HK1228000B - Microanalysis of cellular function - Google Patents

Description

Microanalysis of cell function

This application is a divisional application of the invention patent application with application date of December 5, 2011, application number 201180066877.5, and invention name “Microanalysis of Cell Function”.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/419,377, filed December 3, 2010, entitled "Microanalysis of Cellular Function," which is hereby incorporated by reference in its entirety for all purposes.

Background Art

There is a need for high-throughput, efficient systems for analyzing biological particles, including single living cells, to rapidly and meaningfully identify particles, including cells and molecules, that possess a desired function, such as producing or inducing a desired biological outcome. Such systems are needed for drug discovery, diagnostics, and screening of candidate molecules. In preferred systems, the particles retain their activity and are not significantly damaged by the analytical process.

Existing systems for evaluating single particles typically use dielectrophoresis (DEP) to manipulate the particles. An electric field is imposed on particles, such as cells, to suspend and maintain their position during the induction reaction, and the analysis can damage their activity and disrupt the ability of the particles to perform normal functions. For example, in the case of prolonged use, this damage can lead to lysis and/or cell death. Adhesion of particles to substrates, electrodes, or chamber walls may also require lysis of the cells to remove them from the system after analysis or to capture cell products.

It would be very useful to provide a rapid, high-throughput system for effectively and efficiently capturing, identifying, and analyzing the biological functions of single particles, without causing significant damage to the particles.

Summary of the Invention

Disclosed herein are inverted open microporous systems, devices, and methods of use that allow for rapid and efficient screening and sorting of particles, particularly single particles, including single cells. This screening includes analysis of specific particle characteristics and functional properties of the particles that can indicate whether the particles or agents are useful for therapeutic applications. For example, the particle characteristics can include: the presence and relative amount of specific binding and/or affinity for ligands, such as cancer cell target antigens; cell-to-cell binding reactions and interactions, such as cell lysis, toxicity, biomarker production, electroporation, etc.; induced cellular responses and/or reactions, such as those induced by other cells, drugs, compounds, proteins, antibodies, molecules, enzymes, nucleic acid molecules, cell secretions, etc.; and the induced production of cellular products, such as lytic enzymes, antibodies, biomarkers, etc. Analysis of these characteristics and subsequent particle recovery supports methods for functional cell sorting, which results in the isolation of cells or their byproducts (such as secreted molecules) with proven function and utility, such as therapeutic utility.

In one embodiment, the single cell analysis can include multiple assays that are essentially part of a single response protocol or multiple assays performed sequentially, such as assays where the results of one assay provide information for a subsequent specific assay. For example, cells known to be associated with a specific disease, such as cells obtained from an individual suffering from the disease, can be analyzed in this system to determine the presence of a specific biomarker, such as an antigen, expressed protein, or disease signature. Following a preliminary analysis to determine whether the single cell exhibits the biomarker, the same identified cell can be re-analyzed for its response to a candidate therapeutic drug, compound, or other cell therapy expected to be effective in inducing a desired response in cells expressing the screened biomarker.

Analyses within these inverted, open microwells can be performed on single particles (including single cells) with rapid, efficient timing, without the need to isolate the particle or its products from a complex mixture. Screening methods include screening single cells for functional properties and selecting essentially viable primary cells for subsequent analysis, immortalization, and/or clonal expansion of cells believed to possess useful properties. Cellular components such as DNA, RNA, and proteins can also be isolated from these primary cells.

This approach enables the identification of desired particle reactions and interactions early in the development process. This early understanding of cellular function ultimately enables the development of useful biomaterials based on a better understanding of the material's potential for success and reduces the number of potential candidates earlier in the development process. Multiple, rapid assays can be performed on the same particle, such as a single cell, leading to faster progress in the discovery process.

The inverted open microwell system described herein facilitates the precise delivery of single cells to or removal from microwells, and the precise manipulation of cells and other particles in microchannels and specific microwells, the delivery of reagents, buffers, markers, etc., including other cells for cell-cell interactions. Multiple characteristics of single cells can be evaluated while retaining their activity in the open microwells for optional subsequent testing, immortalization, expansion, etc. Particles (including the original single cells) can be recovered in a very short time frame, using minimal reagents and recovering cells and products in a substantially viable and useful state. In particular, the deposition of one or more particles (including living cells) at the fluid/air interface of the open microwell allows for precise particle-particle interactions that can be effectively monitored and rapidly screened to identify candidate particles such as cells, facilitating subsequent analysis and/or clonal expansion in the microwells.

Surprisingly, when using appropriate geometric constraints and fluid conditions, the fluid filling the inverted open micropores 102 having the lower end 108 open to the air is retained without leaking from the lower open end. In addition, during the fluid wash, the deposited particles 128 are unexpectedly retained in the meniscus 122, while other particles are placed in a position close to the first and analytical procedures. The open micropores act as "microcentrifuges" that can transfer reagents to the deposited particles for rapid analysis, washing, and subsequent analysis of the deposited particles without loss. For example, after analysis and when recovered from the micropores, the cells in the micropores can maintain good activity. For example, they can be recovered from the micropores onto a substrate such as a microtiter plate for, for example, immortalization and/or amplification. In one embodiment, selected single cells can be incubated and amplified in the micropores.

The present invention provides methods and structures for implementing an inverted microwell system comprising a microwell having an upper end open to a microchannel. In one embodiment, the microwell can be sealed at a lower end, preferably using a transparent material such as glass or a transparent polymer to allow viewing of the contents of the microwell. In another embodiment, the microwell is open at a lower end to the atmosphere (e.g., air or other gas) outside the device.

For example, one or more particles can be delivered to the micropores by sedimentation, controlled by cell density, fluid velocity, loading time, and optionally dielectrophoretic forces. The dielectrophoretic forces can be generated by electrodes connected to an appropriate AC voltage and arranged in a microchannel, which is positioned above a micropore that is open to the microchannel and in fluid communication. Also provided herein are methods for effectively aggregating and interacting particles, such as single cells, to generate dielectrophoretic forces that can manipulate particles to desired locations within the micropores, such as by electrodes embedded in open micropores. The methods provide a high-throughput analytical system that uses minimal reagents and allows for high-throughput retrieval of active particles (including cells and cell products) after analysis.

The present invention also provides methods and structures for precisely controlling and sorting particles (including cells and non-cellular particles) to effectively deliver particles to the micropores of the inverted open micropore system. Specific embodiments include configuring electrodes and electrode pairs in microchannels to enable particles to move with little or no damage to the activity of biological materials such as cells; configuring electrodes and electrode pairs in microchannels and close to micropores to allow controlled access of desired particles to the micropores and effectively close the micropores and drive away unwanted particles; and patterns of electrodes and electrode pairs in the micropores to detect and selectively control the position of particles as they enter and pass through the micropores, for example, depositing on the meniscus of the fluid.

The inverted open microwell system includes one ( FIG. 1 ) or more ( FIG. 3 ) microwells, each of which is in fluid communication with one or more microchannels to transfer fluids and particles to the one or more microwells. The microchannels are typically disposed above the microwells, with the microwells being open to the fluid microchannel at the upper end 106 of the microwell and open to the atmosphere (e.g., air or other gas) outside the device at the lower end 108, allowing for control of atmospheric properties (e.g., gas composition, humidity, temperature, and the absence of contaminants). The microwell has a vertical axis 110 (e.g., a central vertical axis) extending between the upper end 106 and the lower end 108 of the microwell 102.

In one embodiment, the vertical walls 112 of the microwell 102 are at least partially formed from a dielectric material 114. The vertical walls can also be at least partially formed from one or more electrodes integrated with the dielectric material, for example, in a laminate forming the microwell in a layered structure perpendicular to the vertical axis of the well, such as shown in FIG2 .

The fluid injected into the microchannel 104 fills the micropore 102 due to capillary action, while surface tension holds the fluid within the open micropore, forming a meniscus 122 at the lower open end 108 of the micropore at the air-fluid interface. The microchannel surface and the micropore surface can be coated with materials having opposite hydrophilic or hydrophobic properties. For example, the micropore can have a hydrophilic coating, while the microchannel surface near the open end of the micropore has a hydrophobic coating, or vice versa.

Typically, particle movement within microchannels and placement of particles within specific microwells is accomplished by limiting dilution, sedimentation, electromagnetic forces, gravity, and combinations thereof. In one embodiment, controllable manipulation of particles within the inverted open microwell system comprises powering one or more electrode arrays appropriately positioned within the microwells and electrodes appropriately positioned within the microchannels. Examples of electrode configurations are shown in Figures 2 and 9-13.

Method disclosed herein includes the method for screening single cell or small cell group, and described single cell or small cell group include the accurate polymer of concrete particle.Usually provide the analysis of single cell to identify the cell that can, for example, produce specific response for the biomaterial of increase, wherein said biomaterial can be different cells, part of cell, protein, nucleic acid molecule, medicine, antibody, enzyme etc. The generation of accurate polymer allows the accurate arrangement of cell, and this accurate arrangement can induce required response together and/or just be used together for its specific characteristic, ability or function to be analyzed.This system makes it possible that for example multiple particles are arranged in polymer, directly contact or approach the arrangement of the cell that carries out functional contact.This cell arrangement in accurate polymer makes it possible that the interaction of particle and particle is detected quickly and efficiently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of an inverted open microwell system showing cells deposited on the meniscus of an open microwell with fluid flowing in the microchannel and particles being transferred from the microchannel to the microwell by gravity.

FIG2 is a cross-sectional view showing a 3-electrode configuration in an inverted open microwell system.

3 is a schematic diagram showing an inverted open microwell system comprising a plurality of microwells connected to a fluidic system for providing fluid into microchannels and an imaging system for supporting optical detection of the contents of the inverted open microwells.

FIG4 is a schematic diagram showing the recovery of microwell contents onto a microtiter plate.

5 is a schematic diagram showing the recovery of microwell contents from a system comprising a plurality of open microwells onto a recovery substrate.

Figure 6 shows photographs of the meniscus of an inverted open microwell observed using an inverted fluorescence microscope, showing: (A) K562 cells randomly arranged on the meniscus during sedimentation when no electric field was applied to aggregate the cells, and (B) K562 cells manipulated by electromagnetic forces to the central perpendicular axis of the microwell during sedimentation and deposited as a cell aggregate near the center of the meniscus.

7 includes a graph and a series of photographs showing the decrease in biomarker fluorescence signal intensity, which is used to measure calcein uptake in active target cells after individual single cells were exposed to activated T lymphocytes for 20 minutes to induce single cell lysis.

Figure 8 is a series of photographs (A, B, C) showing the clonal expansion of single K562 cells recovered from open microwells after dielectrophoretic localization and transfer to V-shaped microtiter plates for 5 days (A = 0 day; B = 3 days; C = 5 days). (D) The graph shows the increase in relative cell number over a 6-day period.

9 is a schematic diagram showing a top view of a representative arrangement of electrodes in a microchannel and in a microwell for controlling dielectrophoretic movement of particles in a microchannel and in a microwell of an inverted open microwell system.

Figure 10 is a cross-sectional view of the schematic diagram of Figure 9 through line A-A', showing the arrangement of electrodes in the microchannel.

Figure 11 is a cross-sectional view of the schematic diagram of Figure 9 through line B-B', showing the arrangement of electrodes in the microchannel.

Figure 12 shows an electrode array centered at the microwell location, viewed through the top of an inverted open microwell system, illustrating an electrode arrangement in a microchannel for trapping particles in the channel at a minimum potential (Δ). This arrangement is most suitable when there is no fluid flow in the channel.

Figure 13 shows an electrode array viewed through the top of an inverted open microwell system, centered upstream of the microwell, showing the arrangement of electrodes in a microchannel used to manipulate the movement of particles (●) toward a minimum potential (Δ), where the minimum potential is established by a specific force applied by the electrodes.

14 is a cross-sectional view of one embodiment showing alternative electrode arrangements in a microchannel, including electrodes disposed along the top of the microchannel.

Figure 15 shows an electrode array shown through the top of an inverted open microwell system, centered upstream of the microwell, and showing the arrangement of electrodes in the microchannel for manipulating particles (●) along F-F’, according to some embodiments.

Figure 16 shows an electrode array shown through the top of an inverted open microwell system, centered upstream of the microwell, and showing the arrangement of electrodes in the microchannel for manipulating particles (●) along F-F’, according to some embodiments.

Detailed description of the preferred embodiment

A. Definition

The following terms and phrases are intended to have the definitions shown below:

Micropores as used herein refer to pores with micron-sized dimensions (less than 1000 microns), including height, cross-sectional area, such as diameter if the micropore is tubular; and volume.

As used herein, microchannel refers to a channel that provides fluid to the micropores and has a cross-sectional area of micrometer size (less than 1000 micrometers).

As used herein, particles include any particle that can be delivered, manipulated, reacted, or analyzed within the microwells of the disclosed inverted open microwell system. The particle can be a cell or cell portion, a microorganism, a biomolecule (e.g., a protein, polynucleotide, antibody, enzyme), or a substrate (e.g., a polymeric particle that can be coated with a reactive substance (e.g., an antigen encapsulated into a spherical shape)).

Meniscus, as used herein, refers to the air/fluid interface formed at the lower end of the inverted open microwell due to surface tension.

As used herein, an electrode is a conductive material, for example, a metal such as gold, copper, nickel-gold, etc. Preferably, the electrode is formed of high-purity gold.

The dielectric material used herein is an electrically insulating substrate. Preferred dielectric materials used in the inverted open microporous system include polyimides such as and

Dielectrophoresis, as used herein, is the force exerted on a particle when it is subjected to an inhomogeneous electric field.

"A" means at least one.

"Majority" means two or more.

"Micro" means having at least one dimension less than 1000 microns.

"Comprises" or "comprising" means including at least the recited elements or steps and is open to inclusion of additional elements or steps.

B. Abbreviations

The following abbreviations are used as indicated:

DEP stands for dielectrophoresis

ASC stands for antibody secreting cell

CTL stands for cytotoxic lymphocyte

NK stands for Natural Killer Cells

LCL stands for lymphoblastoid cell line

ADCC stands for antibody-dependent cellular cytotoxicity

CMC refers to complement-mediated cytotoxicity

PCB refers to printed circuit board

C. Inverted Open Microwell Systems and Devices

The inverted open microwell system comprises a device (100) comprising one ( FIG. 1 ) or more ( FIG. 3 ) microwells, wherein each microwell (102) is in fluid communication with one or more microchannels (104) for transferring fluids and particles to the microwell. The microchannel is typically disposed above the microwell, the microwell being open to the fluid microchannel at an upper end (106) and open to the atmosphere (e.g., air or other gas) outside the device at a lower end (108). The microwell has a vertical axis (110), such as a central axis, extending between the upper and lower ends of the microwell. Fluid injected into the microchannel fills the microwell by capillary action, while surface tension holds the fluid in the open microwell, forming a meniscus (122) at the air/fluid interface.

In this application, the atmosphere outside the device at the lower open end of the microwell is referred to as "air". It should be understood that the atmosphere can be controlled (e.g., in a chamber) to contain a gas other than air, such as a chamber enriched with carbon dioxide, nitrogen, or other desired gas to maintain the particles and/or enable analysis to be performed in the microwell. The chamber can include a system that controls gas composition, humidity, temperature, pressure, and/or other physical parameters and/or maintains the environment in a sterile state.

In one embodiment, a closed chamber is created by placing the substrate 114 of the device on a microtiter plate, with the wells of the microtiter plate aligned with the inverted open microwells. This alignment maintains a gap between the open microwells and the surface of the microtiter plate, thereby separating the contents of the open microwells from the contents of the microtiter plate, such as the fluid contents. For example, the microtiter wells can contain a liquid medium, such as a physiological medium used for cell recovery and cell culture, to receive the contents of the open microwells when they are released. The microtiter wells can include a different medium, such as water or another liquid, to increase the humidity of the closed chamber. The increased humidity of the atmosphere in the closed chamber caused by evaporation of the liquid from the microtiter wells can saturate the vapor pressure within the chamber and prevent evaporation of the liquid from the inverted open microwells within the chamber. In one embodiment, the atmosphere in the closed chamber is fully saturated, such as at or about 100% humidity, to prevent evaporation of the meniscus. To prevent condensation on the substrate surface, the temperature of the substrate 114 can be maintained above the temperature of the humid air contained in the chamber.

The inverted open microwell system can be used to achieve controlled delivery of one or more particles, such as one or more active cells, into the microwells to cause interaction between the delivered particles in the microwells and one or more additional particles delivered into the microwells, and/or to allow high-throughput analysis of biological functions, for example, by inducing a specific reaction in the microwells, analyzing the results of the reaction, and simultaneously identifying and selectively recovering selected particles and/or biological products of the selected particles.

As the wash fluids contained in the channels are changed and the reagents in the open microwells are changed, while the particles disposed on the meniscus are not replaced or lost, the system can be used as a "microcentrifuge." In a specific embodiment, the fluid contained in the microwells is suitable for dielectrophoretic manipulation of the particles. Once the particles are disposed in the microwells, such as on the meniscus, the fluid dielectrophoretic medium can be washed from the microwells and replaced with a medium suitable for the assay or for the growth and amplification of the disposed particles.

The inverted microwell system can provide effective and efficient real-time monitoring for particle function (including the interaction of multiple particles), functional screening and particle (such as living cells) sorting. In specific embodiments, the system can provide high-throughput functional analysis of antibody-secreting cells and rapid identification and selection of high-affinity antibodies, analysis and selection of the lytic activity of desired cytotoxic lymphocytes and natural killer cells, such as by ADCC or CMC assays. The system allows for rapid analysis of the affinity and specificity of molecules (such as monoclonal antibodies) secreted by individual cells, as well as the recovery of identified antibody-secreting cells and/or secreted antibodies, and also allows for the expansion of identified cells in microwells.

To provide optimal functionality, the structural attributes of the device, including relative geometry, coatings, pressures, materials, and the mass of fluids, particles, and reagents to be placed in the microwells, can be optimized for a specific analysis. For example, the coating used on the inner wall of a microwell and the coating used on the inner wall of a microchannel adjacent to the microwell can be designed to have opposite hydrophobic/hydrophilic properties to repel unwanted materials from the well and/or to regulate the entry of desired particles.

The diameter and/or length of the microwell is designed to allow a meniscus to form at the interface between the fluid at the lower end of the microwell and the external environment, such as air, and to allow a particle (a particle) or multiple particles (particles) deposited on the meniscus to be retained with little or no leakage of fluid from the microwell, and to allow fluid and reagent to be exchanged in the microwell while retaining the particles disposed on the meniscus. The width, height and length of the microchannel are designed to create a hydraulic resistance so that under the condition of a specific fluid flow, a pressure is generated in the microchannel, and the pressure is controlled within a range that allows the fluid to fill the microwell while preventing the fluid from leaking out of the bottom of the microwell. These and other features and uses of the inverted open microwell system are described and exemplified herein.

1. Micropores

For example, as shown in FIG1 , the inverted open micropore device 100 includes micropores 102 having dimensions in the micrometer range. The open micropores can be tubular in shape, such as having a circular cross-sectional axis. Other micropore shapes can also be used, such as conical, rectangular, or other geometric shapes. In one embodiment, the micropores are configured such that they extend from a narrow lower end to an upper end of increased width.

1 shows a cross-sectional view of a device 100 including an open microwell 102. The device 100 includes a substrate 114 defining vertical walls 112 of the microwell 102 extending laterally between an upper open end 106 and a lower open end 108 of the open microwell 102.

The substrate 114 may be a moldable plastic, such as polymethyl methacrylate (PMMA), polycarbonate, polyethylene naphthalate (PEN), polyethylene terephthalate (PET), cyclo-olefin polymer, and the like.

Substrate 114 can have a thickness, for example, from about 10 μm to about 500 μm, inclusive, and can be, for example, 12.5 μm, 25 μm, or 50 μm. In some embodiments, particularly those including electrodes, substrate 114 is at least partially formed of a dielectric material, such as polyimide.

2. Electrodes

As shown in Figure 2, the inverted microwell system may include one or more electrodes coupled to a power source that applies a voltage to the electrodes. In various embodiments, electrodes may be disposed within the microwells and microchannels to control the electrical force applied to particles, for example, to push or pull particles within the microchannel toward and/or into or out of the microwell, or to facilitate transport of particles from the entry opening of the microwell to the lower end of the microwell, or any portion thereof.

While the microwell in FIG2 is shown with three electrodes 116, 118, 120, the device 100 may alternatively include more or fewer electrodes. In one embodiment, the microwell is surrounded by at least one annular electrode 116. In another embodiment, a pair of facing electrodes 116, 116A surround the microwell. In one embodiment, the pair of facing electrodes is positioned near the upper end 106 of the open microwell to control the entry of the particle 128 into the microwell and/or control the positioning of the particle along a vertical axis 110, such as a central vertical axis.

Electrodes can also be placed in the microchannels to influence particle transport. For example, Figures 10-11 illustrate possible electrode arrangements in microchannels 155, 156, 153, 154 and in microwells 116, 118. Figures 12-13 illustrate exemplary arrays of electrodes 153-164 arranged in pairs along the microchannels to facilitate particle transport to or from microwell 102.

The electrodes are formed of a conductive material, for example, in the form of a conductive sheet or plate. In some embodiments, the conductive material comprises a biocompatible metal, such as gold, carbon, or aluminum. In the illustrated embodiment, electrodes 116, 118, 120, and 153-164 are embedded in substrate 114 so that adjacent electrodes are separated from each other by portions of substrate 114, for example, in a laminate structure perpendicular to microwell vertical axis 110 (see FIG2 ).

3. Microchannel

For example, as shown in Figures 1, 2, and 3, in an inverted open microwell system 100, 101, the microwell 102 is connected to a microchannel 104 for transporting a fluid 130 that may include one or more particles 128. The microchannel 104 can be connected to one or more microwells 102, such as one or more rows of microwells 102 in the device 100, 101.

The microchannels can be formed from a polymer such as polyimide near the top cover 124, preferably a transparent material such as glass or a suitable plastic such as PMMA, polycarbonate, PEN, PET, or cycloolefin polymer. The formed microchannels can then be adhered to the top of one or more microwells using, for example, a biocompatible adhesive.

The upper end 106 of the microwell is open to the microchannel 104. For example, a pressure differential can be created across the two ends of the microchannel 104 to cause the fluid 130 to flow through the microchannel 104. For example, the microchannel 104 can be connected to a pump, such as a peristaltic pump, or to a compressed air source. By adjusting the pressure differential across the microchannel 104, the flow rate of the fluid 130 in the microchannel can be controlled, thereby controlling the speed at which one or more particles 128 pass through the one or more microwells 102.

4. Multiple microwells or microwell arrays

As shown in Figure 3, the device 101 may include a plurality of micropores 102 arranged in an orderly manner. For example, the device 101 may include a micropore array or a micropore matrix, such as in the form of micropore rows and micropore columns that communicate with one or more microchannels 104. The device 101 can be used to perform multiple parallel operations on multiple micropores 102 at the same time. Although the following discussion relates to a single micropore 102, the described embodiments are applicable to each of the multiple micropores 102, such as can be configured in the device 101 as an array or matrix of micropores 102, and can include micropores 102 and microchannels 104 of different structures. For convenience, the structure of the micropores can be designed, such as to accommodate conversion of traditional systems such as 96-well microtiter plates or 1536-well micro-vial assemblies. This design can also be for specific purposes, such as high throughput.

5. Fabrication of the Inverted Microwell Device

In one example, to form an inverted microwell device, the microwell 102 can be fabricated as a through-hole through a substrate 114 having electrodes 116, 118, and 120 embedded therein. The through-hole can be formed by conventional drilling methods, such as mechanical or laser techniques. A typical fabrication of an inverted microwell system is described in the following example.

In some embodiments, the microwell 102 has a circular or hemispherical cross-section; however, the microwell 102 may have other cross-sectional shapes, such as hexagonal, rectangular, square, or conical. The diameter of the microwell 102 is typically less than 1000 microns and may vary, for example, depending on the diameter of the cells or other particles to be deposited in the microwell and the geometric relationship required to maintain sufficient surface tension to form and maintain the meniscus 122. The diameter of the microwell may be, for example, from about 70 μm to about 150 μm, or, for example, from about 50 μm to about 100 μm. The depth (height) of the microwell is preferably equal to or greater than the diameter and may be, for example, from about 50 μm to 300 μm, such as from about 70 μm to about 200 μm. The ratio of the diameter to the depth (height) may be, for example, about 1:1, 1:1.25, 1:1.5, or 1:2.

In one embodiment, a device was constructed in which the microchannel was 27 mm long, 350 μm wide, and 150 μm high. The micropores had a diameter of 100 μm and a height of approximately 75 μm. No hydrophobic or hydrophilic coatings were used. When the top side of the channel was made of transparent polycarbonate, the polyimide surface in the microchannel and on the bottom side of the device had a moderately hydrophilic behavior, while the roughness of the inner portion of the mechanically drilled micropores was increased, thereby increasing the hydrophilic behavior of the inner portion of the micropores. This difference in surface properties (where the inner portion of the micropores had a higher hydrophilicity than the microchannel and bottom surface) combined with appropriate microchannel and micropore sizes was surprisingly sufficient to allow the fluid from the microchannel to properly fill the micropores without any leakage from the bottom of the open micropores.

6. Hydrophilic/Hydrophobic Surface

To facilitate the introduction and retention of one or more particles 128 within each microwell 102, the devices 100, 101 can be configured so that certain surfaces are hydrophobic while others are hydrophilic. In some embodiments, a surface can be made hydrophobic or hydrophilic by applying a coating to the surface. For example, in embodiments utilizing aqueous fluids, the bottom surface of microchannel 143 and the vertical walls of microwell 112 can be hydrophilic, while the lower surface 142 can be hydrophobic. As another example, in embodiments utilizing grease-based fluids, the bottom surface of microchannel 143 and the vertical walls of microwell 112 can be hydrophobic, while the lower surface 142 can be hydrophilic. Kapton and Polyimide exhibit discontinuous hydrophilic behavior at contact angles in the 20-70 degree range. Kapton and Polyimide surfaces can be rendered more hydrophilic by applying a plasma surface treatment, such as oxygen plasma. Hydrophobic coatings that can be used on Polyimide or Kapton surfaces include FEP, FC-732, or 41-90.

A fluid 130, such as a physiological buffer or culture medium, is injected into the microchannel 104, which is located above one or more micropores 102 and is in fluid communication with the upper open ends of one or more micropores 102. The microscopic size of the micropores 102 and the hydrophobic behavior of their lower open ends 108, in contrast to the hydrophilic nature of the microchannels 104 and the upper ends of the micropores 102, allow the fluid to fill the open-bottomed micropores without leaking out of the lower open ends 108. When using micropores with closed bottoms, a wetting agent, such as ethanol or a mixture of ethanol and water, may be added before adding the physiological medium to aid in filling the micropores. Capillary action and surface tension can hold the fluid 130 in the micropores and form a meniscus 122 at the fluid-air interface, as shown in FIG1 . In one embodiment, the fluid may include a wetting agent to assist in filling the microchannels and/or micropores. Exemplary wetting agents include ethanol, Tween-20, and SDS. Before injecting the cell suspension, the microchannel is properly flushed with a physiological medium such as phosphate buffered saline (PBS) to remove the residue of the wetting agent.

7. Controllable particle delivery and aggregation

7.1 Control Transfer

A fluid containing particles, such as cells, can be delivered to the microchannel 104 at a designed concentration and flow rate to allow a limited number of microparticles, such as a single particle, to be distributed into each microwell 102. Electrodes can be used to transport particles in the microchannel 104, to allow or deny particles 128 from the microchannel 104 to enter the microwell 102, to manipulate one or more particles 128 in the microwell, to cause particles to aggregate or remain at a desired location in the microwell, to induce structural or functional changes in a particle or particles deposited in a microwell, and/or to sense and/or measure the presence, movement, or change of particles in the microwell, or to sense and/or measure the presence of molecules generated by particles in the microwell.

In the embodiments described in Figures 9-11, the flow of fluid in the channel is used to transport the particles of interest from the inlet to a specific microwell. Simultaneously, dielectrophoretic forces can be configured to allow the particles to skip the microwell and follow the microchannel to another microwell. In the embodiments described in Figures 12 and 13, an electrode array is shown. This embodiment does not require fluid flow to transport particles in the microchannel to the microwell, but uses dielectrophoretic forces to achieve the above-mentioned purpose.

7.2 Transport of particles into microchannels in the absence of fluid flow

When precise positioning of each particle is required, precise transport in microchannels is required to deliver the particles precisely to specific microwells. One method of controlling precise delivery is based on a sequence of electrical parameter changes, which can provide very accurate results without relying on fluid flow or similar techniques.

When a particle with a complex dielectric constant is exposed to a spatially varying electric field, it can be propelled by negative dielectrophoresis, depending on the value of the Clausius-Mossotti factor. In this case, the particle will move toward the minimum electric field and be repelled by the maximum electric field.

10 , 11 , 12 , and 13 , electrodes 155 and 156 are positioned above electrodes 153, 154, 157, 158, 159, 160, 161, and 162. Specifically, electrode 155 is positioned above electrodes 153, 157, 159, and 161, while electrode 156 is positioned above electrodes 154, 158, 160, and 162. These electrodes can be used to create an electric field intensity pattern to induce particle (●) 128 to move in a desired direction within microchannel 104.

In one example, each of electrodes 153, 157, 161, and 156 is connected to a sinusoidal voltage source at a specified potential. Each of the facing electrode partners 154, 158, 162, and 155 is connected to a sinusoidal voltage source having the same amplitude as its facing partner but shifted 180 degrees in phase. Electrodes 159 and 160 are connected to ground (G1).

In this embodiment, because electrodes 159 and 160 have the same potential and their thickness creates a groove that can induce a strong minimum electric field, the electric field in microchannel 104 reaches a minimum (Δ) 170 at the distance between electrodes 159 and 160. The electric field in microchannel 104 reaches a maximum when the electrode pairs 153-154, 157-158, 161-162 facing each other are polarized with voltages having a phase difference of 180 degrees. In this case, particles (●) 128 are trapped at the location where the minimum (Δ) 170 between electrodes 159, 160 is surrounded by the location where the electric field reaches its maximum strength.

The position of a trapped particle can change and move. For example, as shown in Figure 13, the position of a trapped particle moves to the left in two stages. In the first stage, the voltage of electrodes 157 and 158, located to the left of the existing static position, is set to ground potential (G2). This creates an electric field with a minimum value (Δ) of the same strength as the electric field between electrodes 159 and 160. In the second stage, electrode 159 is polarized to electrode 161, and electrode 160 is polarized to electrode 162. This polarization creates a strong electric field between electrodes 159 and 160. This electric field pushes the particle trapped in the area closest to the electric field minimum (Δ) and moves it one position to the left.

Therefore, particle displacement obtained in this way does not require a fluid flow.

In another embodiment, as shown in FIG14 , electrodes 155 and 156 are replaced by a single electrode 190 disposed along the top side of the microchannel. Electrode 190 can also be implemented by closing the microchannel at the top with a metallic material or a transparent material with an indium oxide (ITO) coating, such as glass or transparent plastic. This conductive coating provides an electrical connection while maintaining a transparent top cover that does not hinder optical detection or illumination from the top side.

In this example, electrode 190 is connected to ground potential, while each of electrodes 153, 157, and 161 is connected to a sinusoidal voltage source at a specified potential. Each of the facing electrode partners 154, 158, and 162 is connected to a sinusoidal voltage source having the same amplitude as its facing partner but shifted 180 degrees in phase. Electrodes 159 and 160 are connected to a sinusoidal voltage source having the same amplitude as electrodes 153, 157, and 161 but shifted 90 degrees or 270 degrees in phase.

In this embodiment, because electrodes 159 and 160 have the same potential and their thickness creates a groove that can induce a strong minimum electric field, the electric field in microchannel 104 reaches a minimum (Δ) 170 at the distance between electrodes 159 and 160. The electric field in microchannel 104 reaches a maximum when the electrode pairs 153-154, 157-158, 161-162 facing each other are polarized with voltages having a phase difference of 180 degrees. In this case, particles (●) 128 are trapped at the location where the minimum (Δ) 170 between electrodes 159, 160 is surrounded by the location where the electric field reaches its maximum strength.

With this electrode configuration, the position of a trapped particle can be changed and moved, for example, to the left, in two stages. In the first stage, the voltage of electrodes 157 and 158, located to the left of the existing resting position, is set to a sinusoidal voltage source with the same amplitude as electrodes 153, 157, and 161, but shifted by 90 or 270 degrees. This creates an electric field with the same minimum (Δ) strength as the electric field between electrodes 159 and 160. In the second stage, electrode 159 is polarized to become electrode 161, and electrode 160 is polarized to become electrode 162. This polarization creates a strong electric field between electrodes 159 and 160. This electric field pushes the particle trapped in the area closest to the electric field minimum (Δ), moving it one position to the left.

FIG12 shows a structure that can be used to precisely deliver particles in microwell 102 without fluid flow. FIG12 does not show electrode pair 155, 156, but as shown in FIG9 and FIG10, the electrode pair is located above the other electrode pairs. Specifically, electrode 155 is located above electrodes 163, 116, 153, while electrode 156 is located above electrodes 164, 116A, 154.

If electrodes 116 and 116A are polarized to voltages 156 and 155, respectively, while electrodes 153 and 154 have the same ground potential, the particle can be trapped by negative dielectrophoresis at a location (Δ) between 153 and 154. Then, when connected to ground electrodes 116 and 116A while 153 and 154 are polarized to 156 and 155, the particle can be pushed to the electric field minimum (Δ) 170 between 116 and 116A where the micropore is located.

Since electrodes 116, 116A surround the microwell 102 and are connected to ground potential, the presence of electrodes 155 and 156 generates an electric field in the channel below the microwell 102. Following the previous analysis, the particle will now be pushed toward the microwell.

If the particle's sedimentation position in the microwell must be precisely controlled, electrode 118 can be polarized by connecting the electrode to a voltage source having an amplitude substantially smaller than that used to control electrodes 155 and 156 and a phase rotated 90 degrees or 270 degrees relative to the phase of electrodes 155 and 156. This polarization creates a dielectrophoretic force in the microwell that pushes the particle toward the center of the microwell.

If particle transfer in the microwell must be avoided, a different electrode polarization is recommended in the absence of fluid flow. Specifically, assuming a particle is trapped between electrodes 153, 154 connected to ground, particle transfer can be avoided by connecting to grounded electrodes 116, 163, and 164, with electrode 116A polarized to electrode 155, electrode 153 polarized to electrode 156, and electrode 154 polarized to electrode 155. These polarizations can create a temporary particle displacement, causing the particle to move from a position between electrodes 153 and 154 to a position near electrode 116 (not between electrodes 116 and 116A), and ultimately to a stable transfer site between electrodes 163 and 164 where the minimum electric field is located.

In another embodiment, the structure reported in Figure 12 can be used to precisely deliver particles in microwell 102 without fluid flow. As shown in Figure 16, an electrode 190 arranged on the top side of the microchannel replaces the electrode pair 155, 156 and is connected to the ground.

If electrodes 116 and 116A are connected to sinusoidal voltage sources with the same amplitude and phases of 0 and 180 degrees, respectively, while electrodes 153 and 154 are connected to the same sinusoidal voltage with a 90 or 270 degree phase shift, the particle can be trapped by negative dielectrophoresis at a position (Δ) between 153 and 154. Then, when electrodes 116 and 116A are connected to sinusoidal voltages with a 90 or 270 degree phase shift, while 153 and 154 are connected to sinusoidal voltages with the same amplitude and phases of 0 and 180 degrees, respectively, the particle will be pushed to the electric field minimum (Δ) 170 between 116 and 116A where the micropore is located.

Since the electrodes 116, 116A surround the microwell 102 and are connected to the same voltage, the presence of the electrode 190 can induce an electric field in the channel below the microwell 102. Following the previous analysis, the particle will now be pushed towards the microwell.

If the particle's sedimentation position in the microwell must be precisely controlled, electrode 118 can be polarized by connecting it to a voltage source having an amplitude substantially smaller than that used to control electrodes 116 and 116A, and having a phase rotated 90 degrees or 270 degrees relative to the phase of electrodes 116 and 116A. This polarization creates a dielectrophoretic force in the microwell that pushes the particle toward the center of the microwell.

If particle transfer in the micropores must be avoided, a different electrode polarization is recommended in the absence of fluid flow. Specifically, assuming a particle is trapped between electrodes 153 and 154 connected to the same sinusoidal voltage with a 90-degree or 270-degree phase shift, particle transfer can be avoided by connecting electrodes 116, 163, and 164 to ground, while simultaneously connecting electrodes 153, 116A, and 154 to the same sinusoidal voltage with a 0-degree, 90-degree, or 180-degree phase shift, respectively. Electrode 190 remains connected to ground. This polarization creates a temporary particle displacement, causing the particle to move from a position between electrodes 153 and 154 to a position closer to electrode 116 (not between 116 and 116A). Ultimately, by connecting electrode 116 to the same sinusoidal voltage as electrode 153, with a 270-degree phase shift, the particle reaches a stable transfer site between electrodes 163 and 164, where the electric field minimum is located.

The voltage-charged structures proposed in this paper do not rely on the presence of fluid flow for particle displacement and transport. However, fluid flow is beneficial in providing an appropriate cooling structure. These structures can be used to move single cells along microchannels and force them into open micropores, or prevent them from entering open micropores by applying a series of appropriately applied polarization signals.

7.3 Particle Transport in Microchannels and Open Microwells in the Presence of Fluid Flow

Figures 9-11 illustrate a microwell 102 in fluid communication with a microchannel 104, the top of which is enclosed by a surface 124 covering the liquid contained in the microchannel. Particles, such as cells or beads, flowing in the channel fluid are affected by electromagnetic forces, causing particles 128 to be pulled toward or pushed out of the microwell 102. This structure is particularly suitable for operation when there is a significant fluid flow in the channel through which the particles can be transported. For example, see Faenza et al., "Controlled isolation and patterning of K562 leukemia cells using electrically activated microchannels," In: International Conference on Microtechnologies in Medicine and Biology, 2011 (May).

Electrodes are fixed in the structure of the micropores and microchannels to control particle movement. For example, FIG9 shows a micropore 102 surrounded by electrodes 116 shaped in a certain manner so that an opening facing the flow direction from right to left is presented in FIG9 .

The specific embodiments presented in these figures for regulating particle movement include electrodes 116, 118 disposed within microwells 102 and electrodes 153, 154 disposed within microchannels 104. FIG10 shows microwells 102 in fluid communication with microchannels 104, each having a vertical wall 112 formed by a layered structure comprising a dielectric substrate 114 and electrodes. Microchannel electrodes 153-164 are disposed within the microchannels, while electrode pairs 153-154 and 155-156 are disposed near the junction of the microchannel and microwell 102 at the upper open end 108 of the microwell. Microwell electrodes 116 and 118 are disposed near the upper and lower ends of the microwell, respectively.

In a microchannel 104 with fluid flow, particles (●) 128 moving in the microchannel are subject to dielectrophoretic forces if the complex dielectric constant differs from that of the surrounding fluid 130. Particles of interest, such as cells, microbeads, or liposomes, are characterized by negative dielectrophoretic forces that cause them to move toward an electric field minimum (Δ) 170. These particles also have a higher relative density than the surrounding buffer solution. Consequently, they settle under the influence of gravity.

10 and 11 show that particles circulating in microchannel 104 may encounter electrodes 153, 154, 155, and 156 before reaching the microwell and electrode 116. According to the principles of dielectrophoresis, various configurations of applied voltages can cause particles 128 to be captured or expelled by the electric field, as the particles move toward a minimum (Δ) 170 of the electric field and away from a strong electric field.

In one embodiment, electrode 116 is grounded. Electrodes 155 and 156 are polarized by an external power source as a voltage generator. The voltage phase of electrode 155 is rotated 180 degrees relative to the voltage phase of electrode 156. The voltages applied to electrodes 155 and 156 have the same amplitude. The voltages can vary with a sinusoidal signal or a square wave. As a result, a relatively large electric field exists between electrodes 155 and 156.

The polarization of electrodes 153 and 154 will vary depending on the task of trapping or expelling particles. More specifically, in one configuration, referred to as "trapping," electrodes 153 and 154 can be connected to ground. Alternatively, a sinusoidal voltage or square wave can be used to polarize electrodes 153 and 154. When polarized, electrode 153 will be in phase with electrode 156, while electrode 154 will be in phase with electrode 155. This second configuration is referred to as "expelling." The relative positions of the electrodes are shown in Figure 11.

As shown in the electrode configurations of Figures 9-11, when the desired particles 128 flowing in the microchannel 104 are trapped in the micropore 102, electrodes 153 and 154 are set to ground potential. Electrodes 155 and 156 are polarized by an external power supply to form a voltage generator. The voltage phase of electrode 155 is rotated 180 degrees relative to the voltage phase of electrode 156. The voltages applied to electrodes 155 and 156 have the same amplitude. The voltages can be varied with a sinusoidal signal or a square wave. As a result, a relatively large electric field exists between the two electrodes 155 and 156.

In this case, a "trapping" situation occurs because the electric field reaches a strong minimum at location 170 between electrodes 153 and 154. The retention region in the channel is dominated by a strong electric field, pushing the particle toward the minimum (Δ) electric field region. Electrodes 153 and 154, as well as all other electrodes, have a thickness of approximately 5 to 30 microns. This creates a region (Δ) 170 where the electric field reaches a minimum and the particle is approximately the same size as the surrounding electrodes.

Region (Δ) 170 is aligned with the opening of micropore 102. Particles trapped in minimum region (Δ) 170 are pushed horizontally toward the opening of the micropore by the fluid flowing in the microchannel. The distance between region (Δ) 170 and the opening of micropore 102 is set to a value as small as possible based on manufacturing technology. If standard printed circuit board technology is used, a typical distance is about 20 to 100 microns. Since electrode 116 surrounds micropore 102 and is connected to the ground, the presence of electrodes 155 and 156 will generate an electric field in the channel below micropore 102. Based on the previous analysis, the particles will now be pushed toward the micropore.

If the sedimentation position of the particles in the microwell must be precisely controlled, electrode 118 can be polarized by connecting it to a voltage source having an amplitude substantially smaller than that used to control electrodes 155 and 156 and a phase rotated 90 degrees or 270 degrees relative to the phase of electrodes 155 and 156. This polarization creates a dielectrophoretic force in the microwell that pushes the particles toward the center of the microwell.

If the particle is flowing in microchannel 104 and is not intended to enter the microwell, the desired effect can be achieved by polarizing electrodes 153 and 154 in the "ejection" configuration. In this case, the minimum electric field is placed in the middle of microchannel 104. In this position, the particle is exposed to the fluid flow and will not be trapped in microwell 102 if the spacing between microwell 102 and electrodes 153 and 154 is small.

In another embodiment, the movement of particles can be controlled by first trapping the particle between electrodes 153 and 154, and then programming a "loading" or "repelling" configuration. This programming can be achieved by appropriately setting the polarization of electrodes 153, 154, and 116, while maintaining electrode 155 constantly connected to a sinusoidal voltage and electrode 156 connected to the same sinusoidal voltage, shifted 180 degrees, as electrode 155. To first trap the particle, electrodes 153 and 154 are grounded, and electrode 116 is shifted 90 degrees and connected to the same sinusoidal voltage as electrode 155. By configuring the fluid flow from electrodes 153 and 154 to the micropore, a sufficiently low flow rate can be found to trap the particle in the minimum electric field between electrodes 153 and 154 with a drag force that is insufficient to prevent the particle from moving forward and that opposes the electric field established between electrode 116 on one side and electrodes 155 and 156 on the other side. If the particle needs to be transferred to the micropore, electrode 116 is connected to ground. The drag force will cause the particle to move toward the micropore and then enter it. Conversely, if the particle must move toward and around the micropore, electrodes 153 and 116 are connected to the same sinusoidal signal as electrode 156, while electrode 154 is connected to the same sinusoidal voltage as electrode 155. In this configuration, the particle first moves upward and then toward the portion of electrode 116 below electrode 156, where the electric field minimum occurs. In this way, the micropore is circumvented and the particle is not transferred. Now, suppose there is a second micropore along the channel, with the same configuration as shown in Figure 9. A new set of electrodes 153 and 154 can be connected to ground, and electrode 116 of the first micropore can be returned to its initial voltage. In this way, the electric field minimum will be located between the electrode pair 153 and 154 of the second micropore, and the particle will be positioned between these electrodes. This scheme can be repeated for several micropores arranged along the same microchannel.

7.4 Detection of Particles in Microchannels

When the electrodes shown in Figures 9-14 in this configuration manipulate particles in the microchannel 104 to deliver the particles to the target microwell, the electrodes can produce an effect of aligning the particles along the axis F-F' shown in Figures 15-16. In addition, the particles can be pushed vertically toward the bottom side of the microchannel. Assuming that there is a continuous laminar fluid in the microchannel and has the effect of pulling the particles along the F-F' direction, the precise positioning of the particles along the predetermined axis and at a precise vertical position makes them suitable for optical detection or impedance measurement. For example, Duqi et al., 2011 (May), "Automated isolation of a programmable number of cells into microwells using DEP forces and optical detection", an International Conference on Microtechnologies in Medicine and Biology, has demonstrated the benefits of using dielectrophoresis to achieve cell focusing, thereby improving optical detection and enhancing cell delivery in microwells.

In one embodiment, as shown in FIG15 , a structure comprising four electrodes 170, 171, 172, 173 is strategically positioned downstream of the manipulation electrodes 153, 154. The electrodes are designed to allow particles to move in the F-F’ direction and through the gap between the electrodes 170-171 and 172-173. Electrodes 171, 173 are connected to an excitation signal 188 represented by an AC voltage, while electrodes 170, 172 are connected to a readout circuit 186 that amplifies the output currents 180 and 182 and provides a terminal output signal 184 proportional to the difference or ratio between the signals 180, 182. This can be accomplished using known circuit schemes. The presence of the upstream electrodes 153, 154, 155, 156 has the effect of aligning the cells along an axis where the signal-to-noise ratio generated by the channels of a single cell on the output signal 184 is maximized.

In another embodiment, as shown in FIG16 , after individual cells are appropriately arranged and aligned by electrodes 153, 154, 155, 156, single cell detection is performed using a structure comprising three electrodes 174, 175, 176. Electrode 175 is connected to an excitation signal 188 represented by an AC voltage, while electrodes 174, 176 are connected to a readout circuit 186 that amplifies output currents 180 and 182 and provides a terminal output signal 184 proportional to the difference or ratio between signals 180, 182. The presence of upstream electrodes 153, 154, 155, 156 has the effect of aligning the cells along the axis where the signal-to-noise ratio generated by the channels of the individual cells in the output signal 184 is maximized.

In one embodiment, after the single cells are properly arranged and aligned by electrodes 153, 154, 155, and 156, the single cells are detected by optical detection under either fluorescence or normal lighting conditions, by placing an optical excitation and detection system on the top or bottom side of the microwell array and adjusting the alignment of the optical system along the axis F-F'.

In a specific embodiment, once a particle is detected according to one of the aforementioned methods, the structure represented by electrodes 116, 153, 154, 155, 156, 116, 118 and microwell 102 and reported in Figures 9-11 is replicated and arranged downstream of the detection area or electrode. The control of these electrodes is based on the sensed signal 184 obtained by optical or electrical measurement, which is performed in a manner that can selectively pull a single cell into or out of the target microwell upon detection. This embodiment enables the precise positioning of a single cell or a predetermined number of cells in a microwell.

7.5 Particle Aggregation in Micropores

When the particle 128 enters the microwell 102, gravity can cause the particle 128 to fall through the microwell 102 toward the meniscus 122. To ensure that the particle 128 remains substantially in the center of the microwell 102, the microwell electrodes 116, 118, 120 can be powered to push the particle 128 to the center of the diameter of the microwell 102. For example, the voltage applied to the electrodes 116, 118, 120 can be controlled to generate a substantially zero or substantially vertical electric field at or near the vertical axis of symmetry of the microwell 102. This causes the particle 128 to be pushed toward the center of the microwell 102, causing the particle 128 to fall along the central axis of the microwell and be deposited at or near the center of the meniscus 122. Gravity or electromagnetic forces, or a combination of these, can assist in the particle's descent.

In some embodiments, when micropore electrodes 116, 118, and 120 are ring electrodes, the micropores may include paired electrodes, such as facing electrodes, to facilitate particle manipulation and movement within the micropores. Furthermore, the micropores may include additional electrodes, such as electrodes for specific analyses or reactions, such as sensing electrodes and/or measuring electrodes for sensing particles, cell products, and cell changes. For example, the sensing electrodes and/or measuring electrodes may be used to measure changes in electrical signals such as impedance, optical signals, and polarity.

Even after the electromagnetic force (e.g., the voltage applied to the electrodes) is discontinued, the particles 128 deposited on the meniscus 122 can remain stably disposed on the meniscus 122. Exemplary methods of applying voltage to electrodes to manipulate particles using dielectrophoresis are described in published U.S. Patent Application No. 2009/0288963, which is incorporated herein by reference in its entirety.

When particle 128 has entered micropore 102, electrodes 153 and 154 disposed on the bottom side of the microchannel near the junction of micropore 102 and microchannel 104 can be powered to prevent other particles flowing in the microchannel from entering the micropore. For example, the phase of the voltage applied to electrodes 153 and 154 can be controlled to drive away other particles in the fluid, particularly to close micropore 102. This can occur after the particle falls through electrode 116, for example, when electrodes 118 and 120 are used to concentrate the particle at the center of the micropore, allowing the particle to fall down the length of the micropore by gravity and remain substantially in the center even in the absence of an applied electromagnetic force.

In some embodiments, two or more particles 128 are allowed to enter microwell 102 and arrange so as to urge the particles into contact or proximity with one another. For example, after the first particle 128 is arranged on meniscus 122, the voltage applied to electrodes 153 and 154 can be removed, while microwell 102 is opened to receive one or more additional particles from microchannel 104. As discussed above, as one or more additional particles enter microwell 102 from microchannel 104, the additional particles 128 can be concentrated in the center of microwell 102 by applying voltage to electrodes 118, 120. The additional particles 128 fall along the vertical axis of symmetry of the microwell until they are arranged in contact or proximity with particles 128 on meniscus 122. In alternative embodiments, all particles are introduced into the microwell at substantially the same time, and power is applied to electrodes 116, 118, 120 to aggregate the particles into a cell aggregate in the center of the microwell, simultaneously forming a cluster of deposited cells on the meniscus.

8. Observation of deposited cells under a microscope

Due to the open nature of the microwell 102, the particle 128 can be observed, for example, using a microscope, camera, or other optical device, as it rests on the meniscus 122 at the fluid-air interface at the lower end 108 of the microwell 102. The particle 128 can be observed from the upper end 106 of the microwell 102, such as through the microchannel 104 and a top cover made of a transparent material such as glass or plastic, or from the lower end 108 of the microwell, for example, where the view is unobstructed.

9. Stable stay on the meniscus

Surprisingly, when the electric field is deactivated, particles, including living cells, can be stably retained at the meniscus while fluid continues to flow through the microchannel, thereby entering the microwell. It was surprisingly found that, assuming the microwell is of appropriate size, fluid washing or changing the culture medium in the absence of an electric field does not dislodge the cells from the meniscus. This stable retention allows for the removal of harmful liquids, such as buffers and reagents required for DEP procedures, and for the replacement of fresh buffers and culture media more suitable for the maintenance and viability of the bioparticles deposited in the microwells and/or for a particular analytical method.

If the shear stress caused by the horizontal component of the fluid velocity at the bottom level of the microwell is relatively low or absent, stable retention on the meniscus can be achieved. By varying the relative ratio of the diameter to the depth of the closed microwell, the horizontal component of the fluid velocity is also varied, as explained in Han et al., 2010, “Integration of single oocyte trapping, in vitro fertilization and embryo culture in a microwell-structured microfluidic device,” Lab on a Chip, vol. 10, no. 21, pp. 2848-2854. Similar performance is observed in inverted open microwells that feature a meniscus between the air and fluid on the bottom side.

In one embodiment, the micropores are sized to ensure that the depth of the micropores is equal to or greater than the diameter. For example, for a micropore with a diameter of 75 microns, a depth of 100 microns or greater is contemplated. For an average fluid velocity of 2.5 mm/sec in the microchannel, this micropore size maintains a horizontal fluid velocity of less than 1 micron/sec.

In this inverted open microwell system, nutrients from the buffer or culture medium flowing in the microchannels can be continuously supplied, while catabolites produced by the cells can be removed, helping to maintain the vitality of the cells or other particles disposed in the microwells. Furthermore, the lower open ends of the microwells, exposed to the atmosphere outside the device, enable gas exchange, which helps maintain the health and vitality of the particle or particles contained in the open microwells.

Surprisingly, the combination of features in the inverted open microwell system, including small volume, stable residence, efficient exchange of buffer and culture medium, efficient gas exchange, and the ability to manipulate and aggregate cells to desired locations using minimal electromagnetic forces, enables the effective use of this system not only to evaluate the characteristics of single cells, but also to maintain, reuse and recover original cells for clonal expansion.

10. Recovery of micropore contents

In one embodiment, the micropore contents can be recovered, for example, by disrupting the surface tension of the meniscus. For example, disrupting the surface tension of the meniscus can be achieved by applying a pressure pulse, such as from a pump 132 or a compressed air source, to the microchannel 104, wherein the pressure pulse has a pressure range of 0.5 bars to 3 bars and a pulse duration range of 1 millisecond to 100 milliseconds. For example, the micropore contents can be released as droplets containing single particles, cells, or particle or cell clusters as in the micropore fluid into a suitable container 126 (Figures 4, 5) located below the micropore 102, such as a reservoir, a microtiter plate, a collection bottle system, a capture surface, etc. In one embodiment, the container 126 can include a filter or membrane for separating one or more particles from the fluid recovered from the micropore 102.

In one embodiment, the fluid surrounding the particles 128 in the microwells can be concentrated by evaporation before being recovered from the microwells. For example, replacing the fluid in the microchannels with air or a suitable gas can cause the volume in the microwells to decrease because evaporation will occur at the lower ends 108 of the microwells that are open to the air or gas. Applying pressure to the air/gas in the microchannels can cause the concentrated fluid in the microwells to be released into the one or more containers.

In one embodiment, at or near the air/fluid interface at the lower end of the micropore, the micropore comprises a semi-permeable membrane that can retain one or more particles. In this embodiment, fluid can be recovered from each micropore 102 into one or more containers 126.

Where the device 101 comprises a plurality of microwells, the contents of the plurality of wells can be transferred to a set of matching, parallel containers, such as into the wells of a microtiter plate.

During the transfer of cells from the microwells, when the pressure pulse is applied, other cells or particles can be trapped in the microchannel and transferred along with one or more cells in the microwell. To prevent this problem, a bovine serum albumin (BSA) coating or other anti-stiction coating, such as an organic passivation layer, such as a fluorinated fatty acid self-assembled monolayer (SAM) or alkylhalosilane, can be added to the microchannel before use.

Examples

The present invention may be more fully understood by reference to the following examples, which are provided to describe specific exemplary embodiments of the present invention and are not intended to limit the scope of the present invention.

Example 1

Geometric analysis of the fabricated microchannels or microwells

1.1 Production

Devices containing multiple open microwells were created by drilling through-holes from a multilayer flexible printed circuit board (PCB) substrate. The microwell dimensions were varied to analyze the effectiveness of specific microwell geometries. In this study, the diameters of the drilled holes ranged from 70 μm to 150 μm, the PCB thickness ranged from 75 μm to 350 μm, and each device contained either a 6×6 or 8×8 matrix of wells. To directly interface the open microwell array with a standard microtiter plate and facilitate cell retrieval and transfer into microtiter wells, the preferred apparatus was prepared as shown in Table 1 below.

On the top side of the plurality of microwells 102, microchannels 104 with thicknesses ranging from 30 μm to 200 μm and widths ranging from 200 μm to 1 mm were fabricated from polyimide and positioned above the microwell upper ends 106. A channel cover 124 was formed by adhering a 750 μm thick transparent polycarbonate to the top of the microchannels.

Table 1 Microwell array characteristics

Number of micropores Arrange x columns Hole-hole and channel-channel spacing twenty four 6x4 18mm 96 12x8 9mm 384 24x16 4.5mm 1536 48x32 2.25mm

The microchannel walls were made of polyimide and bonded to the top cover using an adhesive that was properly cured at 70°C for 2 hours to overnight to ensure biocompatibility of the adhesive, or an adhesive that was laminated at room temperature and then coated with 1 mM BSA to ensure biocompatibility.

In an alternative embodiment, a 55 μm thick photopolymer film (Ordyl SY550, Elga Europe) was applied to a glass top cover to create microchannels before being applied to a bottom flexible PCB containing drilled microholes. Holes were formed in the top cover to provide input and output fluid connections or to embed reservoirs. The holes had a diameter of approximately 0.45 mm.

In one study, a fluid or fluid containing cells and particles was injected into each microchannel using a peristaltic pump (Watson Marlow 101U/R) connected as shown in Figure 5 and a syringe pump connected to the inlet of another microchannel (KDS-210, KD Scientific, Holliston, MA). The fluid flow rate varied from 1 μL/hour to 20 μL/minute, depending on the cross-section of the channel and the specific operation to be performed on the particles. The capillary forces acting on the microwells prevented the fluid from leaking out of the lower end of the microwells. In some cases, a hydrophobic coating (FC-732) can be added to the bottom surface of the device to increase the resistance to fluid leakage. As a result, the fluid system allowed the fluid in the microchannel to unexpectedly fill the microwells without leaking out of the lower end. 1.2 Analysis of cells deposited by gravity

Live K562 cells (immortalized human myeloid leukemia cells) were suspended in a physiological solution (0.9% w/v NaCl or PBS) at a concentration of 1.6 × 106 cells/mL and injected into the microwells of an inverted microchannel device using a peristaltic pump. The microchannels were 600 μm wide and 55 μm high, with each microwell having a diameter of 70 μm.

The input pump is set to perform a cyclic operation, wherein each cycle includes two stages. During stage 1, the fluid is activated at a speed of 9 microliters per minute, while during stage 2, the pump is removed for 1 minute and 30 seconds. During stage 1, cells flow in the channel along a random trajectory. At the beginning of stage 2, cells are fixed in the channel in a randomly positioned manner. During stage 2, a certain number of cells are allowed to enter each micropore by sedimentation.

An inverted microscope was used to examine the contents of each well and detect the possible presence of cells in the microwells as a result of sedimentation. The results showed that the distribution of cells into the microwells can be controlled by adjusting the cell concentration. Under the conditions described here, we obtained the distribution reported in Table 2.

Table 2 Cell loading efficiency in microwells (41 samples in total)

Number of cells loaded frequency 0 51% 1 42% 2+ 7%

Results from the loading phase showed that single or multiple cells could settle in the microwells, reach the air-fluid interface at the lower open end, and become trapped and survive there without disrupting the surface tension of the meniscus. In those microwells, no forces other than gravity were applied to the cells during their descent into the microwells, and the locations at which the cells settled onto the fluid meniscus were random (see Figure 6A).

1.3 Analysis of cells deposited by aggregation and gravity

As shown in Figure 2, using two or more electrodes, a dielectrophoretic force can be generated that acts on particles in a microwell and, during particle sedimentation, steers the particles downward along the vertical axis of the well from the entrance of the microchannel 104 at the upper open end 106 of the microwell to the lower open end 108. In each pair of adjacent electrodes 116, 118, 120, the first electrode is connected to a sinusoidal signal, while the second electrode is connected to ground or to the same sinusoidal signal with a 180-degree phase rotation.

An inverted microwell system was constructed and used in this study, and the inverted microwell system has the system features shown in Figure 2. The diameter of the microwell 102 is between 100 μm and 120 μm, and the vertical gap between each pair of ring electrodes 116, 118, and 120 is 50 μm.

K562 cells were suspended in a mixture of PBS and glycerol (300 mM) at a ratio of 1:9, and electrodes 116 and 120 were connected to the same sinusoidal voltage with a typical frequency between 80 kHz and 100 kHz. The amplitude of the applied voltage varied from 3.4 V to 15 V. Electrode 118 was grounded. As four cells were loaded into the microwell while the electric field was kept active, each cell was forced to align along the central axis of the microwell. Once it reached the air/fluid meniscus at the lower end of the microwell, each cell was centered and in contact with one or more previously loaded cells. This aggregation resulted from a combination of the vertical gravitational force and the horizontal component of the dielectrophoretic force.

The open microwell is observed under fluorescence conditions using an inverted microscope 140. When the electrodes in the well are properly polarized, one or more cells are aggregated to the central axis of the microwell. During descent along the central axis, the cells thus aligned are each deposited at the center of the microwell meniscus 122, forming a polymer at the air-fluid interface and resulting in cell-to-cell contact.

K562 cells were labeled with fluorescent dye and observed using an inverted microscope. As shown in Figure 6, while cells randomly deposited on the meniscus of the microwell by gravity when no electric field was applied (6A), the same cells deposited by a combination of vertical gravity and dielectrophoretic forces formed a particle aggregate deposited in the center of the meniscus (6B). 1.4 Analysis of microbeads deposited by aggregation and gravity

Polystyrene microbeads with a diameter of 10-25 μm were suspended in deionized water or glycerol at a concentration of 22.5 mM and transferred by gravity into microwells with a diameter of approximately 80 μm. Electrodes 116 and 118 were connected to the same sinusoidal voltage with a frequency of 100 kHz. The electrodes were grounded. The aggregation of the two bead aggregates was analyzed, and the results are reported in Table 3, showing the average bead-to-bead distance and the relative number of bead-to-bead contacts established based on the signal amplitude.

Table 3

Voltage amplitude (V) Average distance (μm) Bead-bead contact ratio 2 7.2 33% 4 2.2 90%

1.5 On-chip labeling of cells captured on the meniscus

The effect of changes in the flow in the microchannel on the fluid surrounding cells trapped on the meniscus at the distal end of the open microwell was analyzed to determine the ability of the inverted open microwell system to provide single cell centrifugation.A calcein staining protocol was performed on K562 cells.

K562 cells were delivered to an inverted open microwell by limiting dilution and sedimentation. After delivery of the cells to the meniscus, the microchannel was flushed with PBS flowing through it for 5 minutes. A buffer containing calcein (1 mM in 0.9% w/v NaCl) was flowed through the microchannel at a constant flow rate of 9 μl/min for 40 minutes. Calcein is a tracer molecule that becomes fluorescent when taken up by cells. Figure 4 shows the dynamics of fluorescence intensity monitored for a single cell in the microwell.

Cell staining was effective, demonstrating diffusion of calcein from the microchannels into the microwells and the stably retained cells. The cells were surprisingly retained on the meniscus after washing and were viable, as demonstrated by calcein uptake. By monitoring calcein uptake in individual cells, different uptake characteristics could be observed. Typically, maximum uptake was achieved after 30-40 minutes. Fluorescence intensities are shown in Table 4.

Table 4

Example 2

2.1 Recovery of K562 cells after delivery and DEP aggregation

Analyzing single cell function using the inverted open microwell system can recover cells that have been determined to possess desired properties. For example, after capturing a single cell and/or particle in each of a plurality of microwells (e.g., a microwell array), the cell function can be analyzed using one or more bioassays. Once a particular cell is determined to possess one or more desired properties or functions, the contents of each microwell can be recovered and transferred to a substrate, such as a microtiter plate.

A device characterized by the features shown in FIG5 was developed to enable the retrieval of cells from the inverted opening. To release the contents of the microwells, a pressurized filtered gas, such as nitrogen or air, is injected into the microchannel 104 in a controlled manner. For example, a pressure pulse of approximately 1 bar is applied by connecting the input of a normally closed electrovalve 172.

An electronic system connected to the control input of the electrovalve 172 generates a voltage or current pulse having a duration of approximately 5 milliseconds. To generate the pulse, the compressed gas enters the microchannel 104 via conduits connected to the microchannel input and output. As a result, a fluid droplet 138 is expelled from each microwell 102 and drips onto a container 126, which can be represented by a capture surface in some experiments or a microtiter plate in other experiments, which is placed below the microwell array and appropriately aligned. One or more particles and/or cells present in the microwell are transferred to the capture surface in the form of fluid droplets 138.

During the transfer of cells from the microwells, when a pressure pulse is applied, other cells or particles can be captured in the microchannel and transferred along with one or more cells in the microwell. To prevent this problem, a bovine serum albumin (BSA) coating was injected into the microchannel for 30 minutes before use to form a protein self-assembled monolayer. Before cell recovery, the microchannel was rinsed with PBS for 20 minutes. All cells were removed from the microchannel so that only the cells contained in the microwells were transferred to the recovery container. 2.2 Viability and growth of recovered cells

One function of the inverted microwell system is to assess the function of a single cell or multiple cells and to recover or expand viable cells that have been determined to have desired functional properties from the microwell. For example, cell viability can be assessed by monitoring the growth and expansion of recovered single cells or small cell clusters under activating or deactivating electric fields and DEP for different time periods, e.g., by counting the number of cells daily. Cell growth can also be monitored by observing the cells under a microscope.

Single K562 cells were resuspended in physiological medium and transferred to the microwells by sedimentation, where they were deposited on the air/fluid meniscus of multiple microwells. Twenty minutes later, the single cells were transferred by delivering a pressure pulse to the fluid in the channel, as described above. This resulted in the transfer of a droplet containing the cells from the microwells into a 96-well microtiter plate with V-shaped wells and RPMI growth medium supplemented with fetal bovine serum. After several hours of incubation, the single cells settled in the V-shaped wells and were observed using an inverted microscope.

The cells were incubated at 37°C, 5% _CO₂ , and cultured for several days. The cell cultures were observed daily to confirm the growth of clonal cell populations from each recovered single cell. After 3-5 days, cell growth and expansion were demonstrated, indicating that a monoclonal cell line had been generated from the single deposited cell. The growth of the single cell or small cell aggregates is shown in a series of time-lapse photographs and reported in the graph of Figure 8. This study demonstrates that cell deposition and cell recovery on the meniscus of an inverted microwell are possible, and that viable cells can be recovered.

Example 3 Cell-cell interaction analysis

3.1 Activity of CTL cells against target LCL cells

The interaction between functional living cells in the inverted microwell system was demonstrated by inducing cytolysis of target tumor cells by T lymphocytes. Target LCL cells were labeled with calcein and delivered to the microwells. T lymphocytes (CTLs) targeting the target cells were activated and delivered to the same microwells. The fluorescence of the target cells was monitored by fluorescence microscopy, and the calcein fluorescence characteristic was determined as a measure of cytolysis.

Compared to the negative control, activated CTL cells induced a sustained decrease in target cell fluorescence. As shown in Figure 7, within 20 minutes, the fluorescence of the active target cells observed in the inverted open microwell system decreased and disappeared, demonstrating effective lysis of the target cells induced by the CTLs (an effective assay for specific cell-cell interactions), and its functional consequences were detected within minutes.

Table 5 reports the fluorescence intensity detected on several LCL targets delivered in inverted open microwells with a diameter of 70 μm. As a control, LCL cells were delivered alone. The CTL-LCL interaction was tested under two conditions: without human papillomavirus (HPV) infection of the target LCLs, and with HPV infection. In the first case, no lysis was expected, while in the second case, the CTL cells were expected to recognize the target and lyse the LCL cells. The results reported in this table show that when the LCLs were infected with HPV (cases e-h), the fluorescence intensity of the CTL-LCL couples decreased significantly within 30 minutes. In contrast, in only one case (d), a-specific lysis was achieved, representing target cell recognition by the CTL cells even in the absence of HPV infection. In all other non-infected cases (a-c), only a physiological decrease in the fluorescence signal was observed.

Table 5

Case Cell type Time (minutes) Intensity (relative) a LCL (control) 30 57.9% b CTL-LCL (without HPV) 20 59.8% c CTL-LCL (without HPV) 20 88.6% d CTL-LCL (without HPV) 20 23.8% e CTL-LCL 1 30 2.3% f CTL-LCL 2 20 6.2% g CTL-LCL 3 15 0.0% h CTL-LCL 4 15 4.5%

Example 4 Controlling Particle Transport in Microwells with Active Fluid Flow

To verify the functionality of the inverted open micropore structure shown in the accompanying figures, an experiment was conducted. A device was fabricated with micropores having a circular diameter of 100 μm and dielectric layer thicknesses of 50 μm and 25 μm. Each electrode had a thickness of 9 μm. The gap 8 between electrodes 153 and 154 was 50 μm. The microchannel had a height of 150 μm and a width of 350 μm.

Polystyrene microbeads with a diameter of 10 μm were resuspended in glycerol (22.5 mM). Glycerol, having a higher density than water, was used to reduce sedimentation velocity and thus limit adhesion of the microbeads to the bottom surface of the microchannel. K562 cells were resuspended in a mixture of PBS and glycerol (300 mM) at a ratio of 1:9. This buffer has a physiological osmotic pressure, while the conductivity is reduced to approximately 0.1 S/m.

In two different experiments, microbeads and cells were introduced into the microchannels, and control electrodes 116, 153, 154, 155, 156, 118 were polarized to either encourage (capture) or prevent (expel) particles from entering the microwells. All signals, except for the ground electrode, were sinusoidal signals with a frequency of 100 kHz and the same amplitude. Table 6 reports the phase shifting scheme. The functionality of the control electrodes was determined by statistically analyzing the number of particles delivered to the microwells for each type of particle and cell delivered, based on particle velocity and applied voltage.

Table 6

When the trapping configuration is activated, the number of particles delivered to the microwell increases due to the higher signal amplitude and lower fluid velocity. Detailed results are reported in Tables 7 and 8, where each reported value considers at least 50 samples.

When the expulsion configuration is activated, we find that the structure functions normally (i.e., no particle transport) for peak-to-peak voltage amplitudes greater than 2 V and for arbitrary particle velocities in the range of 15–150 μm/s.

Flow rates below 15 μm/s are generally not used because they cause particles to adhere to the bottom surface of the microchannel. As shown in calcein release experiments performed on cells flowing through the channel, the expulsion configuration provides higher cell stimulation than the capture configuration. As a result, when working with cells, it is preferred that the peak-to-peak voltage amplitude be below 10 V to limit cell stimulation and maintain cell viability.

Table 7

Table 8

Example 5 Controlling the External Environment of the Inverted Open Microwell to Reduce Evaporation and Enhance Cell Viability

By setting up a system where the humidity outside the microwell is saturated and measuring the continuous decrease in the drag force due to evaporation, the positive effect of controlling the environment around the inverted open microwell can be shown.

The equipment for controlling the evaporation includes a 384-well microtiter plate, wherein each well is filled with 100uL of a fluid stored at a temperature between 4°C and 10°C, such as water, RPMI, PBS or any buffer suitable for cell culture. The inverted open microwell array 114 is placed against the microtiter plate in such a way that each well of the microtiter plate is aligned with the inverted open microwell, thereby creating a closed chamber containing the fluid pre-stored in the microtiter plate, in which the fluid can evaporate, thereby increasing the humidity. Typically, the vertical distance between the meniscus of the inverted open microwell and the pool varies from 0.5mm to 5mm. After a few minutes, the vapor pressure in the chamber reaches a saturation value, thereby preventing any further evaporation from the microtiter plate and the inverted open microwell.

To demonstrate the positive effect of controlling humidity below a microwell on reducing evaporation from the microwell, an apparatus 101 was used featuring a microwell having the electrode configuration shown in Figure 2. Electrodes 116 and 120 were grounded, while electrode 118 was connected to a sinusoidal signal having a frequency of 100 kHz and variable amplitude.

A K562 cell suspension maintained at a temperature between 30°C and 37°C was injected into the microchannel, and the fluid flow was stopped to deposit a single cell in the microchannel above the microwell inlet. In the presence of evaporation, the cell was subjected to a downward drag force _FD from the fluid flow generated by evaporation at the air-fluid interface. Furthermore, the cell was subjected to gravity _FG , buoyancy _FB , and the perpendicular component of the dielectrophoretic force _FDEPy , in the form of _FB - _GF - _FD = _FDEPy .

Due to the relatively high dielectrophoretic force, the cell remains trapped at the entrance of the microchannel, and the force acting on the cell keeps it in a state of equilibrium. As the dielectrophoretic force decreases, the cell's vertical position decreases and it reaches a region where both the electric field and dielectrophoretic forces are higher. This behavior is observed until the amplitude of the electric field reaches a minimum critical value. If the amplitude decreases further, the cell will fall into the microwell because the dielectrophoretic force is not strong enough to offset the other forces acting on the cell.

The vertical position of cells in an inverted open microwell surrounded by air was compared with and without humidity control. The reference value for height is the top side of top electrode 116. Positive values of height correspond to particles remaining in the microchannel outside the microwell, while negative values correspond to particles entering the microwell. As reported in Table 9, cell height was consistently higher when humidity was controlled. This indicates that the presence of a humidity control device beneath the inverted open microwell effectively removes the additional drag force _FD due to evaporation.

Table 9

To maintain an appropriate physiological environment for cells trapped on the meniscus at the air-fluid interface, evaporation needs to be controlled. Indeed, the presence of evaporation will result in an increase in the local concentration of salts and other nutrients contained in the culture medium, and a consequent increase in osmotic pressure. After implementing evaporation control, we measured cell viability using a standard calcein release assay, as reported by Neri et al., 2001, Clin. Diagn. Lab. Immunol., vol. 8, no. 6, pp. 1131-1135, "Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants." In this assay, cells are stained with 1 μM calcein, and the signal loss per hour is approximately 8%, which corresponds to the well-known physiological loss.

This specification contains numerous citations to published references and patent documents, each of which is incorporated herein by reference in its entirety.

Although the present invention has been illustrated and preferred embodiments have been described in the foregoing description and drawings, it will be understood that changes and modifications may be made to the preferred embodiments without departing from the scope and spirit of the invention as embodied in the following claims.

Claims (10)

1. An inverted micropore system for microanalysis of particle function, comprising: a) A microchannel having a first end and a second end, wherein the microchannel is configured to allow fluid to flow in a fluid flow direction defined from the first end to the second end, and wherein the microchannel is defined between a first vertical wall and a second vertical wall, the first vertical wall being disposed on a first sidewall of the microchannel and the second vertical wall being disposed on a second sidewall of the microchannel, the first vertical wall comprising a first substrate portion and the second vertical wall comprising a second substrate portion; b) A micropore having an open upper end and an open or closed lower end, the upper end being open to a microchannel facilitating fluid delivery to the micropore, the micropore including a third vertical wall extending between the upper and lower ends, the third vertical wall comprising a third substrate portion; and c) A controlled electrode array comprising a first electrode (153), a second electrode (154), a third electrode (155), and a fourth electrode (156), wherein at least a portion of each of the first electrode (153) and the third electrode (155) is embedded in a first substrate portion, and at least a portion of each of the second electrode (154) and the fourth electrode (156) is embedded in a second substrate portion. The third and fourth electrodes are disposed adjacent to the upper surface of the microchannel, and the first and second electrodes are respectively disposed below the third and fourth electrodes, such that the first substrate portion is disposed between the first and third electrodes and the second substrate portion is disposed between the second and fourth electrodes. Furthermore, the third electrode (155) is located above the first electrode (153), and the fourth electrode (156) is located above the second electrode (154). The first electrode (153) faces the second electrode (154), and the third electrode (155) faces the fourth electrode (156). The electrode array further includes a fifth electrode (116) at least partially embedded in the third substrate portion and extending at least partially around the micropores, and a sixth electrode (116a) at least partially embedded in the third substrate portion and extending at least partially around the micropores, wherein the third electrode is disposed above the fifth electrode and the fourth electrode is disposed above the sixth electrode. 2. The inverted micropore system according to claim 1, further comprising a detection structure including a first detection electrode (170), a second detection electrode (171), a third detection electrode (172), and a fourth detection electrode (173), wherein the first and second detection electrodes are opposite each other and located on opposite sides of an axis parallel to the direction of liquid flow, such that a first gap is disposed between the first and second detection electrodes, wherein the third and fourth detection electrodes are opposite each other and located on opposite sides of an axis parallel to the direction of liquid flow, such that a second gap is disposed between the third and fourth detection electrodes, and wherein the first, second, third, and fourth detection electrodes are in contact with the bottom surface of the microchannel. 3. The inverted micropore system according to claim 1, further comprising a detection structure, the detection structure comprising a first detection electrode (174) disposed at a first position, a second detection electrode (175) disposed at a second position, and a third detection electrode (176) disposed at a third position, the second position being downstream of the first position with respect to the liquid flow direction, the third position being downstream of the second position with respect to the liquid flow direction, wherein the first, second, and third detection electrodes each partially overlap with an axis parallel to the liquid flow direction, and wherein the first, second, and third detection electrodes are in contact with the bottom surface of the microchannel. 4. The inverted micropore system according to claim 1, wherein the lower end of the micropore is open to the atmosphere outside the micropore. 5. A method for microanalyzing particle function, comprising: a) Providing an inverted micropore system according to any one of claims 1-4, the system comprising one or more micropores open at an upper end to at least one microchannel, wherein electrodes are arranged along the at least one microchannel; b) Incorporating at least one particle into the at least one microchannel; c) wherein the electric field intensity mode causes the particle to move in the at least one microchannel in a desired direction. 6. The method of claim 5, wherein the inverted micropore system is the system claimed in claim 1, and the method comprises: a) The fifth electrode (116) and the sixth electrode (116a) are polarized by the voltages of the third electrode (155) and the fourth electrode (156), respectively, while the first electrode (153) and the second electrode (154) have the same ground voltage, and the at least one particle is captured at a position (Δ) between the first electrode (153) and the second electrode (154). 7. The method according to claim 6, further comprising, after step a),: b) Connect the fifth electrode (116) and the sixth electrode (116a) to ground, and polarize the first electrode (153) and the second electrode (154) to the fourth electrode (156) and the third electrode (155), pushing the at least one particle toward the minimum electric field between the fifth electrode (116) and the sixth electrode (116a) at the location of one or more micropores. 8. The method of claim 5, wherein the inverted micropore system is the system claimed in claim 2, wherein the upstream first, second, third, and fourth electrodes align the particles along an axis, wherein the signal-to-noise ratio of the terminal output signal (184) generated by the passage of the at least one particle is maximized, and the method comprises: a) Connect the second detection electrode (171) and the fourth detection electrode (173) to an excitation signal (188) represented by an AC voltage; b) Connect the first detection electrode (170) and the third detection electrode (172) to a readout circuit (186), which amplifies the first output current (180) and the second output current (182) and provides the terminal output signal (184) proportional to the difference or ratio between the first output current (180) and the second output current (182). 9. The method of claim 5, wherein the inverted micropore system is the system claimed in claim 3, wherein the upstream first, second, third, and fourth electrodes align the particles in a row along the axis, wherein the signal-to-noise ratio of the terminal output signal (184) generated by the passage of at least one particle is maximized, and the method comprises: a) The second detection electrode (175) is connected to an excitation signal (188) represented by an AC voltage, while the first detection electrode (174) and the third detection electrode (176) are connected to a readout circuit (186) that amplifies the first output current (180) and the second output current (182) and provides the terminal output signal (184) that is proportional to the difference or ratio between the first output current (180) and the second output current (182). 10. The method according to any one of claims 5-9, wherein the at least one particle is detected by optical detection after being properly arranged and aligned by the first, second, third and fourth electrodes.