0147] Figure 8CShows an enlarged view of the representative electrical signals of three representative beads for the measurement of the optical position x and their corresponding microscopic optical images (captured simultaneously). The position x measured by the electrical method and the optical method is highly consistent, showing results comparable to the optical estimation. For example, for the 10-μm bead (ii) passing through the relatively middle part or position of the microchannel, the electrical position x of 98.0 μm is comparable to the optical position x of 96.5 μm.

[0148] In various exemplary embodiments, due to the unique design of the bias electrode (e.g., the inclined middle electrode) inclined with respect to the first sensing electrode, if the cell passes through a lateral position relatively close to the lower channel wall, the passing time (t 1 ) of the cell through the first two electrodes (e.g., the first sensing electrode and the bias electrode) is longer than the passing time (t 2 ) through the last two electrodes (e.g., the bias electrode and the second sensing electrode). This may be because at the lower position of the microchannel, the distance between the first two electrodes is longer than that between the last two electrodes. For example, Figure 8C Shows the flowing particle (i), where the particle flows relatively close to the lower channel wall. The corresponding current change (a) (i.e., the amplitude of the first signal peak (e.g., negative peak)) is lower than the corresponding current change (b) (i.e., the amplitude of the second signal peak (e.g., positive peak) from the last two electrodes) because the electric field on the left side is weaker than that on the right side. Conversely, if the cell passes through the upper half of the channel, as shown by the particle (iii), then t 1 is shorter than t 2 , and the corresponding current change (a) (i.e., the amplitude of the first signal peak) is greater than the corresponding current change (b) (i.e., the amplitude of the second signal peak). In the case where the cell passes through the middle position of the channel, as shown by the particle (ii), the two peaks are similar, indicating that t 1 can be equal to t 2 , and the corresponding current change (a) (i.e., the amplitude of the first signal peak) may be equal to the corresponding current change (b) (i.e., the amplitude of the second signal peak).

[0149] Figure 9 Shows a quantitative comparison of the lateral position of the 10-μm bead between the results of the electrical method and the results obtained by the optical method according to various exemplary embodiments. More particularly, Figure 9 Shows the relationship between the electrical position x and the optical position x of the 10-μm bead at the flow rates of (a) 25.4 μl min -1 , (b) 42.4 μl min -1 , and (c) 59.3 μl min -1 . R is obtained for the three flow rates 2A coefficient of determination greater than 0.99 demonstrates a good linear correlation between the electrical method and the optical method according to various exemplary embodiments. The root mean square deviation (RMSD) is calculated for each flow rate. At flow rates of 25.4, 42.4, and 59.3 μl min -1 respectively, the RMSDs of these two measurements are 3.2 μm, 6.9 μm, and 12.7 μm, corresponding to 1.60%, 3.45%, and 6.35% of the channel width respectively.

[0150] In addition to the linear correlation, Bland - Altman analysis is also used to study the agreement between the two measurements. A Bland - Altman plot is a scatter plot of the differences between the two measurements relative to the mean. Figure 10 The Bland - Altman analysis is shown, which compares the lateral positions x obtained by the electrical method and the optical method at flow rates of (d) 25.4 μl min -1 , (e) 42.4 μl min -1 and (f) 59.3 μl min -1 . Most of the values are between the 95% limits of agreement, which are represented by two dashed lines in the figure (i.e., positive and negative deviations). It can be observed that when the particle passes through the lower half of the channel, the electrical position x is higher than the optical position x, resulting in a negative difference. Conversely, when the particle flows relatively close to the upper channel wall, there is a positive difference. This is because the electric field strength inside the two electrodes on the left is different from that of the two electrodes on the right. For the lower half of the N - shaped electrode, the electric field strength on the left is weaker than that on the right because the gap between the two electrodes on the left is larger than that on the right, causing the electrical signal to drop to the baseline before the particle reaches the centerline of the middle electrode. Therefore, the value C used in Equation (4) for the calculation of position x 1 is higher than the true value, and conversely, C 2 is less than the true value, which together results in a higher electrical position x compared to the true position x (i.e., the optical position x). Similarly, when the particle flows through the upper part of the N - shaped electrode, the electric field strength on the left is stronger than that on the right. Therefore, from the electrical method, a smaller C 1 and a higher C 2 result in a smaller position x.

[0151] As Figure 10 shown, when the lateral position x approaches the middle part or position of the channel, the difference between the two measurements decreases. This is because the difference in the electric field strength between the two pairs of electrodes decreases as the position x approaches the middle part of the N - shaped electrode (i.e., if there is no misalignment between the N - shaped electrode and the microfluidic channel, position x is 100 μm). It can be found that the RMSD (as Figure 9 shown) and the maximum difference (as Figure 10increases with the increase in flow rate as shown. This may be due to the increase in the electric field strength difference between the two pairs of electrodes with the increase in flow rate. In other words, a higher flow rate reduces the measurement accuracy of the lateral position of the particles.

[0152] Beads with different diameters were used to study the minimum particle size that can be measured using the microfluidic device according to various exemplary embodiments. Figures 11A to 11B The minimum particle (i.e., 3.6 μm bead) that can be detected by the microfluidic device with good performance is shown. Figure 11A A good linear correlation (R 2 = 0.9616) between the electrical method and the optical method according to various exemplary embodiments is shown, and Figure 11B a Bland - Altman analysis is shown, which demonstrates good agreement between the two measured values. At a flow rate of 25.4 μl min -1 , the root - mean - square deviation (RMSD) of the two measured values is 11.0 μm (i.e., 5.5% of the channel width).

[0153] Human red blood cells (RBCs) were used to verify that the microfluidic device according to various exemplary embodiments can be used for precise lateral position measurement of single cells. Figures 12A to 12B A quantitative comparison of the lateral position of RBCs between the results obtained from the microfluidic device according to various exemplary embodiments and the results obtained by the optical method at a flow rate of 42.4 μl min -1 is shown. Figure 12A A graph showing the electrical position x versus the optical position x is shown, showing a good linear correlation of the coefficient of determination (R 2 of 0.9863) and a high resolution (RMSD of 11.7 μm, i.e., 5.7% of the channel width). Figure 12B A graph showing the Bland - Altman analysis is shown, which compares the lateral position x obtained by the electrical method and the optical method, showing good agreement. Most values (94.6%) are between the 95% limits of agreement, represented by two dashed lines in Figure 12B .

[0154] Measurement of the lateral position and size of a mixture of 5 - μm and 10 - μm beads

[0155] To test whether the microfluidic device according to various exemplary embodiments can distinguish cells or particles with different physical properties (e.g., size) flowing through the same position x, a mixture of 5 - and 10 - μm beads was tested at a flow rate of 42.4 μl min -1 . Figures 13A to 13D Shown is at 42.4 μl min -1Measurement of the lateral position x and electrical diameter of a mixture of 5 and 10 μm beads at a flow rate of Figure 13A shows a comparison between the electrical position x and the optical position x. As Figure 13A shown, the coefficient of determination (R 2 = 0.9895) between the two measurements has a good linear correlation and has a high resolution (RMSD = 10.3 μm, i.e., 5.15% of the channel width).

[0156] Figure 13B shows a Bland - Altman analysis plot that compares the lateral position x obtained by electrical and optical methods. The Bland - Altman analysis demonstrates good agreement between the two methods. Most values are within the 95% limits of agreement, which are represented by two dashed lines. Compared with the results for 10 μm beads at the same flow rate, for mixtures with a majority of 5 μm beads, the RMSD increases from 6.9 μm (3.45% of the channel width) to 10.3 μm (5.15% of the channel width). As described above, the electrical diameter D is calculated according to equation (5). Figure 13C shows a histogram of the electrical diameter. As Figure 13C shown, two distinct distributions are clearly observed, which correspond to 5 μm and 10 μm beads respectively. Figure 13D shows a scatter plot of the electrical diameter versus the electrical position x, indicating that the microfluidic device according to various exemplary embodiments not only measures the lateral position of individual cells / particles but can also simultaneously characterize their physical properties (e.g., size). As Figure 13D shown, even when flowing through the same position x, two different beads can be clearly distinguished, which means that the microfluidic device can not only measure the lateral position x of flowing beads but also characterize their biophysical properties such as the size shown. This enables the evaluation of the efficiency of cell separation, such as calculating the purity and recovery rate of sorted cells with different sizes and lateral positions.

[0157] Monitoring sheath - flow - induced particle focusing

[0158] Particle focusing is usually a necessary step before detecting, counting, and sorting particles or cells. A microfluidic impedance cytometer (microfluidic device) according to various example embodiments is applied to monitor sheath - flow - induced particle focusing, where 7 μm beads are suspended in the sample flow. As Figures 14A to 14B shown, before the particle focusing experiment, a quantitative analysis of the lateral position of 7 μm beads between the results obtained from the microfluidic device according to various example embodiments and the results obtained by optical methods is performed. A good linear correlation (R 2> 0.99) and good consistency (Bland-Altman analysis). The RMSD was 7.0 μm (i.e., 3.5% of the channel width).

[0159] Figure 15A shows at 20 μl min -1 of the total flow rate for monitoring the focusing of 7-μm beads induced by the sheath flow. The image shows the boundary between the sample flow and the sheath flow. The image shows that the sheath flow concentrates the sample flow at the bottom, middle, and top of the channel (lateral direction x). Figure 15B shows Figure 15A a pixel intensity curve graph (gray scale) of the image shown, which reflects the particle focusing region. Figure 15C shows a histogram of the electrical positions x of 7-μm beads focused by the sheath flow in different regions (at the bottom, middle, and top of the channel (lateral direction x)). As Figure 15B and Figure 15C shown by the dashed lines in, the electrical-based results according to various exemplary embodiments are in very good agreement with the optical-based results. For example, for the sample flow focused in the middle of the channel, by both methods, the focusing region is between 80 μm and 125 μm. These results indicate that the microfluidic devices according to various exemplary embodiments can accurately determine the lateral position of particles and are powerful tools for monitoring particle focusing.

[0160] Compared with conventional impedance-based methods, various exemplary embodiments provide microfluidic devices (e.g., microfluidic impedance cytometer devices with N-shaped electrodes) with more accurate single-cell / particle position measurements at the highest flow rates. The position of the cell (e.g., lateral position and / or vertical position) can be directly determined by a simple analytical expression, rather than by a linear mapping with calibration coefficients in conventional techniques. By comparing the electrical lateral positions of beads and RBCs with the optical lateral positions, the functionality of the microfluidic impedance cytometer devices according to various exemplary embodiments was verified. The two methods have good correlation and consistency in all cases. Compared with conventional impedance-based methods, a higher resolution of lateral position measurement can be achieved. The experimental results of the mixture show that the microfluidic devices according to various exemplary embodiments can not only measure the position of a single cell or particle in a fluid channel, but also simultaneously study or provide information related to their physical properties such as size. The experiments on sheath-flow-induced particle focusing show that the microfluidic devices according to various example embodiments are powerful tools for monitoring and evaluating particle or cell focusing. Therefore, the microfluidic devices according to various exemplary embodiments provide great potential for the real-time characterization of cell sorting and separation performance.

[0161] Due to the advantages of fast and precise processing of electrical signals and high throughput of impedance flow cytometers, the various example embodiments described can be easily integrated with other microfluidic platforms, e.g., as a downstream path for real-time measurement of the position (e.g., lateral position, vertical position) and physical properties of single cells and particles.

[0162] While embodiments of the invention have been particularly shown and described with reference to specific embodiments, those skilled in the art should understand that various changes in form and detail may be made without departing from the scope of the invention as defined by the appended claims. Accordingly, the scope of the invention is represented by the appended claims and is thus intended to encompass all variations within the equivalent meaning and scope of the claims.

Claims

1. A microfluidic device for single cell processing, comprising: a substrate; fluid channels provided in the substrate, wherein the fluid channels are configured to form a fluid path for allowing a fluid sample including cells to flow along the channels; and a plurality of electrodes arranged adjacent to the fluid channels for determining the position of the cells in the fluid channels, the plurality of electrodes including: a sensing electrode pair including a first sensing electrode and a second sensing electrode, the sensing electrode pair defining a sensing region overlapping with a sensor portion of the fluid channel, wherein at least the first sensing electrode in the sensing electrode pair extends in a first direction, and the sensing electrode pair is configured to measure a differential electrical signal across the sensing region when the cells flow through the sensor portion of the fluid channel; and a bias electrode arranged between the first sensing electrode and the second sensing electrode, the bias electrode being configured to receive a bias voltage, wherein the second sensing electrode extends in a direction at least substantially parallel to the first sensing electrode and the bias electrode is arranged to have an inclined orientation with respect to the first sensing electrode, or the bias electrode extends in a direction at least substantially parallel to the first sensing electrode and the second sensing electrode is arranged to have an inclined orientation with respect to the first sensing electrode.

2. The device according to claim 1, wherein the electrodes are arranged to form a configuration corresponding to an N shape.

3. The device according to claim 1 or 2, wherein the inclined orientation of the bias electrode is at an angle within a range of 10 degrees to 60 degrees with respect to at least one of the first sensing electrode and the second sensing electrode.

4. The device according to claim 1 or 2, wherein the position of the cells includes a lateral position in the fluid channel, the lateral position being relative to the width direction of the fluid channel and being determined based on the geometric relationship between the cells and the plurality of electrodes.

5. The device according to claim 1, wherein the inclined orientation of the second sensing electrode is at an angle within a range of 10 degrees to 60 degrees with respect to the bias electrode.

6. The device according to claim 1, wherein the plurality of electrodes further includes a floating electrode pair extending in the first direction.

7. The device according to claim 6, wherein the floating electrode pair is arranged between the sensing electrode pair, and the bias electrode is arranged between the floating electrode pair.

8. The device according to claim 1, wherein the bias voltage includes an alternating voltage.

9. The device according to claim 1, wherein the differential electrical signal includes a differential current response across the sensing region.

10. The device according to claim 1, wherein the first direction is along the width direction of the fluid channel.

11. A method of forming a microfluidic device for single cell processing, the method comprising: providing a substrate; providing fluid channels in the substrate, wherein the fluid channels are configured to form a fluid path for allowing a fluid sample including cells to flow along the channels; A plurality of electrodes are formed adjacent to the fluid channel for determining the position of the cells in the fluid channel, the plurality of electrodes comprising: A sensing electrode pair, the sensing electrode pair including a first sensing electrode and a second sensing electrode, the sensing electrode pair defining a sensing region overlapping a sensor portion of the fluid channel, wherein at least the first sensing electrode of the sensing electrode pair extends in a first direction, the sensing electrode pair configured to measure a differential electrical signal across the sensing region when the cells flow through the sensor portion of the fluid channel; and A bias electrode disposed between the first sensing electrode and the second sensing electrode, the bias electrode configured to receive a bias voltage, wherein the second sensing electrode extends in a direction at least substantially parallel to the first sensing electrode and the bias electrode is disposed with an inclined orientation relative to the first sensing electrode, or, the bias electrode extends in a direction at least substantially parallel to the first sensing electrode and the second sensing electrode is disposed with an inclined orientation relative to the first sensing electrode.

12. The method according to claim 11, wherein the first sensing electrode, the second sensing electrode and the bias electrode are arranged to form a configuration corresponding to an N shape.

13. The method according to claim 11 or 12, wherein the inclined orientation of the bias electrode is at an angle within a range of 10 degrees to 60 degrees relative to at least one of the first sensing electrode and the second sensing electrode.

14. The method according to claim 11, wherein the inclined orientation of the second sensing electrode is at an angle within a range of 10 degrees to 60 degrees relative to the bias electrode.

15. The method according to claim 14, wherein the plurality of electrodes further includes a floating electrode pair that extends along the first direction.

16. The method according to claim 15, wherein the floating electrode pair is disposed between the sensing electrode pair, and the bias electrode is disposed between the floating electrode pair.

17. The method according to claim 11, wherein the first direction is along the width direction of the fluid channel.

18. A method for single cell processing using the microfluidic device according to claim 1, the method comprising: Applying a bias voltage to the bias electrode; Obtaining a differential electrical signal based on the first sensing electrode and the second sensing electrode when the cells flow through the sensor portion of the fluid channel corresponding to the sensing region; and Determining the position of the cells in the sensor portion of the fluid channel based on the differential electrical signal.

19. The method according to claim 18, wherein the obtained differential electrical signal includes a plurality of signal peaks corresponding to the situation where the cells flow through the sensing portion of the fluid channel from the first sensing electrode to the second sensing electrode.

20. The method according to claim 18 or 19: The plurality of signal peaks includes a first signal peak corresponding to the cell flowing from the first sensing electrode to the bias electrode in the sensor portion of the fluid channel and a second signal peak corresponding to the cell flowing from the bias electrode to the second sensing electrode in the sensor portion of the fluid channel; and determining the position of the cell in the sensor portion of the fluid channel includes determining a lateral position of the cell in the fluid channel based on a width of the first signal peak and a width of the second signal peak, the lateral position being relative to a width direction of the fluid channel.

21. The method according to claim 20, wherein determining the lateral position of the cell in the fluid channel is further based on a geometric relationship between the cell and the plurality of electrodes.

22. The method according to claim 20, wherein the first signal peak includes a first sub-peak, and determining the position of the cell in the sensor portion of the fluid channel further includes determining a vertical position of the cell in the fluid channel based on a ratio of an amplitude of the first sub-peak to a valley value of the first sub-peak, the vertical position being relative to a height direction of the fluid channel.

23. The method according to claim 20, further comprising determining a size of the cell based on an amplitude of the first signal peak and an amplitude of the second signal peak.

24. A system for single cell processing, the system comprising: a microfluidic device for single cell processing according to claim 1; and a computing system, comprising: a memory; and at least one processor communicatively coupled to the memory and the microfluidic device and configured to: apply a bias voltage to the bias electrode; obtain a differential electrical signal based on the first sensing electrode and the second sensing electrode when the cell flows through the sensor portion of the fluid channel corresponding to the sensing region; and determine the position of the cell in the sensor portion of the fluid channel based on the differential electrical signal.

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