FLOATING ELECTRODE OPTOELECTRONIC TWEEZERS (FEOET) PLATFORM FOR OPTICAL MANIPULATION OF OIL-IMMERSED DROPLETS

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority from U.S. provisional application serial number 60/976,890 filed on October 2, 2007, incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under NSF Career

Award (ECCS-0747950), by the NIH Roadmap for Medical Research Nanomedicine Initiative PN2EY018228, and in part by NIH Grants Nos. CA90571 and CA107300. The government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL

SUBMITTED ON A COMPACT DISC [0003] Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

[0004] A portion of the material in this patent document is subject to copyright protection under the copyright laws of the United States and of other countries. The owner of the copyright rights has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the United States Patent and Trademark Office publicly available file or records, but otherwise reserves all copyright rights whatsoever. The copyright owner does not hereby waive any of its rights to have this patent document maintained in secrecy, including without limitation its rights pursuant to 37 C.F.R. § 1.14.

BACKGROUND OF THE INVENTION 1 . Field of the Invention [0005] This invention pertains generally to microfluidic systems, and more particularly to optically-controlled droplet-based microfluidic systems. 2. Description of Related Art

[0006] Droplet-based microfluidic systems have attracted significant interest for their potential utility in high throughput chemical and biological screening applications. By utilizing small volume droplets, biological and chemical reagents are compartmentalized within an immiscible continuous phase. In response to this compartmentalization, systems of this nature are able to provide benefits in the ability to rapidly mix reagents, control reaction timing, control interfacial properties, and the ability to synthesize and transport solid reagents and products. [0007] Droplet based microfluidic systems using two-phase immiscible flow have demonstrated potential in high-speed diagnosis or chemical synthesis.

However, in this kind of system, the droplets immersed in a liquid (typically oil) are typically guided by physical microfluidic channels, whereby active control of individual droplets as desired is extremely difficult. The use of physically patterned metal electrodes has been put forth as a means for addressing individual droplets in an open oil medium. However, the addressing of a large number of droplets with this method involves solving complex wiring and interconnection issues using active addressing circuit matrices and CMOS techniques. In addition, the need for this complex circuitry dramatically increases the fabrication cost and is highly unfavorable for chemical and biological experiments that frequently require low cost, disposable platforms for the prevention of cross contamination.

[0008] It will be recognized that optoelectronic tweezers (OET) can be utilized to manipulate cells and/or microscopic particles in aqueous mediums using optical images. However, due to impedance matching issues, OET lacks the capability to address particles or droplets in an insulating or low-conductive medium, such as oil.

[0009] Accordingly, a need exists for a system and method of performing optical manipulation of any desired number of droplets while not requiring complex wiring and interconnection. These needs and others are met within the present invention, which overcomes the deficiencies of previously developed microfluidic system and methods.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides a number of methods and apparatus for manipulating aqueous droplets within a fluid medium. Floating electrode optoelectronic tweezers (FEOET), as taught herein, provide a novel optical actuation and addressing mechanism for manipulating aqueous droplets immersed or suspended in electrically insulating or low-conductivity fluid mediums. This optical actuation mechanism enables massively parallel assessment of any desired number (e.g., millions) of individual aqueous droplets immersed in a low conduction medium (e.g., oil) on a low cost substrate, or even a disposable substrate (e.g., silicon coated glass), with optical images patterned by a spatial light modulator, such as a digital micromirror device (DMD) or a liquid crystal display (LCD). When integrated with a flow focusing droplet generation device capable of creating thousands of chemically/biologically controlled droplets per second, the FEOET method and system provides a powerful platform for performing high speed and multiplexed screening for drugs and cells, as well as for use with non-biologic applications. In automated or semi-automatic operations, the flow control device is preferably modulated in response to programming executable on a computer for carrying out the desired manipulation of droplets. [0011] Optical actuation of aqueous droplets is demonstrated in oil medium using a single laser beam according to the instant application. Even using the present proof-of-concept implementations, it has been observed that droplet actuation can be achieved using a light intensity of as low as 1 .6 μWlmm ² . By integrating the device with a million-pixel spatial light modulator and microfluidic flow-focusing high-speed droplet generation system, the FEOET platform has the capability to perform manipulations of millions of droplets in

parallel with a single light emitting diode or halogen lamp. This FEOET platform is extremely well-suited for high throughput, large scale, multiplexed screening of drugs and cells under a variety of physiological and non- physiologic conditions, in addition to non-biologic usages. [0012] The proposed FEOET device has inherited some of the key merits of

OET, including direct optical manipulation on a featureless low cost photosensitive electrode in response to the use of an incoherent light source. FEOET also provides new functionalities that could not be achieved by the use of OET devices. Three of the major advantages of FEOET over OET are as follows. (1 ) FEOET devices enable optical manipulation within an insulating medium like oil. (2) FEOET devices require about 2, 100 times less optical power than the minimum optical power requirement that has ever been demonstrated in OET, which is important when implementing a large optical manipulation platform. In addition, the ability to use low optical power assures less impact to biological constituents and some chemical species. (3) FEOET devices require only one photosensitive electrode and are very compatible with PDMS microfluidic devices fabricated by soft lithography techniques. Consequently, in contrast to OET devices, FEOET devices according to the present invention can be readily integrated with mainstream lab-on-a-chip systems for wide-ranging applications.

[0013] The FEOET platform according to the present invention can open up numerous potential application areas in the field of microfluidics or lab-on-a- chip systems, especially in systems requiring large scale, multiplexed, dynamic, biological, and chemical analysis and synthesis. The FEOET technology provides a platform that allows massively parallel control of millions of droplets individually on an extremely low cost silicon coated glass substrate in response to dynamically reconfiguring optical images, such as controlled from a typical personal computer. The cost of implementing FEOET and its powerful manipulation capability should be extremely attractive for companies in drug screening, chemical synthesis, and biological diagnostics industries.

The following invention description is divided into four general sections,

although the teachings therein overlap to varying degrees. [0014] In the first section a novel floating electrode optoelectronic tweezers

(FEOET) is introduced which enables optical manipulation of oil-immersed droplets on a featureless photoconductive glass layer using direct optical images. Teachings include concepts, device structure, fabrication, numerical simulation, and proof-of-concept demonstration results from the present invention. For example, it is demonstrated that a 44 μW laser beam with an average intensity of 16.55 μW jmm ² is able to transport a 750 μm oil immersed aqueous droplet at a speed of Q2 μm/s on a FEOET device. It will be expected that the necessary average intensity may be decreased and droplet speed increased as the teachings herein are refined. In either case, these results indicate that FEOET provides an effective mechanism for massively parallel droplet manipulation. [0015] In a second section, aqueous droplets containing chemical and biochemical contents are manipulated by optical image-patterned virtual electrodes through dielectrophoretic (DEP) forces to perform various droplet manipulation functions including (1 ) continuous 2D transport, (2) droplet merging, (3) and parallel processing of a plurality (e.g., sixteen) of droplets. Embodiments of the inventive platform promise a low cost, silicon-coated microfluidic system for large scale, multiplexed droplet-based biochemical analysis.

[0016] In the third section, the FEOET is described for light-driven transport of aqueous droplets immersed in electrically insulating oil on a featureless photoconductive glass layer using direct optical images. By way of example, this section demonstrates a 681 μm de-ionized water droplet immersed in a corn oil medium which is actuated by a 3.21 μW laser beam having an average intensity as low as 4.08 μWJ mm ² at a maximum speed of 85.1 μm/s on a FEOET device embodiment. FEOET provides a platform for performing massive parallel droplet manipulation with optical images on low cost, silicon- coated glass. By way of example a FEOET device structure is described

along with fabrication, working principle, numerical simulations, and operational results. [0017] In the fourth section, virtual electrodes are induced by illuminations of optical images onto a photosensitive layer to produce non-uniform electric fields from which arise dielectrophoretic (DEP) forces directing light-driven manipulation of aqueous droplets containing chemical and biochemical reagents (constituents). These photo-adjustable electromechanical forces enable various droplet manipulation functions, including: (1 ) continuous two- dimensional transport with speeds up to 721 μmjs , (2) droplet merging and mixing, (3) droplet delivery to wells (or other microstructures and microfluidic devices), and (4) large-scale parallel processing, such as demonstrated by manipulation of sixteen droplets (although the techniques can be scaled to any desired plurality of droplets). Furthermore, for biochemical demonstration, this section demonstrates successful detection of enhancement of fluorescent signals for fluo-4 preloaded HeLa cells, achieved by optical manipulation of a thmethylin (TMT) chloride droplet for activation of intracellular calcium [ CaJ ⁺ ).

The FEOET system promises a low-cost silicon-coated microfluidic platform which is simple to fabricate (featureless), and programmable to provide flexible and dynamic addressing of optical image patterns when performing large scale multiplexed droplet-based biological and chemical operations and analyses. [0018] The invention is amenable to being embodied in a number of ways, including but not limited to the following descriptions. [0019] One embodiment of the invention is an apparatus for manipulating aqueous droplets within a liquid medium, comprising: (a) a photoconducting layer; (b) a chamber upon the photoconducting layer into which a liquid medium and at least one aqueous droplet can be retained; wherein the liquid medium is substantially less conductive than the aqueous droplets; (c) at least two conductive electrodes electrically coupled to the photoconducting layer and separated by at least a portion of the chamber; (d) means for applying a bias voltage across the electrodes; and (e) means for projecting a dynamic 2D

light pattern onto the photoconducting layer to form virtual electrodes for manipulating the position of aqueous droplets in response to the electric field established about the 2D light pattern based on the conductivity difference between the aqueous droplets and the surrounding liquid medium; (d)(i) wherein the dynamic 2D light pattern breaks the originally symmetric electric field pattern around the aqueous droplets resulting in a non-zero net dielectrophoretic (DEP) force which drives droplets away from nearby lighted portions of the 2D light pattern; and (d)(ii) wherein manipulating of the position of the droplets includes one or more operation, including: trapping, positional translation, merging, mixing, delivery to wells and other microfluidic structures, as well as driving droplets to, or through, other desired operations. [0020] The apparatus can be implemented within a wide range of manual, semi-automatic and automatic applications. At least one embodiment of the invention includes a computer and programming executable on said computer (e.g., computer executes instructions from a memory coupled to the computer) for controlling the output of the dynamic 2D light pattern(s). In automated systems in which the position of the droplets or other structures are unknown or are uncertain, a position registration device, such as an imaging device (i.e., optical imager, sensor array, or similar), can be coupled to the computer to close the feedback loop so that manipulation operations can be accurately performed as desired without manual direction of droplet manipulation. [0021] One embodiment of the invention is a method for manipulating aqueous droplets within a liquid medium, comprising: (a) retaining at least one droplet within a less conductive liquid medium on a photoconductive layer; (b) generating an electric field on the photoconductive layer to induce a balanced dielectrophoretic (DEP) force on the droplets; (c) projecting at least one dynamic 2D light pattern onto the photoconductive layer to alter the electric field of the photoconductive layer based on the conductivity difference between the aqueous droplets and the surrounding less conducting liquid medium, creating a net unbalanced DEP force for moving the droplets; and (d) manipulating of the position of the droplets in response to changing the

position of the dynamic 2D light pattern(s). [0022] The present invention provides a number of beneficial aspects which can be implemented either separately or in any desired combination without departing from the present teachings. [0023] An aspect of the invention is a floating electrode optoelectronic tweezers (FEOET) apparatus and method for manipulating droplets. [0024] Another aspect of the invention is a FEOET system in which an electric field is applied across a portion of a chamber, such as across a photoconductive layer. [0025] Another aspect of the invention is a FEOET system in which a 2D light pattern is projected onto a photoconductive layer to form virtual electrodes which work in combination with an electric field for trapping and moving the position of one or more droplets.

[0026] Another aspect of the invention is an FEOET device in which the dielectrophoretic force arises in response to the conductivity difference between the droplets being manipulated and the less conductive liquid medium (e.g., oil, corn oil, and so forth). [0027] Another aspect of the invention is a FEOET system which utilizes pairs of optical electrodes in combination, such as diamond shaped patterns, or similar (e.g., oval, trapezoidal, elongate and so forth), to optically manipulate droplet position. [0028] Another aspect of the invention is a FEOET system which can be utilized with droplets dispersed in low-conductive and non-conductive fluid mediums. [0029] Another aspect of the invention is a FEOET system which can be utilized with droplets dispersed in oil. [0030] Another aspect of the invention is a FEOET system which includes at least two electrodes between which at least a portion of a chamber is positioned for retaining a liquid medium and droplets is position. [0031] Another aspect of the invention is a FEOET system which utilizes a photoresponsive layer for converting incoming light into an electric field which

interacts with the liquid medium and the droplets retained therein. [0032] Another aspect of the invention is a FEOET system which allows trapping, translating, and merging aqueous droplets which can be directed to any desired wells or other microfluidic structures. [0033] Another aspect of the invention is a FEOET system in which a droplet speed on the order of 0.5mm per second is achieved for a droplet approaching 1 mm in diameter moving through a medium having the viscosity of a corn oil.

[0034] Another aspect of the invention is a FEOET system in which the 2D light patterns are operable even at low light settings on the order of about

[0035] Another aspect of the invention is a FEOET system in which the 2D light patterns for controlling manipulation of the droplets can be generated by conventional light projecting devices and displays, as well as by custom light generation devices.

[0036] Another aspect of the invention is a FEOET system which can perform a wide range of manipulation functions including droplet trapping, positional translation of droplets, droplet merging, droplet mixing, droplet delivery to wells and other microfluidic structures, and other operations to which a droplet must be brought through or delivered to.

[0037] A still further aspect of the invention is a FEOET system which can be implemented to perform simultaneous manual, semi-automatic, or automatic manipulation of any desired plurality of droplets.

[0038] Further aspects of the invention will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the invention without placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS

OF THE DRAWING(S) [0039] The invention will be more fully understood by reference to the following drawings which are for illustrative purposes only:

[0040] FIG. 1 is a cross-section of a floating electrode optoelectronic tweezers

(FEOET) device according to an embodiment of the present invention. [0041] FIG. 2 is a depiction of a two-dimensional electrical field in response to light illumination, according to numerical simulation. [0042] FIG. 3 is a depiction of a two-dimensional electrical field around two droplets without light illumination, according to numerical simulation. [0043] FIG. 4 is a depiction of a two-dimensional electrical field around two droplets in response to light illumination, according to numerical simulation. [0044] FIG. 5A-5D is an image sequence of light induced water droplet motion in an oil medium according to an aspect of the present invention, shown at various times during motion. [0045] FIG. 6A-6E is an image sequence of light induced merging of two water droplets in an oil medium according to an aspect of the present invention, shown at various times leading up to droplet merging. [0046] FIG. 7 is a graph of droplet motion over time within a fluid medium in response to different light intensities. [0047] FIG. 8 is a perspective view of a floating electrode optoelectronic tweezers (FEOET) according to an embodiment of the present invention. [0048] FIG. 9A-9D are schematic illustrations of dielectrophoretic (DEP) forces induced by a circular light spot and virtual electrodes with respect to direction.

[0049] FIG. 10A-10B are graphs of electric field distribution about a droplet positioned with respect to the x direction in response to circular illumination, as determined by numerical simulations according to an aspect of the present invention. [0050] FIG. 1 1 A-1 1 B are graphs of electric field distribution about a droplet positioned with respect to the y direction in response to circular illumination, as determined by numerical simulations according to an aspect of the present invention.

[0051] FIG. 12 is a graph of electric field distribution in response to two closely positioned virtual electrodes as determined by numerical simulations according to an aspect of the present invention.

[0052] FIG. 13A-13B are graphs of electric field distribution about a droplet positioned with respect to the x direction and virtual electrodes as determined by numerical simulations according to an aspect of the present invention. [0053] FIG. 14A-14B are graphs of electric field distribution about a droplet positioned with respect to the y direction and virtual electrodes as determined by numerical simulations according to an aspect of the present invention. [0054] FIG. 15 is an illustration of a fabricated FEOET device according to an embodiment of the present invention, this example having a 6 x 6 cm active area and two aluminum electrodes. [0055] FIG. 16A-16E is an image sequence of light induced optical manipulation of a water droplet by virtual electrodes according to an aspect of the present invention, shown at various times during the trapping and transport sequence.

[0056] FIG. 17A-17E is an image sequence of light induced optical merging of water droplets using virtual electrodes according to an aspect of the present invention, shown at various times during the trapping, transport and merge sequence. [0057] FIG. 18 is a schematic of a micro well structure according to an aspect of the invention, showing an aqueous droplet being directed to a specific well within an array.

[0058] FIG. 19A-19H is an image sequence of delivering aqueous droplets to target wells, such as that shown in FIG. 18, according to an aspect of the present invention.

[0059] FIG. 20A-20D is an image sequence of transporting a desired plurality of aqueous droplets according to an aspect of the present invention, showing droplets being trapped and transported in different directions. [0060] FIG. 21A-21 C are graphs of electric field distribution in the a-Si:H layer of an FEOET device according to an aspect of the invention, shown under different conditions. [0061] FIG. 22A-22C are graphs of electric field distribution on a droplet surface shown without light illumination.

[0062] FIG. 23A-23C are graphs of electric field distribution on a droplet surface shown in response to light illumination. [0063] FIG. 24A-24D is an image sequence of light activated water droplet motion in an oil medium according to an aspect of the present invention. [0064] FIG. 25 is a graph of droplet velocity in response to relative droplet position for different optical intensities. [0065] FIG. 26 is a schematic of an FEOET-based microfluidic platform according to an aspect of the present invention, showing transport to an array of cells and merging in response to optical reconfiguration. [0066] FIG. 27 is a cross-section of a corner portion of the FEOET-based device shown in FIG. 26. [0067] FIG. 28A-28F is an image sequence of two-dimensional optical manipulation of a droplet in response to a projected virtual electrode according to an aspect of the present invention, showing trapping and movement. [0068] FIG. 29A-29E is an image sequence of aqueous droplet delivery to target wells according to an aspect of the present invention. [0069] FIG. 30A-30E is an image sequence of aqueous droplet merging and mixing according to an aspect of the present invention. [0070] FIG. 31A-31 D are images of fluorescent intensity in response to different chemical loading according to aspects of the present invention.

[0071] FIG. 32A-32F is an image sequence of aqueous droplet transport into

Fluo-4 loaded cells in DMEM/CaCb according to an aspect of the present invention.

[0072] FIG. 33A-33B are images showing elevated fluorescence of TMT activated HeLa cells according to an aspect of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0073] Referring more specifically to the drawings, for illustrative purposes the present invention is embodied in the apparatus generally shown in FIG. 1 through FIG. 33B. It will be appreciated that the apparatus may vary as to configuration and as to details of the parts, and that the method may vary as to the specific steps and sequence, without departing from the basic concepts

as disclosed herein. [0074] 1 . FEOET: A Novel Mechanism Enabling Optical Manipulation of Oil

Immersed Aqueous Droplets [0075] 1 .1 Introduction [0076] The desire to manipulate biological cells and micro/nano scale particles has fostered a great deal of interest in microfluidic devices. Optoelectronic tweezers (OET) are one such device that allows for optical manipulation of single cells and microscopic particles sandwiched between an ITO electrode and a photoconductive electrode with direct optical images from incoherent light sources. However, OET devices operate in an ionic medium with a conductivity between 10 ^~3 S/m to 1 S/m (units are Siemens per meter [S/m ] in SI units) but is not able to provide optical modulation of electric fields in electrically insulating biocompatible mediums, having significantly less conductivity (e.g., at least two orders of magnitude less than 10 ^~3 S/m ), such as pure corn oil which has a conductivity around 10 ^~14 S/m or above. It should be noted that typical drinking water has a conductivity of between around .

[0077] In a regular OET device, optical manipulation becomes increasingly difficult as liquid conductivity drops below 10 ^~3 S/m . Although this can be pushed slightly, by reducing the spacing between the top and the bottom electrode this causes other problems for its meager benefits. Thus, OET devices have a practical limit of operation to fluids having a conductivity greater than about 10 ^~5 S/m .

[0078] In contrast, the FEOET device presented here enables, optical manipulation of aqueous droplets in an insulating oil with a low light intensity requirement and promises a powerful platform for parallel manipulation of a large array of oil-immersed aqueous droplets with direct optical images. [0079] 1 .2 Device Design and Working Mechanism

[0080] FIG. 1 illustrates an example embodiment 10 of a FEOET device structure fabricated by applying a photoconductive layer (e.g., amorphous

hydrogenated silicon: a-Si:H, such as 0.5 μm thick) 14 to a light transmissive

(glass) substrate 12. It should be appreciated, however, that a photoresponsive material 14 can be utilized comprising other semiconducting materials, wherein the teaching herein is not limited to silicon. On the periphery above the photoconductive layer are electrodes, for example n+ doped a-Si:H (e.g., 0.1 μm ) 16 over which is a conductive electrode, such as aluminum (0.1 μm Al) 18. It should be appreciated that the n+ doped region of a-Si:H is not necessary in fabricating the electrodes, which may be fabricated in a variety of ways which are well known to those of ordinary skill in the art. An electrical bias voltage is applied at the two edges of this photoconductive electrode, as depicted by the voltage and ground symbols. Over this structure, or otherwise adjacent, is an open chamber 20 (e.g., PDMS) configured for containing a medium, such as oil (e.g., corn oil), into which aqueous droplets 24 are contained or can be introduced. It will be appreciated that means for generating aqueous droplets of a desired composition and size are well known within the art. It should be appreciated that in the equivalent circuit of the FEOET, the oil layer 22 and the photosensitive layer are connected in parallel, instead of the serial connection found within an OET device. The electrical configuration of the FEOET is important toward enabling optical modulation of an electric field in as insulating medium. The device structure shown in FIG. 1 is readily implemented, and is shown fabricated by depositing two featureless a-Si:H layers (doped and undoped) on a sufficiently light transmissive (e.g., glass) substrate to generate the necessary dielectrophoresis (DEP) forces on the droplets. It should be appreciated that light may be directed to the FEOET device from either side, wherein the substrate may be opaque to the transmission wavelength of light. In addition, light may be applied from both sides as desired, insofar as the light patterns are generated cooperatively. In the example of FIG. 1 , two aluminum electrodes deposited at the two edges of the device are separated by a gap

(e.g., 1 cm ). These electrodes may, for instance, be deposited using a lift-off

method enabling electrical contact. The n+ a-Si:H layer is used to interface between the Al electrode and the undoped a-Si:H material, wherein after depositing the metallic electrode the n+ a-Si:H material not covered by the Al electrodes is etched away, such as using a reactive ion etcher (RIE). [0082] The chamber in this example is fabricated from PDMS (poly- dimethylsiloxane) over the undoped a-Si:H photoresponsive layer and filled with corn oil into which aqueous droplets are retained. A sufficient bias voltage (e.g., 600 VDC in this example) is applied between the two aluminum electrodes to provide a lateral electric field across the whole device in parallel with both the oil and the a-Si:H layers. It should be appreciated that the amount of bias voltage which constitutes a sufficient bias voltage depends on the size of the area over which manipulation is being performed, the application, and other factors. The bias voltage is sufficient in a given application if it provides a sufficient motive force for the droplets in response to operation of the device.

[0083] The FEOET system is configured to use the conductivity difference between the aqueous droplets and the surrounding oil. It will be noted that the aqueous droplets are significantly more conductive than the surrounding electrically insulating oil. In response to this difference an electric dipole is induced on each droplet under the application of a lateral electric field.

Although the droplet induces a highly non-uniform electric field around itself, the droplet does not move due to dielectrophoresis (DEP) forces since the symmetric electric field pattern around a droplet results in a zero net force. However, when a light beam illuminates the photoconductive undoped a-Si:H layer at the edge of a droplet, it creates a light patterned virtual electrode which decreases the electric field strength at the illuminated site of the droplet. This phenomenon breaks the originally symmetric electric field pattern around a droplet and results in a non-zero net DEP force, which drives the droplet away from the light beam. [0084] 1 .3 Simulation and Experimental Results

[0085] FIG. 2, FIG. 3 and FIG. 4 illustrate an operating principle of the

inventive FEOET devices shown through numerical simulation of the electric field distribution in the oil medium 22. In FIG. 2 a cross-section is shown of a photoconductive (photosensitive) material 14 at the bottom with biasing electrodes 18 on each end between which is positioned a chamber for retaining an oil medium 22. Optical illumination on the photosensitive layer creates a virtual electrode that decreases the local electric field near the illuminated spot, as seen just right of the lower center. By way of example, the illumination is provided in this case by a 70 μm Gaussian laser beam. Thus, the local electric field near the illuminated spot is greatly decreased due to the light-patterned virtual electrode in the a-Si:H layer.

[0086] Moving down to FIG. 3, upon immersing an aqueous droplet 24 in the oil chamber 22, a symmetric electric dipole is induced around the droplet in response to the water droplet being more conductive than the insulating oil. It should be noted that the conductivity range of aqueous droplets can vary from about IO ^A S/m , for pure water, to about 10 S/m in response to the concentration of dissolved electrolyte. Theoretically, an electric dipole can be induced around any droplet insofar as sufficient conductivity difference exists between the medium and the droplet. Lower levels of electrical conductivity in the oil medium generally provide more ideal FEOET operating characteristics, resulting in lowered levels of FEOET power consumption. To be more specific, non-polar liquid media usually have very low conductivity and can be considered as electrical insulators. In general, materials having electrical conductivity on the order of 10 ^~14 S/m are considered insulators. [0087] Moving on to FIG. 4, when a light beam illuminates the edge of a droplet, the electric field strength at the illuminated side decreases causing a non-uniform electric field distribution around the droplet resulting in a net dielectrophoretic (DEP) force along the direction of the strong electric field, which is in the direction away from the light beam. The net DEP force drives the droplet away from the light beam as shown in FIG. 4. [0088] FIG. 5A-5D depict a demonstration of optically induced droplet manipulation, wherein video snapshots show a water droplet immersed in corn

oil being repelled by a light spot as predicted in our simulation. In the present example, a laser beam (532 nm , 4.35 μW ) was utilized with a spot size of 2 mm , which successfully drove a 750 μm droplet at a speed of approximately 1 16 //m/s through the oil medium. FIG. 5A depicts the droplet prior to light application, upon application of the light spot in FIG. 5B, and at 15 and 30 seconds after introducing the light in FIG. 5C and 5D, respectfully. Although the FEOET is described above manipulating droplets whose size approaches one millimeter, it should be appreciated that the FEOET platform can be used to manipulate droplets over a broad range of sizes, such as from about one micrometer to a few millimeters in diameter, with associated droplet volume ranges from about one femtoliter to tens of microliters. In the examples recited herein, the size of droplets was determined by the available equipment upon which the droplets could be readily prepared, and is not to be considered a limitation on the practice of the invention. [0089] FIG. 6A-6E depicts a demonstration of two droplets driven to merge in response to manipulation by a light beam. The optical power required to drive aqueous droplets on FEOET devices is low. By way of example, the droplets are driven in the figure to a speed of about 82 μm/s even at an average light intensity of only 16.55 μw/mm ² , wherein the FEOET platform promises many benefits for performing massively parallel droplet manipulation. FIG. 6A depicts the droplets prior to light application, upon application of the light spots in FIG. 6B, and at 3 and 6 seconds after introducing the light in FIG. 6C and 6D, respectfully, and finally at merging in FIG. 6E.

[0090] FIG. 7 depicts droplet position with respect to time for two different light intensities. It will be seen from this graph that useful droplet translation speeds can be obtained at low values of light power. [0091] 1 .4 Conclusions

[0092] A floating electrode optoelectronic tweezers (FEOET) has been described which enables optical manipulation of aqueous droplets in an electrically insulating oil medium with a light intensity as low as

16.55 μw/mm ² on a prototype device. The simple and flexible FEOET device allows droplet manipulation to be implemented even with a simple devices structures, such as using an open oil chamber. The simulation results demonstrate the mechanism of successful movement of an aqueous droplet using a light patterned virtual electrode. The virtual electrode decreases the electric field at the illuminated area to disrupt the originally symmetric field pattern around a droplet. The newly non-symmetric electric field induces a net non-zero DEP force thereby repelling the droplet away from the light beam illumination. Experimental results confirm the simulation results and demonstrate droplet movement, of a 750 μm droplet, using a laser beam

(532 nm , 43.5 μW ) with a spot size of 2 mm at a speed of 82 μm/s . This promises FEOET as an effective mechanism for large scale, parallel droplet manipulations. [0093] 2. Optical Image-Based Dynamic Manipulation Of Aqueous Droplets Immersed In Oil Medium

[0094] 2.1 Introduction

[0095] Droplet-based microfluidic systems have the potential for supporting high-throughput and high-speed chemical and biological analysis. Thousands of highly uniform, isolated aqueous droplets containing chemical reagents can be generated in immiscible oil within seconds by flow-focusing devices.

Applications such as fluorescent detection of millisecond chemical kinetics, laser Raman spectroscopic probing, and PCR (polymerase chain reaction) reactions for high throughput DNA amplification have been demonstrated using droplet-based microfluidic devices. A major difficulty associated with a droplet-based microfluidic system is in achieving active control of individual droplets confined in microchannels being driven by a continuous oil flow. It has been shown that multiple droplets can be individually addressed in an open oil chamber using physically patterned electrodes, although complex wiring and interconnection issues arise for addressing numerically large droplet arrays. For example, a microfluidic device integrating a high voltage

CMOS driving circuit recently achieved active and parallel droplet control.

However, this approach increases the fabrication cost of microfluidic devices, which are often preferred to be disposable.

[0096] Alternatively, a light-induced manipulation mechanism has been widely investigated to solve the wiring issues that arise for controlling a large array of electrodes. For example, optoelectronic tweezers (OET) have recently been shown to manipulate single cells and microscopic particles sandwiched between an ITO electrode and a photoconductive electrode created by direct optical images. However, due to impedance mismatch, it is difficult to modulate the electric field strength in electrically insulating mediums, such as oils used in two phase droplet based microfluidic systems, on an OET device since all of the applied voltage drops across the oil layer, because of its lack of conductivity, even without light illumination. [0097] In the preceding section a novel floating electrode optoelectronic tweezers (FEOET) mechanism was described which enables optical actuation of oil-immersed aqueous droplets using a circular laser beam with intensity as low as 4 μWJ mm ² . However, droplet movement in this FEOET device was limited to a linear motion along the direction of the electric field, which may restrict its potentials in chemical and biological applications. Among other things, this section demonstrates two-dimensional droplet manipulation functions on a FEOET platform by utilizing virtual electrodes, such as according to projecting a light pattern, preferably in a diamond-shape, output by a light projection means (spatial light modulator).

[0098] It should be appreciated that although a pair of diamond shaped patterns is described, paired electrodes of any shape can be successfully utilized for capturing and positionally translating a droplet. By way of example and not limitation, preferred variations utilize paired electrodes which are oval, trapezoidal, elongated and so forth. The use of the 2D manipulation opens up the possibility of massively parallel droplet manipulation on featureless (e.g., containing no physical microfluidic structures, for instance channels and walls, for directing the droplet) fluidic platforms (e.g., silicon coated glass substrate) controlled by using dynamic and reconfigurable optical images. Optically

patternable virtual electrodes generate dielectrophoretic (DEP) forces to manipulate aqueous droplets containing chemical and biochemical constituents for performing various microfluidic functions such as two- dimensional transport, droplet merging and parallel processing of a plurality of droplets (e.g., sixteen droplets).

[0099] 2.2 Device Structure And Working Principle

[00100] FIG. 8 illustrates an embodiment of an FEOET 10 device within a system 30. By way of example, this embodiment is fabricated, as in FIG. 1 , by coating a transparent substrate (e.g., glass wafer) 12 with an undoped a-Si:H layer 14 (e.g., Q.b μm ) and an n+ doped (0.1 μm ) region for interconnection.

Two aluminum electrodes 18 with a 6 cm lateral gap are deposited at the two edges of this device by e-beam evaporation and lift-off techniques. The Al electrodes also serve as an etching mask for removing the n+ a-Si:H layer, such as by using a Reactive Ion Etcher (RIE). The n+ a-S ^' wH layer 16 provides ohmic contacts with the Al electrodes. An open poly- dimethylsiloxana (PDMS) chamber 20 retains the aqueous droplets 24 and immiscible oil (corn oil) 22 as affixed upon the layer of undoped a-Si:H. [00101] A means 34 for projecting patterned light 36 onto the photoresponsive layer 14 provides for the generation of virtual electrodes within the chamber for manipulating the droplets therein. It will be appreciated that a number of devices can be used for generating the dynamic patterned light, such as conventional light projectors displaying a mask pattern (e.g., monochrome, or alternatively colored patterns providing different intensities), display devices, LCD shutter arrangements, MEMs mirror driven devices and other light manipulation devices as known to those of ordinary skill in the art. These light pattern projective means are not described in detail herein, as the present invention may be implemented using a wide range of such devices. [00102] The embodiment of FIG. 8 shows additional peripheral aspects of the invention toward integrating the FEOET 10 into a working system 30. It should be appreciated that for the present invention to perform automated droplet manipulation, some form of control device, such as a computer (e.g., at least

one CPU, microcontroller, microprocessor, or similar) 36 is needed to sequence and direct the light patterns being output. An optional user interface 40 is shown coupled to the computer for controlling the operation of the system, in particular when operated in manual, or semi-automatic modes. Computer 36 is shown coupled to memory 38 from which programming is read for execution on computer 36 to dynamically control the operation of the FEOET device, such as by adjusting the light patterns impinging on the photoresponsive area of the FEOET device. [00103] Light projection means 32, shown outputting light pattern 34, is shown controlled via computer 36, which can control the pattern, size, intensity, and motion of the light used to manipulate the droplets within the FEOET. The bias voltage to the FEOET can also be optionally controlled by the system, such as via computer 36 generating commands to a programmable power supply (PS) 42. The droplets used in the FEOET can be optionally generated by a droplet generating device (DG) 44, such as controlled by computer 36. In addition, in many applications the exact position of the constituent elements to be moved, and/or their respective destination targets, are not in fixed positions, wherein the FEOET system can be optionally configured with one or more position registration means (sensors) (C) 46, such as an optical sensor array, microscopic camera, or the like, whose inputs are received and image processed by either a separate device or programming operable on computer 36.

[00104] It will be noted that the FEOET device described can actuate an aqueous droplet by light-induced DEP forces with an intensity as low as A μW/mm ² , which is a sufficiently low intensity to allow large area droplet manipulation platforms to be implemented using regular and commercially available optical projectors and displays, without complex additional optical components. [00105] FIG. 9A-9D illustrate DEP forces induced by a circular light spot string. In the field direction the DEP forces are strong (FIG. 9A), yet are weak in the direction perpendicular to applied field (FIG. 9B). However, DEP forces

induced by two diamond shaped virtual electrodes are strong in both parallel and perpendicular directions as seen in FIG. 9C-9D. [00106] However, the extent of DEP forces induced on droplets within the

FEOET depend upon the direction of applied electric field. The directional force difference is shown in FIG. 9A-9B. This force difference substantially limits droplet motion to one direction, which could be restrictive in a number of applications.

[00107] To provide FEOET two-dimensional manipulation functions, the present invention teaches a method for manipulating droplet position using virtual electrodes, such as trapping a droplet between the gap of two diamond- shaped virtual electrodes as shown in FIG. 9C-9D. In FIG. 9C-9D it will be appreciated that not only the electric field but also the shape, intensity and positioning of optical virtual electrodes alter the DEP forces applied to the droplets. The preferred diamond shape of the virtual electrodes, provides a number of benefits, in particular with regard to submitting a droplet to the same order of magnitude DEP forces in both directions parallel and perpendicular to the applied field. [00108] Although a single electrode pair is described herein, it should be appreciated that a number of electrodes may be utilized in combination having either single or multiple orientations. For example, opposing electrodes may be utilized in both x and y directions upon which electric fields are applied having opposing phases. One of ordinary skill in the art will appreciate that the teachings herein can be modified to implement these variations. [00109] 2.3 Simulation Results [00110] Numerical simulations of a three-dimensional electric field distribution in a FEOET chamber and on a droplet surface are performed using a simulation program, for instance COMSOL Multiphysics 3.2 ®. To reduce the simulation time, the device is simplified to two layers: a b μm thick undoped a-Si:H and a 350 μm thick oil medium. A 100 Volt direct current (DC) bias is applied at the two end planes along the x direction, and a 300 μm diameter aqueous droplet is loaded in a 1 .4 x 0.8 mm oil chamber.

[00111] The dark conductivity of the homogeneous a-Si:H layer (amorphous semiconductor) is assumed to be about 10 ^~8 S/m . However, corn oil is an insulating medium having a typical conductivity lower than about 10 ^~14 S/m . To compare the DEP forces induced by both a circular light beam and two diamond-shaped virtual electrodes, the circular light beam is assumed to have a 200 μm full width at half maximum (FWHM) spot size and it induces a peak photoconductive of 10^ S/m in the center. The same peak photoconductivity is also assumed in the case of diamond-shaped virtual electrodes with a 200 μm separation gap. The Maxwell stress tensor has been applied for calculating DEP forces on droplets.

[00112] 2.3.1 Case of a Circular Laser Beam Illumination

[00113] FIG. 10A-10B and FIG. 1 1A-1 1 B depict numerical simulations of electric field distributions in response to a circular laser beam illumination both parallel to (x-direction) and perpendicular to (y-direction) the applied electric field. In FIG. 10A a droplet is shown located 150 μm away from the center along the x direction. FIG. 10B shows the associated electrical field distribution for FIG. 10A. Similarly, FIG. 1 1A depicts a droplet located 150 μm away from the center along the y direction, with FIG. 1 1 B showing the associated electrical field distribution. The electrical field distributions at z = 170 μm and on a droplet surface are represented along the direction parallel

(FIG. 10B) and perpendicular (FIG. 1 1 B) to the applied electric field, respectively. A circular laser beam illumination is assumed to be a 200 μm full width at half maximum (FWHM) to create a Gaussian photoconductivity distribution with a peak photoconductive of 10 ⁴ S/m in the center of a laser beam.

[00114] Droplet actuation (positional manipulation) on the FEOET arises from the electrostatic dipole-dipole interactions between the droplet and virtual electrodes. Without light illumination, a droplet-induced dipole generates a symmetrical electric field distribution around a droplet surface, resulting in zero net DEP forces for transporting the droplet. To actuate a droplet, one can

illuminate a light beam at one edge of a droplet to break this field symmetry around the droplet. As shown in FIG. 10A-1 OB, the electric field at the illumination side is greatly reduced, resulting in net DEP forces moving the droplet away from the light beam. The calculated DEP force is F _dep = [-9.46, 0.02, -2.87] x 10 ^"9 N , corresponding to the forces in x, y, and z directions, respectively. The 0.02 nN force in the y direction arises in response to numerical errors. [00115] When using a circular light beam, however, the transport of droplets in the y direction is not as effective as in the x direction. Illuminating a droplet edge at the direction perpendicular (y direction) to the electric field fails to transport the droplet since the electric field strength difference between the illuminated and non-illuminated sides on the droplet surface is too small as shown in FIG. 1 1 A-1 1 B. The calculated DEP forces in this situation is F _dep = [-0.00, 0.12, 2.55] ^χ lO ^~9 N . In the example described the y direction force is 78.8 times smaller than that of the x direction force depicted in FIG. 10A-

10B. For this reason, a circular light beam is not suitable for the virtual electrodes used in 2D transportation in FEOET devices. [00116] 2.3.2 Case of a Paired Diamond-Shaped Virtual Electrode

[00117] FIG. 12, FIG. 13A-13B and FIG. 14A-14B collectively illustrate simulation field patterns of a droplet trapped in response to the illumination of two diamond patterns onto the a-Si:H layer previously described. FIG. 12 illustrates electric field distribution induced by two closely positioned diamond- shaped virtual electrodes separated by a 200 μm gap, in which strong electric fields are created. FIG. 13A depicts a droplet located 150 //m away from the center along the x direction, with FIG. 13B showing the unbalanced e-field pattern in the x direction causing the DEP force to drive and trap the droplet to the right. Similarly, FIG. 14A depicts a droplet located 150 μm away from the center along the y direction, with the e-field pattern shown in FIG. 14B which is symmetric in the x direction but asymmetric in the y direction, producing a net DEP force to attract the droplet back to the center.

[00118] It will be noted from the above figures that without loading a droplet, the electric field strength at the gap between these two electrodes is strongly enhanced (FIG. 12). When a droplet is loaded at 150 //m away from the gap center along the x direction (FIG. 13A), the enhanced electric field can generate DEP forces pulling the droplet back to the center of the gap. The calculated DEP forces are F _dep = [1.45, 0.00, -2.48] ^χ lO ^~8 N . The value of the

DEP force in the x direction is similar to the one in case of a circular laser beam. For the situation of positioning a droplet 150 μm away from the gap center along the y direction, the DEP force shows a dramatic increase compared to the y direction force in the case of a circular light beam. As shown in FIG. 14A-14B, the calculated DEP forces in this situation are F _dep = [0.00, -0.64, -2.51] x 10 ^"8 N . The y direction force of FIG. 14A-14B is only

2.26 times smaller than the x direction force in the FIG. 13A-13B situation. [00119] The value of 2.26 times illustrates a dramatic improvement in response to using the illuminated diamond regions, when compared to the 78.8 times smaller value arrived at for the circular light beam case. This y direction force is able to provide effective trapping and transporting of a droplet in the y direction. The following example utilizes the field performance created above toward two-dimensional droplet manipulation functions within FEOET devices. [00120] 2.4 Experimental Demonstrations

[00121] The capability of two-dimensional transport enabled by a paired diamond-shaped electrode has been exemplified by successfully demonstrating several important droplet manipulation functions within the described FEOET platform, including: continuous 2D transport, droplet merging, and parallel processing of a plurality of droplets (e.g., 16 droplets).

[00122] FIG. 15 illustrates an embodiment 10 of a fabricated FEOET device with a 6 x 6 cm active optical manipulation area. Oil medium 22 is shown in a chamber 20 between two Al electrodes 18 to which a 5 kV bias voltage is applied to power the entire device. Although the voltage is high in this example, the requisite power consumption of the whole device is only 0.4 μW

in the dark state and less than A mW in the bright state due to the large electrical impedance of the oil and a-Si:H thin film ( ~ 10 ¹⁴ ω in the dark state). [00123] It should be appreciated that in both OET and FEOET, amorphous silicon is typically utilized as the photoconductor. However, the electric field is applied differently in the two different devices. In an OET, the electric field is applied perpendicular to the thin film, resulting in a dark resistance of 10 ⁶ ω for a 1 cm ^χ 1 cm ^χ 1 μm film. By contrast to the OET, the electric field in an FEOET device is applied in the direction parallel to the amorphous silicon thin film, wherein the resulting resistance is approximately 10 ¹⁴ ω , which is about eight orders of magnitude higher than in the OET. In regards to this difference the unique operating properties of the FEOET are not surprising, and in view of which the electric power consumption of the FEOET can be as low as 0.4 μW even in response to a very high voltage drive. [00124] FIG. 16A-16E depicts video snapshots of two-dimensional optical manipulation on a FEOET device transporting the droplet in both x and y directions by reconfiguring projected optical images. The image sequence shows manipulation of a 1 .47 ^' mm droplet containing the Trypan blue dye. The light and dark circles indicate droplet positions before and after movement. In FIG. 16A-16B two diamond-shaped virtual electrodes create a strong electric field between the gap to trap the droplet shown. In FIG. 16C the trapped droplet is transported to the right hand side along the electric field (e-field) direction. In FIG. 16D the droplet is driven to the top side perpendicular to e- field direction, while in FIG. 16E the droplet is shown being moved to the left hand side again. [00125] The droplet transport speed in the above demonstration was 721 μm/s in the x direction and 3Q3 μm/s in the y direction. The two fold difference between the speeds in the x and y direction comport well with the DEP force difference predicted in the simulation results in FIG. 13A-13B and FIG. 14A- 14B using a 300 μm droplet.

[00126] FIG. 17A-17H illustrate video snapshots of two 1 mm droplets containing different color dyes (e.g., yellow and green food coloring dyes) which are transported and merged. In FIG. 17A the droplets are shown in an initial position with the voltage off. Droplet trapping is then seen in FIG. 17B, with the diamond pattern pairs being moved relative to one another in FIG.

17C at 5 seconds, in FIG. 17D at 14 seconds, in FIG. 17E at 20 seconds, and immediately before to merging in FIG. 17F at 26 seconds. The droplets are merged in FIG. 17G as the diamond pattern pairs align with one another, wherein the droplets mix in FIG. 17H. Thus, according to this aspect of the invention, when two aqueous droplets are driven close and aligned in the electric field direction, dipole-dipole interaction between two droplets induces electrocoalescence and combines two droplets together.

[00127] FIG. 18 illustrates a micro well structure 50 exemplified in a polymer base 52, such as polydimethylsiloxane, generally referred to as PDMS. An aqueous droplet 56 is shown being manipulated 58 to a target well 54, which in this example contains a Trypan blue dye (not shown).

[00128] FIG. 19A-19H depict an image sequence of delivering aqueous droplets to target wells among an array of PDMS wells positioned on an FEOET device. It should be appreciated that although these wells are exemplified as being separate wells, a well as considered herein may comprise any desired structures, microfluidic structures, or droplet interfaces to which one or more droplets can be introduced for a given application. In FIG. 19A-19B a 1 .28 mm de-ionized water droplet is optically trapped between two diamond- shaped images. In FIG. 19C-19H a two-dimensional transport of the droplet is achieved by the corresponding optical image motions which are shown at times of 13, 34, 55, 58 and 60 seconds, respectively. Finally, in FIG. 19H the droplet is merged into the target well containing Trypan blue. [00129] It should be appreciated that the above figure sequence demonstrates the flexibility and compatibility of FEOET devices with other microfluidic structures, for instance micro wells. In the example shown, an array of PDMS micro well structures are positioned on top of a FEOET device. In the

example described, these wells are preloaded with aqueous solutions containing the Trypan blue dye and immersed in an oil environment. A 1 .28 mm de-ionized water droplet is transported by a paired diamond-shaped virtual electrode and delivered to the target well to dilute the dye concentration in that well. The low light intensity requirement of FEOET and the two dimensional droplet transportation function realized by diamond-shaped virtual electrodes promise an FEOET platform capable of massive parallel processing of a large number of droplets for high throughput chemical and biochemical screening applications. [00130] FIG. 20A-20D illustrate an example of transporting a plurality of droplets, (e.g., sixteen as per this example), in parallel. The droplet size in this demonstration varied from about 0.84 mm to about 1 .3 mm in diameter, although it should be appreciated that a droplet may be of a size from about one micrometer on up to a few millimeters in diameter. The black circles indicate the droplet position in the previous step, while the white circles present the droplet position in the current step. In FIG. 20A-20B an array of diamond-shaped optical images are generated to create sixteen traps into whose gaps the droplets are attracted. In FIG. 20C-20D parallel transport of the trapped droplets is performed by translation (relative movement) of the dynamic optical images. In FIG. 2OC the droplets in the first and third rows are driven to the right side, while the droplets in the second and fourth rows are guided to the left, which continues in FIG. 2OD. Accordingly, the figures demonstrate an ability to transport any desired number of droplets even in opposing directions. [00131] 2.5 Conclusions

[00132] This section has taught an optically reconfigurable microfluidic platform enabling parallel processing of oil-immersed aqueous droplets on a photoconductive glass substrate using direct optical images. This platform is realized by using optically patterned virtual electrodes, such as paired diamond-shaped patterns, that allow transporting droplets on a two- dimensional FEOET surface. With this platform, several important droplet

manipulation functions have been demonstrated including continuous 2D droplet transport, droplet merging and parallel processing of any desired number of droplets in parallel. This FEOET system is also compatible with other microfluidic structures, such as an array of micro wells. The inventive platform is capable of providing a low cost, droplet-based, microfluidic system for large scale, multiplexed chemical and biochemical screening applications. [00133] 3. Floating Electrode Optoelectronic Tweezers: Light-Driven

Dielectrophoretic Droplet Manipulation In Electrically Insulating Oil Medium [00134] 3.1 Introduction [00135] Droplet-based microfluidic systems have attracted significant interest for their potential utility in high throughput chemical and biological screening. Using a multiphase flow focusing device, tens of thousands of highly uniform, isolated aqueous droplets can be generated in immiscible oil within seconds. Applications, such as fluorescent detection of millisecond chemical kinetics, laser Raman spectroscopic probing, and polymerase chain reactions for high throughput DNA amplification, and others have been demonstrated using droplet-based microfluidic devices. One challenge associated with a droplet- based microfluidic system is the difficulty in achieving active control of individual droplets confined in micro channels being driven by a continuous oil flow. It has been shown that multiple droplets can be individually addressed in an open oil chamber using physically patterned electrodes, although complex wiring and interconnection issues arise for addressing numerically large droplet arrays. [00136] To solve this problem, a microfluidic device integrating a high voltage complementary metal-oxide semiconductor driving circuit recently achieved active and parallel droplet control. However, this approach increases the fabrication cost of microfluidic devices, which are often preferred to be disposable. Alternatively, a light-induced manipulation mechanism has been widely investigated to solve the wiring issues that arise for controlling a large array of electrodes. For example, optoelectronic tweezers (OET) has recently been shown to manipulate single cells and microscopic particles sandwiched

between an indium tin oxide electrode and a photoconductive electrode created by direct optical images. [00137] However, due to impedance matching, it is difficult to modulate the electric field strength in electrically insulating mediums, such as oils used in two-phase droplet-based microfluidic systems, on an OET device since all of the applied voltage drops across the oil layer even without light illumination. [00138] This section further describes an optically actuated droplet manipulation mechanism called floating electrode optoelectronic tweezers (FEOET) to enable optical modulation of the electric field in electrically insulating oils, from which sufficient dielectrophoretic (DEP) forces are generated to provide aqueous droplet actuation. [00139] 3.2 FEOET Device Structure

[00140] An FEOET device was previously described with reference to FIG. 1 whose implementation was exemplified in response to depositing two a-Si:H layers (Q.b μm undoped and 0.1 μm n+) on a glass substrate. Two aluminum electrodes separated by a 1 cm gap were deposited at the two edges of the device, for instance by using a lift-off method enabling electrical contact. The n+ a-Si:H layer not covered by Al electrodes was then etched away, for example using a reactive ion etcher. The remaining covered n+ a-S ^' wH layer reduces the electrical resistance between the Al electrodes and the undoped a-Si:H layer. An open polydimethylsiloxana (PDMS) chamber, housing aqueous droplets immersed in a corn oil medium, is fixed on top of the undoped a-Si:H layer. [00141] A DC bias is applied across the two aluminum electrodes to provide a lateral electric field across the whole device in both the oil and the undoped a-

Si:H layers. In the absence of light illumination, the electric field uniformly drops across the oil and the silicon layers. When a light beam illuminates the undoped a-Si:H layer, photogenerated electron-hole pairs are created, which locally changes the photoconductivity. [00142] The originally uniform electric field is strongly perturbed near the illuminated area, resulting in two strong electric field regions near the two

edges of the illuminated area parallel to the field direction and a weak field region in the middle of the illuminated area. This effect is similar to the field- induced dipole on a metal ball in a uniform electric field, except that the electric dipole is induced by a light-patterned virtual electrode on a two- dimensional surface. The perturbed field penetrates into the oil layer, which creates nonuniformity of the electric fields necessary for DEP manipulation in the electrically insulating oil. [00143] Since the aqueous droplets are much more conductive than the surrounding electrically insulating oil, an electric dipole is also induced on each droplet under the application of a lateral electric field. Even though a highly nonuniform electric field is induced around a droplet, there is no DEP force actuation on the droplet since the balanced electric field distribution around the droplet results in a zero net force. [00144] However, when a light beam illuminates near the edge of the droplet on the undoped a-Si:H layer, it creates a light-patterned virtual electrode which decreases the electric field strength at the illuminated side of the droplet. This breaks the originally balanced electric field pattern around a droplet and results in a nonzero net positive DEP force, which drives the droplet away from the light beam to the strong electric field region. [00145] This working mechanism is verified by three-dimensional (3D) simulated results of electric field distribution calculated using finite-element software (e.g., COMSOL MULTIPHYSICS 3.2). By way of example and to reduce the simulation time, the device is simplified to the following layers: δ μm a-Si:H, 20 μm PDMS, and 500 μm oil medium. A 300 VDC bias is applied at the two end planes along the x direction, and a 300 μm diameter aqueous droplet is immersed in a 2 X 2 mm oil environment on top of the thin PDMS layer. The dark conductivity of the homogeneous a-Si:H layer is assumed as 10 ^~8 S/m and the optical illumination is assumed to a full width at half maximum to create a Gaussian photoconductivity distribution with its peak conductivity of 10 ^~4 S/m and spot size of 100 μm .

[00146] 3.3 Simulations of Electrical Field Distribution in the a-Si:H layer [00147] FIG. 21 A-21 C depict 3D numerical simulation results of electric field distribution. In FIG. 21 B the light-patterned virtual electrode perturbs the e- field distribution at z=5 μm in the a-Si:H layer. The e-field perturbation shown in FIG. 21 B penetrates into an electrically insulating oil layer at z=55 μm . The electric field perturbation is developed in the y-direction cross-sectional view shown in FIG. 21 C at y=1 mm . Accordingly, the above figures show the 3D numerical simulations of the electric field distribution under the illumination of a circular light beam. The electric strength at the center of the illuminated area greatly decreases, while the field strength at the two edges along the electric field direction is enhanced. The largest electric field gradient exists near the a- Si:H surface as shown in FIG. 21 B and the field perturbation can penetrate into the nearby electrically insulating oil layer as shown in FIG. 21 C, which can be applied to actuate aqueous droplets via DEP forces. [00148] 3.4 Simulations of Electrical Field Distribution on Droplet Surface

[00149] FIG. 22A-22C and FIG. 23A-23C illustrate numerical simulations of the electric field distribution around an ionic aqueous droplet loaded into the oil environment. FIG. 22A-22C depicts the e-field without light illumination, wherein the balanced electric field distribution is exhibited around an electric dipole of a droplet. With no light a symmetrical electric field distribution is developed around a droplet, owing to the droplet induced electric dipole, whereby the balanced electric field pattern produces zero net DEP force on the droplet and thus no movement arises. FIG. 23A-23C depicts the e-field in response to optical illumination at the edge of a droplet which creates an unbalanced electric field distribution near the droplet, wherein the DEP force drives a droplet away from the illuminated spot.

[00150] In the presence of the light beam illumination at the edge of a droplet, the electric field strength at the illuminated side is strongly attenuated, breaking the originally balanced field pattern and giving rise to a net positive DEP force urging the droplet away from the light beam as seen in FIG. 23A-

23C. The 3D electric field strength distributions on the surface of a droplet are

shown in FIG. 22B-22C and FIG. 23B-23C which demonstrate the significant difference in the field patterns with and without light illumination.

[00151] 3.5 DEP Particle Force Estimation

[00152] The DEP force on a particle is usually estimated using F = [pV)E , where p is the induced dipole and E is the electric field. A good approximation can be obtained from this equation only when the particle size is much smaller than the gradient of the electric field, criteria that is not satisfied to estimate the DEP force on a droplet whose size is comparable to the light induced electric field gradient. To precisely calculate the light-induced DEP force exerted on a droplet, the Maxwell stress tensor is integrated over the whole droplet surface,

^F ^ ⁼ Lλ ^εE2ds - ⁽¹⁾ where ε is the permittivity of the medium. The electric field strength acting on all infinitesimal areas of a droplet surface are calculated using the 3D finite element numerical simulation data and their integrations are completed over the entire droplet surface using the spherical coordinates. Based on the droplet situation shown in FIG. 23A-23C, where the center of the droplet is at 100 μm away from the center of the light beam along the x direction, the optically induced DEP force exerted on the droplet is estimated as F _dep = [2.60, -0.01, 1.12] x 10 ^"8 N . This estimated DEP force predominates in the x and z directions, meaning that the light-induced virtual electrode raises a droplet and pushes it away from the illuminated spot along the direction of the electric field. The theoretical value of the y direction force should be zero due to symmetry, and the value in the simulation data comes from numerical errors due to limited computation power for 3D simulation.

[00153] 3.6 Resulting Particle Motion from DEP Forces [00154] FIG. 24A-24D depict video snapshots of a de-ionized water droplet, immersed in corn oil medium, being repelled by a light beam as predicted by the simulation results of the present invention, and shown at 0s, 3s, 6s and 9s, respectively. Light-induced droplet actuation has been experimentally

demonstrated on the FEOET device. For the experimental setup, a laser beam (3.53 m W at 532 nm ) was utilized with a 1 mm output spot size, a charge-coupled device camera (e.g., Sony DFW-SX910) was used with metallic neutral density filters. [00155] FIG. 25 depicts both simulated and experimental velocity profiles depending on the droplet positions at various light intensities. For simulated data, the photoconductivity is assumed in the a-Si:H layer as linearly proportional to a circular light beam with a Gaussian intensity profile. The simulated velocity profiles are determined by the force balance between the DEP and the Stokes' drag. Both simulated and experimental plots show that the maximum droplet velocity and the position where it occurs increase as the light intensity increases. The experimental data also confirms that a light beam with a higher intensity yields a larger actuation range. In the selected corn oil medium, a droplet speed has been found on the order of 0.5 mm per second for a droplet approaching 1 mm in a corn oil liquid medium.

Specifically, a maximum droplet speed of 418.6 μm/s has been achieved on a 748 μm droplet with a light intensity of 4.49 μw/ mm ² . By reducing the light intensity to 4.08 μW/ mm ² , FEOET still allows optical actuation of a 681 μm droplet at a maximum speed of 85.1 μm/s . The maximum velocity is limited by the maximum voltage that can be applied to FEOET devices. The electric field strength applied here is 600 V/cm . Applying a higher voltage could provide larger DEP forces and faster droplet motion. It will also be appreciated that aside from bias voltage increases, droplet motion can be sped up by heating the oil, using lower viscosity oils, and so forth. [00156] 3.7 Conclusion

[00157] In conclusion, a droplet manipulation mechanism FEOET was described which enables optical actuation of aqueous droplets in an electrically insulating media on a plain amorphous silicon coated glass device. FEOET allows light patterned virtual electrodes to perturb a uniform electric field distribution, breaking an originally symmetric electric field pattern around

a droplet for DEP actuation. The use of FEOET promises a large-scale droplet manipulation platform for parallel droplet processing on a low cost substrate using flexible optical addressing.

[00158] 4. Optically Reconfiqurable Droplet-Based Microfluidic Platforms Enabling Effective 2D Addressing of Oil-Immersed Aqueous Droplets and

Biochemical Applications [00159] 4.1 Introduction

[00160] Microfluidic devices or micro total analysis systems (//-TAS) have been developed for providing the prospect to manipulate efficiently the complex protocols in a wide range of applications such as biotechnology, clinical diagnostic, combinatorial chemistry. These systems have the potential to perform various fluidic handling functionalities while offering the advantages of minimal reagent consumption, portability, decreased operating costs, and high throughput. Based on how a small volume of liquid is delivered in microfluidic devices, two types of devices are considered, such as a continuous-flow devices and digitized microfluidic devices. In traditional continuous-flow microfluidic devices, multiple streams of liquids are guided within micro channels by regulating their flow rates. A variety of applications such as detecting, sizing, and sorting biomolecules including protein and DNA, cell- based biosensors, and microfluidic deflection switches have been demonstrated in these systems. [00161] As the scale of reagent volume shrinks, a significant challenge encountered in the traditional continuous-flow microfluidic devices using a single-phase fluid are gaining an ability to suppress dispersion of reacting volumes due to surface absorption and diffusion limitations. One alternative that overcomes these limitations is the encapsulation of reagents in small volume droplets carried by an immiscible fluid, and completing processing of the droplets in digitized microfluidic devices. By isolating biological and chemical reagents in discrete droplets, the issues of surface contamination and concentration change, which arise in response to traditional continuous- flow devices due to diffusion and surface interaction, can be eliminated.

Digitized microfluidic systems have found applications in an expanding number of fields, including enzymatic kinetic assays, protein crystallization, and polymerase chain reactions (PCR) for high throughput DNA amplification. [00162] Several drawbacks associated with such droplet-based microfluidic devices have been pointed out along with the difficulties in flexibility and dynamic reconfigurability. Drawbacks of these conventional microfluidic systems include: (1 ) the need of complex mechanical components, such as pumps, valves, tubing and so forth, to guide digitized droplets within micro channels; and (2) the inefficiency in active control of individual droplets interfered by micro channels being driven by a continuous oil flow. Various actuation mechanisms for effective droplet control have been investigated to overcome these drawbacks, such as thermocapillary, dielectrophoresis (DEP), electrowetting on dielectric (EWOD), surface acoustic wave, and magnetic forces. Among these techniques, electrical methods such as DEP and EWOD are considered to be more compatible with miniaturization. In these electrical- based devices, two-dimensional (2D) digitized and physically patterned metal electrodes have been required for device operation to achieve the effective addressing of multiple droplets in an open chamber or a sandwiched structure. Nevertheless, complex wiring and integration on a chip have still arisen for addressing numerically large droplet arrays. To solve this problem, a microfluidic device integrating a high voltage CMOS driving circuit recently achieved active and parallel droplet control. However, this approach increases the fabrication cost of microfluidic devices, which are often preferred to be disposable. [00163] In order to solve the wiring issues that arises for controlling a large array of electrodes, light-induced manipulation mechanisms have been widely investigated. For example, optoelectronic tweezers (OET) have recently been shown to manipulate single cells and microscopic particles sandwiched between an ITO electrode and a photoconductive electrode created by direct optical images. However, due to impedance mismatch, it is difficult to modulate the electric field strength in electrically insulating mediums, such as

oils used in two phase droplet based microfluidic systems, on an OET device since all of the applied voltage drops across the oil layer even without light illumination. It will be noted that in an OET device the medium is in series connection with the device. [00164] As described in prior sections, the inventor has recently reported a novel floating electrode optoelectronic tweezers (FEOET) mechanism enabling optical actuation of oil-immersed aqueous droplets, using DEP force induced by a circular laser illumination onto a photoconductive layer with an intensity as low as 4 μW/mm ² . To achieve active control of individual droplets, an electrically-based droplet driving mechanisms, such as EWOD or DEP-based techniques require the electrical activation on 2D digitized metal electrodes, while the FEOET device of the present invention uses optically-induced electrical field nonuniformity resulting from the modulation of a lateral electric field (one-directional electric field modulation) applied on electrodes on opposing sides, or ends, of the chamber. In the previous FEOET device, this lateral electric field modulation using circular laser illumination limits the droplet movement to a linear motion along the direction of electric field, which may restrict potential applicability in chemical and biological applications necessary for multiplexed microfluidic functionalities. [00165] In this section an optically reconfigurable droplet-based microfluidic platform is described enabling an effective 2D addressing of aqueous droplets, which is realized by utilizing one or more pairs of diamond-shaped virtual electrodes. The optical illumination of a pair of these images generates the spatial electric field modulation, resulting in continuous 2D droplet manipulation capability on a FEOET platform. This breakthrough opens up the possibility of massively parallel droplet manipulation on a featureless, silicon- coated glass substrate using dynamic and reconfigurable optical images. Optically patternable virtual electrodes produce dielectrophoretic (DEP) forces to manipulate aqueous droplets containing chemical and biochemical contents and to perform various microfluidic functions, such as two-dimensional transport, droplet merging and parallel processing of any desired plurality of

droplets (e.g., sixteen shown by way of example).

[00166] 4.2 Device Structure and Working Principle for 2D Transport [00167] FIG. 26 illustrates an example embodiment 70 of a flexible and reconfigurable FEOET-based microfluidic platform. First, the test sample droplets, which contain biological cells with physiological buffer solutions, are prepared on a PDMS coated substrate, for example a thin flat surface, an array of micro well structures, other microfluidic structures or combination thereof. These sample preparations are very flexible according to biochemical compatibility with a FEOET device. Then the PDMS substrate with the sample cells is loaded on a photoconductive layer and electrically-insulating oil medium covers it in order to prevent such a small volume of the sample droplets from evaporating in air. Multiple droplets containing different biochemical reagents are introduced and mixing can be performed 72, followed by transport 76 through an array of cells 74 to a target cell (sample) 78. The optically-induced DEP force enables actively driving these droplets for 2D transport in response to light 34 from projector 32. Droplets can be merged or otherwise manipulated during transport to each given target cell. The PDMS substrate is taken out from the device and the cells are inspected for biological analysis. It will be appreciated that the FEOET system in this regard can be configured as a laboratory on a chip.

[00168] FIG. 27 depicts the layered structure of the FEOET device shown in FIG. 26, which by way of example depict similar layers as in FIG. 1 with a transparent substrate 12, photoconductive layer 14, electrode 18 and PDMS chamber (substrate) containing wells, or other microfluidic receptacles or structures. It should be appreciated that the fabrication process is relatively simple and featureless when compared to other droplet actuation devices in which the 2D digitized physically deposited metal electrodes are required for electrical activations. In one simple embodiment, the device consists of two layer depositions: a 100 nm a-S ^' wH layer and '] 00 nm aluminum electrodes with 5 cm separation at the two edges on a glass substrate. Test sample droplets and cells are loaded into an oil medium retained in an open poly-

dimethylsiloxana (PDMS) chamber which is fixed on top of the a-Si:H layer. [00169] The FEOET device described in prior sections was able to actuate an aqueous droplet by light-induced DEP forces with an intensity as low as 4 μW/mm ² , an intensity promising the ability to create a large area droplet manipulation platform with commercially available optical projectors or LCD computer monitors, as shown in the lower portion of FIG. 26, without the need of extra optics components. The DEP force induced on the FEOET is definitely dependent upon the direction of applied electric field and shape of optical virtual electrodes. In previous sections, it will be appreciated that the DEP force in the direction parallel to the applied electric field is strong enough to push the droplet away from the light beam to the strong electric field region, but the force in the perpendicular direction is not strong as can be seen from FIG. 9A-9B. This is because a circular laser beam illumination greatly lowers the electric field strength at one side of the droplet when the laser and droplet are aligned in the direction parallel to the electric field, but the field strength is not substantially lowered in the direction perpendicular to the electric field direction. These aspects will be discussed in relation to numerical simulations. [00170] By utilizing a pair of diamond-shaped virtual electrodes, two- dimensional droplet manipulation is efficiently obtained in the FEOET device. The strong electric field region created between a pair of virtual electrodes induces an optical trap and enables 2D active addressing of a droplet into the position of the two-image gap as shown in FIG. 9C-9D. Using the preferred shape of these virtual electrodes, or similar shapes, the device is able to provide a droplet with the same order of magnitude DEP forces in both directions parallel and perpendicular to the applied field. This advancement enables active control of individual droplets and offers the possibility of implementing featureless, low-cost silicon-coated microfluidic platform which are programmable for a flexible and dynamic addressing of optical image patterns in order to perform large scale, multiplexed droplet-based biological and chemical analyses.

[00171] 4.3 Simulated Demonstrations

[00172] In order to see how differently two image patterns, a circular light beam and two diamond-shaped virtual electrodes, have an effect on the droplet actuation in both directions (parallel and perpendicular) to the electric field direction, the DEP forces acting on a droplet are numerically estimated in both situations. The simulations of a three-dimensional (3D) electric field distribution in a FEOET chamber and on a droplet surface are performed using a physics simulation (e.g., COMSOL Multiphysics 3.2) software. Due to computer processing power limitations, the device is simplified to the following two layers: a 5 μm thick undoped a-Si:H and a 350 μm thick oil medium. A

100V DC bias is applied across a portion of the chamber along the x direction, and a 300 μm diameter aqueous droplet is loaded in a 1 .4 x 0.8 mm oil chamber. The conductivity of the homogeneous a-Si:H layer under darkness is assumed to be about 10 ^~8 S/m . [00173] As previously described, the DEP force on a spherical particle is commonly estimated using F = [pV)E where p is the induced dipole and E is the electric field. However, this approximation is obtained only when the particle size is much smaller than the gradient of the electric field, criteria that is not satisfied to estimate the DEP force on a droplet whose size is comparable to the optically induced electric field gradient. To precisely calculate the light-induced DEP force exerted on a droplet, the integration of Maxwell stress tensor has been applied over the whole droplet surface as was shown by Eq. 1 . [00174] The electric field strength acting on all infinitesimal areas of a droplet surface are calculated using the 3D finite element numerical simulation data and their integrations are completed over the entire droplet surface using the spherical coordinates.

[00175] 4.3.1 Case of a Circular Laser Beam Illumination

[00176] Additional tests were performed user a finer mesh within the numerical simulations of electric field distributions as previously described in FIG. 10A-

1 1 B. Accordingly, the data generated in this section is more accurate than

that given in the prior section, which further highlights the advantages of the present invention. The numerical simulations indicate that without light illumination, a field induced dipole generates a symmetrical electric field distribution around a droplet surface, resulting in zero net DEP forces. However, a laser beam illumination at one edge of a droplet decreases the electric field strength and breaks the initial dipole field pattern around a droplet. The resulting imbalance of electric field gives birth to the non-zero net DEP force to move a droplet away from the light beam to the strong electric field region. Such an unbalanced electric field distribution is noticeable in the bottom figure in FIG. 1 OB for x directional transport.

[00177] The DEP force in the x direction is estimated as

F _dep = [-1.07, 0.00, -0.32] x 10 ^"8 N , corresponding to the force components in x, y, and z directions, respectively. Transporting droplets in the y direction, however, is not as effective as transport in the x direction using a circular light beam. Illuminating a droplet edge at the direction perpendicular (y direction) to the electric field fails to transport the droplet since the electric field strength difference between the illuminated and non-illuminated sides on the droplet surface is too small as shown in FIG. 1 1 A-1 1 B. The calculated DEP forces in this situation is F _dep = [-0.00, 0.01, 0.25] x 10 ^"8 N . The y direction force magnitude is 107 times smaller than that of the x direction force in FIG. 10A-

10B. For this reason, a circular light beam is not able to provide the required shape of virtual electrodes for active 2D transportation in FEOET devices. [00178] 4.3.2 Case of a Paired Diamond-Shaped Image Illumination

[00179] As previously discussed, FIG. 12 showed the simulation results of a droplet trapped by a paired diamond-shaped image illumination. A droplet is located at the same location as the situation in a circular laser beam illumination, 150 //m away from the center between two diamond-shaped images along the direction parallel (FIG. 13B) and perpendicular (FIG. 14B) to the applied electric field. To provide consistent simulation conditions, in the previous use of the laser beam, the photoconductivity profile by two diamond- shaped virtual electrodes is assumed to have the same peak

photoconductivity of 10 ⁴ S I m and a 200 μm separation gap. [00180] Prior to loading a droplet, the electric field strength is strongly enhanced at the gap between these two electrodes, as was shown in FIG. 12. This strong electric field region enables the optical trap pattern between the images to attract a droplet. When a droplet is loaded at 150 μm away from the gap center along the x direction (FIG. 13A-13B), the enhanced electric field can generate DEP forces trapping the droplet into the center of the gap. The calculated DEP force is F _dep = [1.62, 0.00, -2.75] ^χ lO ^~8 N . The value of the DEP force in the x direction is similar to the one in case of a circular laser beam. [00181] For the situation of a droplet positioned 150 μm away from the gap center along the y direction, however, the DEP force shows a dramatic increase compared to the y direction force in the case of a circular light beam. As shown in FIG. 14B, the calculated DEP forces in this situation are F _dep = [0.00, -0.67, -2.65] x 10 ^"8 N . The y direction force is only 2.41 times smaller than the x direction force in the FIG. 13B situation. This value shows a dramatic improvement compared to the 107 times smaller in the circular light beam case. This y direction force is able to provide effective trapping and transporting of a droplet in the y direction, and also realizes effective 2D droplet manipulation functions on FEOET devices. [00182] 4.4 Experimental Results

[00183] With the capability of two-dimensional transport enabled by using paired electrodes, several important droplet manipulation functions on the FEOET platform have been successfully demonstrated, including continuous 2D transport, droplet merging and mixing, and parallel processing of sixteen droplets. Additionally, chemical loading of HeIa cells and calcium flux assay on FEOET have been demonstrated by optical manipulation of trimethyltin (TMT) chloride droplets for activation of intracellular calcium ( Ca ₂ ²⁺ ) with HeLa cells. An FEOET device with a 5 * 5cm active optical manipulation area was fabricated, having a similar structure as the 6 * 6cm device shown previously in FIG. 15. A few kV of bias across the two aluminum electrodes was applied

in this example to power the entire device. Although the voltage is high, the power consumption of the whole device is a couple of hundred μW in the dark state and less than a few m W in the bright state due to the large electrical impedance of the oil and a-Si:H thin film ( - 1014 ω in the dark state) in the lateral direction.

[00184] 4.4.1 FEOET 2D Transport and Parallel Processing

[00185] Video snapshots of 2D transport of a 1 .47 mm aqueous droplet containing the Trypan blue dye were shown in FIG. 16A-16E. Initially the droplet is located at 4.5 mm away from the center of two projected diamond- shaped virtual electrodes as shown in FIG. 16A. These images indicate the strong electric field region produced in the center of the images which cause the optically-induced droplet trap in response to applied bias voltage. Then the droplet is attracted to such an optically-induced trap and the continuous 2D transport of the droplet in both x and y directions is now achieved by following the motion of paired diamond-shaped images as shown in FIG. 16C through

FIG. 16E. The droplet transport speed is 721 μm/s in the x direction and

363 μm/s in the y direction. The two times difference between the speeds in the x and y directions matches well with the DEP force difference predicted in the simulation results in FIG. 13A-13B and FIG. 14A-14B using a 300 μm droplet.

[00186] FIG. 28A-28F illustrates images from a demonstration similar to the above, however, in this case the pattern images were generated by an LCD computer monitor upon which the FEOET device was positioned. After gently positioning the FEOET device on the LCD monitor, images are directly patterned to the device without any additional optical components. Because of the pixilated LCD images, their intensity and contrast are greatly lower than the one from the projector illumination. Nevertheless, continuous 2D transport of a 1 .54 mm de-ionized (Dl) water droplet was achieved by LCD image motions of 5\ 5 μmfs in the x direction and 221 μm/s in the y direction. [00187] In this sequence, FIG. 28A depicts the initial position of the droplet before trapping. In FIG. 28B the droplet is seen trapped at time = 0 seconds.

In FIG. 28C at time = 20 seconds, the dashed lines show the current position of the droplet which has moved, in response to the motion of the diamond pattern, from the solid circle. In FIG. 28D, at time equal to 38 seconds, the droplet has been moved upward in response to the diamond pattern, then in FIG. 28E the droplet was moved back to the right, and in FIG. 28F the droplet was moved back down to its original position.

[00188] The low light intensity requirement of FEOET and the two dimensional droplet transportation function realized by diamond-shaped virtual electrodes promise an FEOET platform capable of massively parallel processing of a large number of droplets for high throughput chemical and biochemical screening applications. An example of parallel transporting of 16 droplets was previously described in FIG. 20A-20D. The droplet size in these tests varied from 0.84 mm to 1 .3 mm in diameter. The diamond-shaped virtual electrodes generate sixteen traps into which the droplets are positioned in the center between their gaps to perform parallel processing. The droplets in the first and third rows are driven to the right side, while the droplets in the second and fourth rows are guided to the left as per FIG. 2OC. This process is repeated in the opposite direction in FIG. 2OD. [00189] 4.4.2 FEOET Compatibility with Biochemical Applications [00190] It will be appreciated that the flexibility and compatibility of FEOET devices can be readily combined with other microfluidic structures to address a number of biochemical applications. FIG. 18 as previously described, illustrated a simple example of a 2 x 3 array of PDMS-fabricated micro well structures. It should be appreciated that these wells can contain any desired molecular or biological specie, for example adherent cells pre-grown in these micro wells with physiological buffer solutions and loaded into the FEOET device according to the present invention.

[00191] FIG. 29A-29E demonstrates operation of the FEOET wherein Trypan blue dye droplets are confined in the micro wells, with the PDMS structure positioned on top of a FEOET device, being immersed in an oil environment.

A 1 .28 mm de-ionized water droplet is transported by a paired diamond-

shaped virtual electrode and delivered to the target well to dilute the dye concentration in that well. In this sequence, FIG. 29A depicts the initial position of the droplet with the bias voltage off. In FIG. 29B the bias voltage is turned on, and the droplet is seen trapped by the electrodes in FIG. 29C, shown at 45 seconds, then in FIG. 29D the droplet is depicted immediately prior to merging into the target well at 60 seconds, and finally in FIG. 29E the droplet is shown upon delivery to the well.

[00192] Another example of droplet manipulation is the case of directly retaining aqueous droplets in oil without an additional PDMS structures. Aqueous droplets may involve non-adherent cells in buffer solutions. As previously described, FIG. 17A-17H depicts an example of two 1 mm droplets containing red and green food coloring dyes being individually manipulated and merged together. When two aqueous droplets are driven closely and aligned in the electric field direction, dipole-dipole interaction between two droplets induces electrocoalescence and combines two droplets together.

[00193] FIG. 30A-30E depicts a sequence of images showing that mixing inside the droplet is achieved by a simple movement of the droplet. The friction with the solid surface induces the shear flow inside the droplet for mixing while optical transport is taking place within the FEOET. FIG. 3OA shows the two droplets prior to merging and coalescing, then in FIG. 3OB after droplet merging. In FIG. 3OC with the patterned illumination activated, a DEP force drives the merged droplet and as seen in FIG. 3OD at a time of 4s, wherein the shearing flow inside the droplet causes mixing within the droplet which is substantially intermixed by FIG. 3OE shown at a time of 8 seconds. [00194] 4.4.3 HeLa Cells and Calcium Flux Assay

[00195] It will be appreciated that the FEOET provides effective capabilities of 2D transport as well as flexibility and compatibility with biochemical applications using reconfigurable and dynamic image modulation. [00196] FIG. 31 A-31 D illustrate a successful demonstration of the enhancement of fluorescent signal for HeLa cell detection using a FEOET device. This process is accomplished by optical manipulation of the droplet of trimethyltin

(TMT) chloride for activation of intracellular calcium [ CaJ ⁺ ) with HeLa cells.

Before the demonstration began, the fluorescent intensities of HeLa cells with different chemicals under Zeiss microscope excitation at Alb nm were as shown in FIG. 31 A. The intensity increase of fluo-4 preloaded HeLa cells corresponds with the level of CaJ ⁺ flux into cells as depicted in FIG. 31 B. The cells are shown with both fluo-4 and TMT in FIG. 31 C. No photobleaching was observed for up to a few hours. Then in FIG. 31 D HeLa cells are shown loaded with fluo-4, CaJ ⁺ , and TMT in which a greatly enhanced fluorescent signal can be seen. This greatly enhanced signal arises because the application of TMT elevates CaJ ⁺ flux, resulting in reactions with fluo-4.

[00197] HeLa cells in this example were maintained at 37°C and 5% CO2 in

DMEM supplemented with 10% FBS, 1 % L-glutamine, penicillin/streptomycin, and non-essential amino acids. For the cell attachment to a flat PDMS substrate, cells at 3.0 ^χ l0 ⁴ /m/ were plated (1 μl ) with a 2 x 2 array on the substrate and incubated for twelve hours. The media was replaced with 1 μl of Tyrode's buffer (1 mM MgCI ₂ , 1 O mM glucose, 1 O mM HEPES, 5 mM KCI, 14O mM NaCI, pH 7.2). The cells on the PDMS substrate were incubated for thirty minutes with 1 μl of 2 M fluo-4 dye (Molecular Probes, Invitrogen Life Technologies, Carlsbad, CA), followed by washing twice with 1 x PBS, pH 7.4, and DMEM media (1 μl ) was placed in cells. Cellular fluo-4 dye uptake was confirmed by an increased background fluorescence intensity, which was compensated for prior to FEOET actuation. Cells on a PDMS substrate received 1 μl of 2.O mM CaCI ₂ thirty minutes prior to FEOET manipulation. [00198] FIG. 32A-32E demonstrates TMT droplet transport into Fluo-4 loaded cells in DMEM/CaCI ₂ . A 20 μl droplet containing 250 μM trimethyltin chloride

(TMT, Sigma Aldrich, St. Louis, MO) was placed on the PDMS substrate to be optically transported and delivered into the droplets containing fluo-4 loaded cells in DMEM/CaCb. Then the PDMS substrate is taken and loaded under a fluorescent microscope for detection of increased Ca-flux by the TMT

activator. FIG. 32A depicts TMT loading, wherein a TMT droplet is seen, as well as a Fluo-4 preloaded HeLa cell and a target cell. In FIG. 32B the optical images are activated with a TMT droplet trapped in FIG. 32C and moved in FIG. 32D (t=10s) and FIG. 32E (t=13s) to the target cell as combined in FIG. 32F.

[00199] FIG. 33A-33B demonstrate the positive result of droplet manipulation as clearly indicated by the noticeable fluorescent intensity elevation observed between TMT activated (FIG. 33A) and non-activated droplets (FIG. 33B). [00200] 4.5 Conclusions [00201] An optically reconfigurable microfluidic platform has been demonstrated which enables parallel processing of oil-immersed aqueous droplets on a photoconductive glass substrate manipulated using direct optical images. This platform is realized by using a manipulation pattern for a virtual electrode, such as a paired diamond-shaped virtual electrode, which are controllable moved to transport droplets on a two-dimensional FEOET surface. With this

FEOET platform, several important droplet manipulation functions have been demonstrated, including: continuous 2D droplet transport, droplet merging and parallel processing of any desired plurality of droplets. The FEOET system of the present invention is also compatible with other microfluidic structures, such as micro wells arrays. This inventive platform promises a low cost, droplet- based, microfluidic system for large scale, multiplexed chemical and biochemical screening applications.

[00202] Although the description above contains many details, these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the presently preferred embodiments of this invention.

Therefore, it will be appreciated that the scope of the present invention fully encompasses other embodiments which may become obvious to those skilled in the art, and that the scope of the present invention is accordingly to be limited by nothing other than the appended claims, in which reference to an element in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." All structural, chemical, and

functional equivalents to the elements of the above-described preferred embodiment that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Moreover, it is not necessary for a device or method to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed under the provisions of 35 U. S. C. 1 12, sixth paragraph, unless the element is expressly recited using the phrase "means for."