HK1228000A1

HK1228000A1 - Microanalysis of cellular function

Info

Publication number: HK1228000A1
Application number: HK17101434.3A
Authority: HK
Inventors: 马西莫．博基; 罗伯托．圭列里
Original assignee: 塞普莱有限公司
Priority date: 2010-12-03
Filing date: 2017-02-09
Publication date: 2017-10-27

Abstract

An inverted microwell (102) provides rapid and efficient microanalysis system (100) and method for screening of biological particles (128), particularly functional analysis of cells on a single cell basis. The use of an inverted open microwell system (102) permits identification of particles, cells, and biomolecules that may be combined to produce a desired functional effect also functional screening of secreted antibody therapeutic activity as well as the potential to recover cells and fluid, and optionally expand cells, such as antibody secreting cells, within the same microwell.

Description

Microanalysis of cellular function

The present application is a divisional application of an invention patent application having an application date of 2011, 12/5, and an application number of 201180066877.5, entitled "micro-analysis of cell function".

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 61/419,377 entitled "micro-analysis of cellular function" filed on 3.12.2010, which is incorporated herein by reference in its entirety for all purposes.

Background

There is a need for a high throughput, efficient system for analyzing biological particles, including single living cells, to enable rapid and valuable identification of particles, including cells, molecules, etc., that have desired functions, such as producing or inducing desired biological results. Such systems are needed for new drug development, diagnostics, screening of candidate molecules, etc. In a preferred system, the particles may retain their activity without significant damage by the assay process.

Existing systems that evaluate individual particles typically use Dielectrophoresis (DEP) to manipulate the particles. An electric field is imposed on a particle, such as a cell, to suspend the particle and maintain the position of the particle during the induction reaction, and the assay can impair its activity and destroy the ability of the particle to function properly. For example, in the case of prolonged use, such damage may lead to lysis and/or cell death. Adhesion of particles to the substrate, electrodes or chamber walls may also require lysis of the cells to remove the cells from the system after analysis or to capture cellular products.

It would be very useful to provide a rapid, high throughput system for efficiently and effectively capturing, identifying, and analyzing biological functions of individual particles, wherein the system does not significantly damage the particles.

Disclosure of Invention

Inverted open microwell systems, devices, and methods of use are disclosed that allow for rapid, efficient screening and sorting of particles, particularly single particles, including single cells. Such screening includes analysis of specific particle characteristics and functional properties of the particles that may indicate whether the particles or agent are useful for therapeutic purposes. For example, the particle characteristics may include: the presence and relative amount of specific binding and/or affinity of a ligand, such as a cancer cell target antigen; cell-to-cell binding reactions and interactions, such as cytolysis, toxicity, biomarker production, electroporation, and the like; induced cellular responses and/or responses, as induced by other cells, drugs, compounds, proteins, antibodies, molecules, enzymes, nucleic acid molecules, cellular secretions, and the like; induced production of cellular products such as lytic enzymes, antibodies, biomarkers, etc. Analysis of these characteristics and subsequent particle recapturing supports a method of functional cell sorting that results in the isolation of cells or their byproducts (e.g., secreted molecules) with proven function and utility, such as therapeutic utility.

In one embodiment, the single cell analysis may comprise a plurality of assays belonging substantially to one reaction protocol or a plurality of assays in series, e.g., resulting in one assay providing information for a particular assay to follow. For example, cells known to be associated with a particular disease, e.g., cells obtained from an individual suffering from the disease, can be analyzed in the system to determine the presence of a particular biomarker, e.g., an antigen, an expressed protein, or a characteristic of the disease. After a preliminary analysis to identify whether the individual cells exhibit the biomarker, the same identified cells can be used to re-analyze their response to treatment with a candidate therapeutic drug, compound, or other cell expected to effectively induce the desired response in the cells exhibiting the selected biomarker.

Analysis in the inverted open microwell can be done on a single particle (including a single cell) with fast, efficient timing without the need to separate the particle or the product of the particle from a complex mixture. Screening methods include screening for individual cells with functional properties and selecting for substantially viable primary cells for subsequent analysis, immortalization and/or clonal expansion that is believed to have useful properties. Cellular components such as DNA, RNA, proteins, etc., may also be isolated from the original cells.

Early in the development process, the method enables identification of the desired particle reactions and particle interactions. Early understanding of cellular function makes it possible to ultimately develop useful biomaterials with a better understanding of the potential for success of the material, and may reduce the number of potential candidates for the development process earlier. Methods for performing multiple, rapid assays on the same particle, e.g., on a single cell, and achieve more rapid progress in the search process.

The inverted open microwell system described herein facilitates precise delivery of individual cells to or from microwells and precise manipulation of cells and other particles in microchannels and in specific microwells, delivery of reagents, buffers, labels, and the like, including other cells for cell-cell interactions. Multiple characteristics of a single cell can be evaluated while retaining its activity in open microwells for optional subsequent assays, immortalization, amplification, and the like. Particles can be recovered in a short time frame (including recovery of the original individual cells), with minimal use of reagents, and recovery of cells and products in a substantially viable and useful condition. In particular, the deposition of one or more particles (including living cells) at the fluid/air interface of an open microwell allows for accurate particle-to-particle interactions that can be effectively monitored and rapidly screened to identify candidate particles, such as cells, for subsequent analysis and/or clonal expansion in the microwell.

Surprisingly, when appropriate geometric constraints and fluid conditions are used, fluid filling the inverted open pores 102 having the lower end 108 open to air is retained without leaking from the lower open end. Furthermore, during fluid washing, the deposited particles 128 are unexpectedly retained at the meniscus 122, while other particles are placed in close proximity to the first one and the analysis procedure. The open microwells function as "microcentrifuges" that can deliver reagents to deposited particles for rapid analysis, washing, and subsequent analysis of the deposited particles without loss. For example, the cells in the microwells may retain better activity after analysis and when recovered from the microwells. For example, a substrate, such as a microtiter plate, can be recovered from the microwells for, e.g., immortalization and/or amplification. In one embodiment, selected individual cells may be incubated and expanded in microwells.

The present invention provides methods and structures for implementing an inverted microwell system comprising microwells with upper terminals open to microchannels. In one embodiment, the microwells may be closed at the lower end, preferably using a transparent material such as glass or a transparent polymer to allow the contents of the microwells to be seen. In another embodiment, the microwells are open at the lower end to the atmosphere (e.g., air or other gas) outside the device.

For example, one or more particles may be delivered to a microwell by sedimentation, controlled by cell density, fluid velocity, loading time, and optionally dielectrophoretic force. The dielectrophoretic force may be generated by electrodes connected to a suitable alternating voltage and arranged in microchannels positioned above micro-wells open and in fluid communication with the microchannels. Also provided herein are methods of efficient particle aggregation and interaction, such as controlled by electrodes embedded within open microwells, to produce dielectrophoresis capable of manipulating particles, such as single cells, into desired locations within microwells. The method provides a high throughput assay system that uses minimal amounts of reagents and allows high throughput recovery of assayed active particles, including cells and cell products.

The present invention also provides methods and structures for precisely controlling and sorting particles, including cellular and non-cellular particles, to efficiently deliver particles to the microwells of the inverted open microwell system. Particular embodiments include the configuration of electrodes and electrode pairs in microchannels that allow particles to move with little or no harm to the activity of biological materials such as cells; a structure that disposes electrodes and electrode pairs in the microchannel and in proximity to the microwells, allowing controlled access of desired particles to the microwells and effectively closing the microwells and driving away undesired particles; and a pattern of electrodes and electrode pairs in the microwells for detecting and optionally controlling the position of particles as they enter and pass through the microwells, e.g., deposited on a fluid meniscus.

The inverted open microwell system comprises one (fig. 1) or more (fig. 3) microwells, wherein each microwell is in fluid communication with one or more microchannels to deliver fluids and particles to the one or more microwells. The microchannel is generally disposed above the microwells, which are open to the fluid microchannel at the upper end 106 of the microwells and open to the atmosphere (e.g., air or other gas) outside the device at the lower end 108, allowing control of atmospheric properties (e.g., gas composition, humidity, temperature, and 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, at least a portion of the vertical walls 112 of the micro-vias 102 are formed from a dielectric material 114. The vertical walls may also be formed at least in part by one or more electrodes integrated with the dielectric material, for example, in a layered structure perpendicular to the vertical axis of the hole, the laminate forming the micro-hole, for example, as shown in fig. 2.

Fluid injected into microchannel 104 fills pores 102 under capillary action while surface tension holds the fluid in the open pores, forming a meniscus 122 at the pore lower open end 108 of the air-fluid interface. The microchannel and microporous surfaces may be coated with materials having opposite hydrophilicity or hydrophobicity. For example, the microwells may have a hydrophilic coating and the microchannel surfaces near the open ends of the microwells have a hydrophobic coating, or vice versa.

Typically, the movement of particles in microchannels and the placement of particles in specific micropores is accomplished by limiting dilution, sedimentation, electromagnetic forces, gravity and combinations of these. In one embodiment, the controllable manipulation of particles in the inverted open microwell system comprises powering one or more electrode arrays suitably arranged in microwells and those electrodes suitably placed in microchannels. Examples of electrode configurations are shown in fig. 2 and 9-13.

The methods disclosed herein include methods of screening individual cells or small groups of cells that include precise polymers of specific particles. Analysis of individual cells is often provided to identify cells that are capable of producing a particular response, for example, to an increase in biological material, which may be a different cell, a portion of a cell, a protein, a nucleic acid molecule, a drug, an antibody, an enzyme, and the like. The production of precise polymers allows for precise alignment of cells that together can induce a desired response and/or are merely used together to analyze their specific characteristics, capabilities, or functions. The system enables, for example, the alignment of multiple particles in a polymer, directly contacting or in close proximity to cells for functional contact. This arrangement of cells in the precision polymer makes it possible to detect particle-to-particle interactions quickly and efficiently.

Drawings

FIG. 1 is a schematic diagram of an inverted open microwell system showing cells deposited on the meniscus of an open microwell in the presence of a fluid flowing in the microchannel and particles transferred from the microchannel to the microwell by gravity.

Figure 2 is a cross-sectional view showing a 3-electrode configuration in an inverted open-pore system.

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

FIG. 4 is a schematic showing the recovery of the contents of a microwell onto a microtiter plate.

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

FIG. 6 shows a photograph of the meniscus of an inverted open pore observed with an inverted fluorescence microscope showing: (A) k562 cells randomly arranged on the meniscus during sedimentation when no electric field is applied to aggregate the cells, and (B) K562 cells manipulated by electromagnetic force to the central vertical axis of the microwells and deposited near the center of the meniscus as a cellular polymer during sedimentation.

Fig. 7 includes a graph and a series of photographs showing the decrease in biomarker fluorescence signal intensity as a measure of calcein uptake in active target cells after exposure of individual cells of an individual to activated T lymphocytes for 20 minutes to induce lysis of the individual cells.

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

Fig. 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 the microchannel and in the microwell of an inverted open microwell system.

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

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

Figure 12 shows an electrode array centered at a microwell location shown through the top of an inverted open microwell system showing the electrode arrangement in the microchannel for trapping particles in the channel at a minimum potential (Δ). This arrangement is most suitable when the fluid stream is not flowing in the channel.

Fig. 13 shows an array of electrodes centered upstream of a microwell, shown through the top of an inverted open microwell system, showing an arrangement of electrodes in a microchannel for manipulating particles (●) to move to a minimum potential (Δ), where the minimum potential is established by a specific force applied by the electrodes.

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

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

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

Detailed description of the preferred embodiments

A. Definition of

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

as used herein, microporous, refers to micron-sized (less than 1000 microns) pores, including height, cross-sectional area, e.g., the diameter of the microporous in the case of a tube; and volume.

As used herein, microchannel refers to a channel that provides fluid to the micropores, having a cross-sectional area of micron-sized dimensions (less than 1000 microns).

As used herein, particles include any particle that can be transported, manipulated, reacted, or analyzed in a microwell of the disclosed inverted open microwell system. The particle may be a cell or a part of a cell, a microorganism, a biomolecule (e.g. a protein, polynucleotide, antibody, enzyme) or a substrate (e.g. a polymeric particle which may be coated with a reactive substance (e.g. an antigen which is encapsulated in a sphere, etc.).

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

As used herein, an electrode is an electrically conductive material, e.g., a metal such as gold, copper, nickel-gold, etc. Preferably, the electrodes are formed of high purity gold.

The dielectric material, as used herein, is an electrically insulating substrate. Preferred dielectric materials for use in the inverted open pore system include polyimides, such asAnd

dielectrophoresis, as used herein, is the force applied to a particle when the particle is subjected to a non-uniform electric field.

"A" means at least one.

"majority" means two or more.

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

"comprising" or "Comprises" 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 follows:

DEP refers to dielectrophoresis

ASC refers to antibody-secreting cells

CTL means cytotoxic lymphocytes

NK refers to natural killer cell

LCL refers to the lymphoblastoid cell line

ADCC refers to antibody-dependent cellular cytotoxicity

CMC means complement mediated cytotoxicity

PCB refers to a printed circuit board

C. Inverted open microwell systems and devices

The inverted open microwell system comprises a device (100), the device (100) comprising one (fig. 1) or a plurality (fig. 3) of microwells, wherein each microwell (102) is in fluid communication with one or with a plurality of microchannels (104) for delivering fluids and particles to the microwell. The microchannel is generally disposed above the microwell, which is open at an upper end (106) to the fluid microchannel and at a lower end (108) to the atmosphere (e.g., air or other gas) outside the device. The microwell has a vertical axis (110), such as a central axis, extending between an upper end and a lower end of the microwell. Fluid injected into the microchannel fills the pores by capillary action while surface tension holds the fluid in the open pores, 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 shown as "air". It will be appreciated that the atmosphere may be controlled (e.g. in a chamber) to contain a gas other than air, for example a chamber enriched in carbon dioxide, nitrogen or other desired gas, for maintaining the particles and/or enabling analysis to be performed in the microwells. The chamber may include a system that controls the gas composition, humidity, temperature, pressure, and/or other physical parameters and/or maintains the environment in a sterile state.

In one embodiment, the closed chamber is created by placing the substrate 114 of the device on a microtiter plate with wells aligned with the inverted open microwells. This alignment leaves a void between the open microwell and the surface of the microtiter plate to separate the contents of the open microwell from the contents of the microtiter plate, such as fluid contents. For example, the microtiter wells may contain a liquid medium, such as physiological medium for cell recovery and cell culture, so that it is accepted when the contents of the open microwells are released. The microtiter wells may contain a different medium, such as water or other liquid, for example, to increase the humidity of the closed chamber, which may saturate the vapor pressure within the chamber due to increased atmospheric humidity of the closed chamber caused by evaporation of the liquid in the microtiter wells, and prevent evaporation of the liquid from the inverted open microwells within the chamber. In one embodiment, the atmosphere of the closed chamber is fully saturated, e.g. with a humidity of up to or about 100%, to prevent evaporation of the meniscus. To prevent condensation on the substrate surface, the temperature of the substrate 114 may be maintained higher than the temperature of the humid air included in the chamber.

The inverted open microwell system may be used to achieve control of the delivery of one or more particles, such as one or more active cells, to a microwell to cause interaction of the particles already delivered in the microwell with one or more additional particles delivered to the microwell and/or to allow high throughput analysis of biological functions, e.g., by inducing specific reactions in the microwell, analyzing the results of the reactions, while identifying and selectively recovering selected particles and/or biological products of the selected particles.

The system may be used as a "microcentrifuge" when the particles disposed on the meniscus are not replaced or absent as the wash-providing fluid included in the channel is changed and the reagents in the open wells are changed. In a particular embodiment, the fluid comprised in the micro-wells is adapted for dielectrophoretic manipulation of the particles. Once the particles are disposed in the microwells, as on the meniscus, the fluid dielectrophoretic medium can be washed from the microwells and replaced with a medium suitable for testing or for growth and amplification of the disposed particles.

The inverted microwell system can provide efficient and effective real-time monitoring for particle function (including interaction of multiple particles), function screening, and particle (e.g., living cell) sorting. In particular embodiments, the system can provide high throughput functional analysis of antibody secreting cells and rapid identification and selection of high affinity antibodies, and analysis and selection of 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 (e.g., monoclonal antibodies) secreted by individual cells, as well as recovery of the identified antibody-secreting cells and/or secreted antibodies, and also allows for expansion of the identified cells in microwells.

To provide optimal functionality, the structural properties of the device, including relative geometry, coatings, pressure, materials, and the quality of fluids, particles, and reagents intended to be placed in the microwells, can be optimized for specific analyses. For example, the coating for the interior walls of the micropores may be designed to have opposite hydrophobic/hydrophilic characteristics to the coating for the interior walls of the microchannels adjacent to the micropores to drive off unwanted materials from the pores and/or mediate the entry of desired particles.

The diameter and/or length of the microwells are designed to allow a meniscus to form at the interface of the fluid at the lower end of the microwells with the external environment, such as air, and to allow the particle(s) (a particle) or particles (particles) deposited on the meniscus to be retained with little or no leakage of fluid from the microwells, and to allow exchange of fluid and reagents in the microwells while retaining the particles disposed on the meniscus. The width, height and length of the microchannel are designed to create a hydraulic resistance such that, in the case of a particular fluid flow, a pressure is generated in the microchannel, the magnitude of the pressure being controlled within a range that allows the fluid to fill the micropores while preventing the fluid from leaking out of the bottoms of the micropores. These and other features and uses of the inverted open microporous system are described and exemplified herein.

1. Micro-pores

For example, as shown in FIG. 1, the inverted open microwell device 100 comprises microwells 102 having dimensions in the micron range. The open cells may be tubular in shape, such as with a circular cross-sectional axis. Other shapes of the pores, such as conical, rectangular or other geometries, may also be used. In one embodiment, the microwells are structures that extend from a narrow lower end to an extended width upper end.

Fig. 1 shows a cross-sectional view of a device 100 including open microwells 102. The device 100 includes a substrate 114 defining vertical walls 112 of the open 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 Polymethylmethacrylate (PMMA), polycarbonate, polyethylene naphthalate (PEN), polyethylene terephthalate (PET), cyclo-olefin polymers, and the like.

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

2. Electrode for electrochemical cell

As shown in fig. 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 provided in the microwells and microchannels to control the electrical force applied to the particles, for example to push or pull the particles in the microchannels towards and/or into or out of the microwells, or to facilitate the transport of the particles from entering the open microwells to the lower end of the microwells or any part of these.

Although the microwell of FIG. 2 is shown with three electrodes 116,118,120, the device 100 may alternatively include more or fewer electrodes. In one embodiment, the microwells are surrounded by at least one ring electrode 116. In another embodiment, a pair of facing electrodes 116,116A surround the microwells. In one embodiment, a pair of facing electrodes is disposed near the upper end 106 of the open microwell to manipulate the particle 128 into the microwell and/or to manipulate the particle for positioning along a vertical axis 110, such as a central vertical axis.

Electrodes may also be disposed in the microchannels to affect transport of the particles. For example, FIGS. 10-11 show possible electrode arrangements in microchannel 155,156,153,154 and in microwells 116, 118. Fig. 12-13 show exemplary arrays of electrodes 153-164 arranged in pairs along a microchannel to facilitate transport of particles to microwells 102 or out of microwells 102.

The electrodes are formed of an electrically conductive material, which may be in the form of an electrically conductive sheet or plate, for example. In some embodiments, the conductive material comprises a biocompatible metal, such as gold, carbon, or aluminum. In the illustrated embodiment, the electrodes 116,118,120, and 153-164 are embedded in the substrate 114 such that adjacent electrodes are separated from each other by portions of the substrate 114 (see FIG. 2), for example, in a laminated structure perpendicular to the microwell vertical axis 110.

3. Micro-channel

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

Near the top cover 124, the microchannels may be formed of a polymer such as polyimide, preferably a transparent material such as glass or a suitable plastic such as PMMA, polycarbonate, PEN, PET, cyclo-olefin polymer. The formed microchannel may then be adhered to the tip of one or more microwells, for example, using a biocompatible adhesive.

The upper end 106 of the microwell is open to the microchannel 104. For example, fluid 130 may be caused to flow through microchannel 104 by creating a pressure differential at the two ends of microchannel 104. For example, the microchannel 104 may be connected to a pump, such as a peristaltic pump, or to a source of compressed air, or the like. The velocity of the one or more particles 128 through the one or more micropores 102 may be controlled by adjusting the pressure differential across the microchannel 104 to control the flow rate of the fluid 130 in the microchannel.

4. Microwell or microwell array

As shown in fig. 3, device 101 may include an ordered arrangement of a plurality of microwells 102. For example, device 101 may include an array or matrix of microwells, e.g., in the form of rows and columns of microwells, in communication with one or more microchannels 104. Device 101 may be used to perform multiple parallel operations simultaneously on multiple microwells 102. Although the following discussion refers to a single microwell 102, the described embodiments are applicable to each of the microwells 102, as may be configured in the device 101 as an array or matrix of microwells 102, and may include microwells 102 and microchannels 104 of different configurations. For convenience, the structure of the microwells may be designed, for example, to accommodate conversion of conventional systems such as 96-well microtiter plates or 1536-well micro-vial assemblies (micro-visual assembly). This design may also be used for specific purposes, such as high throughput.

5. Device for manufacturing inverted micropores

In one example, to form an inverted microwell device, the microwell 102 may be fabricated as a through-hole through a substrate 114, the substrate 114 embedding electrodes 116,118, 120. The vias may be formed by conventional drilling methods using, for example, mechanical or laser techniques. Typical fabrication of inverted microwell systems is described in the examples below.

In some embodiments, microwells 102 have a circular or hemispherical cross-section, however microwells 102 may have other cross-sectional shapes such as hexagonal, rectangular, square, conical, and the like. The diameter of microwells 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 microwells and on the geometric relationship needed to retain sufficient surface tension to form and maintain meniscus 122. The diameter of the micropores 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 micropores 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 had a length of 27mm, a width of 350 μm, and a height of 150 μm. The micropores had a diameter of 100 μm and a height of about 75 μm. No hydrophobic or hydrophilic coating is used. When the top side of the channel is made of transparent polycarbonate, the polyimide surface in the microchannel and at the bottom side of the device has a moderately hydrophilic behavior, while the roughness of the inner part of the mechanically drilled microwell increases, thereby improving the hydrophilic behavior of the inner part of the microwell. This difference in surface properties, where the inner portion of the microwell is more hydrophilic than the microchannel and bottom surface, in combination with appropriate microchannel and microwell sizes, is unexpectedly sufficient for the fluid from the microchannel to properly fill the microwell without any leakage from the bottom of the open microwell.

6. Hydrophilic/hydrophobic surfaces

To facilitate the introduction and retention of one or more particles 128 in each microwell 102, the devices 100,101 may be configured such that some surfaces are hydrophobic while other surfaces are hydrophilic. In some embodiments, the 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 the microchannel 143 and the vertical walls of the microwells 112 may be hydrophilic, while the lower surface 142 may be hydrophobic. As another example, in embodiments utilizing a grease fluid, the bottom surface of the micro-channel 143 and the vertical walls of the micro-wells 112 may be hydrophobic, while the lower surface 142 may be hydrophilic. Kapton and Polyimide exhibit a discontinuous hydrophilic behavior in the contact angle range of 20-70 degrees. By applying a plasma surface treatment, such as oxygen plasma, the Kapton and Polyimide surfaces become more hydrophilic. Hydrophobic coatings that may be used on Polyimide or Kapton surfaces include FEP,FC-732 or41-90。

A fluid 130, such as a physiological buffer or culture medium, is injected into the microchannel 104, which is positioned above the one or more microwells 102 and is in fluid communication with the upper open end of the one or more microwells 102. The micro-size of the pores of microwells 102 and the hydrophobic behavior of their lower open ends 108, in contrast to the hydrophilicity of microchannels 104 and the upper ends of microwells 102, allow fluids to fill microwells with open bottoms without leaking out of lower open ends 108. When using microwells with closed bottoms, to help fill the microwells, a wetting agent, such as ethanol or a mixture of ethanol and water, may be added first before the physiological medium is added. Capillary action and surface tension can hold the fluid 130 in the pores and form a meniscus 122 at the fluid-air interface, as shown in fig. 1. In one embodiment, the fluid may contain a wetting agent to assist in filling the microchannels and/or micropores. Exemplary wetting agents include, for example, ethanol, tween-20 and SDS. Prior to injection of the cell suspension, the microchannels are suitably washed with a physiological medium, such as Phosphate Buffered Saline (PBS), to remove any residual wetting agent.

7. Controlled particle transport and aggregation

7.1 controlled delivery

A fluid containing particles, such as cells, may be delivered to microchannel 104 at a concentration and flow rate designed to allow a limited number of microparticles, such as individual particles, to be distributed into each microwell 102. The electrodes may be used to transport particles in microchannel 104, to allow or reject particles 128 from microchannel 104 into microwell 102, to manipulate one or more particles 128 in a microwell, to aggregate or hold particles in a desired location in a microwell, to induce a structural or functional change in the particle or particles deposited in a microwell, and/or to sense and/or measure the presence, movement or change of particles in a microwell, or to sense and/or measure the presence of molecules produced by particles in a microwell.

As with the embodiments described in fig. 9-11, the fluid flow in the channel is used to transport particles of interest from the inlet to a particular microwell, while the dielectrophoretic force can also be configured so that particles can jump over a microwell and follow the microchannel to another microwell. An electrode array is shown in the embodiment depicted in fig. 12 and 13, which does not require fluid flow to transport particles in microchannels to microwells, but uses dielectrophoretic forces to achieve the above.

7.2 transport of particles to microchannels in absence of fluid flow

When precise positioning of each particle is required, precise transport in the microchannel is required to deliver the particle precisely to a specific microwell. One method of controlling precise delivery is based on a sequence of electrical parameter changes that can provide very precise results independent of fluid flow or similar techniques.

When particles with complex dielectric constants are exposed to spatially varying electric fields, the particles can be pushed by negative dielectrophoresis, depending on the value of the Clausius-Mossotti factor. In this case, the particles will move towards the smallest electric field and be driven away by the largest electric field.

For example, as shown in fig. 10, 11, 12, and 13, electrodes 155 and 156 are disposed above electrodes 153,154,157,158,159,160,161 and 162. In particular, electrode 155 is disposed above electrodes 153,157,159, and 161, while electrode 156 is disposed above electrodes 154,158,160 and 162. These electrodes may be used to create a pattern of electric field strength to cause particles (●)128 to move in a desired direction in 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 that is shifted 180 degrees out of phase with its facing partner by the same amplitude. The electrodes 159 and 160 are connected to a ground (G1).

In this scenario, the electric field in the microchannel 104 reaches a minimum value (Δ)170 between the electrodes 159 and 160 because the electrodes 159 and 160 have the same potential and their thickness creates a groove that induces a strong minimum electric field. The electric field in the microchannel 104 is maximized in the case where the electrode pairs 153-. In this case, the particle (●)128 is trapped at a location where the minimum (Δ)170 between the electrodes 159,160 is surrounded by the electric field where it reaches its maximum intensity.

The position of the trapping particles can be changed and moved, for example, to the left in two stages as shown in FIG. 13. In a first phase, the voltage of the electrodes 157,158, which are located to the left of the existing rest position, is set to ground (G2). This creates a minimum (Δ) electric field that is the same as the electric field strength between electrodes 159 and 160. In the second stage, electrode 159 is polarized as electrode 161 and electrode 160 is polarized as electrode 162. This polarization creates a strong electric field between electrodes 159 and 160. This field will push the particle that is trapped at that point that is about to fall within the field closest to the minimum (Δ) and move the particle one position to its left.

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

In another embodiment, as shown in FIG. 14, electrodes 155 and 156 are replaced by a single electrode 190 disposed along the top side of the microchannel. The electrodes 190 may also be realized by closing the microchannels at the top with a metallic material or a transparent material with an indium oxide (ITO) coating, such as glass or transparent plastic. Such a conductive coating provides an electrical connection while preserving a transparent top cover that does not interfere with 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 supply at a specified potential. Each of the facing electrode tabs 154,158,162 is connected to a sinusoidal voltage source that is shifted 180 degrees out of phase with its facing tab by the same amplitude. Electrodes 159 and 160 are connected to a sinusoidal voltage source of the same amplitude but shifted by 90 or 270 degrees out of phase with electrodes 153,157 and 161.

With this electrode configuration, the position of the trapping particles can be changed and moved, for example, to the left in two stages. In the first phase, the voltage of the electrodes 157,158, which are to the left of the existing rest position, is set to a sinusoidal voltage source with the same amplitude as the electrodes 153,157 and 161 but shifted by a phase difference of 90 degrees or 270 degrees. This creates a minimum (Δ) electric field that is the same as the electric field strength between electrodes 159 and 160. In the second stage, electrode 159 is polarized as electrode 161 and electrode 160 is polarized as electrode 162. This polarization creates a strong electric field between electrodes 159 and 160. This field will push the particle that is trapped at that point that is about to fall within the field closest to the minimum (Δ) and move the particle one position to its left.

Figure 12 shows a structure that can be used to accurately transfer particles in microwells 102 without fluid flow. The electrode pair 155,156 is not shown in fig. 12, but as shown in fig. 9 and 10, is positioned above the other electrode pair, specifically electrode 155 is positioned above electrode 163,116,153 while electrode 156 is positioned above electrodes 164,116a, 154.

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

Since electrodes 116,116A surround microwell 102 and are connected to ground potential, the presence of electrodes 155 and 156 creates an electric field in the channel below microwell 102. Through the previous analysis, the particles will now be pushed towards the micropores.

If the position of the particles settling in the microwells must be precisely controlled, electrode 118 can be polarized by connecting the electrode to a voltage source having a magnitude substantially less 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 may create dielectrophoretic forces in the microwells that push the particles toward the center of the microwells.

If transfer of particles in the micropores must be avoided, a different electrode polarization is recommended in the absence of fluid flow. In particular, assuming that particles are trapped between electrodes 153,154 connected to ground, particle transfer can be avoided by connecting to ground electrode 116,163,164 while electrode 116A is polarized as electrode 155, electrode 153 is polarized as electrode 156, and electrode 154 is polarized as electrode 155. These polarizations may create a temporary particle shift, causing the particle to shift from a position between 153 and 154 to a position close to electrode 116 (not between 116 and 116A) and eventually to a stable transfer site between electrodes 163 and 164 where the minimum electric field is located.

In another embodiment, the structure reported in FIG. 12 can be used to accurately transfer particles in microwells 102 in the absence of fluid flow. As shown in FIG. 16, an electrode 190 disposed on the top side of the microchannel replaces the electrode pair 155,156 and is connected to ground.

If electrodes 116 and 116A are connected to sinusoidal voltage sources with the same amplitude and phase 0 degrees and 180 degrees, respectively, while electrodes 153 and 154 are connected to the same sinusoidal voltage with a phase shift of 90 degrees or 270 degrees, the particles 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 phase shifted by 90 or 270 degrees while 153 and 154 are connected to sinusoidal voltages having the same amplitude and phase of 0 and 180 degrees, respectively, the particles will be pushed towards the minimum (Δ)170 electric field between 116 and 116A where the microwells are located.

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

If the position of settling of the particles in the microwells has to be precisely controlled, electrode 118 can be polarized by connecting electrode 118 to a voltage source whose amplitude is substantially smaller than the amplitude used to control electrodes 116 and 116A and whose phase is rotated by 90 degrees or 270 degrees with respect to the phase of electrodes 116 and 116A. This polarization may create dielectrophoretic forces in the microwells that may push the particles toward the center of the microwells.

If transfer of particles in the micropores must be avoided, a different electrode polarization is recommended in the absence of fluid flow. In particular, assuming that the particles are trapped between electrodes 153,154 connected to the same sinusoidal voltage with a phase shift of 90 degrees or 270 degrees, particle transfer can be avoided by connecting electrode 116,163,164 to ground while connecting electrodes 153,116a and 154 to the same sinusoidal voltage with a phase shift of 0 degrees, 90 degrees or 180 degrees, respectively. The electrode 190 is always kept connected to ground. These polarizations may create a temporary displacement of the particles such that the particles are displaced from a position between 153 and 154 to a position proximate to electrode 116 (not a position between 116 and 116A). Finally, by connecting electrode 116 to the same sinusoidal voltage with a 270 degree phase shift as electrode 153, the particles are made to reach a stable transfer site between electrodes 163 and 164 where the minimum electric field is located.

These charged structures presented herein do not rely on the presence of a fluid stream for the displacement and transport of particles. However, the fluid flow is advantageous to provide a suitable cooling structure. These structures can be used to move individual cells along microchannels and drive them into open microwells, or to block them from entering open microwells by appropriate application of a series of polarization signals.

7.3 transport of particles in microchannels and open micropores in the presence of a fluid flow

Fig. 9-11 illustrate a microporous 102 structure in fluid communication with a microchannel 104, the top of which is closed by a surface 124 covered with liquid contained in the microchannel. Particles, such as cells or beads (beads), flowing in the channel fluid are subjected to electromagnetic forces, causing particles 128 to be pulled toward microwell 102 or pushed out of microwell 102. This configuration is particularly suitable for operation when there is a significant flow of fluid in the particle-conveying channel. See, for example, Faenza et al, 2011 (May), "Regulation and modeling of k562 leukemia cells Using electrically activated microchannels" ("Controlled isolation and characterization of k562 leukemia cells using electrically activated microchannels"), In: International Conference on Microtechnologies In Medicine and Biology.

Electrodes are fixed in the structure of the micropores and microchannels to control particle movement. For example, fig. 9 shows a pore 102 surrounded by a shaped electrode 116, such that an opening facing the flow direction from right to left is present in fig. 9.

The specific embodiments presented in these figures for modulating particle movement include those electrodes 116,118 disposed in microwells 102, and those electrodes 153,154 disposed in microchannels 104. Fig. 10 shows micro-wells 102 in fluid communication with micro-channels 104, each having a vertical wall 112 formed by a layered structure comprising a dielectric substrate 114 and electrodes. The microchannel electrodes 153-164 are disposed in the microchannels, while the electrode pairs 153-154 and 155-156 are disposed near the junction of the microchannels at the upper open end 108 of the microwells and the microwells 102. Microwell electrodes 116 and 118 are disposed near the upper and lower ends of the microwells, respectively.

In a microchannel 104 in which a fluid flows, a particle (●)128 moving in the microchannel may be affected by dielectrophoretic forces if the dielectrophoresis is different from the complex permittivity of the surrounding fluid 130. Particles of interest, such as cells, microbeads or liposomes, can be driven by negative dielectrophoresis to encourage these particles to move towards a minimum (Δ)170 electric field. The relative density of these particles is also higher than the relative density of the surrounding buffer. For this reason, they can settle under the action of gravity.

Fig. 10 and 11 show that particles flowing in microchannel 104 may encounter electrode 153,154,155,156 before reaching the microwell and electrode 116. In accordance with the principles of dielectrophoresis, application of voltages of various configurations can cause particles 128 to be trapped or repelled by the electric field where the particles move toward a minimum (Δ)170 electric field and away from very strong electric fields.

In one embodiment, the electrode 116 is set to ground. Electrodes 155 and 156 are polarized by an external power source to become voltage generators. The voltage phase of electrode 155 is rotated 180 degrees with respect to the voltage phase of electrode 156. The voltages applied to the electrodes 155 and 156 have the same magnitude. The voltage may vary with a sinusoidal signal or a square wave. For this reason, a relatively large electric field exists between the two electrodes 155 and 156.

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

As with the electrode configurations shown in fig. 9-11, electrodes 153 and 154 are set to ground potential when desired particles 128 flowing in microchannel 104 are trapped in micropores 102. Electrodes 155 and 156 are polarized by an external power source to become voltage generators. The voltage phase of electrode 155 is rotated 180 degrees with respect to the voltage phase of electrode 156. The voltages applied to the electrodes 155 and 156 have the same magnitude. The voltage may vary with a sinusoidal signal or a square wave. For this reason, a relatively large electric field exists between the two electrodes 155 and 156.

In this case, a "trapping" situation will occur due to the electric field reaching a strong minimum in the position 170 placed between the electrodes 153 and 154. The reserved area in the channel is dominated by the stronger electric field, pushing the particles towards the minimum (Δ) electric field area. The thickness of electrodes 153 and 154, and all other electrodes, is about 5 to 30 microns. This will create a region (a) 170 where the electric field reaches a minimum and where the particle has almost the same size as its surrounding electrodes.

Region (Δ)170 is aligned with the opening of microwell 102. The particles trapped in the minimum value region (Δ)170 are pushed along a horizontal path toward the opening of the micro-pore by the fluid flowing in the micro-channel. The distance between the area (Δ)170 and the opening of the micro-hole 102 is set to a value as small as possible as is feasible in terms of manufacturing technology. A typical distance is about 20 to 100 microns if standard printed circuit board technology is used. Since electrode 116 surrounds microwell 102 and is connected to ground, the presence of electrodes 155 and 156 will create an electric field in the channel below microwell 102. After the previous analysis, the particles will now be pushed towards the micropores.

If the position of the settling of the particles in the microwells has to be precisely controlled, electrode 118 can be polarized by connecting electrode 118 to a voltage source whose amplitude is substantially smaller than the amplitude used to control electrodes 155 and 156 and whose phase is rotated by 90 degrees or 270 degrees with respect to the phase of electrodes 155 and 156. This polarization may create dielectrophoretic forces in the microwells that push the particles toward the center of the microwells.

If particles flow in the microchannel 104 and are not intended to enter the micropores, the desired effect can be obtained by polarizing the electrodes 153 and 154 in a "repelled" configuration. In this case, the minimum electric field is placed in the middle of the microchannel 104. In this position, the particles are exposed to the fluid flow and, if microwell 102 is spaced less from electrodes 153 and 154, the particles are not trapped by microwell 102.

In another embodiment, particle movement may be controlled by first trapping the particles between electrodes 153 and 154, and then programming the "load" or "eject" configuration by appropriately setting the polarization of electrodes 153,154 and 116 while keeping electrode 155 constantly connected at a sinusoidal voltage, and electrode 156 phase shifted by 180 degrees connected at the same sinusoidal voltage as electrode 155. To capture the particles first, electrodes 153 and 154 are grounded and electrode 116 is connected to the same sinusoidal voltage as electrode 155, shifted 90 degrees. By setting the fluid flow from the electrodes 153 and 154 to the micropores, a sufficiently low flow value can be found that particles in the minimum electric field between the electrodes 153 and 154 can be trapped with a drag force that is insufficient to prevent the particles from moving forward and that opposes the electric field established between the electrode 116 on the one hand and the electrodes 155 and 156 on the other hand. If it is desired to transfer the particles to the microwells, the electrodes 116 are connected to ground and the drag force will move the particles towards and then into the microwells. Conversely, if the particles have to move towards and around the microwells, 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 particles first move upward and then move to the portion of electrode 116 below electrode 156 where the minimum electric field is also located. In this way, micropores are circumvented and particles are not transferred. Assuming now that there is a second pore along the channel and having the same configuration as shown in fig. 9, a new set of electrodes 153 and 154 may be set to ground and the electrode 116 of the first pore may be brought back to the original voltage. In this way, the minimum electric field will be disposed between the pair of electrodes 153,154 of the second microwell, and the particles will be disposed between these electrodes. This scheme can be repeated in several wells arranged along the same microchannel.

7.4 detection of particles in microchannels

When the electrodes shown in fig. 9-14 in such a configuration manipulate the particles in the microchannel 104 to pass the particles to the target microwell, the electrodes can have the effect of aligning the particles along the axis F-F' shown in fig. 15-16. Furthermore, the particles may be pushed vertically towards the bottom side of the microchannel. Given that a continuous laminar flow is present in the microchannel and has the effect of pulling the particles in the direction F-F', the precise positioning of the particles along the predetermined axis and at the precise vertical position makes them suitable for optical detection or impedance measurement detection. For example, Duqi et al, 2011 (May), "automatically separate a programmed number of cells into microwells using DEP force and optical detection" ("Automated isolation of a programmable number of cells in micro wells using DEP force and optical detection"), International Conference on micro technology in medicine and Biology (International Conference on micro technologies in medicine and Biology) has demonstrated the benefits of improved optical detection and improved cell transfer in microwells by dielectrophoresis to achieve cell focusing.

In one embodiment, as shown in FIG. 15, a structure comprising four electrodes 170,171,172,173 is arranged downstream of the steering electrodes 153, 154. The electrodes are designed to allow particles to move in the direction F-F' and pass through the gap between electrodes 170 and 171 and 172 and 173. Electrodes 171,173 are connected to an excitation signal 188 represented by an alternating voltage, while electrodes 170,172 are connected to a readout circuit 186, which readout circuit 186 can amplify output currents 180 and 182 and provide a terminal output signal 184 proportional to the difference or ratio between signals 180,182. This can be done using known circuit schemes. The presence of upstream electrode 153,154,155,156 has the effect of aligning the cells along the axis on which the signal-to-noise ratio produced by the channel of a single cell on output signal 184 is greatest.

In another embodiment, as shown in FIG. 16, after the single cells are properly arranged and aligned by electrode 153,154,155,156, detection of the single cells is performed by a structure comprising three electrodes 174,175, 176. Electrode 175 is connected to an excitation signal 188 represented by an alternating voltage, while electrodes 174,176 are connected to a sensing circuit 186, which sensing circuit 186 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 electrode 153,154,155,156 has the effect of aligning the cells along the axis on which the signal-to-noise ratio produced by the channel of a single cell on output signal 184 is greatest.

In one embodiment, after the individual cells are properly arranged and aligned by electrodes 153,154,155,156, under either fluorescent or normal lighting conditions, the individual cells are detected by optical detection 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 axis F-F'.

In a particular embodiment, the structure represented by electrode 116,153,154,155,156,116,118 and microwell 102 and reported by FIGS. 9-11 will be replicated and placed downstream of the detection region or electrode upon detection of a particle according to one of the methods previously described. The control of these electrodes depends on the sensing signal 184 obtained from optical or electrical measurements in a manner that, upon detection, individual cells can be selectively pulled into or pushed out of the target microwell. Such an embodiment enables a precise positioning of a single cell or a predetermined number of cells in a microwell.

7.5 aggregation of particles in micropores

When a particle 128 enters a micro-pore 102, gravity may cause the particle 128 to fall through the micro-pore 102 towards the meniscus 122. To ensure that the particles 128 remain substantially centered in the microwell 102, the microwell electrodes 116,118,120 may be energized to push the particles 128 to a position centered on the diameter of the microwell 102. For example, the voltages applied to electrodes 116,118,120 may be controlled to generate a substantially zero or substantially vertical electric field at or near the vertical axis of symmetry of microwell 102. This causes particle 128 to be pushed toward the center of micro-hole 102, causing particle 128 to fall the length of the micro-hole along the central axis and deposit at or near the center of meniscus 122. Gravity or electromagnetic forces or a combination of these may assist in the dropping of the particles.

In some embodiments, when the microwell electrodes 116,118,120 are ring electrodes, the microwells may include paired electrodes, such as facing electrodes, for particle manipulation and movement within the microwells. In addition, the microwell may contain additional electrodes, including, e.g., electrodes for a particular assay or reaction, such as sensing electrodes and/or measurement electrodes for sensing particles, cell products, and cell changes. For example, sensing electrodes and/or measuring electrodes may be used to measure changes in electrical signals such as impedance, optical signals, polarity, and the like.

Particles 128 deposited on meniscus 122 remain stably disposed on meniscus 122 even after the electromagnetic force (e.g., voltage applied to the electrodes) is discontinued. Published U.S. patent application No. 2009/0288963, the entire contents of which are incorporated herein by reference, describes an exemplary method of applying voltages to electrodes to manipulate particles with dielectrophoresis.

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

In some embodiments, two or more particles 128 are allowed to enter micropores 102 and are arranged so as to urge the particles into contact or proximity with each other. For example, after first particle 128 is disposed at meniscus 122, the voltage applied to electrodes 153 and 154 may be removed, while microwell 102 is opened to receive one or more other particles from microchannel 104. As discussed above, when one or more other particles enter the microwell 102 from the microchannel 104, the other particles 128 may be concentrated in the center of the microwell 102 by applying a voltage to the electrodes 118, 120. The other particle 128 falls along the vertical axis of symmetry of the micro-pore until the other particle is disposed in contact with or in close proximity to the particle 128 on the meniscus 122. In an alternative embodiment, all of the particles are introduced into the micro-wells at substantially the same time, and the electrodes 116,118,120 are energized to concentrate the particles in the center of the micro-wells as a population of cellular polymers, while forming a cluster of deposited cells at the meniscus.

8. Microscopic observation of deposited cells

Due to the open nature of microwell 102, the anchoring of particles 128 on meniscus 122 at the fluid-air interface of lower end 108 of microwell 102 can be observed, for example, using a microscope, camera, or other optical device. The particle 128 may be viewed from the upper end 106 of the microwell 102, e.g., through the microchannel 104 and a cover made of a transparent material, e.g., glass or plastic, or from the lower end 108 of the microwell, e.g., where there is no obstruction to the field of view.

9. Stable dwell at meniscus

Surprisingly, when the electric field is deactivated, the particles, including living cells, can stay stably at the meniscus while the fluid continuously flows in the microchannel, entering the microwells. It was surprisingly found that neither fluid washing nor changing the medium could move the cells on the meniscus in the absence of an electric field, provided the microwells were of appropriate size. This stable residence allows for the removal of harmful liquids, such as buffers and reagents required for DEP operation, and allows for the renewal of fresh buffers and media more suitable for the maintenance and viability of the biological particles deposited in the microwells and/or more suitable for the particular analytical method.

A stable dwell on the meniscus is obtained if the shear stress induced by the horizontal part of the fluid velocity at the bottom level of the micro-pores is relatively low or absent. By varying the relative ratio of the diameter to the depth of the closed pores, the horizontal component of the fluid velocity is also varied, as explained in Han et al, 2010, "Integration of single pore tracking, in vitro transduction and embryo culture in a microwell-structured device," Lab on a Chip, vol.10, No.21, pp.2848-2854. Similar performance can also be observed in inverted open pores featuring a meniscus between 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 pore of 75 microns in diameter, a depth of 100 microns or more is contemplated. This pore size is capable of maintaining a horizontal fluid velocity of less than 1 micron/second for an average fluid velocity of 2.5 mm/second in the microchannel.

In the inverted open microwell system, nutrients in a buffer or a culture medium flowing in a microchannel and catabolites produced by the removal of cells can be continuously supplied and help maintain the viability of the cells or other particles disposed in the microwells. Furthermore, exposure to the atmosphere outside the device at the lower open ends of the micropores enables gas exchange, which helps to maintain the health and viability of the particle or particles contained in the open micropores.

Surprisingly, in the inverted open microwell system, a combination of features including small volume, stable residence, efficient exchange of buffer and culture media, efficient gas exchange, and the ability to manipulate and aggregate cells to desired sites using minimal electromagnetic forces, allows for efficient use of the system not only to assess the characteristics of individual cells, but also to maintain, reuse, and recover the original cells for clonal expansion.

10. Recovery of microporous contents

In one embodiment, the microwell contents can be retrieved, for example, by breaking 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 pump 132 or a compressed air source, to the microchannel 104, the pressure pulse having a pressure in the range of 0.5bars to 3bars and a pulse duration in the range of 1 millisecond to 100 milliseconds. For example, the contents of a microwell can be released as droplets containing, for example, individual particles, cells, or clusters of particles or cells in a microwell fluid, into a suitable container 126 (fig. 4,5) located below the microwell 102, such as a reservoir, microtiter plate, collection vial system, capture surface, or the like. In one embodiment, the vessel 126 may include a filter or membrane for separating one or more particles from the fluid recovered from the microwells 102.

In one embodiment, the fluid surrounding the particles 128 in the micropores may be concentrated by evaporation prior to recovery from the micropores. For example, replacing the fluid in the microchannel with air or a suitable gas may result in a reduction in volume in the microwells, as evaporation will occur at the lower end 108 of the microwells that are open to air or gas. Applying pressure to the air/gas in the microchannel may cause the concentrated fluid in the micropores to be released and into the one or more containers.

In one embodiment, at or near the air/fluid interface at the lower end of the microwell, the microwell contains a semi-permeable membrane 134 that can retain one or more particles. In this embodiment, fluid may be recovered from each microwell 102 into one or more containers 126.

In the case where the device 101 comprises a plurality of microwells, the contents of the wells can be transferred to a set of matched, parallel, multiple containers, such as to the wells of a microtiter plate.

During transfer of the cells from the microwells, when the pressure pulse is applied, other cells or particles may be captured in the microchannel and transferred along with one or more cells in the microwells. To combat this problem, a coating of Bovine Serum Albumin (BSA) or other anti-stiction coating, such as an organic passivation layer, such as a fluorinated fatty acid self-assembled monolayer (SAM) or an alkylhalosilane, may be added to the microchannels prior to use.

Examples of the invention

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

Example 1

Geometric analysis of fabricated microchannels or microwells

1.1 preparation of

A device comprising a plurality of open micro vias is created by drilling through holes from a substrate of a multi-layer flexible Printed Circuit Board (PCB). The dimensions of the micropores were varied to analyze the effectiveness of the micropores for a particular geometry. In this study, the drilled holes varied in diameter from 70 μm to 150 μm, the thickness of the PCB was between 75 μm and 350 μm, and each device included either a 6 × 6 or 8 × 8 matrix of holes. To interface the open microwell array directly to a standard microtiter plate and to perform cell recovery and transfer easily into microtiter wells, preferred instruments were prepared as shown in table 1 below.

On the top side of the plurality of micropores 102, microchannels 104 having a thickness varying from 30 μm to 200 μm and a width varying from 200 μm to 1mm are made of polyimide and arranged above the upper ends 106 of the micropores. The channel cover 124 is formed by adhering a transparent polycarbonate having a thickness of 750 μm to the top of the microchannel.

TABLE 1 microwell array characterization

Number of micropores	Arranged in x rows	Pore-pore and channel-channel spacing
			24	6x4	18mm
96	12x8	9mm
			384	24x16	4.5mm
1536	48x32	2.25mm

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

In an alternative embodiment, a photosensitive polymer film (Ordyl SY550, ElgaEurope) having a thickness of 55 μm is attached to a glass top cover to create the micro-channels before it is attached to the bottom flexible PCB containing the drilled micro-holes. An aperture is formed in the top cover to provide fluid connection for input and output or to embed a reservoir. The hole diameter is about 0.45 mm.

In one study, a peristaltic pump (Watson Marlow 101U/R) connected as shown in FIG. 5 and an inlet to another microchannel (KDS-210, KD Scientific, Holli) was usedston, MA) connected syringe pumps that inject fluid or fluid containing cells and particles into each microchannel. The fluid flow varies from 1 microliter/hour to 20 microliter/minute depending on the cross-section of the channel and the particular operation to be performed on the particle. Capillary forces acting on the micropores may prevent fluid leakage from the lower ends of the micropores. In some cases, a hydrophobic coating may be applied to the bottom surface of the device(s) ((FC-732) to increase the resistance to fluid leakage. As a result, the fluidic system allows fluid in the microchannel to accidentally fill the micropores without leaking out of the lower end. 1.2 analysis of cells sedimented by gravity

Viable K562 cells (immortalized human granulocytic leukemia cells) were suspended in physiological solution (NaCl 0.9% w/v or PBS) at a concentration of 1.6 × 10e6 cells/ml and injected into the wells of an inverted microwell device using a peristaltic pump. The width of the microchannel was 600 μm, the height was 55 μm, and the diameter of each micropore was 70 μm.

The input pump is arranged to operate in cycles, wherein each cycle comprises two phases. During phase 1, the fluid was activated at a rate of 9 microliters per minute, while during phase 2, the pump was removed for 1 minute and 30 seconds. During phase 1, cells flow in the channel along random trajectories. At the beginning of stage 2, cells are fixed in the channel in a randomly localized manner. During phase 2, a certain number of cells are allowed to enter each microwell by sedimentation.

The contents of each well were examined using an inverted microscope and the possible presence of cells in the microwells was detected as a result of sedimentation. The results show 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 efficiency of cell loading in microwells (41 samples in total)

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

The results of the loading phase show that a single cell or multiple cells can be deposited in the microwell, reach the air-fluid interface at the lower open end, and can be captured and survive there without destroying the surface tension of the meniscus. In those microwells, there was no other force applied to the cells than gravity during their descent into the microwells, and the position of the cell deposition onto the fluid meniscus was random (see fig. 6A).

1.3 analysis of cells sedimented by aggregation arrangement and gravity

Using two or more electrodes, it is possible to generate dielectrophoretic forces acting on particles in a microwell and to manipulate the particles 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 during particle settling, as shown in fig. 2. In each pair of adjacent electrodes 116,118,120, a first electrode is connected to a sinusoidal signal while a second electrode is connected to ground or the same sinusoidal signal with a phase rotation of 180 degrees.

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

K562 cells were suspended in a 1:9 ratio of PBS to glycerol (300mM) and electrodes 116 and 120 were connected to the same sinusoidal voltage with a typical frequency comprised between 80KHz and 100 KHz. The magnitude of the applied voltage varies from 3.4V to 15V. The electrode 118 is grounded. Since four cells are loaded into a microwell while keeping the electric field activated, each cell is forced to align along the central axis of the microwell. Once the air/fluid meniscus at the lower end of the microwell is reached, each cell is centered and in contact with the previously loaded cell or cells. This polymerization results from a combination of vertical gravitational forces and the horizontal portion of the dielectrophoretic forces.

The open microwells were observed under fluorescent conditions using an inverted microscope 140. When the electrodes in the well are appropriately polarized, one or more cells are concentrated to the central axis of the microwell. During the descent along the central axis, the cells thus aligned each deposit at the center of microporous meniscus 122, forming a polymer at the air-fluid interface and causing the cells to contact the cells.

K562 cells were labeled with fluorescent dye and observed using an inverted microscope. As shown in fig. 6, although cells are randomly deposited by gravity at the meniscus of the microwell (6A) when no electric field is applied, the same cells deposited by the combination of vertical gravity and dielectrophoretic forces can form a polymer of particles deposited at the central portion of the meniscus (6B). 1.4 analysis of microbeads deposited by aggregation alignment and gravity

Polystyrene microbeads 10-25 μm in diameter are suspended in deionized water or glycerol at a concentration of 22.5mM and transferred by gravity into microwells having a diameter of about 80 μm. The electrodes 116 and 118 are connected to the same sinusoidal voltage with a frequency of 100 KHz. The electrode 117 is grounded. Aggregation of 2 bead polymers 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 from the signal amplitude.

TABLE 3

Magnitude of voltage (V)	Average distance (μm)	Bead-to-bead contact ratio
			2	7.2	33％
4	2.2	90％

1.5 on-chip labeling of cells trapped in the meniscus

Analyzing the effect of changes in fluid in the microchannel on fluid surrounding the captured cells on the open-microwell distal end meniscus to determine the ability of the inverted open microwell system to provide a single cell centrifugation function. Calcein staining protocol was performed on K562 cells.

K562 cells were delivered to inverted open microwells by limiting dilution and sedimentation. After transferring the cells to the meniscus, the microchannel was rinsed with PBS flowing in the microchannel for 5 minutes. A buffer containing calcein (1mM in NaCl 0.9% w.v), a tracer molecule that becomes fluorescent when taken up by cells, was flowed continuously in the microchannel for 40 minutes at a constant flow rate of 9 microliters per minute. FIG. 4 shows the dynamics of the fluorescence intensity monitored on individual cells in a microwell.

Cell staining was effective showing diffusion of calcein from the microchannel to the microwells and the stably retained cells. The cells were unexpectedly retained on the meniscus after washing and were in a viable state as shown by calcein uptake. By monitoring the uptake of calcein in individual cells, different uptake characteristics can be observed. Typically, maximum uptake is achieved after 30-40 minutes. The fluorescence intensities are shown in Table 4.

TABLE 4

Example 2

2.1 recovery of K562 cells following delivery and DEP aggregation

Analysis of single cell function using the inverted open microwell system allows recovery of cells that have been determined to have the desired properties. For example, after a single cell and/or particle is captured by each microwell of a plurality of microwells (e.g., a microwell array), cell function can be analyzed by one or more biological assays. When a particular cell is considered to have one or more desired characteristics or functions, the contents of each microwell can be retrieved and transferred to a substrate, such as a microtiter plate.

Devices of the character shown in figure 5 were developed to enable cell recovery from the inverted open. To release the contents of the microwells, a pressurized filtering gas such as nitrogen or air is injected into the microchannel 104 in a controlled manner. For example, by connecting the input of a normally closed electrovalve 172, a pressure pulse of about 1bar is applied.

An electronic system connected to the control input of the electrovalve 172 generates a voltage or current pulse having a duration of about 5 milliseconds. With respect to the generation of the pulse, the compressed gas enters the microchannel 104 via tubing connected to the microchannel input and output ends, as a result of which fluid droplets 138 are expelled from each microwell 102 and drip onto a receptacle 126, represented by a capture surface in some experiments or a microtiter plate in other experiments, wherein the plate is placed under the microwell array and properly aligned. One or more particles and/or cells present in the microwell are transferred to the capture surface in the form of a fluid droplet 138.

During cell transfer from a microwell, 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 circumvent this problem, a coating of Bovine Serum Albumin (BSA) was injected into the microchannel for 30 minutes to form a protein self-assembled monolayer prior to use. The microchannels were washed with PBS for 20 min before cell recovery. All cells have been removed from the microchannel so that only the cells contained in the microwells are transferred to the recovery vessel. 2.2 rejuvenation of cell viability and growth

One of the functions of the inverted microwell system is to assess the function of a single cell (a single cell) or multiple cells (cells) and to recover from or expand in the microwells living cells that have been determined to have the desired functional properties. For example, cell viability under activating or deactivating electric fields and DEP and for varying lengths of time can be assessed by monitoring the growth and expansion of recovered individual cells or small cell clusters, e.g., counting the number of cells per day. The growth of the cells can also be monitored by observing the cells under a microscope.

The individual K562 cells were resuspended in physiological medium, transferred to microwells by sedimentation, and deposited on the air/fluid meniscus of multiple microwells. After 20 minutes, single cells were transferred by delivering a pressure pulse to the fluid in the channel, as described above, resulting in transfer of droplets 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 individual cells were pelleted in V-shaped wells and observed with an inverted microscope.

At 37 ℃ 5% CO₂The cells were incubated and cultured for several days. The cell culture was observed daily to confirm the growth of the clonal cell population from each single cell recovered. Growth and expansion of cells was shown after 3-5 days, and a monoclonal cell line had been generated from the single sedimented cells. The growth of the single cell or small cell polymer is shown in a series of timed photographs and reported in the graph of fig. 8. This study showed that deposition of cells on the meniscus of an inverted microwell and recovery of said cells is possible and enables recovery of live cells.

Example 3 analysis of cell-cell interactions

3.1 Activity of CTL cells against target LCL cells

The functional, living cell-to-cell interactions in the inverted microwell system are shown by T lymphocytes inducing cytolysis of the target tumor cells. Target cell LCL cells were labeled with calcein and delivered to microwells. T Lymphocytes (CTL) against the target cells are activated and delivered into the same microwells. The fluorescence of the target cells was monitored by fluorescence microscopy and calcein fluorescence properties were determined as a measure of cell lysis.

Activated CTL cells induce a sustained decrease in fluorescence of the target cells compared to negative controls. As shown in fig. 7, the decrease and disappearance of the fluorescence of the active target cells observed in the inverted open microwell system within 20 minutes shows the effective lysis of the target cells induced by the CTLs (an effective analysis of the interaction induced between a specific cell and a cell) and the functional results thereof are detected within several minutes.

Table 5 reports the fluorescence intensity detected on some LCL targets delivered in inverted open microwells of 70 μm diameter. As a control, we delivered LCL cells alone. The CTL-LCL interaction was detected under two conditions: there was no Human Papillomavirus (HPV) infection of the target LCL, and there was HPV infection. In the first case, no lysis is expected, while in the second case, the CTL cells are expected to recognize the target and lyse the LCL cells. The results reported in this table show a strong decrease in fluorescence intensity of CTL-LCL conjugates (couples) within 30 minutes when the LCL is infected with HPV (cases e-h). In contrast, in only one case (d), a-specific (a-specific) lysis can be obtained, representing the recognition of the target cells by the CTL cells even in the absence of HPV infection. In all other non-infectious cases (a-c), only a physiological decrease in the fluorescence signal was observed.

TABLE 5

Case(s)	Cell type	Time (minutes)	Strength (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 control of particle transport in micropores with active fluid flow

Experiments were performed to verify the function of the inverted open microporous structure as shown in the figure. Devices were made with circular pores having a diameter of 100 μm and dielectric layer thicknesses of 50 μm and 25 μm. The thickness of each electrode was 9 μm. The gap 8 between the electrodes 153 and 154 was 50 μm. The microchannel has a height of 150 μm and a width of 350 μm.

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

In two different experiments, microbeads and cells were introduced into the microchannel and the control electrodes 116,153,154,155,156,118 were polarized to encourage (capture) or discourage (repel) particles into the microwells. All signals, except the ground electrode, were sinusoidal signals with a frequency of 100kHz and the same amplitude. Table 6 reports the phase shift scheme. The function of the control electrodes is determined by statistical analysis of the number of particles delivered to the microwells for each particle and cell delivered according to particle velocity and applied voltage.

TABLE 6

When the trapping configuration is activated, the number of particles delivered to the microwells increases due to higher signal amplitude and lower fluid velocity. Specific results are reported in tables 7 and 8, where at least 50 samples were considered for each numerical value reported.

When the expelling configuration is activated, we find that the structure works properly (i.e. no particle transfer) for the case where the peak-to-peak voltage amplitude is greater than 2V and the velocity of any particle is in the range of 15-150 μm/s.

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

TABLE 7

TABLE 8

Example 5 controlling the external environment of the inverted open microwells to reduce evaporation and enhance cell viability

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

The equipment used to control the evaporation comprises a 384 well microtiter plate, wherein each well contains 100uL of a fluid such as water, RPMI, PBS or any buffer suitable for cell culture at a storage temperature between 4 ℃ and 10 ℃. The array of inverted open microwells 114 is rested on the microtiter plate in such a way that each well of the microtiter plate is aligned with an inverted open microwell, thereby creating a closed chamber containing the fluid pre-stored in the microtiter plate, which can be evaporated in the closed chamber, thereby increasing the humidity. Typically, the perpendicular distance between the meniscus of the inverted open microwell and the well varies from 0.5mm to 5 mm. After a few minutes, the vapor pressure in the chamber reaches a saturation value, preventing any further evaporation from the microtiter plate and inverted open microwells.

To show the positive effect of controlling the humidity under the microwells on reducing evaporation in the microwells, a device 101 featuring microwells with the electrode configuration shown in fig. 2 was used. Electrodes 116 and 120 are grounded while electrode 118 is connected to a sinusoidal signal having a frequency of 100kHz and variable amplitude.

K562 cell suspension maintained at a temperature between 30 ℃ and 37 ℃ was injected into the microchannel and fluid flow was stopped to configure individual cells in the microchannel above the inlet of the microwells. In the presence of evaporation, the cells are subjected to a drag force F directed downwards and by the fluid flow generated by evaporation at the air-fluid interface_D. In addition, cells were treated with F_B-F_G-F_D＝F_DEPyBy the gravity force F_GBuoyancy F_BAnd vertical part of dielectrophoretic force F_DEPy。

Due to the relatively high value of dielectrophoretic forces, the cell is still trapped at the entrance of the microchannel, the forces acting on the cell bringing it to equilibrium. When the dielectrophoretic force is reduced, the vertical position of the cell is lowered and reaches a region where the electric and dielectrophoretic forces are higher. This behavior is observed until the magnitude of the electric field reaches a minimum threshold. If the amplitude is further reduced, the cell will fall into the microwell because the dielectrophoretic force is not strong enough to counteract the other forces acting on the cell.

The vertical position of cells of inverted open microwells surrounded by air with or without humidity control was compared. The reference value for the height is the top side of the top electrode 116. Positive values of the 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, the cell height was always higher when controlling humidity. This shows that the presence of the equipment for controlling humidity under the inverted open micropores can effectively remove the additional drag force F due to evaporation_D。

TABLE 9

To maintain a proper physiological environment for cells trapped on the meniscus at the air-fluid interface, evaporation needs to be controlled. In fact, the presence of evaporation will cause an increase in the local concentration of salts and other nutrients contained in the culture medium, and a consequent increase in the osmotic pressure. After evaporation control was performed, 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, "calcium-acetyl ethylene cytotoxin Assay: Standardization of a Method with added amplification on modified efficiency Cells and supermatants", in which Cells were stained with 1 μ M Calcein and the signal loss per hour was approximately 8%, which corresponds to a well-known physiological loss.

This specification includes a number of citations for published references and patent documents, each of which is incorporated herein by reference in its entirety.

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

Claims

1. An inverted microwell system for microanalysis of particle function, comprising:

a) a microchannel having a fluid inlet and a fluid outlet, wherein the microchannel is configured to allow fluid to flow between the fluid inlet and the fluid outlet;

b) at least one microwell having open upper and lower ends, the upper end being open to the microchannel, the microchannel facilitating fluid delivery to the microwell;

c) a controlled electrode array arranged along the microchannel between the fluid inlet and the at least one microwell, wherein the electrode array comprises a plurality of upstream electrodes positioned along an axis (B-B') perpendicular to fluid flow in the microchannel, the plurality of upstream electrodes comprising at least a first upstream electrode (153), a second upstream electrode (154), a third upstream electrode (155), and a fourth upstream electrode (156) at least partially embedded in a substrate (114) such that adjacent electrodes of the plurality of upstream electrodes are separated from each other by a portion of the substrate (114), wherein the third upstream electrode (155) is positioned above the first upstream electrode (153), the fourth upstream electrode (156) is positioned above the second upstream electrode (154), and the first upstream electrode (153) faces the second upstream electrode (154), and the third upstream electrode (155) faces the fourth upstream electrode (156).

2. The inverted microwell system of claim 1, further comprising a pair of facing electrodes (116,116A) at least partially surrounding the at least one microwell, the third electrode (155) being at a position above a first facing electrode (116) of the pair of facing electrodes, and the fourth upstream electrode (156) being at a position above a second facing electrode (116A) of the pair of facing electrodes.

3. The inverted microwell system of claim 1, further comprising at least one additional electrode (116) at least partially surrounding the at least one microwell, shaped such that it provides an opening facing the fluid inlet, the third and fourth upstream electrodes (155) and (156) being at a position above the additional electrode (116).

4. The inverted microporous system according to any of claims 1 to 3 further comprising along the microchannel: a structure comprising four downstream electrodes (170,171,172,173) located downstream of the first and second upstream electrodes (153,154) with respect to fluid flow along the microchannel, wherein the downstream electrodes and the first and second upstream electrodes are arranged parallel to fluid flow in the microchannel, and the downstream electrodes and the first and second upstream electrodes are located on the bottom of the microchannel.

5. The inverted microporous system according to any of claims 1 to 3 further comprising along the microchannel: a structure comprising three detection electrodes (174,175,176) located downstream of the first and second upstream electrodes (153,154) with respect to fluid flow along the microchannel, wherein the detection electrodes and the first and second upstream electrodes (174,175,176,153,154) are placed on an axis (F-F') parallel to fluid flow in the microchannel, and the detection electrodes and the first and second upstream electrodes (174,175,176,153,154) are located on the bottom of the microchannel.

6. The inverted microwell system of claim 1, wherein the at least one microwell has an open lower end and comprises a semi-permeable membrane (134) at the lower end.

7. The inverted microwell system of claim 1, wherein the lower end of the at least one microwell is open to the atmosphere outside of the at least one microwell.

8. A method for precisely transporting at least one particle in a microchannel, comprising:

a) providing an inverted microwell system comprising at least one microwell open at an upper end to at least one microchannel, wherein an electrode is disposed along the at least one microchannel;

b) adding at least one particle to the at least one microchannel;

c) creating a pattern of electric field strength in the at least one microchannel to cause the at least one particle to move in a desired direction in the at least one microchannel.

9. The method of claim 8, wherein the inverted microwell system is the system claimed in claim 2, and the method comprises:

a) polarizing the first and second facing electrodes (116,116a) at voltages of the third and fourth upstream electrodes (156,155), respectively, while the first and second upstream electrodes (153) and (154) are at the same ground voltage, and trapping the at least one particle at a location (Δ) between the first and second upstream electrodes (153) and (154).

10. The method of claim 9, further comprising after step a):

b) connecting the facing electrodes (116,116a) to ground and polarizing the first and second upstream electrodes (153) and (154) to fifth and fourth upstream electrodes (156,155) urges the at least one particle towards a minimum (170) of the electric field between the facing electrodes (116,116a) at which the at least one microwell is located.

11. The method of claim 8, wherein the inverted microwell system is the system claimed in claim 4, wherein an upstream electrode (153,154,155,156) lines the particles along an axis, wherein a signal-to-noise ratio of an output signal (184) resulting from passage of the at least one particle is maximized, and the method comprises:

a) connecting first and second downstream electrodes (171,173) of the downstream electrodes to an excitation signal (188) represented by an alternating voltage;

b) connecting third and fourth downstream electrodes (170,172) of the downstream electrodes to a readout circuit (186) that amplifies first (180) and second (182) output currents, the first and second output currents corresponding to the third and fourth downstream electrodes, and wherein the readout circuit further provides the terminal output signal (184) proportional to a difference or ratio between the first and second output currents (180, 182).

12. The method of claim 8, wherein the inverted microwell system is the system claimed in claim 5, wherein an upstream electrode (153,154,155,156) lines the particles along an axis, wherein a signal-to-noise ratio of an output signal (184) resulting from passage of the at least one particle is maximized, and the method comprises:

a) the electrodes (175) are connected to an excitation signal (188) represented by an alternating voltage (186), while the detection electrodes (174,176) are connected to a readout circuit (186) that amplifies the first and second output currents (180) and (182) and provides the terminal output signal (184) proportional to the difference or ratio between the first and second output currents (180, 182).

13. The method of any one of claims 8-12, further comprising: the at least one particle is detected by optical detection after proper placement and alignment.