WO2024116093A1

WO2024116093A1 - Hydroelectric macrodevice for 3-dimensional analysis of electrophoretic and dielectrophoretic behavior of cancer cells

Info

Publication number: WO2024116093A1
Application number: PCT/IB2023/062027
Authority: WO
Inventors: Mohammad ABDOLAHAD; Shima MOHARAMIPOUR; Pooya FARANOOSH; Mohammadreza FOROUGHI GILVAEE
Original assignee: Abdolahad Mohammad; Moharamipour Shima
Priority date: 2022-11-30
Filing date: 2023-11-29
Publication date: 2024-06-06

Abstract

Disclosed herein is a three-dimensional macrodevice for cancer cells detection based on electrophoretic and dielectrophoretic behavior of cells. The three-dimensional macrodevice includes a fluid container with macroscale dimensions and a pair of electrodes. The pair of electrodes includes two electrically conductive transparent electrodes at the top and bottom of the fluid container. A sample including a plurality of cells is placed inside the fluid container. The pair of electrodes defines a visible path through a height of the fluid container configured to optically being monitored by capturing images/videos from motion of the plurality of cells alongside. A presence of cancer cells in the plurality of cells is detectable based on a settling speed of a first cell of the plurality of cells on the bottom electrode due to an AC/DC generated electric field between the pair of electrodes using an electrical device connected to the pair of electrodes.

Description

HYDROELECTRIC MACRODEVICE FOR 3-DIMENSIONAL ANALYSIS OF ELECTROPHORETIC AND DIELECTROPHORETIC BEHAVIOR OF CANCER CELLS

TECHNICAL FIELD

[0001] The present disclosure relates to systems, devices, and methods for diagnosis of cancer, and particularly, to a hydro-electric three-dimensional macrodevice for cancer diagnosis and detection of invasion grade of cancer cells based on analysis of electrophoretic and dielectrophoretic behavior of cancer cells. More particularly, the present disclosure relates to a system including an exemplary macrodevice and a method utilizing thereof for detection of cancer cells and their invasion grade.

BACKGROUND ART

[0002] Various cell biological parameters, including biochemical, biomechanical, and bioelectrical properties, can be effectively recruited for diagnostic approaches. Among those, electrical properties are of particular interest due to non-destructive ability of electric field in cell stimulation and response recording by advanced electrode designation. As an example, reduced membrane potential to -15 mv, highly negative membrane charge, increased electrical permeability of the membrane, and drastic cellular polarization upon external fields were essential methods of detecting cancer cells in cytologic samples which all are carried out by various sensors with different electrode and chip designation. Movement of cancer cells in cytological samples is affected by membrane electrical charge and their polarization which are different between normal and cancer cells even from the same tissue.

[0003] Cancerous cells carry greater negative electricity charges than normal ones. Alternating current (AC) electric fields have been used to analyze the cell surfaces charges, such as Electrophoresis (EP) and Dielectrophoresis (DEP). EP-based systems were rarely used to separate cancer cells, while the difference between surface electricity charges of cancerous and normal cells can be a perfect diagnostic pattern. This would be especially promising in cytological samples, such as pap smear, which a pathologist uses for detecting single cervical neoplastic or preneoplastic cells among normal cervical and blood cells. Moreover, no label- free electrical 3D method was presented to enrich and relocate the cancerous cells from liquid cytology samples, such as smears and aspirations, on the slide for cytological staining. [0004] Other research has been in the field of electrophoretic motion of cells in micro dimensions and stimulation of DEP and EP of cells with microelectrodes. For example, Sh. Shalileh et al. in a paper entitled as “Label-free mechanoelectrical investigation of single cancer cells by dielectrophoretic-induced stretch assay” published in Sensors and Actuators B: Chemical, Vol. 346, 2021 reported a label-free method for detection of cancer cells from normal cells by applying an AC electric field to a sample of cells using a group of transparent ITO electrodes coated on glass and analysis of cells’ mechanical deformation. The system designed in this research consists of a dielectric microchip placed on a printed circuit board in order to make the chip more stable under a microscope and an imaging system. The system designed in this research included a dielectric microchip placed on a printed circuit board in order to make the chip more stable under the microscope and an imaging system. In this method, cytological samples were used to detect, where cells isolated from a tumor sample were suspended in a buffer solution and injected into the microfluidic channel. By applying an electric field, cancerous cells are mechanically stretched compared to healthy cells, which has been determined by microscopic imaging of the microchannel surface and image analysis in order to determine whether the cell is cancerous or healthy.

[0005] There are two complications in analysis of the effect of EP and DEP on movement of cells in a microchip with miniaturized two-dimensional degrees of freedom; firstly, due to a limited movement in two dimensions and a limited width of a channel where cells are flown, excess cells would be accumulated and physically entrapped together, disrupting a test, and secondly, a backflow effect due to limited space in a fluidic chip would make turbulence which perturbs guided movement of cells from field direction to non-estimated paths.

[0006] Hence, there is a need in the art to overcome the problems and drawbacks of two- dimensional and micro-scale devices, systems, and methods in electrical investigations of cells. There is a need in the art for three-dimensional macro devices, systems, and methods for analysis of electrical behavior of cells, specifically, detection of cancer cells.

SUMMARY OF THE DISCLOSURE

[0007] This summary is intended to provide an overview of the subject matter of this patent, and is not intended to identify essential elements or key elements of the subject matter, nor is intended to be used to determine the scope of the claimed implementations. The proper scope of this patent may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0008] In one general aspect, the present disclosure is directed to a three-dimensional macrodevice for cancer cells detection. In an exemplary embodiment, the three-dimensional macrodevice may include a fluid container, a pair of electrodes, and a pair of electrical connectors. In an exemplary embodiment, the fluid container may include a cylindrical container with macroscale dimensions. In an exemplary embodiment, the fluid container may be used to put a fluid including a plurality of cells therein. In an exemplary embodiment, the pair of electrodes may include two electrically conductive transparent electrodes. In an exemplary embodiment, the pair of electrodes may be used to generate an electric field there between inside the fluid container. In an exemplary embodiment, the pair of electrodes may define a visible path through a height of the cylindrical container. In an exemplary embodiment, the visible path may be optically monitored and images and/or videos may be captured there through from motion of the plurality of cells along the height of the cylindrical container. In an exemplary embodiment, the pair of electrodes may include a first electrode mounted on a top side of the fluid container and a second electrode adhered to a bottom side of the fluid container. In an exemplary embodiment, the second electrode may be used to settle the plurality of cells thereon. In an exemplary embodiment, each electrical connector of the pair of electrical connectors may include an electrically conductive wire with a distal end and a proximal end. In an exemplary embodiment, the distal end may be attached to an electrode of the pair of electrodes and the proximal end may be attached to an electrical device. In an exemplary embodiment, the three-dimensional macrodevice may be utilized to detect a presence of cancer cells among the plurality of cells based on a settling speed of a first settled cell of the plurality of cells on the second electrode due to the generated electric field.

[0009] In another general aspect of the present disclosure, a system for cancer cells detection via three-dimensional macro-scale hydro-actuating analysis of electrophoretic and dielectrophoretic behavior of cells is disclosed. In an exemplary embodiment, the system may include the three-dimensional macrodevice, an electrical device connected to the pair of electrodes of the three-dimensional macrodevice, an optical microscope where the three- dimensional macrodevice may be placed onto a stage of the optical microscope and the second electrode may be located in front of a lens of the optical microscope, an image-capturing device attached to the optical microscope where a lens of the image-capturing device may be adjusted in front of the first electrode aligned to the visible path, and a processing unit electrically connected to the electrical device, the optical microscope, and the image-capturing device. In an exemplary embodiment, a fluid including a plurality of cells therein may be placed inside the cylindrical container.

[0010] In an exemplary embodiment, the processing unit may include a memory having processor-readable instructions stored therein and a processor. In an exemplary embodiment, the processor may access the memory and execute the processor-readable instructions. In an exemplary embodiment, the processor may be utilized to perform a method when the processor-readable instructions are executed by the processor. In an exemplary embodiment, the method may include generating an electric field between the pair of electrodes utilizing the electrical device, recording a first settling time of the first settled cell of the plurality of cells on the second electrode by monitoring motion of the plurality of cells along the fluid container utilizing the microscope, recording a second settling time of the first settled cell by capturing at least one of time-lapse images, videos, and combinations thereof from the second electrode utilizing the image-capturing device, calculating an average settling time of the first settled cell by calculating an average of the first settling time and the second settling time, and detecting the plurality of cells being cancerous responsive to the average settling time being less than a threshold time.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.

[0012] FIG. 1A schematically shows an exemplary three-dimensional macrodevice for cancer cells detection, consistent with one or more exemplary embodiments of the present disclosure. [0013] FIG. IB shows an exploded view of an exemplary three-dimensional macrodevice, consistent with one or more exemplary embodiments of the present disclosure.

[0014] FIG. 1C schematically shows a view of an exemplary fluid container, consistent with one or more exemplary embodiments of the present disclosure.

[0015] FIG. ID schematically shows a view of an exemplary fluid container without a top lid thereon, consistent with one or more exemplary embodiments of the present disclosure. [0016] FIG. IE schematically shows an exemplary mounting support, consistent with one or more exemplary embodiments of the present disclosure.

[0017] FIG. 2 shows an exemplary system for cancer cells detection via three-dimensional macro-scale hydro-actuating analysis of electrophoretic (EP) and dielectrophoretic (DEP) behavior of cells, consistent with one or more exemplary embodiments of the present disclosure.

[0018] FIG. 3 shows an exemplary method for cancer cells detection via three-dimensional macro-scale hydro-actuating analysis of EP and DEP behavior of cells, consistent with one or more exemplary embodiments of the present disclosure.

[0019] FIG. 4 shows an example computer system in which an embodiment of the present disclosure, or portions thereof, may be implemented as computer-readable code, consistent with one or more exemplary embodiments of the present disclosure.

[0020] FIG. 5 shows atomic force microscopy (AFM) analysis of topography of fluorinedoped tin oxide (FTO) in 3D view scale 10 pm, normal view scale 10 pm, 3D view scale 2 pm, normal view scale 2 pm, a marked direction of height checking, and height profile in 2 pm according to the marked direction, consistent with one or more exemplary embodiments of the present disclosure.

[0021] FIG. 6A shows zeta potential test diagrams of 5 % and 7 % agarose evaluated at -0.9 mV and -4.1 mV, consistent with one or more exemplary embodiments of the present disclosure.

[0022] FIG. 6B shows a graph of comparing settling time of an exemplary first settled cell on an exemplary bottom electrode in hydrostatic ambient of an exemplary three-dimensional macrodevice under various DC (4 V/cm) and AC (4 V/cm, 2 MHz) field intensities for MDA- MB-231 cancer cells, HUVEC cells, MCF-10A cells, and WBC in DMEM, 5 % and 7 % agarose during 10 min of each test, consistent with one or more exemplary embodiments of the present disclosure.

[0023] FIG. 7 shows optic images taken from HUVEC cells incubated for 48 to 72 hours and their function started, MDA-MB-231 cells settled next to HUVEC cells, MDA-MB-231 cells attacked HUVEC cells, and MDA-MB-231 cells eliminated HUVEC cells, consistent with one or more exemplary embodiments of the present disclosure. [0024] FIG. 8 shows speed of first MDA-MB-231 cells settling next to HUVEC cells and speed of first invasion of MDA-MB-231 cells to HUVECs and subsequent invasions, consistent with one or more exemplary embodiments of the present disclosure.

[0025] FIG. 9 shows fluorescence microscopic images associated with intracellular ROS measurement by DCFH-DA staining, consistent with one or more exemplary embodiments of the present disclosure.

[0026] FIG. 10 shows histopathological samples of invasive ductal carcinoma patients’ tumors indicating various Nottingham scores (NS), consistent with one or more exemplary embodiments of the present disclosure.

[0027] FIG. 11 shows comparative graphs for settling time of first settled cell of tumor samples versus tubule formation, nuclear pleomorphism, mitotic rate (p-value = 0.0001), and Nottingham score (p-value = 0.0001), consistent with one or more exemplary embodiments of the present disclosure.

DESCRIPTION OF EMBODIMENTS

[0028] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0029] Herein, methods, systems, and devices in three-dimensions and macroscale are disclosed for cancer detection and/or determination of cancer grade of a tumor or cancerous cells based on electrophoretic (EP) and dielectrophoretic (DEP) behavior of cells. An exemplary three-dimensional macroscale device with transparent electrodes may be designed to apply AC and DC electric fields and analytically evaluate electrostatic behavior of cells to distinguish cancer cells from normal ones. Two parameters were presented for such distinction; first, a time required for settling the first cell in both negative and positive electric poles of a bottom electrode of an exemplary three-dimensional macroscale device, and second, a time needed to observe the first attack of cancer cells on a HUVECs layer seeded on an exemplary bottom electrode in both electric pole situations. The latter parameter may show a valuable data about faster settling time of cancer cells in a meaningful correlation with their Nottingham scoring assayed on patients-derived breast cancer cells.

[0030] FIG. 1A schematically shows a three-dimensional macrodevice 100 for cancer cells detection, consistent with one or more exemplary embodiments of the present disclosure. In addition, FIG. IB shows an exploded view of three-dimensional macrodevice 100, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, three-dimensional macrodevice 100 may include a fluid container 101, a pair of electrodes 104 and 106, and a pair of electrical connectors 108 and 110.

[0031] FIGs. 1C and ID schematically shows two views of fluid container 101, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, fluid container 101 may include a container with a flat base for put a fluid there inside. In an exemplary embodiment, fluid container 101 may have any geometric shapes, for example, cylinder, cube, triangular prism, rectangular prism, etc. In an exemplary embodiment, fluid container 101 may include a cylindrical container 102. In an exemplary embodiment, fluid container 101 may have macroscale (essentially not microscale) dimensions.

[0032] Referring to FIG. ID, cylindrical container 102 may have a height 105 in a range of about 2 cm to about 5 cm. In an exemplary embodiment, cylindrical container 102 may have an outer diameter 107 in a range of about 1 cm to about 3 cm. In an exemplary embodiment, cylindrical container 102 may have an inner diameter 109 in a range of about 0.5 cm to about 2.5 cm.

[0033] In an exemplary embodiment, cylindrical container 102 may include a cylinder made of a non-toxic material which may have no effect on an exemplary fluid placed therein. In an exemplary embodiment, cylindrical container 102 may include a cylinder made of a biocompatible material. In an exemplary embodiment, cylindrical container 102 may include a cylinder made of a cylinder made of polylactic acid (PLA).

[0034] In an exemplary embodiment, cylindrical container 102 may be used to put a fluid therein. In an exemplary embodiment, an exemplary fluid may be injected or poured into cylindrical container 102 through an opening 103. In an exemplary embodiment, an exemplary fluid may include a sample to be tested using three-dimensional macrodevice 100 for detection of a presence of cancer cells therein. In an exemplary embodiment, an exemplary fluid may include a plurality of cells therein. [0035] In an exemplary embodiment, fluid container 101 may further include a top lid 112 fixed on top side 111 of cylindrical container 102 and a bottom base 114 fixed around a bottom potion 113 of cylindrical container 102. In an exemplary embodiment, a first electrode 104 of pair of electrodes 104 and 106 may be adhered onto top lid 112. In an exemplary embodiment, a second electrode 106 of pair of electrodes 104 and 106 may be adhered to a bottom side (not illustrated) of cylindrical container 102. In an exemplary embodiment, bottom base 114 may protect second electrode 106 as well as firmly hold cylindrical container 102 upright. In an exemplary embodiment, connection of pair of electrodes 104 and 106 to fluid container 101 may be insulated entirely with silicone adhesive.

[0036] In an exemplary embodiment, three-dimensional macrodevice 100 may further include a mounting support 116. FIG. IE schematically shows mounting support 116, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, mounting support 116 may include an opening 118. In an exemplary embodiment, opening 118 may be used to fix fluid container 101 therein. In an exemplary embodiment, mounting support 116 may include a shape resembling a shape of a stage of a microscope; allowing for fixing fluid container 101 onto an exemplary stage of an exemplary microscope while imaging and monitoring motion of an exemplary fluid inside fluid container 101 along cylindrical container 102. In an exemplary embodiment, mounting support 116 may allow for adjusting pair of electrodes 104 and 106 at proper locations in front of a lens of an exemplary microscope. In an exemplary embodiment, second electrode 106 may be adjusted in the vicinity of a bottom lens of an exemplary microscope using mounting support 116. In an exemplary embodiment, a thickness 120 of mounting support 116 may be selected in a range that the lowest distance between second electrode 106 and an exemplary bottom lens of an exemplary microscope may be obtained. In an exemplary embodiment, mounting support 116 may be made of PLA.

[0037] In an exemplary embodiment, each electrode of pair of electrodes 104 and 106 may include an electrically conductive transparent electrode. In an exemplary embodiment, pair of electrodes 104 and 106 may be used to generate an electric field there between inside cylindrical container 102; thereby, resulting in electrically affected motion of an exemplary plurality of cells along cylindrical container 102. In an exemplary embodiment, pair of transparent electrodes 104 and 106 may form a visible path through height 105 of cylindrical container 102 for optically monitoring and capturing images from motion of an exemplary plurality of cells along height 105 of cylindrical container 102. In an exemplary embodiment, an exemplary plurality of cells may settle on second electrode 106. In an exemplary embodiment, settling time of an exemplary plurality of cells may affected by an exemplary electric field generated between pair of electrodes 104 and 106. In an exemplary embodiment, cancer cells may settle faster on second electrode 106 due to their electrical behavior in an exemplary generated electric field in comparison with normal (healthy) cells.

[0038] In an exemplary embodiment, each electrode of pair of electrodes 104 and 106 may include a glass substrate and a layer of a transparent highly electrical conductive material deposited on an exemplary glass substrate. In an exemplary embodiment, a surface of the glass substrate coated with an exemplary layer of transparent highly electrical conductive material may include a roughened surface. In an exemplary embodiment, an average roughness of surface of each of first electrode 104 and second electrode 106 may include a peak-to-peak distance in a range of about 100 nm to about 500 nm and a peak height in a range of about 4 nm to about 60 nm. In an exemplary embodiment, an exemplary transparent highly electrical conductive material may include at least one of fluorine-doped tin oxide (FTO), indium tin oxide (ITO), and combinations thereof. In an exemplary embodiment, an exemplary transparent highly electrical conductive material may include FTO.

[0039] In an exemplary embodiment, a structure of each electrode of pair of electrodes 104 and 106 including an exemplary glass substrate with an exemplary transparent highly electrical conductive material may allow for transparency of pair of electrodes 104 and 106 in the visible light spectrum, so that optical monitoring and/or image capturing from both sides of each electrode of pair of electrodes 104 and 106 and an exemplary visible path along pair of electrodes 104 and 106 may be possible. In an exemplary embodiment, as light needs to pass through cylindrical container 102 to allow for optical monitoring and/or image capturing, colorless pair of electrodes 104 and 106 may prevent color reflection, increases light, and minimize fog to optimize clear vision.

[0040] In an exemplary embodiment, three-dimensional macrodevice 100 may be used to detect a cancerous grade of cancer cells. In an exemplary embodiment, an exemplary cancerous grade of cancer cells may be detected based on an invasion grade of cancer cells to normal cells. In an exemplary embodiment, second electrode 106 may further include a plurality of normal cells attached thereon. In an exemplary embodiment, second electrode 106 may include a plurality of normal cells seeded onto an exemplary layer of an exemplary transparent highly electrical conductive material. In an exemplary embodiment, metastatic cancer cells or more invasive cancer cells may have shorter settling time onto second electrode 106 due to their tendency to attack normal cells on second electrode 106 in comparison with non-metastatic cancer cells. In an exemplary embodiment, second electrode 106 may include a plurality of Human umbilical vein endothelial cells (HUVECs) seeded onto an exemplary layer of an exemplary transparent highly electrical conductive material. In an exemplary embodiment, an exemplary plurality of HUVECs may form a target trap for metastatic cancer cells to attack thereon.

[0041] Referring back to FIGs. 1A and IB, pair of electrical connectors 108 and 110 may be attached to pair of electrodes 104 and 106. In an exemplary embodiment, each electrical connector 108 or 110 may include an electrically conductive wire. In an exemplary embodiment, pair of electrical connectors 108 and 110 may be connected to respective pair of electrodes 104 and 106 using silver paste. In an exemplary embodiment, electrical connector 108 may include a distal end 108a and a proximal end 108b. In an exemplary embodiment, distal end 108a may be connected to first electrode 104 and proximal end 108b may be connected to an electrical device for applying an exemplary electric field between first electrode 104 and second electrode 106. In an exemplary embodiment, electrical connector 110 may include a distal end 110a and a proximal end 110b. In an exemplary embodiment, distal end 110a may be connected to second electrode 106 and proximal end 110b may be connected to an exemplary electrical device for applying an exemplary electric field between first electrode 104 and second electrode 106. Referring to FIG. 1C, two apertures 115 and 117 may be formed in top lid 112 and bottom base 114, correspondingly for passing electrical connectors 108 and 110.

[0042] In an exemplary embodiment of the present disclosure, a method and system for detecting a cancerous state of a sample based on electrophoretic (EP) and dielectrophoretic (DEP) behavior of cancer cells may be disclosed. In an exemplary embodiment, an exemplary method and system may utilize three-dimensional macrodevice 100 for cancer status detection. [0043] FIG. 2 shows a system 200 for cancer cells detection via three-dimensional macroscale hydro-actuating analysis of electrophoretic and dielectrophoretic behavior of cells, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, system 200 may include three-dimensional macrodevice 100, an electrical device 202, an optical microscope 204, an image-capturing device 206, and a processing unit 208. [0044] In an exemplary embodiment, three-dimensional macrodevice 100 may be placed onto a stage 205 of optical microscope 204. In an exemplary embodiment, second electrode 106 of three-dimensional macrodevice 100 may be fixed in front of a bottom lens 207 of optical microscope 204. In an exemplary embodiment, a location of image-capturing device 206 may be adjusted so that first electrode 104 of three-dimensional macrodevice 100 may be placed in front of a lens 211 of image-capturing device 206. In an exemplary embodiment, an exemplary visible path along height 105 of cylindrical container 102 of three-dimensional macrodevice 100 may be monitored using optical microscope 204 and/or image-capturing device 206. In an exemplary embodiment, image-capturing device 206 may be coupled to optical microscope 204. In an exemplary embodiment, image-capturing device 206 may be mounted on optical microscope 204 so that lens 211 of image-capturing device 206 may be placed on a top lens 209 of optical microscope 204.

[0045] In an exemplary embodiment, proximal ends 108b and 110b of pair of electrically conductive connectors 108 and 110 may be respectively connected to two poles 202a and 202b of electrical device 202. In an exemplary embodiment, electrical device 202 may include an ohmmeter, an electric signal generator, and an oscilloscope. In an exemplary embodiment, electrical device 202 may be used to generate at least one of an alternating current (AC) electric field, a direct current (DC) electric field, and combinations thereof. In an exemplary embodiment, electrical device 202 may be used to generate an exemplary electric field inside cylindrical container 102 via pair of electrodes 104 and 106.

[0046] In an exemplary embodiment, processing unit 208 may be electrically connected to at least one of electrical device 202, optical microscope 204, image-capturing device 206, and combination thereof via respective wireless connections or utilizing respective electrically conductive wires. In an exemplary embodiment, processing unit 208 may include a memory having processor-readable instructions stored therein and a processor. In an exemplary embodiment, an exemplary processor may be utilized to access an exemplary memory and execute exemplary processor-readable instructions. In an exemplary embodiment, executing exemplary processor-readable instructions by an exemplary processor may configure an exemplary processor to perform a method. In an exemplary method, an exemplary sample may be put inside cylindrical container 102 of three-dimensional macrodevice 100. In an exemplary embodiment, an exemplary sample may include an exemplary fluid containing an exemplary plurality of cells. In an exemplary embodiment, electrical device 202 may be used to apply an electric field between pair of electrodes 104 and 106 of three-dimensional macrodevice 100. In an exemplary embodiment, an exemplary plurality of cells may settle down towards second electrode 106 along an exemplary visible path inside three-dimensional macrodevice 100. In an exemplary embodiment, a motion and a settling speed of an exemplary plurality of cells may be affected by an exemplary generated electric field between pair of electrodes 104 and 106. In an exemplary embodiment, cancer cells may settle faster than normal cells due to their different electrical properties. In an exemplary embodiment, optical microscope 204 may be used to observe motion of an exemplary plurality of cells of an exemplary fluid along an exemplary visible path. In an exemplary embodiment, image-capturing device 206 may be used to capture images from an exemplary visible path. In an exemplary embodiment, imagecapturing device 206 may be used to capture time-lapse images from second electrode 106. In an exemplary embodiment, a time required for settling a first cell of an exemplary plurality of cells on second electrode 106 may be recorded and measured using at least one of optical microscope 204, image-capturing device 206, and combinations thereof. In an exemplary embodiment, a settling time of an exemplary first settled cell may be used to detect a cancerous state of an exemplary plurality of cells.

[0047] FIG. 3 shows a method 300 for cancer cells detection via three-dimensional macroscale hydro-actuating analysis of electrophoretic (EP) and dielectrophoretic (DEP) behavior of cells, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, method 300 may include putting a sample including a plurality of cells inside a fluid container of a three-dimensional macrodevice (step 302), generating an electric field between pair of electrodes of an exemplary three-dimensional macrodevice (step 304), recording a first settling time of a first settled cell of an exemplary plurality of cells on an electrode of an exemplary pairs of electrodes at the bottom of an exemplary fluid container using a microscope (step 306), recording a second settling time of an exemplary first settled cell by capturing images and/or videos from an exemplary electrode at the bottom of an exemplary fluid container utilizing an image-capturing device (step 308), calculating an average settling time of an exemplary first settled cell by calculating an average magnitude of an exemplary first settling time and an exemplary second settling time (step 310), and detecting a cancerous state of an exemplary plurality of cells based on an exemplary calculated average settling time (step 312). In an exemplary embodiment, method 300 may be carried out utilizing three-dimensional macrodevice 100 and system 200. So, method 300 may be described herein below in connection with FIGs. 1A-1E and FIG. 2.

[0048] In further detail with respect to step 302, step 302 may include putting a sample including a plurality of cells inside fluid container 101 of a three-dimensional macrodevice 100. In an exemplary embodiment, an exemplary sample may include a fluid comprising a plurality of cells. In an exemplary embodiment, an exemplary sample may include at least one of a fluid containing a cell line, a fluid containing a plurality of cells isolated from a tumor in a living body, a fluid containing a plurality of cells isolated from bloodstream, a fluid containing a plurality of cells isolated from a tumor margin, a fluid containing a plurality of cells isolated from a tissue of a living body, a fluid containing a plurality of cells isolated from a resected tumor, a fluid containing a plurality of cells of a biopsied sample from a living body. In an exemplary embodiment, putting an exemplary sample inside fluid container 101 may include putting an exemplary sample inside cylindrical container 102. In an exemplary embodiment, an exemplary sampled may be put inside cylindrical container 102 through opening 103. In an exemplary embodiment, putting an exemplary sample inside fluid container 101 may include releasing droplets of an exemplary sample into cylindrical container 102 without causing an exemplary sample to move or accelerate. In an exemplary embodiment, putting an exemplary sample inside fluid container 101 may include putting an exemplary sample inside cylindrical container 102 using a syringe and/or a pipette.

[0049] In further detail with respect to step 304, step 304 may include generating an electric field between pair of electrodes 104 and 106 of three-dimensional macrodevice 100. In an exemplary embodiment, generating an exemplary electric field between pair of electrodes 104 and 106 may include generating an exemplary electric field inside an exemplary sample in fluid container 101. In an exemplary embodiment, generating an exemplary electric field between pair of electrodes 104 and 106 may include generating at least one of an alternating current (AC) electric field, a direct current (DC) electric field, and combinations thereof between pair of electrodes 104 and 106. In an exemplary embodiment, generating an exemplary electric field between pair of electrodes 104 and 106 may include connecting first electrode 104 to a first pole 202a of electrical device 202, connecting second electrode 106 to a second pole 202b of electrical device 202, and applying an electrical voltage between pair of electrodes 104 and 106. In an exemplary embodiment, generating an exemplary electric field between pair of electrodes 104 and 106 may include applying an AC electric field with a magnitude in a range of 3 V/cm to 8 V/cm and a frequency in a range of 1 MHz to 3 MHz between pair of electrodes 104 and 106. In an exemplary embodiment, generating an exemplary electric field between pair of electrodes 104 and 106 may include applying an AC electric field with a magnitude of 4 V/cm and a frequency of 2 MHz between pair of electrodes 104 and 106. In an exemplary embodiment, generating an exemplary electric field between pair of electrodes 104 and 106 may include applying a DC electric field with a magnitude of 4 V/cm between pair of electrodes 104 and 106. In an exemplary embodiment, generating an exemplary electric field between pair of electrodes 104 and 106 may include applying a DC- electric signal to first electrode 104 and applying a DC+ electric signal to second electrode 106 by connecting a negative pole of poles 202a and 202b of electrical device 202 to first electrode 104 and connecting a positive pole of poles 202a and 202b of electrical device 202 to second electrode 106.

[0050] In further detail with respect to step 306, step 306 may include recording a first settling time of a first settled cell of an exemplary plurality of cells on second electrode 106 using microscope 204. In an exemplary embodiment, recording an exemplary first settling time may include monitoring motion of cells towards second electrode 106 through lens 207 of microscope 204 and recording a time at which an exemplary first cell may be settled on second electrode 106. In an exemplary embodiment, monitoring motion of cells towards second electrode 106 may include monitoring or observing a surface of second electrode 106.

[0051] In further detail with respect to step 308, step 308 may include recording a second settling time of an exemplary first settled cell by at least one of capturing time-lapse images from second electrode 106, capturing a video from second electrode 106, and combinations thereof utilizing image-capturing device 206. In an exemplary embodiment, recording an exemplary second settling time of an exemplary first settled cell may include adjusting lens 211 of image-capturing device 206 in front of second electrode 106, capturing at least one of time-lapse images, a video, and combinations thereof from surface of second electrode 106, and recording a time at which an exemplary first cell is settled on second electrode 106. In an exemplary embodiment, adjusting lens 211 of image-capturing device 206 in front of second electrode 106 may include mounting image-capturing device 206 on microscope 204 so that lens 211 of image-capturing device 206 may be adjusted along lens 209 of microscope 204 along an exemplary visible path from first electrode 104 to second electrode 106. In an exemplary embodiment, capturing time-lapse images may include capturing a set of images from surface of second electrode 106 at a corresponding set of time intervals. In an exemplary embodiment, each time interval of an exemplary set of time intervals may include a time step in a range of about 1 seconds to about 1 minute.

[0052] In further detail with respect to step 310, step 310 may include calculating an average settling time of an exemplary first settled cell by calculating an average of an exemplary first settling time and an exemplary second settling time. In an exemplary embodiment, an exemplary second settling time may be equal to an exemplary first settling time or not.

[0053] In further detail with respect to step 312, step 312 may include detecting a cancerous state of an exemplary plurality of cells based on an exemplary calculated average settling time. In an exemplary embodiment, detecting an exemplary cancerous state of an exemplary plurality of cells may include detecting an exemplary an exemplary plurality of cells being cancerous if an exemplary average settling time is less than a threshold time. In an exemplary embodiment, an exemplary threshold time may be about 30 seconds if an exemplary AC electric field with an exemplary magnitude in an exemplary range of 3 V/cm to 8 V/cm and an exemplary frequency in a range of 1 MHz to 3 MHz is generated between pair of electrodes 104 and 106 in step 304. In an exemplary embodiment, an exemplary threshold time may be about 65 seconds if an exemplary DC electric field with an exemplary magnitude in an exemplary range of 3 V/cm to 8 V/cm is generated between pair of electrodes 104 and 106 in step 304 while a positive pole of poles 202a and 202b of electrical device 202 is connected to second electrode 106.

[0054] In an exemplary embodiment, method 300 may further include detecting a grade of metastasis of cancer cells. In an exemplary embodiment, step 312 of detecting an exemplary cancerous state of an exemplary plurality of cells may further include detecting an exemplary grade of metastasis of detected cancer cells. In an exemplary embodiment, detecting an exemplary grade of metastasis of cancer cells may include detecting a metastatic cancerous state for an exemplary plurality of cells if an exemplary average settling time is less than about 18 seconds when an exemplary AC electric field with an exemplary magnitude in an exemplary range of 3 V/cm to 8 V/cm and an exemplary frequency in a range of 1 MHz to 3 MHz is generated between pair of electrodes 104 and 106 in step 304 while second electrode 106 comprises an exemplary plurality of normal cells, such as HUVECs thereon. In another exemplary embodiment, detecting an exemplary grade of metastasis of cancer cells may include detecting a metastatic cancerous state for an exemplary plurality of cells if an exemplary average settling time is less than about 31 seconds if an exemplary DC electric field with an exemplary magnitude in an exemplary range of 3 V/cm to 8 V/cm is generated between pair of electrodes 104 and 106 in step 304 while second electrode 106 with an exemplary plurality of normal cells, such as HUVECs thereon is connected to the positive pole of poles 202a and 202b of electrical device 202.

[0055] FIG. 4 shows an example computer system 400 in which an embodiment of the present disclosure, or portions thereof, may be implemented as computer-readable code, consistent with one or more exemplary embodiments of the present disclosure. For example, computer system 400 may include an example of processing unit 208, and steps 304-312 of flowchart presented in FIG. 3 may be implemented in computer system 400 using hardware, software, firmware, tangible computer readable media having instructions stored thereon, or a combination thereof and may be implemented in one or more computer systems or other processing systems. Hardware, software, or any combination of such may embody any of the modules and components in FIGs. 1A-1E and 2.

[0056] If programmable logic is used, such logic may execute on a commercially available processing platform or a special purpose device. One ordinary skill in the art may appreciate that an embodiment of the disclosed subject matter can be practiced with various computer system configurations, including multi-core multiprocessor systems, minicomputers, mainframe computers, computers linked or clustered with distributed functions, as well as pervasive or miniature computers that may be embedded into virtually any device.

[0057] For instance, a computing device having at least one processor device and a memory may be used to implement the above-described embodiments. A processor device may be a single processor, a plurality of processors, or combinations thereof. Processor devices may have one or more processor “cores”.

[0058] An embodiment of the present disclosure is described in terms of this example computer system 400. After reading this description, it will become apparent to a person skilled in the relevant art how to implement the invention using other computer systems and/or computer architectures. Although operations may be described as a sequential process, some of the operations may in fact be performed in parallel, concurrently, and/or in a distributed environment, and with program code stored locally or remotely for access by single or multiprocessor machines. In addition, in some embodiments the order of operations may be rearranged without departing from the spirit of the disclosed subject matter. [0059] Processor device 404 may be a special purpose or a general-purpose processor device. As will be appreciated by persons skilled in the relevant art, processor device 404 may also be a single processor in a multi-core/multiprocessor system, such system operating alone, or in a cluster of computing devices operating in a cluster or server farm. Processor device 404 may be connected to a communication infrastructure 406, for example, a bus, message queue, network, or multi-core message -passing scheme.

[0060] In an exemplary embodiment, computer system 400 may include a display interface 402, for example a video connector, to transfer data to a display unit 430, for example, a monitor. Computer system 400 may also include a main memory 408, for example, random access memory (RAM), and may also include a secondary memory 410. Secondary memory 410 may include, for example, a hard disk drive 412, and a removable storage drive 414. Removable storage drive 414 may include a floppy disk drive, a magnetic tape drive, an optical disk drive, a flash memory, or the like. Removable storage drive 414 may read from and/or write to a removable storage unit 418 in a well-known manner. Removable storage unit 418 may include a floppy disk, a magnetic tape, an optical disk, etc., which may be read by and written to by removable storage drive 414. As will be appreciated by persons skilled in the relevant art, removable storage unit 418 may include a computer usable storage medium having stored therein computer software and/or data.

[0061] In alternative embodiments, secondary memory 410 may include other similar means for allowing computer programs or other instructions to be loaded into computer system 400. Such means may include, for example, a removable storage unit 422 and an interface 420. Examples of such means may include a program cartridge and cartridge interface (such as that found in video game devices), a removable memory chip (such as an EPROM, or PROM) and associated socket, and other removable storage units 422 and interfaces 420 which allow software and data to be transferred from removable storage unit 422 to computer system 400. [0062] Computer system 400 may also include a communications interface 424. Communications interface 424 allows software and data to be transferred between computer system 400 and external devices. Communications interface 424 may include a modem, a network interface (such as an Ethernet card), a communications port, a PCMCIA slot and card, or the like. Software and data transferred via communications interface 424 may be in the form of signals, which may be electronic, electromagnetic, optical, or other signals capable of being received by communications interface 424. These signals may be provided to communications interface 424 via a communications path 426. Communications path 426 carries signals and may be implemented using wire or cable, fiber optics, a phone line, a cellular phone link, an RF link or other communications channels.

[0063] In this document, the terms “computer program medium” and “computer usable medium” are used to generally refer to media such as removable storage unit 418, removable storage unit 422, and a hard disk installed in hard disk drive 412. Computer program medium and computer usable medium may also refer to memories, such as main memory 408 and secondary memory 410, which may be memory semiconductors (e.g. DRAMs, etc.).

[0064] Computer programs (also called computer control logic) are stored in main memory 508 and/or secondary memory 410. Computer programs may also be received via communications interface 424. Such computer programs, when executed, enable computer system 400 to implement different embodiments of the present disclosure as discussed herein. In particular, the computer programs, when executed, enable processor device 404 to implement the processes of the present disclosure, such as the operations in method 300 illustrated by FIG. 3, discussed above. Accordingly, such computer programs represent controllers of computer system 400. Where an exemplary embodiment of method 300 is implemented using software, the software may be stored in a computer program product and loaded into computer system 400 using removable storage drive 414, interface 420, and hard disk drive 412, or communications interface 424.

[0065] Embodiments of the present disclosure also may be directed to computer program products including software stored on any computer useable medium. Such software, when executed in one or more data processing device, causes a data processing device to operate as described herein. An embodiment of the present disclosure may employ any computer useable or readable medium. Examples of computer useable mediums include, but are not limited to, primary storage devices (e.g., any type of random access memory), secondary storage devices (e.g., hard drives, floppy disks, CD ROMS, ZIP disks, tapes, magnetic storage devices, and optical storage devices, MEMS, nanotechnological storage device, etc.).

[0066] EXAMPLE 1 : Three-dimensional macrodevice fabrication

[0067] In this example, a three-dimensional macrodevice similar to three-dimensional macrodevice 100 was fabricated. A three-dimensional (3D) cylindrical body with a circular cross-section with a diameter of 1 cm and a cylindrical height of 3 cm was made of polylactic acid (PLA) with a 3D printer. Two FTO electrodes deposited on a glass substrate were used as a top and a bottom electrodes. The glass substrate has a thickness of about 1.1 mm and a low electrical resistance of about 8 ( xcm). Connection of the electrodes to the body was insulated entirely with silicone adhesive. Silver paste was used for electrical connections of two wires to the electrodes. A leak test was performed for 24 hours to prevent leakage of a fluid from the three-dimensional macrodevice.

[0068] An effective contact area and electrical interaction due to average roughness of surface of FTO electrodes were measured with atomic force microscopy (AFM) study. FIG. 5 shows AFM analysis of topography of FTO in 3D view scale 10 pm (image 502), normal view scale 10 pm (image 504), 3D view scale 2 pm (image 506), normal view scale 2 pm (image 508), a direction 511 of height checking (image 510), and height profile in 2 pm according to direction 511 marked in image 510 (image 512), consistent with one or more exemplary embodiments of the present disclosure. FTO coated electrodes may provide a platform for transparent slides with an effective electrical adhesion of cells thereon. An average roughness of the tested electrode was about 4.71 nm, and peak-to-peak was about 45 nm.

[0069] EXAMPLE 2: Differentiation of cancer cells from normal ones based on their electrophoretic (EP) and dielectrophoretic (DEP) behavior in an electric field (DC/AC)

[0070] In this example, a system similar to system 200 was prepared for differentiating between cancer cells and normal cells using the fabricated three-dimensional macrodevice according to EXAMPLE 1. Thereafter, a method similar to method 300 described herein above was conducted utilizing the prepared system. To control the temperature and CO2, and prevent effects of their changes on test results and system performance, the optical microscope was placed inside a chamber, where the temperature was controlled at about 37 °C and a concentration of CO2 was maintained at about 5 %. A 3D printed plate structurally similar to mounting support 116 was used to fix a location of pair of electrodes on the fabricated three- dimensional macrodevice and the mechanical protection of the bottom electrode. Droplet of each sample was released slowly from a pipette into an exemplary fluid container of the fabricated three-dimensional macrodevice, without causing the sample to move or accelerate. Cells of each sample were imaged every one and thirty seconds for measuring a settling time of the cells and an effect of invasion to HUVEC cells and hypoxia in an generated electrical field between the pair of electrodes, respectively, with a camera attached to the optical microscope. All observations and control processes were done using a computer device similar to processing unit 208. A main parameter in performed tests was a time required for settling a first cell on the bottom electrode.

[0071] To prepare samples, MDA-MB-231 , HUVEC, and MCF-10A were purchased and were cultured in DMEM (serum-free cell media) supplemented with 1 % antibiotics (penicillinstreptomycin) and 10 % fetal bovine serum (FBS). Cells were cultured in a standard incubator at 37 °C with 5 % CO2 and 90-95 % humidity. Additionally, blood samples were collected from healthy volunteers and anti-coagulated to prevent clotting. The sample was then mixed with Ficoll solution to create a density gradient. Ficoll solution usually consists of a buffered salt solution and Ficoll, a synthetic polymer with a high molecular weight. The mixture was then centrifuged at a low speed between 400 and 600 rpm for several minutes to separate the blood into separate layers, including plasma on top, white blood cells and Ficoll in the middle, and red blood cells at the bottom. The layer containing WBCs and Ficoll was carefully collected and transferred to a new tube. The WBCs were then collected by centrifugation at a higher speed between 1000 and 1500 rpm for about 10 tol5 minutes. WBCs formed a pellet at the bottom of the tube, while Ficoll and other blood components remained in the supernatant. The supernatant was carefully removed and the WBC pellet was resuspended in a buffer or medium for downstream applications by density gradient centrifugation. Different types of blood cells may be separated based on their density. CTC and white blood cells have a lower density than red blood cells, so they can be separated by centrifugation through a density gradient.

[0072] In a first set of tests, zeta potential as an important parameter in cells setting time was investigated. It should be noted that zeta potential may act as a functional part of electrical charge of cells’ membrane surface. Media of the cells, number of ions, and pH of the media may affect a magnitude of zeta potential. Many substances may exhibit various levels of zeta potential when exposed to different liquids or water. In cells’ membranes, fluid environments may affect membrane activity. As a result, a concentration of nearby ions on surface of the membrane may differ from total ions in the environment. Zeta potential may affect electroosmotic motion of a particle. Accordingly, an effect of viscosity of different cell media, including lymphatic nodes’ electrophoretic mobility, were used to evaluate an effect of concentration on cell motility, surface charge, and agarose viscosity on a first cell settling. The zeta potential tests were performed in different cell cultures’ viscosity, including 5 % and 7 % agarose to investigate effect of concentration on mobility of cells under an electric field since less than 5 % concentration did not significantly affect cell mobility and cells did not show significant mobility under an electric field at more than 7 % agarose concentrations. The effect of agarose viscosity on settling time of the first settled cell and control of the cells by an electric field was also tested. To prepare 5 % and 7 % agarose, distilled water was added to agarose powder and dissolved with heat until a clear solution with a 50 % concentration was obtained. The DMEM was added to the solution at room temperature until the concentration reached 5 % (or 7 %). Agarose gel preparation was bubble-free because bubbles in the gel can trap cells and affect an accuracy of test results.

[0073] FIG. 6A shows zeta potential test diagrams of 5 % and 7 % agarose evaluated at -0.9 mV (diagram 602) and -4.1 mV (diagram 604), consistent with one or more exemplary embodiments of the present disclosure. The zeta potential of MDA-MB231 cells suspended in agarose was -0.9 mv for 5 % agarose and -4.1 mv for 7 % agarose. According to the results, zeta potential is negative for both agarose concentrations, which means that it acts as hardness for speed of movement of the cells. Breast cancer cells maintained their negative surface charges in agarose ambient.

[0074] FIG. 6B shows a graph 606 of comparing settling time of the first cells on the bottom electrode in hydrostatic ambient of the three-dimensional macrodevice under various DC (4 V/cm) and AC (4 V/cm, 2 MHz) field intensities for MDA-MB-231 cancer cells, HUVEC cells, MCF-10A cells, and WBC in DMEM, 5 % and 7 % agarose during 10 min of each test, consistent with one or more exemplary embodiments of the present disclosure. In this figure, effect of viscosity on gravity (without applying any electric field) was reported by recording the settling times of the first settled cell of MDA-MB-231 cancer cells, HUVEC cells, and MCF- 10A cells, and WBC in pure DMEM (48, 44, 45, and 32 s), DMEM contained 5 % agarose (122, 318, 93, and 250 s), and DMEM contained 7 % agarose (127, 341, 123, and 300 s). So, a correlation was demonstrated between an increasing viscosity of media and reduced mobility rate of cells. Adding agarose may increase the viscosity of media and decreases the mobility of suspended cells.

[0075] Referring to FIG. 6B, an effect of the hydroelectric actuator three-dimensional macrodevice on the cells was evaluated by applying a DC electric field with a magnitude of 4 V/cm to the suspended cells in individual chambers with pure DMEM, DMEM containing 5 %, and DMEM containing 7 % agarose. All types of assayed cells were tested under two different DC polarities (DC+ up and DC+ bottom). The time required to observe the seeding of the first cell on the bottom electrode was recorded. Meaningful differences in the time required for the settling time of the first cell on the bottom electrode in all types of cell lines were captured by time-dependent video recording. Effects of attraction and repulsion of electrical stimulation according to the time required for the adhesion of MDA-MB-231 cells, in DC+ bottom mode, without applying an electric field and DC+ up mode in DMEM were evaluated which were 27 s,48 s, and 100 s, respectively. It was shown that cancer cells settled faster in the DC+ bottom mode and slower in the DC+ up mode, respectively. Increased viscosity of the culture media showed its stopping effect on the mobility of the cells even in the presence of an electric field. Comparative presentation of the settling time results of the various types of the cells in the different biases of electric field and different viscosities of the media confirmed the supportive effect of the electric field in the motility of normal and cancer cells in contrast bias and similar suppressive effect of media viscosity on the motility of both normal and cancer cells. According to the mathematical relations of the EP effect, the mobility of the cancer cell is similar to a particle with a negative electric charge. MCF-10A, HUVEC cells, and WBC (as normal cells) showed meaningful differences in tendency between negative and positive electric poles. The settling time of MCF-10A, HUVEC cells, and WBC in the presence of a more robust positive DC electric field was 70, 66, and 82 s, and in the presence of a more substantial negative DC electric field was 20, 14, and 20 s, respectively. Hence, they showed a mobility such as particles with minor surface positive charges in contrast to the MDA-MB-231 cell. Entrapment of cancer cells showed a strong correlation with bias and intensity of DC electric field while this evidence was so weaker in normal cells, which may be due to lower surface charges (in any polarity) of normal cells concerning strong negative surface charges in cancer cells.

[0076] Moreover, DEP response characterization of the cells in AC fields may be analyzed according to the results shown in FIG. 6B. Herein, DEP used as an electrokinetic method for separating and discriminating suspended cells based on their intrinsic dielectric properties. DEP may also separate circulating tumor cells (CTCs) in bloodstream in their epithelial, mesenchymal, or epithelial-mesenchymal phenotypes. Herein, DEP was applied at the frequency of 2 MHz with the intensity of 4 V/cm on normal and cancer cells suspended in a medium with different viscosities. The first settling time of MDA-MB-231 cells was measured at 15, 38, and 55 s, MCF-10A cells were measured at 60, 112, and 180 s, WBC cells were measured at 48, 98, and 150 s, and HUVECs cells were measured at 50, 324, and 375 s in pure DMEM, DMEM contained 5 % and DMEM contained 7 % agarose, respectively. Based on the dielectric properties of the cells, in all three viscosities, MDA-MB-231 cancer cells settled significantly faster than the normal HUVEC, MCF-10A cell lines, and WBC.

[0077] In a second set of tests, invasive ability of cancer cells in the fabricated three- dimensional macrodevice was analyzed. The invasive ability of malignant cells were investigated as their invasion to vascular endothelial cells. An effect of electrophoretic parameters of an exemplary system and method on invasive ability of cancer cells via the invasion of the cancer cells (MDA-MB-231) to the HUVECs under various EP parameters was tested. This process may be important in detection of metastasis. HUVECs were seeded on the bottom electrode, and cancer cells were suspended in the medium. So, the invasion assay of cancer cells to the endothelial layer was investigated under a supportive electric field with an intensity of 4 V/cm. Moreover, an effect of hypoxia on electrical behavior of MDA-MB-231 cancer cells was investigated. To induce hypoxia in MDA-MB-231 cancer cell, lid of cell flask was blocked with wax. After about 8 hours (one cell division cycle), pH and color of culture solution were monitored. The cylindrical container of the fabricated three-dimensional macrodevice was filled with culture solution, where about 2 x 10⁴ of HUVEC cells were poured onto the bottom electrode and incubated for 48 hours to 72 hours until the cells reached function. So, invasion assay of cancer cells was investigated under supportive and preventive electric fields. After that, 3 x 10⁴ MDA-MB-231 cells were released into the cylindrical container and controlled for 12 hours. The microscope was placed inside the chamber to control the temperature and the concentration of CO2 on the test results and cell performance. Timelapse image capturing was activated every 30 seconds.

[0078] FIG. 7 shows optic images taken from HUVEC cells 701 incubated for 48 to 72 hours and their function started (image 702), MDA-MB-231 cells 703 settled next to HUVEC cells 701 (image 704), MDA-MB-231 cells 703 attacked HUVEC cells 701 (image 706), and MDA- MB-231 cells 703 eliminated HUVEC cells 701 (image 708), consistent with one or more exemplary embodiments of the present disclosure. Additionally, FIG. 8 shows speed of first MDA-MB-231 cells 703 settling next to HUVEC cells 701 (diagram 802) and speed of first invasion of MDA-MB-231 cells 703 to HUVECs 701 and subsequent invasions (diagram 804), consistent with one or more exemplary embodiments of the present disclosure.

[0079] Furthermore, settling time of first settled cell of MDA-MB-231 cells 703 and first invasion time of MDA-MB-231 cells 703 to HUVECs 701 are presented in Tables 1 and 2 herein below, respectively. [0080] Table 1. First settled time of MDA-MB-231 cells next to HUVECs at different applied conditions

Settling Time None DC DC

Without hypoxia (min) 96 36 6

Hypoxic (min) 6 12 6

[0081] Table 2. First invasion time of MDA-MB-231 cells to HUVECs at different applied conditions

First Invasion None C DC⁺

Without Hypoxia (min) 96 ..............210 114

528 318

Hypoxic (min) 54 66 12

228

102 90

438

[0082] Recorded time of the first attack and subsequent attacks of MDA-MB-231 cells 703 on target cells (HUVECs 701) were about 114 and 210 s when HUVECs 701 seeded bottom electrode was individually connected to positive and negative electric poles, respectively. Drastic reduction/enhancement in velocity of invasion was observed as a consequence of suppressive/supportive effect of the DC +/- field on the motility of cancer cells. This observation indicated the impact of EP in controlling the activities of cancer cells for both diagnostic and theranostic approaches. The negative charge of the MDA-MB-231 cells 703 increases/decreases their gradual motility to endothelial cells 701 due to the electrophoresis effect when the electrical pole of the slide is positive/negative and induces attractive/repulsive electrical force to cancer cells.

[0083] Effect of hypoxia on hydroelectric response of cancer cells has been analyzed based on obtained results shown in FIG. 8 and Tables 1 and 2. It should be noted that hypoxia may be one of the crucial factors in tumor environment and may stimulate tumor invasion. Hypoxia in cells and tissues may lead to induction of transcription of a series of genes that participate in angiogenesis, glucose metabolism, cell proliferation, and migration. HIF (Hypoxia-inducible factor) is the first factor that mediates this response. During hypoxia, overactivation in the factor (la) signaling pathway (HIF- la) mitochondrial overproduction of reactive oxygen species (ROS) has been reported. In many types of cancers, an increase in ROS levels was reported as a mediation agent of Epithelial-to-mesenchymal transition (EMT) and metastasis. Herein, inducing hypoxic media was carried out and its effect on settling time and invasion ability of cancer cell line was evaluated. Florescent ROS imaging as an indication for the effect of hypoxic media on cellular invasive functions was done as shown in FIG. 9. For conducting fluorescence imaging of intracellular ROS generation, DCFH diacetate form (DCFH-DA) was prepared to evaluate the net intracellular generation levels of ROS. The MDA-MB-231 cell line was cultured on a 48-well plate. After it reached 70 to 80 % of the total well capacity, the lid of the well plate was blocked with parafilm to induce hypoxia. DCFH-DA was added to the well and incubated for 30 min. The wells were washed twice with PBS and imaged in the epifluorescence mode with a blue filter. As may be seen in FIG. 9, strong correlation between the expression of ROS-associated green fluorescent markers and incubation of the MD-MB-231 cells in hypoxic media was observed.

[0084] One of the main results of hypoxia may be entering a cell to metabolic pathways, which increases an amount of sialic acid in membrane of cells. Carboxyl groups of sialic acid may significantly increase negative surface charges of cancer cells membrane. So, the time of the first attack to HUVECs between MDA-MB-231 cells incubated in non-hypoxic and hypoxic conditions was compared. The first settling time of non-electrically biased MDA-MB-231 cells that had been incubated in hypoxic media was 6 s, which was the same as the hypoxic cells under the DC+ electric field (intensity of 4 V/cm). Hence, the electric field didn’t show any additive supportive effect on the settling time of hypoxic cancer cells. However, in the negative bias of the field (DC- with the same intensity as DC+), the settling time of hypoxic cells was 12 s, revealing the electric field’s suppressive effect on the settling time of hypoxic cancer cells. The results showed that hypoxia might lead to saturation of motility velocity of cancer cells and the supportive effect of EP couldn’t be recordable in hypoxic ambient, but its suppressive effect was significantly observed. The invasion of MDA-MB-231 to HUVECs cells was faster in a hypoxic environment with a DC+ field than in a non-biased chip (12 and 54 s, respectively). Moreover, in DC- field, a slower attack on HUVECs was observed than on non-biased chips (66 and 54 s, respectively) (diagram 804 of FIG. 8).

[0085] Invasion assay is co-affected by both hypoxic media and EP bias in either supportive or suppressive regimes. However, settling time in the presence of hypoxia reached a saturation value that was not co-affected by the supportive EP field. Although, the suppressive effect of EP was deduced even in the presence of hypoxia (diagram 802 of FIG. 8). A mechanism behind such evidence may be the saturated invasive function of Triple-negative breast cancer cells in hypoxic media. If those cancer cells reach their highest invasive activity (which may be expectable in hypoxic media), a supportive electric field may not make a sensible increase in the time of their first invasion to endothelial cells. On the other hand, the contrast bias of the electric field can reduce this saturation level.

[0086] In a third set of tests, an exemplary method was applied to cytologically separated cancer cells from non-used residue of resected tumors after surgery of some breast cancer patients due to permission of patients, surgeons, and pathologists of the patients using an exemplary system including the fabricated three-dimensional macrodevice. For cell isolation from patients’ tissue, a sample of patients with 70 % or more cells with desired specific characteristics was selected according to initial core needle biopsy report results and diagnosis of a pathologist. To investigate the effect of cancer grade on the first cell settling, 14 patient samples were obtained. Tumor samples were stored with PBS and Pen-strep on ice and incubated for 5 hours with a serum-free medium containing type 1 collagenase. Then, cells were placed in 0.25 % trypsin-EDTA solution, and after 5 min, they were neutralized with a culture medium. Finally, the cells were passed through a 44 pm cell strainer. The cells were kept under a constant positive and negative electric field of 4 V/cm; then, the settling time of the first cell was recorded with a camera attached to the optical microscope.

[0087] The electrophoretic motion (p) was directly related to the net electric charge of the cancer cells. The pathological parameters that had been declared for the tumors by the pathologist included tubule formation, nuclear pleomorphism, mitotic rates, and histologic grade, which all were co-evaluated as “Nottingham histologic score”. This scoring ranged from 3/9 to 9/9 due to a progressive state of the tumor. Tumors in Nottingham scores of 6/9 to 9/9 were used as may be seen in FIG. 10. Table 3 shows the settling times of the first cell isolated from the patient’ s sample due to type of DC electric field.

[0088] Table 3. Settling times of the first cell isolated from the patient’s sample x N,ottingh , am . T..u .bu .lar Nuc .lear Mitotic The settling time under

Patient _ score formation p

^Kleomorp Khism rate „ „ DC+(S) Non(S) DC-(S) easel 9 3 3 3 44 123 184 case2 9 3 3 3 41 114 173 case3 8 3 3 2 64 98 120 case4 8 3 3 2 53 74 128 case5 8 3 3 2 54 70 100 case6 8 3 3 2 66 98 120 case? 8 3 3 2 68 100 169 case8 7 3 3 1 82 70 110 case9 7 3 3 1 90 79 106 caselO 7 3 3 1 80 102 135 casell 6 2 2 2 84 95 102 casel2 6 3 2 1 98 78 84 casel3 6 3 2 1 103 77 82 casel4 - - - - 123 86 76

[0089] Moreover, FIG. 11 shows comparative graphs for settling time of first settled cell of tumor samples versus tubule formation, nuclear pleomorphism, mitotic rate (p-value = 0.0001), and Nottingham score (p-value = 0.0001), consistent with one or more exemplary embodiments of the present disclosure. According to Table 3 and FIG. 11, a significant correlation between the Nottingham score, mitotic rate, and DC was demonstrated. As it is seen, the cell settling time in the DC+ decreased with the tumor grade increasing. It was also observed that this phenomenon is the same in the mitotic rate study. Furthermore, case 14 was pre-cancer (DIN- 3), showing a slower settling time in DC+ compared to other patients.

[0090] Notably, cells derived from tumors with higher Nottingham scores showed faster settling time on the bottom electrode connected to DC positive electric pole (case ID# 1 in 44 s with 9/9 Nottingham score and case ID# 13 in 103 s with 6/9 Nottingham score). On the other hand, applying the DC negative electric pole to the bottom electrode showed a slower settling time with higher Nottingham scores (Case ID#1 in 184 s with 9/9 Nottingham score and case ID# 13 in 82 s with 6/9 Nottingham score).

[0091] It seems that a constructive correlation exists between the tumor cells’ electrophoretic activity and their Nottingham scoring state. Also, by individuals examining the effect of nuclear pleomorphism, mitotic rates, and tubule formation, a correlation was observed between the electrophoretic response of tumor cells and mitotic rates, similar to Nottingham scoring (case ID# 2 in 41 s with 3 mitotic rates and Case ID#12 in 98 s with 1 mitotic rate). However, no correlation was observed between cancer cells’ electrophoretic responses and two other pathologic parameters their nuclear pleomorphism and tubule formation scores (Case ID#10 in 80 s with nuclear pleomorphism 3 and Case ID#11 in 84 s with 2 nuclear pleomorphism scores). This may be revealed that a hydroelectrically actuated chip could be a complementary helpful device for scoring the aggressiveness of breast cancer tumors. Similar to the cell line analysis results, it was observed that the breast cancer cells of the patients meaningfully respond to an external DC electric field, which confirms their faster settling time dependent on negative surface charges.

Industrial Applicability

[0092] Disclosed herein is a device, system, and method using thereof for cancer detection and determination of cancer grade. An exemplary device, system, and method may be marker-free and capable of fast application which may not only applicable for diagnostic approaches but also useful for cell study purposes. An exemplary device, system, and method may shed new light in the field of bioelectro nic diagnosis based on both EP and DEP phenomena.

[0093] While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0094] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0095] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0096] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0097] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0098] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0099] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. A three-dimensional macrodevice for cancer cells detection, the three-dimensional macrodevice comprising: a fluid container, comprising a cylindrical container with macroscale dimensions, the fluid container configured to put a fluid comprising a plurality of cells therein; a pair of electrodes, comprising two electrically conductive transparent electrodes, the pair of electrodes configured to generate an electric field there between inside the fluid container, the pair of electrodes defining a visible path through a height of the cylindrical container, the visible path configured to be optically monitored and capture images and/or videos there through from motion of the plurality of cells along the height of the cylindrical container, the pair of electrodes comprising: a first electrode mounted on a top side of the fluid container; and a second electrode adhered to a bottom side of the fluid container, the second electrode configured to settle the plurality of cells thereon; and a pair of electrical connectors, each respective electrical connector comprising an electrically conductive wire comprising a distal end and a proximal end, the distal end being attached to an electrode of the pair of electrodes, the proximal end configured to being attached to an electrical device, wherein the three-dimensional macrodevice is configured to detect a presence of cancer cells among the plurality of cells based on a settling speed of a first settled cell of the plurality of cells on the second electrode due to the generated electric field.

2. The three-dimensional macrodevice of claim 1, wherein the height of the cylindrical container is in a range of 2 cm to 5 cm.

3. The three-dimensional macrodevice of claim 1, wherein the cylindrical container has an outer diameter in a range of 1 cm to 3 cm and an inner diameter in a range of 0.5 cm to 2.5 cm.

4. The three-dimensional macrodevice of claim 1, wherein the cylindrical container comprises a cylinder made of a biocompatible material.

5. The three-dimensional macrodevice of claim 4, wherein the cylindrical container comprises a cylinder made of polylactic acid (PLA).

6. The three-dimensional macrodevice of claim 1, wherein the fluid container further comprises: a top lid fixed on the top side of the cylindrical container, the first electrode adhered onto the top lid; and a bottom base fixed around a bottom potion of the cylindrical container, the bottom base defining a protective element around the second electrode and a holding member for the cylindrical container on a stage of a microscope.

7. The three-dimensional macrodevice of claim 1, further comprising a mounting support made of PLA, the mounting support comprising an opening configured to fix the fluid container therein, the mounting support comprising a shape fixed onto a stage of a microscope while imaging and monitoring motion of the fluid along the cylindrical container.

8. The three-dimensional macrodevice of claim 1, wherein each of the first electrode and the second electrode comprises: a glass substrate; and a layer of a transparent highly electrical conductive material deposited on the glass substrate forming a roughened surface on the glass substrate, the transparent highly electrical conductive material comprising at least one of fluorine-doped tin oxide (FTO), indium tin oxide (ITO), and combinations thereof.

9. The three-dimensional macrodevice of claim 8, wherein an average roughness of surface of each of the first electrode and the second electrode comprises a peak-to-peak distance in a range of 100 run to 500 nm and a peak height in a range of 4 run to 60 run.

10. The three-dimensional macrodevice of claim 8, wherein the second electrode further comprises a plurality of Human umbilical vein endothelial cells (HUVECs) seeded onto the respective layer of the transparent highly electrical conductive material, the plurality of HUVECs configured to be a target trap for metastatic cells to attack thereon.

11. A system for cancer cells detection via three-dimensional macro-scale hydro-actuating analysis of electrophoretic and dielectrophoretic behavior of cells, the system comprising: a three-dimensional macrodevice, comprising: a fluid container, comprising a cylindrical container configured to put a fluid comprising a plurality of cells therein; a pair of electrodes, comprising two electrically conductive transparent electrodes, the pair of electrodes defining a visible path through a height of the cylindrical container, the pair of electrodes comprising: a first electrode mounted on a top side of the fluid container; a second electrode mounted on a bottom side of the fluid container; and a pair of electrical connectors, each respective electrical connector comprising a distal end and a proximal end, the distal end being attached to an electrode of the pair of electrodes; an electrical device configured to generate an electric field between the pair of electrodes, the proximal end of each electrical connector of the pair of electrical connectors being connected to a different pole of two poles of the electrical device; an optical microscope, the three-dimensional macrodevice being placed onto a stage of the optical microscope, the second electrode being in front of a lens of the optical microscope, the optical microscope configured to observe motion of the plurality of cells of the fluid along the visible path, the optical microscope configured to detect settling of a first cell of the plurality of cells on the second electrode; an image-capturing device attached to the optical microscope, a lens of the image-capturing device being adjusted in front of the first electrode aligned to the visible path, the image-capturing device configured to capture at least one of time-lapse images, videos, and combinations thereof from the second electrode; and a processing unit electrically connected to the electrical device, the optical microscope, and the image-capturing device, the processing unit comprising: a memory having processor-readable instructions stored therein; and a processor configured to access the memory and execute the processor- readable instructions, which, when executed by the processor configures the processor to perform a method, the method comprising: generating, utilizing the electrical device, an electric field between the pair of electrodes; recording a first settling time of the first settled cell of the plurality of cells on the second electrode by monitoring motion of the plurality of cells along the fluid container utilizing the microscope; recording a second settling time of the first settled cell by capturing at least one of time-lapse images, videos, and combinations thereof from the second electrode utilizing the image-capturing device, capturing time-lapse images from the second electrode comprising recording an image from the second electrode with the first settled cell thereon at the second settling time; calculating an average settling time of the first settled cell by calculating an average of the first settling time and the second settling time; and detecting the plurality of cells being cancerous responsive to the average settling time being less than a threshold time.

12. The system of claim 11, wherein generating the electric field between the pair of electrodes comprises applying an alternating current (AC) electric field with a magnitude in a range of 3 V/cm to 8 V/cm and a frequency in a range of 1 MHz to 3 MHz between the pair of electrodes.

13. The system of claim 12, wherein the threshold time comprises 30 seconds.

14. The system of claim 11, wherein generating the electric field between the pair of electrodes comprises applying a direct current (DC) electric field with a magnitude in a range of 3 V/cm to 8 V/cm while a positive pole of the electrical device being connected to the second electrode.

15. The system of claim 14, wherein the threshold time comprises 65 seconds.

16. The system of claim 11, wherein each of the first electrode and the second electrode comprises: a glass substrate; and a layer of a transparent highly electrical conductive material deposited on the glass substrate forming a roughened surface on the glass substrate, the transparent highly electrical conductive material comprising at least one of fluorine-doped tin oxide (FTO), indium tin oxide (ITO), and combinations thereof.

17. The system of claim 16, wherein an average roughness of surface of each of the first electrode and the second electrode comprises a peak-to-peak distance in a range of 100 nm to 500 nm and a peak height in a range of 4 nm to 60 nm.

18. The system of claim 11, wherein the cylindrical container comprises a cylinder made of polylactic acid (PLA) with the height in a range of 2 cm to 5 cm, wherein an outer diameter of the cylindrical container is in a range of 1 cm to 3 cm and an inner diameter of the cylindrical container is in a range of 0.5 cm to 2.5 cm.

19. The system of claim 11, wherein the second electrode further comprises a plurality of Human umbilical vein endothelial cells (HUVECs) seeded onto the respective layer of the transparent highly electrical conductive material, the plurality of HUVECs configured to be a target trap for metastatic cells to attack thereon.

20. The system of claim 19, wherein the method further comprises detecting a grade of metastasis of cancer cells, comprising one of: detecting a metastatic cancerous state for the plurality of cells responsive to the average settling time being less than 18 seconds responsive to generating an AC electric field with a magnitude in a range of 3 V/cm to 8 V/cm and a frequency in a range of 1 MHz to 3 MHz between the pair of electrodes; or detecting a metastatic cancerous state for the plurality of cells responsive to the average settling time being less than 31 seconds responsive to generating a DC electric field with a magnitude in a range of 3 V/cm to 8 V/cm while a positive pole of the electrical device being connected to the second electrode.