WO2012178095A1

WO2012178095A1 - Electromotive system for high-throughput screening

Info

Publication number: WO2012178095A1
Application number: PCT/US2012/043861
Authority: WO
Inventors: Igor Mezic
Original assignee: Ifluidics, Inc.
Priority date: 2011-06-22
Filing date: 2012-06-22
Publication date: 2012-12-27

Abstract

The present application discloses a system for high-throughput screening experiments consisting of a function generator, controller and a high-lhroughput screening device, such as a microplate, microslide or microarray.

Description

T U 2012/043861

ELECTROMOTIVE SYSTEM FOR HIGH-THROUGHPUT SCREENING

FIELD OF THE DISCLOSURE

[0001] The present invention relates a s stem for high-throughput screening experiments

consisting of a function generator, controller and a high-throughput screening device, such as a micro-plate, micros!lde or microarray.

RELATED APPLICATIONS

[0002 The following related applications are herein incorporated by reference in their entireties; U.S. Patent Application No. i 1/561 ,248, filed ! [- 37-2006, published as U.S.

Publication No, 2007-0175755; U.S. Patent Application No, 12/1 18,343, filed 05-09-2008,

published as U.S. Publ ication No, 2009-0038938; and U.S. Patent Application No.

12/120,620, filed 05-34-2008, published as U.S. Publication No. 2009-0032398,

BACKGROUND OF THE DISCLOSURE

[0003] High -throughput screening (HTS) is a method for scientific experimentation

especially used in drug discovery and relevant to the fields of biology and chemistry. Using

robotics, data processing and control software, liquid handling devices, and sensitive

detectors, High-Throughput Screening or FITS allows a researcher to quickly conduct

millions of biochemical, genetic or pharmacological tests. Through this process one can

rapidly identify active compounds, antibodies or genes which modulate a particular

biomolecuiar pathway. The results of these experiments provide starting points for drug

design and for understanding the interaction or role of a particular biochemical process in

biology. The key labware or testing vessel of HTS is the microliter plate: a small container, usually disposable and made of plastic, that features a grid of small, open divots called wells.

Modem (circa 2008) microplates for HTS generally have either 384, 1536, or 3456 welts.

These are all multiples of 96, reflecting the original 96 well microplaie with 8 x 12 9mm

spaced wells. Most of the welts contain experimentally useful matter, often an aqueous

solution of dimethyl sulfoxide (DMSO) and some other chemical compound, the latter of

which is different for each well across the plate. Current methods for high-throughput screening in large-well number (e.g. 1536 and 3456) vessels, as well as rnicrostides and microarrays are static: no dynamic liquid or particle manipulation is done prior to deploying 3 detection method, This leads to long processing times and problems with accuracy and sensitivity,

[0004] Devices using eleetrokinetic properties (electrophoresis, ^electrophoresis, electroosn-iosts and electrothermal convection) alongside with thermal convection have been used to manipulate fluids and particles in small liquid volumes [29], Electric fields induce a force on charged particles in solutions, moving the particles towards either the cathode or the anode depending on the sign of the charged particles [22] ,

|^'0005{ Electrophoresis is a technique for manipulating components of a mixture of charged molecules (proteins, DNAs, or RNAs) in an electric field within a gel or liquid. Under AC electric field, uncharged particles suspended in a dielectric media can be polarized and further manipulated, if the field is spatially inhomogeneous, it exerts a net force on the- polarized particle known as dielectrophoretic (DEP) force [ I ]. This force depends upon the temporal frequency and spatial configuration of the field as well as on the dielectric^, properties of both the medium and the particles. Single frequency electric fields can be used to transport and separate particles.

[0006] Fluid motion can also be induced by appiying an electric field onto a solution. The force driving the fluid thus originates m ^" the bulk (buoyancy, electrothermal effect) or at the interface between the fluid and the device containing the fluid (electroosmosis). 0007] The buoyancy generates a flow because of a density gradient. It can be produced by internal or external heating- An electric field is often used as internal energy source. Appl ied to a solution, part of the electric energy dissipates in the fluid by Joule effect and locally heats the fluid. Furthermore, local heating creates gradients of conductivity and permittivity. The fluid can then move under the influence, of an electrothermal Sow [2, 3, 4], fOOOS] Under certain conditions (material properties, conductivity and permeability of the fluid and the device containing the fluid), ion. layers develop at the fluid-surface interface due to chemical associations or dissociations and physical adsorption on or desorption from the solid surface. Ion layers can also be generated at the surface of electrodes where a potential is externally imposed. Applying an electric field with a tangential component to the layers moves the ions which carry the fluid along by viscous force. This process produces a bulk flow [2, 3, 4].

[0009] Coupled with an elec-trohydrodynamic flow, several electrode geometries have been designed as a tool to manipulate fluids and particles, interdigitatsd castellated electrodes are, for instance, designed to trap and separate particles [5, 6], Polynomial electrodes {?], planar electrodes [8, 9}. quadri polar electrodes [27] or more complex geometries [10] have also been proposed. Θ0ΪΘ] To be able to execute multiple forces described above, a system needs to be presented for high-throughput screening that is capable of inducing such forces inside a vessel utilizing different amplitude, frequencies and signal shapes delivered to a specifically designed working volume such as a mtcropiate, rrticrosiide or microarray.

[0011] Multiple reports have shown that rnierornixing, transport or concentration improves hybridization reaction [14- 16,17.18]. Micronrixing can be achieved by ultrasonic agitation (the nucleation of bubbles creates small jets that enhance the mixing) [3.9] or by vortexing or agitating the solution and creating convection [20]. Micromixing can also be produced by surface wave generation [23 ] for instance. However, all of these methods and other methods for mixing, concentration, separation, transport are presented with challenges when the operating volume is lowered to small microl iter or nanoliter scale.

(0012] What is needed then is a high-throughput system to efficiently and accurately mix, separate, concentrate, arid transport small volume of fluids with or without particles (e.g., atoms, molecules, ceils in biological and chemical assays] using scalable physical forces.

[0013] The. foregoing description of related art is not intended in an way as an admission that any of the documents described therein, including pending United States patent applications, are prior art lo embodiments of the present disclosure. Moreover, the description herein of any disadvantages associated with the described products, methods, and/or apparatus, ia not intended to limit, the disclosed embodiments, indeed, embodiments of the present disclosure may include certain features of the described products, methods, and/or apparatus without suffering from their described disadvantages. [0014! This application references a number of different publications as indicated throughout the specification by one or more reference numbers within brackets, e.g., [xj. A list of these different publications ordered according to these reference numbers caa bs found below in the section entitled "Reference!;." Each of these publications is incorporated by reference herein.

SUMMARY OF THE DISCLOSURE

[0015 According to some embodiments, system is provided for dynamic manipulation of^* fluid flow and/or particles such as molecules and cells in micropiates, on microslides and on microarrays. Micropiates are defined as multi-well containers of liquid. Microslides are defined as slides on which a thin (under 1 mm) layer of liquid is deposited. Microarrays are defined as slides on whose surface molecules are deposited.

[0016] in some embodiments., the system for high-throughput screening enables separation, concentration, transport, dispersion, reaction and mixing on high-throughput screening devices such as micropiates, tnicroslides ana microarrays. In some embodiments, the system enables improved results of high-throughput screening experiments by supplying different functional shape and different frequency AC electric signals to move liquids and panicles in micropiates, on microslides and on microarrays.

[0017] According to some embodiments, an apparatus and methods to achieve

manipulation of particles and/or solutions for high-throughput screening applications are provided. According to some embodiments, a system is provided for manipulation of fluid and particles inside micropiates, on microslides or microarrays wells using electrokinellc properties resulting from applied electric fields, comprising of: 1 ) elecirode-enabled volume (microp!ate, microsSide or microarray) with at leas': one pair of electrodes on parr of the volume, made of electrically conductive materials, for manipulating a fluid and particles using e!eetroldnetie properties resulting from applied electric fields generated by the electrodes; and connected to a function generator used to generate a single electrical waveform or a multiplicity of electrical waveforms supplied to the microplate- electrodes. jf!OlS] According to some embodiments, methods are provided for producing micropiates consisting of gluing or bonding a bottomless microplate with a printed circuit board. According to some embodiments, methods are. provided for producing microsiides for high- Shroughpui screening using printed circuit board technology. According to some embodiments, methods are provided to achieve manipulation of particles and/or solutions risin such microsiides or microptates within the system of the present embodiments,

[0019] in some embodiments, the system, devices and method of the present disclosure uses eiectrckinetic properties. The manipulation may be performed by bringing the active part of the device into contact with the fluidic solution. The eiectrodes (made of my applicable material) may be inserted inside one or more vessels containing one or more fluids or one or more fluids and one or more types of particles for tire purpose of manipulating fiuid(s) and/or parti cies. The manipulations can include concentration, separation, transport, mixing or ceil eiectroporation.

[0020J IK some embodiments, the system include a device containing electrodes capable of inducing eiectrokinetic (including electroosmotic and electrothermal) fluid How inside vessels (including micropiates or well-plates). The device may be either applied externally by inserting electrodes inside the vessel, or the device may be built into the vessel itself, for example, when flow is generated by forces other than single-frequency electroosmosis. The device can be used for general manipulation of fluids and particles inside the vessel, including concentration, separation, transport, mixing or cell eiectroporation, in some embodiments, the device may be tunable, so that by applying different DC and/or AC voltages, frequencies and shape forms different flow effects can be induced and adapted to efficiently manipulate the fluids and particles contained inside the vessel. In some embodiments, the device can perform one or more particle manipulation operations, in some embodiments, the device may include a function generator that is used to deliver signals of needed shape to the device just described, thus enabling improvements in accuracy, sensitivity and speed of high-throughput screening.

[0021 ] According to some embodiments, a system is provided for manipulation of fluid and particles inside microslats wells using eiectrokinetic properties resulting from applied electric fields, in some embodiments, the system comprising an electrode-enabled microplate with at least one pair of electrodes within a well for manipulating a fluid and particles using eiectrokinetic properties resulting from applied electric fields generated by the electrodes, wherein the electrodes are made of electrically conductive materials; and wherein the electrode-enabled rnicropiate is connected to a function generator used to generate a single electrical waveform or a multiplicity of electrical waveforms supplied to the rnicropiate electrodes. See e.g., figure ί .

[0022] According to some embodiments, a system for manipulation of fluid and particles inside on micro array slides or chamber using electrokinetic properties resulting from applied electric fields. In some embodiments, comprising of: an electrode-enabled slide with at least one pair of electrodes on the slide for manipulating a .fluid and particles using eiecirokinetic properties resulting from appl ied electric fields generated by t e electrodes, wherein the electrodes are made of electrically conductive materials: and wherein the eiectrode-enabled rnicropiate is connected to a function generator used to generate a single electrical waveform or a multiplicity of electrical waveforms supplied to the rnicropiate electrodes. See e.g.,, figure 1 ,

[0023] in some embodiments, the function generator is controlled by an external multipurpose device capable of generating control schedules, such as a digital computer, setting the schedule for application of different electrical waveforms. In some embodiments, the function generator is controlled by a dedicated internal or external device capable of generating control schedules, such as an embedded system, setting the schedule for appiicaiion of differerst electrical waveforms, in some embodiments, the function generator is capable of producing one or multiple outputs of harmonic, square, triangular, puise voltage waveform or any combination thereof.

[0024] in some embodiments, the electrodes inside inicroplates are controlled independently or jointly, in some embodiments, the electrodes on the electrode-enabled slide are controlled independently or jointly. In some embodiments, the electrodes ate arranged in an interdigitated array format, See figure 2, In some embodiments, the electrodes are arranged in an spiral format. See figure 2, in some embodiments, the- electrodes are coated with an insulating layer. [0025] In some embodiments, the at least three sets of electrodes are controlled independently, and the amount of voltage supplied to different eiectrodes is varied in time, therefore enabling chaotic adveciion-based mixing,

[0026] In seme embodiments, the plate consists of a bottomless top part and a printed circuit board attached to she bottom by gluing, bonding but not restricted to such methods of attachment. In sortie embodiments, the pi ate consists of a bottomless top pari and a glass or plastic siide wiih eiectrodes printed on it attached to the bottom by gluing, bonding bat not restricted to such methods of attachment. In some embodiments, the plate consists of a bottomless top part and a glass or plastic slide with electrodes deposited or etched on it, attached to the bottom,

[0027] In some embodiments, the fluid a liquid, or a liquid with at least one organic or inorganic, charged or neutral particle, in some embodiments, the fluid is mixed or dispersed by applying a time-dependent elec rohydrodynamic fluid flow using a fluid motivating force, and the fluid motivating force is an electromechanical, mechanical or electrochemical force.

[G0.28j in some embodiments, the particles are concentrated, separated, transported, mixed or dispersed by applying a time- dependent electrohydrodynamic fluid flow together with a particle .motivating force, and the particle motivating force is an electromechanical, mechanical or electrochemical force, irt sotne embodiments, the particles are detected ahd collected.

[0029] In some embodiments, a polymerase chain reaction (PGR) is performed within the vessel, by energizing the electrodes, In order to perform thermocycling and enhance a readout signal.

[0030] in some embodiments, a kina-se-based or ELiS A assay is performed within the vessel, by energizing the electrodes, such that, the- concentration needed to obtain a sufficient sigxial is reduced.

[0031] In some embodiments, an elsctroporation process is performed within the device, by applying an electromagnetic field using the eiectrodes, in order to simultaneously enable opening of pores in a ceil membrane using an electric pulse and to enhance efficient transport of the particles that pass through the- pores by cell suspension, mixing or concentration.

BRIEF DKsouFTioN OF THE DRAWINGS

[0032 ] For a better understanding of the present invention, reference is made to the following description, taken in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout.

[0033] FIG. J shows the components of the disclosed system: the bottomless wei (plate is equipped with the plate with a layers of electrodes (whose possible designs are shown in figure 2) and placed onto a connector pad. The connector pad is connected to the electrical function generator that is in turn connected to a controller. The depiction of possible rnicropiale well arrangement on top of the electrodes is shown on the bottom left of the figure.

[0034] FIG. 2(a)-2(f) are diagrams that Illustrate some examples of the arrangement of electrode arrays used in an embodiment of the present invention, wherein FIG. 2(a) is a drawing ofa planar electrode array where electrodes are interdigitated at the same physical layer, FIG. 2(b) is a drawing ofa planar electrode array where electrodes are interdigitated at ihe different physical layer, PIG. 2(c) is s drawing ofa square spiral electrode array, where electrodes are interdigitated at the same physical layer. FIG. 2(d) is a drawing of a square spirai electrode array, where electrodes are interdigitated at different physical layers, FIG. 2(e) is a drawing of a cylindscal spiral electrode array, where electrodes are interdigitated at the same physical layer, FIG. 2(f) is a drawing ofa cylindrical spiral electrode array, where electrodes are interdigitated at different physical layers,

[0035] In the following description of the preferred embodiment, reference is made to the accompanying drawings which form a part hereof; and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] According to some embodiments, a system is provided for high-throughput screening experiments consisting of a function generator, controller and a high-throughput screening device, such as a rnicropi te, micros!ide or microarray. In some embodiments, the high-throughput screening device is a rnicropiate and comprises at leas; the following two parts: a bottomless frame and an electrode-enabled bottom. The bottomless frame and electrode-enabled bottom may be joined together (e.g., by gluing or bonding).

[0037] The impact of the manipulation of fluids and/or particles induced by electric fields is described theoretically and experimentally herein. By means of a micro flutdic device comprising a periodic array of rnicroeiectrodes, fiuid(s) and/or particles manipulations are shown including concentration, separation, transport or mixing using eleetrokmetic properties. The theoretically predicted dynamical phenomena are demonstrated experimentally.

[0038] According to some embodiments, a system, devices, and methods are provided to improve the mixing of microliter or nana) iter volume protein solutions analyzed in high throughput screening assays. Electrokinetic micremixiag improves the time and reliability for protein expression by rapidly homogenizing the small volume solution. Current methods require extensive human or robotic operations and generally lack the requited sensitivity to meet reliability testing standards.

[0939] The system, devices, and methods of the present disclosure may be useful in the separation and detection of small populations of pre-cancerous cells from body fluids (blood, sputum, urine), the concentration of ON A particles inside a Polymerase Chain Reaction (PGR) apparatus for improved DNA detection or enhancement in quality and duration of BUS A assays, Another possible application is cell electroporation, Eiectroporation, or elect rope rmeabilizat ion, is a significant increase in the electrical conductivity and permeability of the ceil plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drag that can change the cell's function., or a piece of coding DMA [28], The current invention can serve to provide electric fields for

eiectroporation while keeping the ceils in suspension by induced fluid flow. This can be done, fix example, by bringing an external assembly of electrodes in contact with the solution, or by embedding the electrodes inside the well-plate wails, specifically for standard 1 536-wel! plate, 3456-weiS plats and higher we!l-plaie formats, not excluding the plates with any other number of wells.

[0040] According to some embodiments, a system Is provided to achieve manipulation of particles and/ot solution in m icro- to nanotiter volumes. FIG. 1 shows the components of the disclosed system according to some embodiments. The bottomless wel!piaie is equipped with the plate that has layers of electrodes (whose possible designs are exemplified in figure 2) and placed onto a connector pad. The connector pad is connected to the electrical function generator thai is in turn connected to a controller. The depiction of possible micrcplate well arrangement on top of the electrodes is shown on the bottom left of the figure.

[0041] In some embodiments, the function generator may be controlled by either an external computing device equipped with appropriate software or an embedded controller.

[0042] In some embodiments, a high-thtougbput screening microplate is produced by attaching the top frame of a bottomless microplate to a bottom part that is enabled by electrodes. The attachment may be achieved using any method known in the art, for example, by gl uing or bonding (including high- temperature bonding). This process may be used to lead to an electrode-enabled microplate shown m the middle of Figure 1, for example.

[0043] The bottom part of the raicrop!ate with electrodes can be produced using methods for manufacturing printed circuit boards, The electrodes can be produced out of gold, silicon, palladium, platinum, aluminum or any other suitable conductive material,

[0044] A preferred embodiment of a microplate with electrode-enabled bottom is shown in bottom left of figure 1 , where the positio of electrodes Is shown with respect to individual wells. Different embodiments o electrode layering are shown in figure 2. (0045] in some embodiments, the high-throughput screening device is a microslide. Here, the hi h throughput screening may be performed using free- droplets as the test volume, electrodes can be layered on the microslide using methods for manufacturing printed circuit boards. The electrodes can be produced out of gold, silicon, palladium, platinum, aluminum or any other suitable conductive materia!.

[0046] In some embodiments, the high-throughput screening device is a micro-array., Here, the high throughput screening may be performed using a thin layer of liquid as the test volume, eiecirodes can be layered on the rcucroarray using methods for manufacturing printed circuit boards, The electrodes can be produced out of gold, si licon, palladium, platinum, aluminum or any other suitable conductive material,

[0047] In some embodiments, the a connector pad with connecting electrodes may be placed on the high-throughput screening stage in order to enable communication between the function generator and the plate. The connector pad has a minimum of two connector electrodes op. it, and is connected to the function generator using wires or direct e!eotrode-to- eiectrode connection, hi another embodiment, the miercplate can be positioned directly on the function generator that is equipped with connector electrode pads on top of its surface.

[ .04SJ in some embodiments, the controller past of the system possesses information on specific process (assay) to be performed in the; high-throughput screening device. The contolier instructs the function generator to send an electric, signal (DC or AC) of specific amplitude, frequency and shape to the electrodes on the connector pad. Once electrode connectors on the well-plate are in contact with the connector electrodes on the high- throughput screening device, the induced potential on the electrodes induces electrokinetic physical effects thai move the liquid and particles inside the liquid.

[0049] In some embodiments, the high-throughput device uses electrokinetic physicaS effects. The manipulation may be performed by bringing the active part of the device (i.e., the electrode arrays) into contact with the fluidic solution considered and applying precise and carefully chosen electric fields combinations. [0050] En some embodiments, the general purpose of the active part of the device is to manipulate the flow and/or particles using an electric field, including actively changing properties of particles by use of electromagnetic fields, Dielectrophoresis and fluid flow precisely combined make possible the manipulation of submicron particles [25]. Experiments and numeric;;! simulations of the coupled electro4bermo-hydrodynamic problem in devices with interdigitaied arrays of electrodes [32, 13. 1 ] or electrode poles [26] show that both electrothermal and AC-e eetroosmotlc Hows consist of corrective roils centered at the. electrode edges and provide good estimates for their strength and frequency dependence.

Near the electrodes, the fluid velocity ug ranges from ί to 1 000 «?».„·^'"' decaying exponentially with the transversal distance to the electrodes.

[0051] in some embodiments, the electric Held induces heating inside the solution induces buoyancy flow effects. These are caused by gravity acting on nonhomogeneities in densities inside the liquid soiuiion to induce flow. These are possibly used in the device in conjunction with eiectrokinetic/eleetrothermal effects to provide mixing, concentration, separation and transport effects.

[0052] in some embodiments, the apparatus of the present disclosure contains electrodes capable of producing any of the physical properties described in the sections above, in some embodiments, the device is capable of inducing eiectrokinetic fluid flow inside liquid volumes. This may include eiectiroosmotic and electrothermal, where the latter appears to be due to a non-unifonn Joule heating of the fluid which leads to gradients of its permittivity and conductivity. The applied electric fields acting on the permittivity and conductivity gradients generate electrical body forces that induce the flow [13]. The former is caused by electrical stresses in the diffuse double layer of charges accumulated above the electrodes (AC- electroosmosis) [14] or at the wails (electroosnsosis) [24].

[0053] In some embodiments, the electrode arrays are designed to fit microliter size (or smaller) vessels as well as microliter (or smaller) droplets. The. electrodes are generally micron sized wires shaped like, or ileposiied layers of metal on a substrate, as show in FIGS, 2(a)-2(f), These are diagrams that illustrate some examples of the arrangement of electrode arrays used in an embodiment of the present invention., wherein FIG, 2(a) is a drawing of a piaaar electrode array where electrodes are interdigitated at the same physical layer, FIG. 2(b) is a drawi ng of a planar electrode array where electrodes are interdigitated at the different physical layer, For such electrode arrangements, a closed-form solution of the electric Held and the DEP force was derived in [23]. FIG. 2(c) is a drawing of a square spiral electrode array, where electrodes are interdigitated at the same physical layer, FIG. 2(d) is a drawing of a square spiral electrode array, whe e electrodes are interdigitated at different physical layers. FIG, 2(e) is a drawing of a eylir:dis;a! spiral electrode array, where electrodes are interdigitated ai the same physical layer, FIG. 2(f) is a drawing of a cylindrical spi ral electrode array, where electrodes are interdigitated at di fferent physical layers.

[O0S4] One particularly important application of the system is for inducing mixing hi small liquid volumes using the process named chaotic advection [30]. As shewn in [3 1 ] inducing unsteady flows at specific frequencies can tremendously Improve the mixing quali y.

Definitions

[0055] Unless otherwise defined, ail technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. AU publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control, In addition, the materials, methods, and examples are illitsirative only not intended to be limiting- Other features and advantages of the invention will be apparent from the following detailed description and claims.

[0056] For the purposes of promoting an understanding of the embodiments described herein, reference will be made to preferred embodiments and specific language wiii be used to describe the same. The terminology used herein is for the purpose o descrsbttig particular embodiments only, and is not intended to limit the scope of the present invention. As used throughout this disclosure, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "3

composition" includes a plurality of sucf; compositions, as weli as a single composition, and a reference to "a therapeutic agent" is a reference to one or more therapeutic and/or pharmaceutical agents and equivalents thereof known to those skilled in the art, and so forth.

[0057] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the val ue.

[0058] The use of the term "or" in the claims is used to mean "and/or" unless explicitly- indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[0059| As used in this specification and ciaim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Examples

[0060] it is understood that, modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the disclosed examples are intended to illustrate but not limit the present invention. While the claimed invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of rdinar skill in the art that various changes arid modifications can be made to the claimed invention without departing from the spirit and scope thereof Thus, for example, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims. REFERENCES

[0061] The following references are incorporated by reference herein.

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Claims

WHAT IS CLAIMED JS :

1 . A system for manipulation of fluid and particles inside microplate wells using eleetrokinetic properties resulting from appiled electric fields, the system comprising an electrode-enabled microplate with at least one pair of electrodes within a well for manipulating a fluid and particles using eleetrokinetic properties resulting from applied electric fieids generated by the electrodes., wherein the electrodes are made of electrically conductive materials; and wherein the electrode-enabled microplate is connected to a function generator used to generate a single electrical waveform or a multiplicity of electrical waveforms supplied to the microplate electrodes.

2. A system for manipulation of fluid and particles inside on inicroarray slides or chamber using slectrokinetic properties resulting from appiled electric fields, comprising of: an electrode-enabled sl ide with at least ens pair of electrodes on the slide for manipulating a fluid and particles using eleetrokinetic properties resulting from applied electric fields- generated by the. electrode^'s, wherein the electrodes are. made of electrically conductive materials; and wherein the electrode-enabled microplate is connected to a function generator used So generate a single electrical waveform or a multiplicity of electrical waveforms supplied to the microplate electrodes.

3. The system of claim 1 or 2, 'wherein the function, generator is controlled by an externa! multi-purpose device capable of generating control schedules, such as a digital computer, setting the schedule for application of different electrical waveforms.

A . The system of claim 1 or 2, wherein the function generator is controlled by a dedicated internal or external device capable of generating control schedule:; setting the schedule for application of different electrical waveforms.

5, The system of claim 1 or 2 wherein the function generator is capable of producing one or multiple outputs of harmonic, square, triangular, pulse voltage waveform or any combination thereof.

5. The system of claim I , wherein the electrodes inside roicroplates are controlled independently or jointly. 6, The system of ciaim 2, wherein the electrodes or. the eiectrode-enabied slide are controlled independently or jointly.

7. The system of ciaim 1 or 2 wherein the electrodes are arranged in an interdigitaied array format.

8, The system of ciaim 1 or 2 wherein the. electrodes are arranged in an spiral format.

9. The system of claim 1 or 2 wherein at least three sets of electrodes are controlled independently, and the amount of voltage supplied to different electrodes is varied in time, therefore enabling chaotic advection-based mixing,

30. T e system of claim 1 , wherein the plate consists of a bottomless top part and a printed circuit board attached to the bottom by gluing, bonding but not resin eted to such methods of attachment. i 1. The system of claim 1. wherein the plate consists of a bottomless top part and a glass or plastic slide with electrodes printed on it attached to the bottom by gluing, bonding but not restricted to such methods of attachment.

S2. The system of claim 1, where the plate consists of a bottomless top pari and a glass or plastic slide with electrodes deposited or etched on it, attached to the bottom.

13. The system of claim t, where the electrodes are coated with an insulating layer,

14. The system of claim 1 , wherein the fluid a liquid, cr a liquid with at least one- organic or inorganic, charged or neutral particle,

15. The system of claim 1. wherein the fluid is mixed or dispersed by applying a time- dependent electrohycirodynaniic fluid flow using a fluid motivating force, and the fluid motivating force is an electromechanical, mechanical or electrochemical force.

16. The system o f claim L wherein the particles are concentrated, separated, transported, mixed or dispersed by applying a iirne-depeadent electrohydrodynarnic fluid flow together with a particle motivating force, and the particle motivating force is an electromechanical, mechanical or electrochemical force, I S. The system ofclsim I , wherein She pertidss are detected and eoHested.

19. Ths system ofcisim I , wherein a polymerase chain reaction (P€ ) is performed wiihS« she vessel, by energizing the electrodes, is order so perforin thennoeyclissg a d enhance & readout signal

20, The system of claim 1 , wherein a kinase-bssed or EUSA assay is performed iihfrt she yssssi, by energising she electrodes, soch thai she concentration needed So obtain » sufficient signs! is redneed.

2 t , The s stem of claim I , whe ein an eieclroporation process is psrfbnrsed within she device, by applying electromagnetic Held using the decide , in order So simultaneously enabis epeoirsg of pores in s ceil membrane using an electric pulse and is enhance efficient rraris oil of dre pisrSides that pass thimsgh the pores by eeii suspension, m ning or concentration.