A system and method for determining cellular properties of red blood cells (RBCS)

Abstract

The present invention relates to an electro-fluidic system (100) for determining cellular properties of Red Blood Cells (RBCs). The system (100) comprises a micropore assembly (116), an amplifier, and a processing unit. The micropore assembly comprises a micropore device (122) and a syringe pump (124) for applying force. The micropore device (122) comprises an orifice and a set of capillaries. The amplifier is configured for applying voltage across the micropore assembly. The system (100) applies a constant electric voltage in order to ensure constant baseline current flow across micropore channel of the micropore device. The system (100) induces the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump. Further, the system (100) estimates the cellular properties based on change in electrical pulses during passage of the cells through the orifice.

Description

[0001] A SYSTEM AND METHOD FOR DETERMINING CELLULAR PROPERTIES OF RED BLOOD CELLS (RBCs)

[0002] TECHNICAL FIELD

[0003] [1] The present disclosure relates to field of studying or estimating mechanical or morphological properties of soft materials using electro-fluidics. Particularly, but not exclusively, the present disclosure is directed towards an electro-fluidic system and method for determining cellular properties of Red Blood Cells (RBCs).

[0004] BACKGROUND

[0005] [2] Understanding the mechanical properties of soft materials, either biological or non- biological (natural and synthetic) has been an interest of research. In order to quantify the mechanics of various soft materials, they have to be probed. There are different probing tools used by the scientific community and industries to measure these mechanical properties. Among them, the most used ones are atomic force microscopy (AFM), magnetic twisting cytometry, particle-tracking Micro-rheology, fast imaging cytometry, parallel-plate rheometers, monolayer rheology, and optical stretching using a dual optical tweezer.

[0006] [3] Further, understanding of mechanical properties becomes all the more critical in case of biological substances such as cell. The gargantuous interest in understanding the cellular mechanics has been explosive with scientists, doctors and pharmaceutical companies. This is due to cell mechanics being a key marker for cell development and functioning. Biomechanics plays an important role in cell metabolism, regulation of mechanotransduction pathways and so on. The mechanical properties of biological samples are tightly linked to normal and pathological functions. The mechanical properties of cells are determined with the use of many different factors, including but not limited to cytoskeleton elasticity, membrane tension, cell-substrate adhesion, etc. Literature study has established the mechanical interplay of these factors to be very complex in nature.

[0007] [4] When studying biological substances, one of the most utilized probing tools for cells is AFM measurement. AFM measurements are performed on cells adhered to a solid surface with a throughput of 1-20 cells / hr. The default AFM analysis models primarily utilize contact mechanics theories that assume a large thickness of the sample relative to the indentation. However, a major disadvantage of AFM for cellular mechanics analysis is the time-consuming and low-throughput nature of single-cell experiments and the inability to probe beneath the cell surface. The results of measurements of the AFM models also vary with the geometry of the cantilever and loading rate. Magnetic twisting cytometry measurements are also performed on cells adhered to the surface, with magnetic beads attached to cells, giving a throughput of 2000 cells / hr. However, magnetic twisting cytometry is a cell micromechanics technique which requires a strong magnetic field and a sensitive magnetometer which is expensive and difficult to operate. It may even induce unwanted effects on the cells, such as heating, magnetization, or membrane damage. For particle-tracking Micro-rheology, cells are adhered to the surface and beads were injected into the cell. The throughput of this method is only 30 cells / hr, and the results vary if the measurement shear frequencies are changed.

[0008] [5] In specific studies, where the understanding of the cellular mechanics is the key to track the intracellular dynamics, the interpretation of the physical parameters becomes important. It is well understood that the changes in the physical properties of a cell can predominantly result in several-fold increases or decreases in cell stiffness. These changes to cell stiffness are signatures of severe pathology and a potential breakdown of bodily functions. Disease biomechanics provide for not only an understanding of the mechanics of the underlying disease progression but also a tool to analyse the corresponding data to detect the cell status. The same is advantageous in diseases such as cancer which are difficult to detect at an early stage. Some cell characteristics can be detected using the Coulter principle which is a technique known for resistive pulse sensing. The coulter principle provides for having micro channels where two separate sections or chambers are provided containing electrolytes. As the particles or cells pass through the micro channels, the passage of the particles or cells causes a brief change in the resistance of the liquid.

[0009] [6] The existing techniques for studying mechanical or morphological properties of soft materials require expensive high-speed cameras as well as huge data storage spaces for big sequential images. The data storage limitation also bounds these measurements to span only small intervals. The voltage signals are smaller in space, faster & efficient in analysis. Further, conventional techniques require dyes or surface coatings, or adhesion for sample preparation or for measurement. However, none of the techniques known in the art provide for a label-free, high-throughput electro-fluidic technique to study the mechanical or morphological characteristics of soft materials.

[0010] [7] Further, all the soft materials, including hydrogel, lipid vesicles, and various biological systems, are incredibly complex in constitution. Their morphology, as well as mechanics, are highly sensitive to their local environment (pH, Tonicity, Temperature). These soft systems often deform even by small thermal fluctuations and external forces. In preparation for the growth evolution of cells or industry-related hydrogels, the incubation conditions (like time, temperature, humidity, and suspension chemical compositions) can cause a considerable change in their mechanical properties which, in turn, affects their functionality. To understand the role and further optimize these soft materials for various applications, a careful measurement of their mechanical and morphological properties is important. Most mechanical studies use the means of adherence of the material to substrates, which could provide different mechanical numbers for the same population. In order to avoid the involvement of different parameters, such as the use of substrates, the measurements must either be done in In-vivo-like conditions (in suspension) or the mechanical probing method must make sure that the substrate effects are not present / do not interfere in the measurement.

[0011] [8] Thus, there is a need in the art for a technique that is capable of studying soft materials, either biological or non-biological (natural or synthetic), without any need for labels / tags, a fast camera, a microscope, or expensive device fabrication methodologies. The present invention overcomes the limitations in the prior art and provides a technique which does not require labels / tags for the material and does not require expensive devices, etc. The present invention provides a high-throughput technique to study the material.

[0012] SUMMARY

[0013] [9] One or more shortcomings of the prior art are overcome, and additional advantages are provided through the present disclosure. Additional features and advantages are realized through the techniques of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered a part of the claimed disclosure.

[0014]

[0010] In one embodiment, the present disclosure relates to an electro-fluidic system for determining cellular properties of Red Blood Cells (RBCs). The system comprises a micropore assembly, an amplifier, and a processing unit. The micropore assembly comprises a micropore device and a syringe pump for applying force. The micropore device comprises an orifice and a set of capillaries acting as micropore channels. The amplifier is configured for applying voltage across the micropore assembly. The processing unit is communicably coupled with the amplifier, the micropore assembly, and a memory. The processor is configured to apply a constant electric voltage in order to ensure constant baseline current flow across micropore channel of the micropore device. The processor induces the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump. Further, the processor is configured to estimate the cellular properties based on change in electrical pulses during passage of the cells through the orifice, and determine cellular mechanical or morphological properties.

[0015]

[0011] In another embodiment, the present invention discloses a method of determining cellular properties of Red Blood Cells (RBCs). The method comprises the steps of applying a constant electric voltage across a micropore device in order to ensure constant baseline current flow through the micropore channel of the micropore device, wherein the micropore device comprises an orifice and a set of capillaries as micropore channels. The method includes inducing the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of a syringe pump. The cells are considered to be in room temperature and having a defined cell volume. The method further comprises estimating the cellular properties based on change in electrical pulses during passage of the cells through the orifice, and determine cellular mechanical or morphological properties.

[0016]

[0012] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0017] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0018]

[0013] The novel features and characteristics of the disclosure are set forth in the appended claims. The disclosure itself, however, as well as a preferred mode of use, further objectives, and advantages thereof, will best be understood by reference to the following detailed description of an illustrative embodiment when read in conjunction with the accompanying figures. One or more embodiments are now described, by way of example only, with reference to the accompanying figures wherein like reference numerals represent like elements and in which:

[0019]

[0014] Figure 1 illustrates a schematic block diagram of an electro-fluidic system for determining cellular properties of Red Blood Cells (RBCs), in accordance with an embodiment of the present disclosure;

[0015] Figure 1A illustrates a schematic block diagram of the micropore device, in accordance with an embodiment of the present disclosure;

[0020]

[0016] Figure 2(A - B) illustrate a method of determining cellular properties of Red Blood Cells (RBCs), in accordance with an embodiment of the present disclosure;

[0021]

[0017] Figure 3 illustrates a method of fabrication of a micropore device, in accordance with an embodiment of the present disclosure;

[0022]

[0018] Figure 4A illustrates graphical representation of detection of electrical signal when a cell translocates through the constricted micropore showing the dwell time (At) and conductance drop (AG), in accordance with an embodiment of the present disclosure;

[0023]

[0019] Figure 4B illustrates schematic version of the detection region of the micropore, showing the applied voltage across the electrodes, and soft (Red) and stiff (Blue) cells, in accordance with an embodiment of the present disclosure;

[0024]

[0020] Figure 4C illustrates graphical representation of detection of electrical signals when a stiff (blue) and soft (red) cells are detected through the constricted micropore device, in accordance with an embodiment of the present disclosure;

[0025]

[0021] Figure 4D illustrates an exemplary mapping of electro-fluidic measurements with the elasticity of the cells as measured with the AFM control, in accordance with an embodiment of the present disclosure;

[0026]

[0022] Figure 5(a - c) illustrate baseline conductance of an open micropore, electrical signals showing the translocation of cells through the constricted micropore, and dwell time (At) histogram of the series of electrical pulses recorded, in accordance with an embodiment of the present disclosure;

[0027]

[0023] Figure 6 illustrates the normalized data from the control AFM experiment is plotted alongside with the data from 5 different micropore devices showing the overlapping all the trends when the RBCs were treated with different concentrations of Latrunculin-A drug mimicking the cancerous state of cells, in accordance with an embodiment of the present disclosure;

[0028]

[0024] Figure 7 illustrates steps involved in forging a micropore from a glass capillary, in accordance with an embodiment of the present disclosure;

[0029]

[0025] Figure 8 illustrates AFM Sample Fluid Cell, in accordance with an embodiment of the present disclosure;

[0030]

[0026] Figure 9 illustrates effect of incubation temperature on translocation experiment, in accordance with an embodiment of the present disclosure;

[0031]

[0027] Figure 10 illustrates schematic of electro-fluidic device detection principle and signals for measurement of stiffness and size through the micropore device, in accordance with an embodiment of the present disclosure;

[0032]

[0028] Figure 11 illustrates effect of Amplifier Bandwidth on TL Data, in accordance with an embodiment of the present disclosure;

[0033]

[0029] Figure 12 illustrates microscopic images (with 60X objective) of the same cell (marked with red arrow) treated with different concentration of Lat-A and probed with the spherical cantilever (pointed with red arrow), in accordance with an embodiment of the present disclosure;

[0034]

[0030] Figure 13 illustrates schematic of AFM force-indentation experiment, in accordance with an embodiment of the present disclosure;

[0035]

[0031] Figure 14 illustrates demonstration of cell volume and stiffness detection using micropore devices;

[0036]

[0032] Figure 15 illustrates effect of the actin inhibitor drug (Latrunculin-A) on the stiffness of red blood cells., in accordance with an embodiment of the present disclosure;

[0037]

[0033] Figure 16 illustrates effect of Lat-A on the free-flight AG of RBCs, in accordance with an embodiment of the present disclosure;

[0038]

[0034] Figure 17 illustrates empirical formulation for modeling the cellular stiffness using the electro-fluidic experimental data, in accordance with an embodiment of the present disclosure;

[0039]

[0035] Figure 18 illustrates graphical representation of Effect of flow rate on the At values of RBCs treated with different concentrations of Lat-A, in accordance with an embodiment of the present disclosure;

[0040]

[0036] Figure 19 illustrates graphical representation of At histogram with the Gaussian fit for RBCs treated with different concentrations of Lat-A at different fluid flow, in accordance with an embodiment of the present disclosure;

[0037] Figure 20 illustrates graphical representation of cellular stiffness estimation of sickled RBCs of the SCD patients using microfluidic devices;

[0041]

[0038] Figure 21 illustrates images of Red blood cells acquired from healthy donors and SCD patients, in accordance with an embodiment of the present disclosure;

[0042]

[0039] Figure 22 illustrates summary of SCD patient data, in accordance with an embodiment of the present disclosure; and

[0043]

[0040] Figure 23 illustrates graphical representation of summary of At values of the constricted micropore data acquired for healthy donors and SCD patients, in accordance with an embodiment of the present disclosure.

[0044] DETAILED DESCRIPTION

[0045]

[0041] In the present document, the word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment or implementation of the present subject matter described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0046]

[0042] While the disclosure is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however, that it is not intended to limit the disclosure to the forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the disclosure.

[0047]

[0043] The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a setup, device, or process that comprises a list of components or steps does not include only those components or steps but may include other components or steps not expressly listed or inherent to such setup or device or process. In other words, one or more elements in a system or apparatus proceeded by “comprises... a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or apparatus.

[0048]

[0044] As used herein, the term soft material means, but is not limiting to, materials which can be deformed by applying external forces, these materials usually have elasticity ranging from a few pascals to a hundred pascals As used herein the term “mechanical properties” means, the physical behavior of materials in response to an external force (or a probe), example of these properties are, but are not limited to, elasticity, stiffness, plasticity, bulk modulus, shear modulus etc. As used herein the term “morphological properties” means, the visual properties of the material, in other words properties associated with the material’s appearance like, but are not limited to, it’s shape, size, texture, color, volume, cross sectional area etc. Further, the definitions of other terminologies are as the following.

[0049] Micropore: A small orifice made of glass capillary used here to probe the cells.

[0050] Latrunculin-A: It is a toxin from the red sea sponge Latrunculia magnifica which binds to actin monomers near the nucleotide binding cleft with 1 : 1 stoichiometry and prevents them from polymerizing

[0051] Dwell time (At): The amount of time spent by the cells inside the sensing region of our micropore device

[0052] Conductance Drop (At): The change in the conductance of the entire electro-fluidic system caused when a sample translocates through and blocks the conducting ions

[0053] Resistive pulse sensing: Technique using the change in the channel resistance (or conductance) to detect particles in the form of electrical pulses.

[0054]

[0045] Embodiments of the present disclosure provide an electro-fluidic system for determining cellular properties of Red Blood Cells (RBCs). The system comprises a micropore assembly, an amplifier, and a processing unit. The micropore assembly comprises a micropore device and a syringe pump for applying force. The micropore device comprises an orifice and a set of capillaries acting as micropore channels. The amplifier is configured for applying voltage across the micropore assembly. The processing unit is communicably coupled with the amplifier, the micropore assembly, and a memory. The processor is configured to apply a constant electric voltage in order to ensure constant baseline current flow across micropore channel of the micropore device. The processor induces the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump. Further, the processor is configured to estimate the cellular properties based on change in electrical pulses during passage of the cells through the orifice, and determine cellular mechanical or morphological properties.

[0055]

[0046] In the following detailed description of the embodiments of the disclosure, reference is made to the accompanying drawings that form a part hereof, and which are shown by way of illustration-specific embodiments in which the disclosure may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosure, and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0056]

[0047] Figure 1 illustrates a schematic block diagram of an electro-fluidic system for determining cellular properties of Red Blood Cells (RBCs), in accordance with an embodiment of the present disclosure. As shown in Figure 1, the system (100) comprises an electro-fluidic computation unit (102), a micropore assembly (116), an amplifier (118), a noise eliminator (120), and a data repository (114), wherein the electro-fluidic computation unit (102) is communicably coupled with the amplifier (118), the micropore assembly (116), the noise eliminator (120), and the data repository (114).

[0057]

[0048] The micropore assembly (116) comprises a micropore device (122) and a syringe pump (124) for applying force as illustrated in Figure 1A. The micropore device (122) comprises an orifice and a set of capillaries acting as micropore channels. In one embodiment, shape and size of the orifice of the micropore device (122) is designed to be narrower with respect to diameter of cell in context. The micropore device is the primary component in microfluidics, filtration, and biomedical applications, where precise control over fluid flow is essential. The orifice serves as the main entry or exit point for the RBC cells, allowing controlled passage based on pressure differentials and fluid properties. The micropore channels as capillary network, characterized by its minute dimensions, enhances the regulation of flow due to capillary action, surface tension, and pressure-driven mechanisms. The micropore orifice comprises an ‘omega-shaped’ (Q) heating coil which shapes the constricted micropore within a borosilicate capillary, wherein the opening of the orifice is flame polished in order to make entry of cells smooth, and the micropore orifice is made by specially designed tool.

[0058]

[0049] The micropore device operates based on the principles of fluid dynamics and microfluidics. The orifice determines the initial flow characteristics, while the capillary channels influence the rate and distribution of the flow through capillary action and pressure differences. The interplay of forces such as viscous drag, surface tension, and pressure gradients determines the overall efficiency of the micropore device.

[0059]

[0050] In one embodiment, a constant electric voltage is applied on the micropore device in order to ensure constant baseline current flow across micropore channel of the micropore device. Such application of electric voltage induces the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump (124).

[0060]

[0051] The syringe pump (124) is a device designed to deliver precise and controlled amounts of fluids containing RBC cells at a set rate. When coupled with a micropore device consisting of an orifice and micropore channels, it enhances fluid regulation at the microscale, enabling accurate manipulation of liquid flow through capillary action and pressure differentials. A motor-driven system pushes the plunger of a syringe, controlling the flow of liquid with high precision. The flow rate can be adjusted to match the requirements of the micropore device.

[0061]

[0052] The present invention works on the principle of the Resistive pulse technique. A constant baseline current flows across the micropore channel when a constant voltage is applied across the device. As the cells are forced through the micropore, the cells block some conducting ions in the sensing region of the device, and a momentarily decrease in the conductance is observed in the form of the electrical pulse (See Figure 2b). The width of these electrical pulses attributes to the time spent by the cells inside the sensing region of the micropore, i.e. stiffer the cell more is the width of the pulse, which we refer to as dwell time (At). A series of such electrical pulses are recorded and the dwell time histogram gives us the statistical information of the cellular stiffness.

[0062]

[0053] In another embodiment, the amplifier (118) is configured to apply voltage across the micropore assembly (116). Applying a voltage across the micropore assembly, by means of the amplifier (118) drive electrophoretic transport, regulate ion flow, or induce electroosmotic effects. The amplifier (118) provides stable and precise voltage control, ensuring minimal fluctuations, and further amplifies low-voltage signals to higher, well- regulated outputs. The amplifier enables low-noise operation for sensitive measurements, such as nanopore sensing or microfluidic applications.

[0063]

[0054] The noise eliminator (120) is configured to eliminate line frequency interferences in the electrical signals. Such interference can significantly affect precision measurements, especially in applications such as nanopore sensing, electrophysiology, and electrochemical detection. The noise eliminator (120) suppresses AC mains hum (50 / 60 Hz) that can corrupt low-level signals. The noise eliminator (120) enhances signal clarity and accuracy in micropore-based measurements.

[0064]

[0055] The electro-fluidic computation unit (102) comprises at least a processing unit (104) and a memory (106) coupled with the processing unit (104). The processor (104) may be for example, a specialized processing unit, a micro processing unit, a typical graphics processing unit (GPU) or any other processing unit capable of processing the information regarding physiological changes of cells and determining cellular properties of Red Blood Cells (RBCs). The electro-fluidic computation unit (102) further comprises a data acquisition module (108), a data analysis module (110), and a cellular properties determination module (112).

[0065]

[0056] The data acquisition module (108) is configured to receive information related to plurality of AMF based measurements including specific physical, mechanical, and electrical information of cells etc. from a plurality of data sources. In an example, such information includes but not limited to fluid flow rate, applied voltage, fluid viscosity and temperature as well as geometrical parameters of the cell in context. In one embodiment, the data acquisition module stores the received information in the data repository (114). In another embodiment, the data acquisition module (108) is further configured to stores the information generated from the real time execution of the present system so that such data can aid in estimation of the morphological parameters of cells more accurately.

[0066]

[0057] The data analysis module (110) retrieves the historical information related to physical, mechanical, and electrical information of cells and computes dwell time, conductance variation for each of the historical instances. Thereafter, the data analysis module (110) further determines a correlation between such dwell time and conductance variation, and morphological properties of cells. In an example, the dwell time is higher in case of higher stiffness of the cells in context, and the dwell time is also dependent on fluid flow rate, applied voltage, fluid viscosity, sample temperature, and cell volume as well as geometrical parameters including length and diameter of the micropore. In another example, longer dwell times indicate stronger interactions or slower translocation due to size, shape, or adhesion properties, wherein shorter dwell times suggest rapid translocation or weaker interactions. In another embodiment, the data processing module (110) stores such correlation information in the data repository (114).

[0067]

[0058] The cellular properties determination module (112) applies a constant electric voltage by means of the amplifier in order to ensure constant baseline current flow across micropore channel of the micropore device. The cellular properties determination module (112) further induces the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump.

[0068]

[0059] In one embodiment, the cellular properties determination module (112) determines change in conductance in the form of change in the detected electrical pulses and stores series of the detected electrical pulses in a data repository (114). The cellular properties determination module (112) analyzes a dwell time and a conductance drop by correlating the stored electrical pulses with respect to physiological changes of the cells in context, wherein such correlation information is retrieved from the data repository (114) as stored by the data analysis module (110). The time period of stay of the cell in the orifice is defined as dwell time, and momentary decrease in conductance in the form of electrical pulse is considered as conductance drop. The change in the conductance of the system is caused when a cell translocates through and blocks conducting ions. Thus, the cellular properties determination module (112) estimates the cellular properties based on change in electrical pulses during passage of the cells through the orifice, and determines cellular mechanical or morphological properties.

[0069]

[0060] In one embodiment, the system is configured to determine cellular properties of Red Blood Cells for prognosis of sickle cells anemia. The system is configured to determine stiffness of Red Blood Cells.

[0070]

[0061] As illustrated in Figure 2A, the method (200) comprises one or more blocks implemented by the system (100) while in operation. The order in which the method (200) is described is not intended to be construed as a limitation, and any number of the described method blocks can be combined in any order to implement the method. Additionally, individual blocks may be deleted from the methods without departing from the spirit and scope of the subject matter described herein. Furthermore, the method can be implemented in any suitable hardware, software, firmware, or combination thereof.

[0071]

[0062] At block (202), a constant electric voltage is applied across a micropore device. In one embodiment, the processing unit is configured to apply, a constant electric voltage across a micropore device by means of the amplifier in order to ensure constant baseline current flow through the micropore channel of the micropore device. The micropore device comprises an orifice and a set of capillaries as micropore channels.

[0072]

[0063] At block (204), the constant voltage is induced across a plurality of cells of a material in context. In one embodiment, the processing unit is configured to induce the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump. The cells are considered to be in room temperature and having a defined cell volume.

[0073] [1] At block (206), the cellular properties are estimated based on change in electrical pulses. In one embodiment, the processing unit estimates the cellular properties based on change in electrical pulses during passage of the cells through the orifice, and determines cellular mechanical or morphological properties.

[0074] [2] As illustrated in Figure 2B, the method (206) comprises one or more blocks implemented by the system (100) while in operation for estimating the cellular properties. The order in which the method (300) is described is not intended to be construed as a limitation, and any number of the described method blocks can be combined in any order to implement the method.

[0075] [3] At block (252), change in conductance is determined. In one embodiment, the processing unit determines the change in conductance in the form of change in the detected electrical pulses and stores series of the detected electrical pulses in the data repository (H4).

[0076] [4] At block (254), a dwell time and a conductance drop are analyzed. In one embodiment, the processing unit analyzes a dwell time and a conductance drop by correlating the stored electrical pulses with respect to physiological changes of the cells in context. The time period of stay of the cell in the orifice is defined as dwell time, and momentary decrease in conductance in the form of electrical pulse is considered as conductance drop.

[0077] [5] In one embodiment, cellular properties of Red Blood Cells are determined for studying the stiffness of the cells. Such cellular properties of Red Blood Cells are determined for prognosis of Sickle Cell Anemia.

[0078]

[0064] As illustrated in Figure 3, the method (300) comprises one or more blocks implemented while fabrication of a micropore device. The order in which the method (300) is described is not intended to be construed as a limitation, and any number of the described method blocks can be combined in any order to implement the method. Furthermore, the method can be implemented in any suitable hardware, software, firmware, or combination thereof.

[0079]

[0065] At block (302), a borosilicate capillary is cleaned by ultrasonication. In one embodiment, a borosilicate capillary is cleaned by ultrasonication in ethanol, acetone, and then ethanol for 2 mins - 5 mins each.

[0080]

[0066] At block (304), a micropipette puller is configured with presetting of parameters. In one embodiment, a micropipette puller is configured with presetting of parameters including heat, filament, velocity, delay, and pull to pull the cleaned capillary.

[0081]

[0067] . t block (306), a omega- shaped (f ) filament is mounted on a flame polisher instrument. In one embodiment, a custom designed ‘omega-shaped’ (Q) filament is mounted on a flame polisher instrument to shape the pulled capillary to a desired micropore dimensions that is lesser than the cell size for obtaining a long-constricted micropore. The mouth of the micropore is also flame polished to aid in smooth entry of the cells.

[0082]

[0068] At block (308), a set of capillaries are engaged as micropore channel. In one embodiment, a set of capillaries are engaged as micropore channel around the micropore for enabling application of constant electric voltage across the cells.

[0083]

[0069] Figure 4A illustrates graphical representation of detection of electrical signal when a cell translocates through the constricted micropore showing the dwell time (At) and conductance drop (AG), wherein Figure 4B illustrates schematic version of the detection region of the micropore, showing the applied voltage across the electrodes, and soft (Red) and stiff (Blue) cells. Further, Figure 4C illustrates graphical representation of detection of electrical signals when a stiff (blue) and soft (red) cells are detected through the constricted micropore device, and Figure 4D illustrates an exemplary mapping of electro-fluidic measurements with the elasticity of the cells as measured with the AFM control.

[0084]

[0070] Figure 5(a) illustrates baseline conductance of an open micropore. Figure 5(b) illustrates electrical signals showing the translocation of cells through the constricted micropore. Inset shows the zoom version of a single cell detection electrical pulse. Figure 5(c) illustrates dwell time (At) histogram of the series of electrical pulses recorded.

[0085]

[0071] Furthermore, Figure 6 illustrates the normalized data from the control AFM experiment is plotted alongside with the data from 5 different micropore devices showing the overlapping all the trends when the RBCs were treated with different concentrations of Latrunculin-A drug mimicking the cancerous state of cells. Experimental Details:

[0086]

[0072] In an experiment, borosilicate capillaries having outer diameter of 1 mm, inner diameter of 0.5 mm, and length of 75 mm were used to fabricate micropores. Two types of devices were fabricated in this work: first type of devices with large micropores (pore diameter (DP) > cell size) for free-flight experiments and the second type with small micropores (pore diameter < cells size) for constricted-flight experiments. The glass capillaries were cleaned by ultrasonication in ethanol, acetone, and then ethanol for 2 mins each. Thereafter, a micropipette puller (Model P - 2000, Sutter Instrument) is used with the parameters as Heat: 350, Filament: 0, Velocity: 25, Delay: 150, Pull: 200 to pull the cleaned capillaries. For a free-flight micropore a conventional ‘V-shaped’ filament is mounted on a flame polisher instrument (MF - 900, Micro Forge, Narshige), to heat-shrink the pulled capillaries further to a desired dimension (DP> cell size). For devices with a long-constricted micropore, an ‘omega-shaped’ (Q) filament was mounted on the flame polisher instrument to shrink the pulled capillaries to our desired micropore dimensions (DP< cell size). Then the omega-shaped (Q) filament is replaced with the conventional ‘V-shaped’ filament to cut the glass capillary's extra front region and, flame polish the cut region. Figure 7 illustrates the images of the two types of filaments and the steps involved in fabricating these micropores. Figure 7 illustrates steps involved in forging a micropore from a glass capillary. Figure 7(a) is the image of the glass capillary pulled using the Shutter Puller instrument is shown here. Figure 7(b - g) are images of a ‘V’ shaped filament and the steps involved in forging a free- flight micropore are shown. Figure 7(h - n) are images of an omega-shaped (Q) filament and the steps involved in forging a constricted micropore are shown. After flame polishing, it was also ensured that the micropores remained circularly symmetric before mounting them in a glass-bottom Teflon fluid chamber (-250 pL) using curable silicone glue. The pore diameter (DP) was estimated from the smallest opening in the side-view optical image of the pore cross-section. The micropore was then connected to a syringe pump, using a PTFE tubing to generate stable fluid flow.

[0087]

[0073] The blood samples for the study were acquired from human volunteers and sickle cell disease (SCD) patients with approval from institutional ethics committee. For experiments, about 10 pL of the whole blood (from a finger prick) was diluted by adding 500 pL of RPMI-1640 (RPMI (Roswell Park Memorial Institute) buffer, Sigma #SLBT0197) (pH = 7.4). The solution was then centrifuged three times at 600 ref for 3 min at 4°C, and the RBCs were isolated from blood plasma and other cells, followed by resuspension of the RBC pellets in 1500 pL of RPMI (Sample stock concentration). After the isolation step for every experiment, RBCs were checked for health and debris under an optical microscope.

[0088]

[0074] To measure drug-dependent changes in cell stiffness, aliquots of the actin depolymerizing drug Latrunculin A (Lat-A) (Cat# L5163, Sigma) were made in DMSO buffer to 1 mM stock concentration. An appropriate concentration of this aliquot was then used to treat the RBCs for different experiments. The cells are incubated in Lat-A for at least 5 minutes at room temperature before performing the experiments.

[0089]

[0075] For Atomic Force Microscope (AFM) based force spectroscopy experiments on RBC cells, a 100 pL circular fluid cell is made using silicone glue on a glass slide as illustrated in Figure 8. This glass slide was cleaned by ultra-sonication in 20% Extran (Part# 34022090- 5L Merck), ultrapure water twice (Milli-Q, Millipore), acetone, and ethanol for 5 min each. The glass slide was finally rinsed with ultrapure water to make sure there is no leftover ethanol from the last cleaning step. Then Nitrogen is used to dry the fluid cell completely and immediately added 50 pL drop of 0.001% PLL for 30 min incubation on the glass surface. Thereafter, the PLL is removed using vacuum line and immediately added 50 pL of RBC sample stock and incubated it for 30 min. The fluid cell was then gently washed with 3-5 mL of fresh RPMI buffer to make sure that all the floating cells were removed. After all these steps, the fluid cell is checked for RBC adherence under an optical microscope and then force spectroscopy measurements are performed using the AFM. Since the RBC stiffness was found to be dependent on the storage temperature and storage duration as illustrated in Figure 9. All experiments mentioned in this work were performed within 150 minutes of sample preparation.

[0090]

[0076] Briefly, microcapillary with the micropore at its tip was filled with RPMI buffer and glued on the fluid chamber. A PTFE tubing was connected at the back of the capillary to a syringe pump. RPMI buffer was filled in the glass bottom fluid chamber, micro-capillary, and microfluidic PTFE tubing avoiding air bubbles everywhere. The RPMI buffer works as the electrolyte for ionic current measurements through our device. A pair of Ag / AgCl electrodes, on either side of the micropore, were used to acquire the current signals, one dipped in the teflon fluid chamber and the other inserted in the tubing. A syringe pump (in withdraw mode) was used to pull samples into the micropore device maintaining a constant fluid flow (500 nL / min). The device was kept inside an aluminum Faraday cage to reduce electrical noise as illustrated in Figure 1A. Low noise amplifier AM Systems (Model 2400), with 40 kHz bandwidth, and Dagan Chem-Clamp with 10 kHz bandwidth are used in this experiment, to apply a voltage across the devices and record the open pore current and translocation events. A noise eliminator (Hum-Bug, Quesst Scientific) was also used to eliminate the input source line frequencies in the signal. Data acquisition cards from National Instruments were used to acquire and store the signals using a custom-written Lab VIEW code. All the experiments performed in the lab and the hospital, used NI PCIe- 6363 and NI myDAQ data acquisition cards respectively, with data sampling done at 100 kHz. The recorded data was then low pass filtered at 10 kHz and analyzed using an offline custom-written Lab VIEW code. A representative quantification of an event showing the drop-in conductance (AG) and the dwell time (At) of a recorded electrical event is shown in Figure 10(c). The AG values in free-flight micropores are used to quantify any physiological changes in RBCs as shown in the schematic Figure 10(d). In contrast, the At values from the constricted micropores indicate a change in the cellular stiffness as represented in Figure 10(e). A more detailed quantitative estimation of the cellular stiffness is later in the text. The constricted-flight and free-flight experiments were performed on patient RBC samples simultaneously to avoid any aging effects. The equivalence of the two separate amplifiers used in this study was independently confirmed as illustrated in Figure 11. All datasets shown in this experiment are measured in triplicate on same pore and same sample and their mean and error of mean values of AG and At histograms is plotted.

[0091]

[0077] For experiments with controlled changes in RBC cell stiffness, the cells were incubated in RPMI buffer with appropriate concentration of the Latrunculin-A (Lat-A) drug. All stiffness comparison experiments were performed on the same device. The sample fluid chamber was washed thoroughly with 3 - 5 mL of filtered RPMI in between experiments with different drug concentrations ensuring no RBCs were left from the previous experiment.

[0092]

[0078] An MFP-3D Infinity Bio (Asylum Research AFM, Oxford Instruments) AFM system mounted on an 1X73 inverted Olympus microscope is used for cell stiffness measurements of RBCs. Contact mode silicon nitride cantilevers attached with a spherical polystyrene bead of 4.5 pm diameter (PT.PS.SN.4.5, Novascan) were used for taking F-X curves in this study. RBCs were adhered on the glass substrate fluid cells as briefed previously and the AFM tip was optically centered on the RBC for every measurement. The RPMI buffer in the fluid cell was then gently washed with 1 mL (20 steps of 50 pL each) RPMI with the appropriate Lat- A concentration needed for the study. This step was repeated for all the different Lat-A concentrations. It is important to note that another camera with a 60X objective was used to visually ensure the same cell was probed for all the Lat-A concentrations as illustrated in Figure 12. A schematic of a spherical bead attached to a cantilever probing a soft sample is shown in Figure 13 along with the cantilever’s piezo position (z), deflection of the cantilever (d), and the indentation into the sample (x). The cantilever had a resonant frequency of 15 kHz, length of 225 pm, width of 25 pm, and spring constant of 0.03 N / m. Each cantilever was calibrated for the spring constant (k, pN / nm) and sensitivity (P, nm / V) using the thermal fluctuation mode of the AFM and taking an F-X curve on the glass surface in RPMI, respectively. On each cell, at least 10 F-X curves were recorded with a dwell time of 2 seconds between consequent curves. The data was recorded at a sample rate of 10 kHz and low pass filtered at 5 kHz, with a total vertical travel distance of 500 nm. During the approach curve, the trigger in deflection value to stop and retrace was set at 100 nm. For F- X curve analysis and stiffness estimation, a custom-written MATLAB code was used to find the cantilever-sample contact point from the force-distance curve, a representative schematic of the contact point is shown in Figure 13. To ensure that our AFM experiments are well within the hertz model limit and to achieve reproducible fits, an indentation depth of 50 nm was fixed for all curve fits.

[0093]

[0079] As a result, it is first shown that the two modes (free-flight and constricted-flight) of micropore experiments which detect changes in cell volume and whole-cell stiffness, respectively. Figure 14(a) shows electrical events corresponding to free-flight translocation of model cells (spherical polystyrene beads) of diameter 6.0 (blue) and 4.3 pm (red), through a micropore of 7.0 pm diameter. While the dwell time (At) corresponds to duration of translocation, the event depth (AGff) of these events correspond to the volume of the translocating model cells. The translocation events, scatter plot (AGff vs At) of events of a population of model cells and the AGff histograms are shown in Figures 14(a), 14(b), and 14(c) respectively. It is observed that model cells of different cell volumes correspond to distinct AGff histograms as illustrated in Figure 10(d), 14(a), and 14(c). In constricted-flight experiments, red blood cells (typical diameter (~ 8 pm) are translocated through a micropore of smaller diameter, using a syringe pump. In Figure 14(d) we show concatenated translocation events corresponding to native (blue) and soft (red, LatA treated) RBCs, translocating through a 3.6 pm micropore. The changes in event depths (AGCf) and event duration (At) is shown as a scatter plot in Figure 14(e). We see that in constricted-flight experiments, the event duration (At) corresponds to whole cell stiffness that allows the cells to squeeze through the constriction. Cells of different stiffness, as shown in Figure 14(f), are identified with their distinct At histograms.

[0094]

[0080] Next, constricted-flight measurement of RBCs is shown that are softened to varying degree using the Lat-A drug. Latrunculin A is known to soften cells, in a concentration dependent manner, by binding to actin monomers near the nucleotide-binding cleft and thus sequestering them from actin polymerization in cells. This actin-spectrin scaffolding inside the inner membrane of RBCs gets affected due to the depolymerization of actin, which results in the softening of the entire cell. Red blood cells from a healthy donor were incubated in different concentrations of Lat-A and the effect of the drug on RBC sample was measured with constricted-flight experiments using micropores. Note, to avoid devicedependent artifacts, experiments with all Lat-A concentrations were performed, back-to- back, on the same device, with thorough washing of fluid-cell between experiments and randomizing the order of Lat-A concentrations. The translocation events on a 3.6 pm diameter constricted micropore are shown in Figure 15(a). It shows that the effect of reduced whole cell stiffness by incubation in Lat-A, translates to longer translocation times (At). Since Lat-A treated RBCs show no change in cell size / volume as illustrated in Figure 16, the increase in constricted-flight translocation times is attributed to reduction in cell stiffness. This result is confirmed with constricted flight experiments done on multiple devices. The constricted flight At data for RBCs treated with five different Lat-A concentrations and measured using 3.0, 3.2, 3.6, 3.8, and 3.9 pm diameter micropores is shown in Figure 15(b). A systematic decrease is observed in the At with the increasing Lat- A concentrations.

[0095]

[0081] However, to compare the cell stiffness changes in the cell population being measured using the micropore, we directly quantify the change in their elastic modulus using an atomic force microscope (AFM) mounted on an inverted optical microscope. The AFM cantilever with a microsphere attached to the tip was optically aligned on top of the red blood cell and force-indentation curves were obtained by pressing the microsphere on the AFM tip into the cell as illustrated in Figure 13. From the force indentation curves as illustrated in, the Figure 15(c)elastic modulus of RBCs was estimated using the Hertz Model given by:

[0082] Here, F, Rc, E, u, and 6 are force, the radius of the spherical probe, the elastic modulus of the sample, Poisson’s ratio of RBC and indentation respectively. In Figure 15(c), the force-indentation curves with the respective Hertz model fits for untreated RBC (black) and then same cell treated with 0.50 pM (green) and 1.0 pM (red) Lat-A drug, successively, are shown. The experiment is repeated for all Lat-A concentrations and the resulting changes in the elastic modulus of that same red blood cell is plotted in Figure 15(d) inset. These experiments are repeated on multiple cells. The Lat-A concentration-wise average (overall measured cells) of the AFM measurement of cell elasticity is plotted in Figure 15(d). As expected, AFM measurements show systematic decrease in elastic modulus (E, kPa) with increasing Lat-A concentrations.

[0096]

[0083] Next, qualitative dependence of dwell times (At, ms) of constricted-flight micropore experiments of RBCs with AFM-based elastic modulus (E, kPa) measurements is established. It is noted that both the micropore At measurements as illustrated in Figure 15(b), as well as their elastic modulus as illustrated in Figure 15(d), decrease similarly with different concentrations of Lat-A. This direct dependence is plotted in Figure 17(a). It is found, empirically, linear dependence of constricted-flight translocation time, At, on the elastic modulus, E, of the translocating cell. This dependence is confirmed for multiple devices with different micropore diameters (3.0, 3.2, 3.6, 3.8, and 3.9 pm) as illustrated in Figure 17(a) inset). The values of the linear fits are summarized in Table 1, with an average slope of 1.15 ± 0.12.

[0097] Table 1

[0098]

[0084] This suggests that the RBCs elastic modulus (E) and the constricted-flight micropore dwell times are directly proportional: At oc E (2)

[0099]

[0085] The constricted-flight dwell times may depend on the flow rate (Q, nL / min) (as illustrated in Figure 18 and Figure 19). Figure 18(a) illustrates constricted At for RBCs treated with different concentrations of RBCs translocating through a 3.8 pm micropore device at 500 (blue), 750 (black), 1000 (green), 1250 (cyan), and 1500 (red) nL / min constant fluid flow is shown here. Figure 18(b) represents the normalized At values for different flow rates. The overlapping values of the normalized data imply that although the At values are decreasing with an increase in the fluid flow rates, but the effective stiffness changes inherited due the different concentrations of Lat-A remains same at all flow rates. Figure 18(c) illustrates the linear relationship between the elasticity of RBCs treated with different Lat-A concentrations with the constricted At measurements. Figure 18(d) shows the slope of the linear relationship between elasticity and At for different constant fluid flow values. In Figure 19(a), 19(b), 19(c), 19(d), and 19(e) the histogram of constricted At measured for RBCs treated with different concentration of Lat-A translocating through a 3.8 pm micropore decice with 500 (blue), 750 (black), 1000 (green), 1250 (cyan) and 1500 (red) nL / min constant fluid flows are shown. Note that all the histograms are Gaussian fitted. It is found that the linear dependence of At on the elastic modulus (E) is maintained across multiple Q values as illustrated in Figure 17(b). The dwell time for constricted-flight experiments may also depend on the cell size / volume. Although Lat-A doesn’t change the cell volume as illustrated in Figure 16, this dependence should be considered when RBC from different donors (see below) is compared. To establish this dependence, red blood cells are acquired from different healthy donors, with different mean cell volumes (as measured from pathology reports) and compared their free-flight AGff (corresponds to cell volume) values to their constricted-flight At values. 4.3 and 8.0 pm micropore devices are used for constricted and free flight experiments respectively and the measured data is shown in Figure 17(c). The dotted red line is a power-law fit giving the following relationship:

[0100] At oc (AGFF)2'37(3)

[0101]

[0086] Here, since all RBC samples are from different healthy donors, we assume they differ only in their cell volume. The constricted-flight dwell times, At, also depend on the experimental parameters (such as fluid flow rate (Q), applied voltage (Vm), fluid viscosity (p) and temperature (T)) as well as geometrical parameters (Length (Lp) and Diameter (Dp) of the micropore).

[0102]

[0087] On combining all the parameters discussed above, an empirical relation is determined

[0103]

[0088] The expression for At can now be written as:

[0104]

[0089] Here, k is the proportionality constant. Note, that experiments that compare control and sample cells are performed using the same micropore (i.e. same LPand DP), a constant fluid flow (i.e. same Q and p), and experimental and sample incubation temperature (T). This allows all the terms in f Lp, Dp, Vm, Q, iJ., T^ to be absorbed into the proportionality constant k in equation (5). Now, the change in the elastic modulus of unknown samples can be directly measured, relative to the elastic modulus of a control sample:

[0105] 2.37

[0106] (6)

[0107]

[0090] Here, Ereiativeis the ratio of the elasticities of the sample and the control cells, whereas At and GFFare the constricted-flight dwell times and free-flight conductance change values respectively. The above empirical equation is used to estimate the relative change in RBC cell stiffness upon Lat-A treatment. The Ereiative estimated for Lat-A treated RBC cells is shown in Figure 17(d). In the same plot, the change in elastic modulus (measured relative to the native RBC sample) as measured by the AFM for RBCs treated with the same concentrations of Lat-A is also shown. The excellent agreement of our micropore empirical approach with the AFM data shown in Figure 17(d) provides strength to the experimental procedure to estimate changes in the elastic modulus of a sample relative to a control.

[0108]

[0091] Finally, the micropore device is applied and analysis to compare translocation experiments is performed on RBC samples drawn from a healthy donor and sickle cell disease patients, at the hospital. The two samples, when measured using the same device, showed a clear distinction that the SCD patient RBCs translocated much slowly than the healthy RBCs. This indicates that the SCD RBCs are stiffer than the native healthy RBC cells. Representative plots of the raw electrical translocation signals, the AGCf vs At scatter plots, and the At histograms (as illustrated in Figures 20(a), 20(b), 20(c) respectively), show a clear distinction between the RBC samples from a healthy donor (blue) and an SCD patient (red) when detected using a constricted micropore device (4.6 pm). RBC stiffness is measured for 10 SCD patients. For these experiments, the samples were collected from patients and a healthy donor and the respective RBCs were suspended in RPMI buffer. This was to ensure that every SCD patient’s RBC data had in-situ control of the healthy donor RBCs on the same device and experimental conditions. The RBCs were imaged, as illustrated in Figure 21 and split into two batches for simultaneous free-flight (to compare cell volumes) and constricted-flight (to compare cell stiffness) experiments. A complete summary of all the micropore experiments performed in the hospital and relevant pathology data from patients and the healthy donor is provided in Figure 22. Figure 22 shows the summary of electrofluidic (both free flight and constricted) and pathology (HPLC) data collected for healthy donor and SCD patients used in this experiment. It is noted that, in general, RBCs from SCD patients have a larger distribution in cell volume (as measured by AGff) and take a longer time to translocate through the constricted geometry of the micropore (as measured by constricted-flight dwell times, At). This is shown in Figure 20(d). Figure 20(a), 20(b) and 20(c) show translocation electrical signals, AG vs At scatter plot, and the At histograms for RBCs from a healthy donor (blue) and an SCD patient (red) recorded using a 4.6 pm micropore device respectively. Figure 20(d) represents scatter plot of Normalized At and AG from Constricted and free flight experiments respectively, showing distinct signals for SCD patients (red circles), Healthy donors (blue squares), and Control donors (black triangles). Figure 20(e) shows the bar plot of relative elasticity empirically estimated according to equation 6 respectively. Figure 20(f) shows the relative elasticity of the SCD patient’s samples plotted with their respective HbS % values obtained from the HPLC test. Inset shows an increasing trend of relative elasticity for SCD patients with higher HbS% values. Here, the scatter plot of normalized constricted-flight At and AGff is showing distinct populations for SCD patients (red dotted region) and healthy donors (blue dotted region). The data shown in Figure 20(d) is normalized with a single control (healthy) donor (black triangle). The summary of the constricted-flight At values and the normalized At estimated for each patient with respect to the control is shown in Figure 23. Figure 23(a) and 23(b) show the bar plot of the At values and normalized At values acquired in the constricted micropore translocation experiments performed on samples acquired from healthy donors and SCD patients normalized with respect to the healthy donor’s data respectively. The relative elastic modulus (Ereiative) of patient RBCs is estimated using equation (6) and the data is shown in Table 2 and Figure 20(e).

[0109] Table 2

[0110]

[0092] The Ereiative plot demonstrates that the SCD patient RBCs are, in general, always stiffer compared to the healthy donor RBCs by a factor of 2 - 4 and the proposed electro-fluidic device detects the SCD patient RBCs with high signal-to-noise ratio and throughput. Since HbS% values of the SCD patients are available from the HPLC -based pathology tests, the relative elastic modulus of the RBCs of all the SCD patients of this experiment is compared with their HbS % values in Figure 5f. It is noted that other than two data points (possibly belonging to Sickle Cell Trait patients, HbAS trait) the inset of Figure 20(f) shows a linear relationship of Ereiativewith the HbS% content in the cell. It is also noted that given the low patient number of this study, this linear relationship is only empirical.

[0111] Advantages of the invention

[0112]

[0093] The technique of the invention is label-free. Therefore, the suspension samples can be in their physiologically relevant environment for the measurements. No dyes or surface coatings or adhesion are required for sample preparation or for measurement.

[0113]

[0094] The signals used for the purpose of the invention are only electro-fluidic and independent of microscopic imaging. However, the system and devices of the present invention are compatible to be complemented with imaging and visual inspection of samples.

[0095] The voltage signals of the present invention are smaller in data storage space, faster and efficient in analysis.

[0114]

[0096] Finally, the language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based here on. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention, which is set forth in the following claims.

[0115]

[0097] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0116]

[0098] While various aspects and embodiments have been disclosed herein, other aspects and embodiment will be apparent to those skilled in the art.

[0117]

[0099] In the detailed description of the embodiments of the disclosure, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration specific embodiments in which the disclosure may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosure, and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the present disclosure. The description is, therefore, not to be taken in a limiting sense.

Claims

We Claim:1) An electro-fluidic system (100) for determining cellular properties of Red Blood Cells (RBCs), the system comprises: a micropore assembly (116) comprising a micropore device (122) and a syringe pump (124) for applying force, wherein the micropore device (122) comprises an orifice and a set of capillaries acting as micropore channels; an amplifier (118) for applying voltage across the micropore assembly; a processing unit (104) communicably coupled with the amplifier (118), the micropore assembly (116), and a memory (106), wherein the processor (104) is configured to: apply a constant electric voltage in order to ensure constant baseline current flow across micropore channel of the micropore device; induce the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump; and estimate the cellular properties based on change in electrical pulses during passage of the cells through the orifice, and determine cellular mechanical or morphological properties.2) The system (100) as claimed in claim 1, wherein the processor (104) is configured to estimate the cellular properties by: determining change in conductance in the form of change in the detected electrical pulses; storing series of the detected electrical pulses in a data repository (114); and analyzing a dwell time and a conductance drop by correlating the stored electrical pulses with respect to physiological changes of the cells in context, wherein time period of stay of the cell in the orifice is defined as dwell time, and momentary decrease in conductance in the form of electrical pulse is considered as conductance drop.3) The system (100) as claimed in claim 1, wherein shape and size of the orifice of the micropore device (122) is designed to be narrower with respect to diameter of cell in context.4) The system (100) as claimed in claim 1, wherein the system further comprises a noise eliminator (120) for eliminating line frequencies in the electrical signals.5) The system (100) as claimed in claim 2, wherein the dwell time is higher in case of higher stiffness of the cells in context, and the dwell time is also dependent on fluid flow rate, applied voltage, fluid viscosity, sample temperature, and cell volume as well as geometrical parameters including length and diameter of the micropore.6) The system (100) as claimed in claim 2, wherein change in the conductance of the system is caused when a cell translocates through and blocks conducting ions.7) The system (100) as claimed in claim 1, wherein the system is configured to determine cellular properties of Red Blood Cells for prognosis of sickle cells anemia.8) The system as claimed in claim 1, wherein the system is configured to determine stiffness of Red Blood Cells.9) A method (200) of determining cellular properties of Red Blood Cells (RBCs) said method comprising the steps of: applying (202), by a processing unit, a constant electric voltage across a micropore device in order to ensure constant baseline current flow through the micropore channel of the micropore device, wherein the micropore device comprises an orifice and a set of capillaries as micropore channels; inducing (204), by the processing unit, the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of a syringe pump, wherein the cells are considered to be in room temperature and having a defined cell volume; and estimating (206), by the processing unit, the cellular properties based on change in electrical pulses during passage of the cells through the orifice, and determine cellular mechanical or morphological properties.10) The method (200) as claimed in claim 9, wherein the cellular properties are estimated by the processing unit by the steps of: determining (252) change in conductance in the form of change in the detected electrical pulses; storing series of the detected electrical pulses in a data repository (114); andanalyzing (254) a dwell time and a conductance drop by correlating the stored electrical pulses with respect to physiological changes of the cells in context, wherein time period of stay of the cell in the orifice is defined as dwell time, and momentary decrease in conductance in the form of electrical pulse is considered as conductance drop.11) The method (200) as claimed in claim 9, wherein cellular properties of Red Blood Cells are determined for studying the stiffness of the cells.12) The method (200) as claimed in claim 9, wherein cellular properties of Red Blood Cells are determined for prognosis of Sickle Cell Anemia.13) A micropore assembly for enabling determination of cellular properties, the assembly comprises: a micropore device comprising an orifice and a set of capillaries as micropore channels; and a syringe pump for applying force to cells in context for which mechanical or morphological properties are to be determined.14) The micropore assembly as claimed in claim 13, wherein a constant electric voltage is applied on the micropore device in order to ensure constant baseline current flow across micropore channel of the micropore device, wherein such application of electric voltage induces the constant voltage across a plurality of cells of a material in context, upon probing the cells by forcing them through the orifice by means of the syringe pump.15) The micropore assembly as claimed in claim 13, wherein an amplifier is configured to apply voltage across the micropore assembly.16) A method (300) of fabrication of a micropore device, the method comprises steps of: cleaning (302) a borosilicate capillary by ultrasonication in ethanol, acetone, and then ethanol for 2 mins - 5 mins each; configuring (304) a micropipette puller with presetting of parameters including heat, filament, velocity, delay, and pull to pull the cleaned capillary; mounting (306) a custom designed ‘omega-shaped’ (Q) filament on a flame polisher instrument to shape the pulled capillary to a desired micropore dimensionsthat is lesser than the cell size for obtaining a long-constricted micropore, wherein the mouth of the micropore is also flame polished to aid in smooth entry of the cells; and engaging (308) a set of capillaries as micropore channel around the micropore for enabling application of constant electric voltage across the cells.17) The method (300) as claimed in claim 16, wherein the borosilicate capillaries is having a dimension of outer diameter as 1 mm, inner diameter as 0.5 mm, and length of 75 mm.18) A micropore orifice comprising of an ‘omega-shaped’ (Q) heating coil which shapes the constricted micropore within a borosilicate capillary, wherein the opening of the orifice is flame polished in order to make entry of cells smooth, and the micropore orifice is made by specially designed tool.

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