Liquids

Field of the Invention

This invention relates to the field of microfluidics. Specifically, it relates to devices and methods for the manipulation of particles in liquids based on dielectrophoresis.

Background of the Invention

Recently, microfluidic devices (also known as Lab-on-a-Chip devices or Micro Total Analysis Systems) have gained interests from many fields due to their advantages such as small sample consumption, fast measurement, low experiment cost, automation easiness, and high measurement repeatability and excellent data quality, etc.

Traditional liquid handling typically involves large sample quantity and multiple complex steps. An Electro wetting-on-dielectric (EWOD) based digital microfluidic (DMF) device controls liquid samples in a discrete fashion - in the format of droplets. The capability of being able to control droplets individually offers a great flexibility such as parallel sample processing and multiplexed detections of multiple samples. Liquid operations such as droplet transportation, merging of multiple droplets, splitting a droplet into multiple smaller droplets, incubation, mixing, reaction, and waste droplet storage, etc., can now be achieved electronically by controlling the electrodes in a DMF device. The elimination of dependency on mechanical parts for operating liquids dramatically increases the system's stability and reliability.

In terms of DMF designs, the multi-layer-control-electrode design presented in patent WO 2008/147568 (by the same inventor) not only makes it practical to design universal format digital microfluidic devices but also makes a great step forward in manufacturing low cost and high quality microfluidic devices. It also greatly simplifies the controls and operations in microfluidics. However, patent WO 2008/147568, while concerning primarily with the operations of droplets, does not address the control of particles suspended in the droplets, which is very important in sample preparation and biochemical analyses.

Dielectrophoresis (DEP) is a phenomenon in which a neutral particle experiences a force when it is subjected to a non-uniform electric field. When a particle suspended in a liquid medium is exposed to a non-uniform electric field, it experiences a force that can cause it move to a region of higher electric field (positive dielectrophoresis) or to a region of lower electric field (negative dielectrophoresis). Unlike electrophoresis, the dielectrophoretic force does not require the particle to have charge. Also the dielectrophoretic force is insensitive to the polarity of the electric field. The effect of dielectrophoresis can occur in both AC (time varying) and DC (non-time varying) electric fields. All particles exhibit dielectrophoretic activity in the presence of non-uniform electric fields. The strength of the dielectrophoretic force depends on the particle's size and shape, the medium and the particle's electrical properties, as well as the frequency of the electric field. As an easy-to-control experiment parameter, the electric field frequency is often adjusted for the manipulation of different particles of interest.

The underlying theory and application of utilizing a non-uniform electric field to control the motion of a dielectric media have been known for quite some time. More than 100 years ago, Pellat demonstrated that a non-uniform electric field could dramatically influence the hydrostatic equilibrium of a dielectric liquid. Pohl originally used the term "dielectrophoresis" to describe the phenomenon in which a force is exerted on a dielectric particle when it is subjected to a nonuniform electric field. A description of the theory of dielectrophoresis has been published by H. A. Pohl in "Dielectrophoresis the Behavior of Neutral Matter in Nonuniform Electric Fields", Cambridge University Press, Cambridge 1978.

Without being bound by theory, here is a calculation of the time-averaged dielectrophoresis force for a homogeneous spherical particle of radius r:

— Eqn 1 where H?iis the absolute permittivity of the suspending medium, rms is root-mean- square value of the local electric field (assuming a sinusoidal time dependence), y is the del operator, ^j ^r s! ^" signifies the gradient of the electric field, and Re {/CM} is the real part of the Clausius-Mossotti factor, defined as:

Where *-p and m are the complex permittivities of the particle and the medium respectively. Furthermore, the complex dielectric constant is defined as: ε ^* = — Eqn 3 where g is the real permittivity constant, & is the electrical conductivity, is the frequency of the electric field, and j is the imaginary unit (the square root of -1). In Eqn 2, the Clausius-Mossotti factor † M contains all the frequency dependence of the DEP force. When the electric frequency is very high ( » 0), Eqn 2 becomes

For very low frequency ( ¾s 8 ), Eqn 2 becomes

This indicates that at high frequency, permittivity is the predominant factor in determining the

Clausius-Mossotti factor †CM, and at low frequency, conductivity is the predominant factor.

So, the dielectrophoretic effects can be designed in two ways. The first one is to choose medium to have greater conductivity (than the particle to be manipulated) but smaller permittivity, i.e., &m ^':> &p, and E _m < £p . In this case, the particle shows negative DEP at low frequency and positive DEP at high frequency and, at a frequency (known as cross-over frequency) somewhere in the middle, zero DEP. The second one is to choose medium to have smaller conductivity (than the particle to be manipulated) but greater permittivity, i.e., £¾. &p, and £ _m > £.p . In this case, the particle shows positive DEP at low frequency and negative DEP at high frequency and, at the cross-over frequency, zero DEP.

For particles suspended in a uniform aqueous electrolyte, in the typical frequency range used for particle manipulation 100Hz to 100MHz, the permittivity and conductivity of a suspending medium usually remains fairly constant, while for the particles themselves these parameters can vary significantly. The term R&{† _CM ) (the real part of Clausius-Mossotti factor ¾ _. ) can therefore be positive or negative, and thus over an extended frequency range a particle can exhibit both positive DEP (pDEP) and negative DEP (nDEP). So, by selecting medium with different permittivity constant and electrical conductivity and choosing a proper electric field frequency, dielectrophoresis can be utilized to selectively manipulate any particles suspended in the medium.

It should be mentioned that Eqn 1 only takes into account the electric dipole formed and not higher order polarization. When the electric field gradients are large, higher order terms become relevant, and can result in higher forces. As elaborated above, depending on the frequency dependencies of the permittivities and the conductivities of the particle and the medium, the dielectrophoresis force may be positive (positive DEP or pDEP) or negative (negative DEP or nDEP) at a particular frequency. The configuration and the geometry of the control electrodes can be quite different for the effectiveness of particle manipulations. Also, as can be seen from Eqn 1, the dielectrophoretic force is proportional to the volume of the particle being manipulated. So, if all other parameters are the same, bigger particles moves faster than smaller ones under a dielectrophoretic force.

One important application of dielectrophoresis is particle separation in aqueous solutions. It relies on the fact that one particular sub-population of particles has unique frequency-dependent dielectric properties, which is different from any other population. The relative magnitude and direction of the dielectrophoretic force exerted on a given population of particles depends on the conductivity and permittivity of the suspending medium, together with the frequency and magnitude of the applied field. Therefore, differences in the dielectric properties of particles manifest themselves as variations in the dielectrophoretic force magnitude or direction, resulting in separation of particles.

For example, depending on the polarizability of the medium, the cells undergo either positive or negative dielectrophoresis. The motion of different cells in different directions can be controlled by varying the frequency. For instance, it has been seen at lower frequencies that red blood cells undergo negative dielectrophoresis, but undergo positive dielectrophoresis at higher frequencies (see reference Electrophoresis 2008, 29, 2272-2279).

One of the biggest advantages of DEP is that it can be used for the manipulation of objects of various sizes (such as cells, virus, DNA, proteins, nanoparticles, and even single) simultaneously, (DEP works with both neutral and charged particles). Dielectrophoresis has been used to separate cells (WO 98/04355 by Pethig, et al), to determine cell viability (Pethig etal. WO 94/22583), to manipulate DNAs (Choi etal. WO 2008/094980), viruses (Morgan etal, Biophysical Journal, Vol77, p516-525), and nano-particles (J. Phys. D: Appl. Phys. 30 (1997) L41-L84.), etc.

Summary of the Invention

The present invention provides apparatuses and methods for manipulating particles in aqueous solutions.

To achieve the above mentioned goal, the present invention provides microfluidic apparatuses based on using dielectrophoresis effect to manipulate particles in liquids, which include at least: The first substrate and the second substrate;

The first electrode layer deposited on the first substrate and the second electrode layer deposited on top of the first electrode layer, and the third electrode layer deposited on the second substrate. The electrodes on the first substrate and the electrodes on the second substrate are aligned and separated to have enough space to contain liquid;

Said first electrode layer contains, at least, the first set of narrow electrodes, the first subset of electrodes, and a dielectric layer to electrically separate the electrodes in the first electrode layer and the second layer. The widths and spacings of the first set of narrow electrodes range from 0.1 um to 100 um. The widths and the spacings of the first subset of electrodes range from 100 um to 20 mm and from 1 um to 2 mm, respectively. In said second electrode layer, there are regions where at least some of the electrodes overlap with at least some of the narrow electrodes in the first electrode layer and regions where no overlapping occurs, and these overlapping and non-overlapping regions alternate in space.

A method for manipulating particles in aqueous solution via dielectrophoresis comprising the steps of:

a For the above mentioned apparatus, apply AC voltages of a specified frequency to at least some of the narrow electrodes, and least one electrode in the third electrode layer, wherein the phase of the AC voltages applied on some of the narrow electrodes is the same as the phase of the AC voltage applied to the electrodes(s) in the third electrodes, but different from the phase of the AC voltages applied on the other narrow electrodes of the same electrode layer. This is to form dielectrophoretic potential cage(s) in the space between the narrow electrodes and the third electrode layer, and to trap a particle(s) contained in the droplet of interest.

Furthermore, the above mentioned method includes:

b. Adjust the AC voltage phases at least once to change the position(s) of the dielectrophoretic potential cage(s), which changes the position(s) of the particle(s) trapped within the said dielectrophoretic potential cage(s).

From the discussions above, it can be seen that, similar to the multi-layer-electrode apparatuses presented in invention WO 2008/147568, the present invention provides apparatuses and methods for manipulating particles in aqueous solutions. Without being bound to the theory, the mechanisms of the invention allow the particles suspended in liquids to be redistributed and/or separated. While it has applications of its own, this invention makes the functions of a DMF device more complete, especially when combined with the inventions by the same inventor (WO 2008/147568, WO 2009/003184, and PCT/CN2012/070594). With these four inventions together, liquid samples can be dispensed, transported, merged, mixed, separated, position and volume measured, incubated and thermal processed, etc., and individual particle populations within the liquid sample can be redistributed or isolated for further processing and/or analysis. This invention enables the possibility of using a DMF device to separate and identify biomarkers (antibodies or other proteins, DNA, or RNA, etc.), virus, bacteria, and cells in complex liquid samples (such as blood, serum, plasma, sweat, saliva, and urine, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A and IB are the cross-sectional views, 90 degrees relative to each other, of a DMF device related to the present invention;

Figure 1C is a top plan view of control electrodes in the two electrode layers deposited on the first substrate surface;

Figure ID is a top plan view of the electrodes embedded on the second substrate surface;

Figure 2A shows a two dimensional sketch of the basic principle of the establishment of dielectrophoretic potential cages;

Figures 2B to 2D shows the movement, from left to right, of a DEP cage when varying the electric field by changing the voltage patterns on the electrodes;

Figures 3 A to 3D show a three dimensional sketch of the basic principle of the establishment of DEP cages and their movement when varying the electric field by changing the voltage patterns on the electrodes.

Figures 4A to 4L show a simplified demonstration of using dielectrophoresis to redistribute the particles in the droplet;

Figures 5A to 5E show a demonstration of the separation of different types of particles by splitting the droplet into two daughter droplets utilizing electrowetting mechanism following the redistribution of particles as shown in Figure 4L;

Figures 6A to 6E are the same as Figures 5A to 5E, respectively, except the overall plan view of the device is displayed to indicate the location of the droplets on the microfluidic device;

Figure 7 shows an example of a heterogeneous immunoassay on a DMF device with separation step being performed on the device itself;

Figure 8 shows an example of extracting DNA sample from a whole blood running real-time PCR on the extracted DNA on the device. All the steps, from sample preparation, to sample manipulation (heating, mixing and moving), to signal detection, are executed on the device;

Figure 9 shows an example of the physiology study of cells on a DMF device.

DETAILED DESCRIPTION OF THE INVENTION Presented in the following are examples to describe some of the implementations of this invention. From these examples, people in this field should be able to easily understand other advantages and effects of this invention. While the preferred embodiments of the invention have been illustrated and described, various different implementations can be made therein without departing from the spirit and scope of the invention

It should be mentioned that Figures 1A through 9 are only for illustration purposes of this invention. The numbers, shapes, and dimensions of different parts in the Figures do not typically represent a real implementation of an apparatus, which can have different shapes and numbers of the parts and different combinations; and the layout of a real apparatus can be more complex.

The following are the definition and/or explanation of some of the terminologies used in this patent application.

In this invention, the term "particles" is used to indicate micrometric or nanometric entities, either natural ones or artificial ones, such as cells, subcelluars components, viruses, liposomes, nanospheres, and micropheres, or even smaller entities, such as macro-molecules, proteins, DNAs, R As, etc., as well as droplets of liquid immiscible with the suspension medium, or bubbles of gas in liquid. The sizes of the "particles" range from a few nanometers to hundreds of micrometers.

By "dielectrophoretic potential" what is meant is a three-dimensional (3D) scalar function whose gradient is equal to the dielectrophoretic force. By "equal-potential surface" what is meant is a surface defined in the 3D space whose points have the same dielectrophoretic potential; the dielectrophoretic force is always perpendicular to said surface. By "potential cage" what is meant is a portion of space enclosed by an equal-potential surface and containing a local minimum of the dielectrophoretic potential. By "particle trapped inside a potential cage" what is meant is a particle is subject to the dielectrophoretic force and is located inside the said cage. At equilibrium, if the particle is subject to the dielectrophoretic force only, then it will be located at a position corresponding to the said dielectrophoretic potential minimum, otherwise it will be positioned at a displacement from that minimum given by the balance of forces.

The term "electrowetting" is used to indicate the effect that the change of the contact angle between a liquid and a solid surface due to an applied electric field. It should be pointed out that, when AC voltages or electric fields are applied, both the electrowetting effect and the dielectrophoretic effect exist. As the frequency of the AC voltages or electric fields increases, the dielectrophoretic effect will be more pronounced compared to the electrowetting effect. It is not the intent of this invention to strictly differentiate the electrowetting effect and the dielectrophoretic effect.

By "manipulation" what is meant, in particular, consists of one of the following operations and/or combinations thereof:

1. Selection, which consists of the isolation of a particular type of particles from a sample containing a multiplicity types of particles;

2. Reordering, which consists of the arrangement of the particles in an order different from the beginning.

3. Union, which consists of selecting two or more types of particles and bringing them closer together until they are forced against one another, for the purpose of bringing them into contact or of merging them or of including them one within the other.

4. Separation, which consists of separating particles that initially were in contact with one another, within certain distance from one another, or uniformly distributing in the media.

5. Trapping (or focusing), which consists of moving particles to a specific location on the device, and keeping the particles at said location for a specified amount of time.

In one embodiment of this invention, dielectrophoresis uses a non-uniform field of force through which to attract (one, a few, or a few groups of) particle(s) towards position(s) of stable equilibrium (dielectrophoretic potential cage). Said dielectrophoresis can be either negative dielectrophoresis (nDEP) or positive dielectrophoresis (pDEP).

For purposes of the present disclosure, the term "microfluidic" refers to a device or a system having the capability of manipulating liquid with at least one cross-sectional dimension in the range of from a few micrometers to about a few hundred micrometers.

For purposes of the present disclosure, the term "droplet" is used to indicate one type (or a few types mixed together) of liquid of limited volume is separated from other parts of liquid of the same type by air (or other gases), other liquids (typically not immiscible ones), or solid surfaces (such as inner surfaces of a DMF device), etc. The volume of a droplet can have a huge range - from a few femtoliters to hundreds of microliters. A droplet can take any arbitrary shape, such as sphere, semi- dome, flattened round, or irregular, etc.

Apparatuses and methods are provided by this invention to detect target analytes in a sample solution. As will be appreciated by those in the art, the sample solution may include, but not limited to, bodily fluids (including, but not limited to, blood, serum, saliva, urine, etc.), purified samples (such as purified DNA, RNA, proteins, etc.), environmental samples (including, but not limited to, water, air, agricultural samples, etc.), and biological warfare agent samples, etc. While the bodily fluids can be from any biological entities, the present invention is more interested in the bodily fluids from mammals, especially that from human.

For purposes of the present disclosure, the term "analyte" is a substance or a chemical constituent that undergoes measurement or analysis. Suitable analytes include organic and inorganic molecules. It may be biomolecules (including proteins, lipids, cytokines, hormones, carbohydrates, etc.), viruses (including herpesviruses, retroviruses, adenoviruses, lentiviruses, etc.), whole cells (including prokaryotic and eukaryotic cells), environmental pollutant (including toxins, pesticides, insecticides, etc.), therapeutic molecules (including antibiotics, therapeutic and abused drugs, etc.), nuclei, spores, etc.

For purposes of the present disclosure, the term "reagent" describes any material useful for reacting with, diluting, solvating, suspending, emulsifying, encapsulating, interacting with, or adding to a sample material.

For purposes of the present disclosure, the term "biomarker" refers to something that can be used as an indicator of a particular disease state or some other physiological state of an organism, or the body's response to therapy. A biomarker can be, a protein measured in (but not limited to) blood (whose concentration reflects the presence or severity of a disease), a DNA sequence, a traceable substance that is introduced into an organism as a means to examine organ function or other aspects of health, etc.

For purposes of the present disclosure, the term "amplification" refers to a process that can increase the quantity or concentration of a target analyte. Examples include, but not limited to, Polymerase Chain Reaction (PCR) and its variations (such as quantitative competitive PCR, immune -PCR, reverse transcriptase PCR, etc.), Strand Displacement Amplification (SDA), Nucleic Acid Sequence Based amplification (NASBA), Loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), etc.

For purposes of the present disclosure, the terms "layer" and "film" are used interchangeably to denote a structure of body that is typically but not necessarily planar or substantially planar, and is typically deposited on, formed on, coated, treated, or is otherwise disposed on another structure.

For purposes of the present disclosure, the term "electronic selector" describes any electronic device capable to set or change the output signal to different voltage or current levels with or without intervening electronic devices. As a non- limiting example, a microprocessor along with some driver chips can be used to set different electrodes at different voltage potentials at different times.

As used herein, the term "ground" (e.g., example, "ground electrode" or "ground voltage") indicates the voltage of corresponding electrode(s) is set to zero or substantially close to zero. All other voltage values should be, although typically less than 300 volts in amplitude, high enough so that substantially electrophoretic, dielectrophoretic, and electrowetting effect can be observed.

It should be pointed out that the spaces between adjacent electrodes at the same layer are generally filled with the dielectric material when the covering dielectric layer is disposed. These spaces can also be left empty or filled with gas such as air, nitrogen, helium, and argon. All the electrodes at the same layer, as well as electrodes at different layers, are preferably electrically isolated.

For purposes of the present disclosure, the term "communicate" (e.g., a first component "communicates with" or "is in communication with" a second component) is used herein to indicate a structural, functional, mechanical, electrical, optical, or fluidic relationship, or any combination thereof, between two or more components or elements. As such, the fact that one component is said to communicate with a second component is not intended to exclude the possibility that additional components may be present between, and/or operatively associated or engaged with, the first and the second components.

For purposes of the present disclosure, it will be understood that when a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being "on", "at", or "over" an electrode, array, matrix or surface, such liquid could be either in direct contact with electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface.

For purposes of the present disclosure, it will be understood that when a given component such as a layer, region or substrate is referred to herein as being disposed or formed "on", "in" or "at" another component, that given component can be directly on the other component or, alternatively, intervening components (e.g., one or more buffer layers, interlayers, electrodes or contacts) can also be present. It will be further understood that the terms "disposed on" and "formed on" are used interchangeably to describe how a given component is positioned or situated in relation to another component. Hence, the terms "disposed on" and "formed on" are not intended to introduce any limitations relating particular methods of material transport, deposition, or fabrication.

For purposes of the present disclosure, the terms "detection" and "measurement" are used interchangeably to denote a process of determining a physical quantity (such as position, charge, temperature, concentration, pH, luminance, and fluorescence, etc.). Normally at least one detector (or sensor) is used to measure a physical quantity and convert it into a signal or information which can be read by an instrument or a human. One or more components may be used between the object being measured and the sensor, such as lenses, mirrors, and filters in optical measurements, or resistors, capacitors, and transistors in electronic measurements. Also, other apparatuses or components may be used to make it easier or possible to measure a physical quantity. For example, when using the fluorescence intensity is used to deduce the particle concentration of, a light source, such as a laser or laser diode, may be used to excite the particles to the electronic excited states from their electronic ground state, which emits fluorescence light when returning to their ground states. The sensors can be a CCD (Charge Coupled Device), a photodiode, and a photomultiplier tube, etc., in optical measurements, or operational amplifier, analog-to-digital convertor, thermocouple, and thermistor, etc., in electronic measurements.

Detection or measurement can be done to a plurality of signals from a plurality of products, either simultaneously or sequentially. For example, a photodiode can be used to measure of the fiuorescence intensity from a particular type of particles in a droplet, while the position of the said droplet is sensing by a capacitance measurement at the same time. Also, a detector (or sensor) can include or be operably linked to a computer, e.g., which has software for converting detector signal to information that a human or other machine can understand. For example, the fluorescence intensity information is used to deduce the concentration of can be converted to particle concentration.

For purposes of the present disclosure, the term "elongated electrode" is used to indicate an electrode with a length that is at least 3 times or more than its width; preferably, 5 times or more; and even more preferably, 10 times or more.

As non-limiting examples, optical measurements include laser induced fluorescence measurement, infrared spectroscopy, Raman spectroscopy, chemiluminescence measurement, surface plasmon resonance measurement, absorption spectroscopy, etc. Electronic measurements include, but are not limited to, amperometry, voltammetry, photoelectrochemistry, coulometry, capacitance measurement, and AC impedance measurement, etc.

DETAILED DESCRIPTION OF THE DRAWINGS

The droplet-based methods and apparatus provided by the present invention will now be described in detail, with reference being made as necessary to the accompanying figures (Figures 1 A to 9). It should be understood that examples used herein are for the purposes of illustration and are not intended to limit the spirit of the invention.

Figures 1A and IB are the cross-sectional views of a digital micro fluidic device 100 used for droplet operations and the manipulation of particles suspended in said droplet. In this embodiment, droplet D is sandwiched between a bottom plate, generally designated 102, and a top plate, generally designated 104. Preferably, the gap between plate 102 and plate 104 is less than 1mm; more preferably, the gap is less than 0.3 mm. The terms "bottom" and "top" are used in the present context only to distinguish these two planes 102 and 104, and not as a limitation on the orientation of the planes 102 and 104 with respect to the horizontal. Plate 102 comprises the first electrode layer and the second electrode layer, and plate 104 comprises the third electrode layer. Deposited on the first substrate 101, the first electrode layer comprises the first subset of elongated electrodes El, the first narrow electrodes EID and dielectric layer 103 A. Deposited on dielectric layer, the second electrode layer comprises the second subset electrodes E2, the second narrow electrodes E2D and dielectric layer 103B. Deposited on the second substrate 105, the third electrode layer comprise electrodes L and dielectric layer 107.

Preferably, the electrodes contained in both the first electrode layer and the second electrode layer described above are elongated electrodes.

The widths and the spacings of the narrow electrodes EID range from 0.1 um tolOOum. The widths and spacings of the first subset electrodes El range from 100 um to 20 mm and 1 um to 2 mm, respectively.

Preferably, the widths and the spacings of the narrow electrodes EID range from 1 um to 50um; the widths and spacings of the first subset electrodes El range from 200 um to 5 mm and 5 um to 500 um, respectively. More preferably, the widths and the spacings of the narrow electrodes EID range from 1 um to 50um. The widths and spacings of the first subset electrodes El range from 200 um to 2 mm and 5 um to 100 um, respectively.

The widths and the spacings of the electrodes in the second electrode layer range from 0.1 um to 20mm.

Preferably, the widths and the spacings of the second set of narrow electrodes E2D range from 0.1 um to 100 um, and the widths and spacings of the second subset electrodes E2 range from 100 um to 20 mm. More preferably, the widths and the spacings of the second set of narrow electrodes E2D range from 1 um to 50 um, and the widths and spacings of the second subset electrodes E2 range from 200 um to 10 mm. Even more preferably, the widths and the spacings of the second set of narrow electrodes E2D range from 1 um to 50 um, and the widths and spacings of the second subset electrodes E2 range from 200 um to 2 mm.

The narrow electrodes EID (or E2D) can be utilized to generate electro wetting effect to operate the droplets above them, and they can also be utilized to generate dielectrophoretic effect to operate the particles contained within said droplets. Of course, the primary purposes of the control electrodes El and E2 are used to generate electrowetting effect. It should be understood that in the construction of devices benefiting from the present invention, control electrode arrays El, EID, E2 and E2D will typically be part of a larger number of control electrodes that collectively form a two- dimensional electrode array or grid.

Figure 1C is a top plan view of the control electrodes embedded in the bottom plate (designated 102) of a DMF device used in Figures 1A and IB. Droplet D is also shown for illustration purposes. The electrodes in the first electrode layer (including the first narrow electrodes E1D and the first subset electrodes El) are perpendicular to the electrodes in the second electrode layer (including the second narrow electrodes E2D and the second subset electrodes E2). This results in space alternation of regions where some of the electrodes in the second electrode layer overlap with the first electrodes E1D and regions where no such overlap occurs. It should be noted that the geometry of the electrodes in Figure 1C is not proportional to the ones in Figures 1A and IB with the purpose to make them easier to visualize (especially E1D and E2D electrodes). Figure ID is a top plan view of the control electrodes embedded in the up plate of the said devices used in this invention, designated 104 in Figures 1A and IB. The geometries of electrodes in Figure 1C and ID are proportional. Electrode L2 overlaps with electrode E1D (in Figure 1C) in space. L2 can be connected to ground, alone or together with LI and L3, when electro wetting effect is desired in the space between L2 and E1D. L2 can also be connected to an AC voltage source to have specified phase differences, which may vary with time, with control electrodes E1D (or E2D) in order to generate dielectrophoretic effects in the same space. Droplet D is also shown here for position reference purposes

The material for making the substrate or the cover plate is not important so long as the surface where the electrodes are disposed is (or is made) electrically non-conductive. The material should also be rigid enough so that the substrate and/or the cover plate can substantially keep their original shape once made, and the spacing between the two plates does not change substantially. The substrate and/or the cover plate can be made of (not limited to) quartz, glass, or polymers such as polycarbonate (PC) and cyclic olefin copolymer (COC).

The primary function of electrode arrays El and E2 is to generate electro wetting effect for droplet operations, although particles within the droplets will be effected inevitably. The electrode arrays E1D and E2D offer two main functions. First, when each electrode in E1D (or E2D) array is individually connected to an AC voltage source and its voltage is configured with specified phase offset relative to the others along with the corresponding the electrode(s) in up substrate being connected to an AC voltage, dielectrophoretic effect is designed to dominate, which will be used mainly to manipulate the particles suspended within the droplet. It should be mentioned that, although not always required, the frequencies of substantially the same value are typically chosen for all the electrodes in order for a significant dielectrophoretic effect is generated. (At the same time, electrowetting will be generated too.) Second, when the electrodes in array E1D (or E2D) are connected to the same DC or low frequency AC voltage source (or different low frequency AC voltage sources with similar voltage amplitudes and phases), while the corresponding electrode(s) in the up substrate is/are connected to ground, they will act together to generate electrowetting effect with purpose of manipulating the droplet. The number of electrodes in subsets El and E2 can range from 1 to 10,000, but preferably from 2 to 1,000, and more preferably from 2 to 200, respectively. The number of electrodes in narrow electrodes EID and E2D can range from 1 to 10,000, but preferably from 1 to 1,000, and more preferably from 1 to 500, respectively. The number of electrodes L in up substrate 104 can range from 1 to 10,000, but preferably from 2 to 1,000, and more preferably from 2 to 200, respectively. The spacings between adjacent electrodes L can range from 0.1 um to 20 mm, but preferably from lum to 2mm.

Control electrode arrays El, EID, E2 and E2D are placed in electrical communication with suitable voltages sources, which can be DC voltage sources or AC voltage sources, through conventional conductive lead lines. These voltage sources are independently controllable, but could also be connected to the same voltage source, in which case mechanisms like switches will be needed to make sure at least some of the electrodes can be selectively energized. Typically, the voltage amplitude is less than 300 volts. The frequency of the AC voltages for generating electrowetting effect is normally less than 10 KHz (10,000 Hz). When trying to achieve dielectrophoretic, each electrode in control electrode arrays EID and E2D is individually placed in electrical communication with a suitable AC voltage source through a conventional conductive lead line. Typically, the frequency of the AC voltages range from 1 Hz to 1 GHz (1,000,000,000 Hz), but preferably from 100 Hz to 100 MHz (100,000,000 Hz), and more preferably from 1 KHz to 10 MHz.

The electrodes can be made of any electrically conductive material such as copper, chrome and indium-tin-oxide (ITO), and the like. The shapes of the electrodes illustrated in Figures 1A to ID are displayed as rectangles for convenience. However, the electrodes can take many other shapes to have electrowetting or electrophoretic effects. As a matter of fact, the shape, width and spacing of E2D (or EID) electrodes can be purposely made different at different regions on the device, so that different types of particles of different sizes and shapes can be manipulated more efficiently at different locations.

The materials for making the dielectric layers 103 A, 103B and 107 can be (but not limited to) Teflon, Parylene C, silicon nitride, silicon dioxide, and the like. Preferably, the surfaces of layers 103B and 107 are hydrophobic. This can be achieved by coating layers 103B and 107 with a thin layer of Teflon, Cytop or other hydrophobic materials.

It should be pointed out that although the electrowetting and dielectrophoresis effects described in this invention are achieved using electrodes in two layers, substantially similar effects can be achieved using electrodes in more layers. As a non- limiting example, the electrodes El or EID in the first electrode layer can be made in two different layers separated by a thin layer by a dielectric layer while keeping the horizontal spacing between the adjacent electrodes substantially the same; the resulting dielectrophoretic and electrowetting effects are substantially similar.

Control electrode arrays El , E1D, E2 and E2D are embedded in or formed on a suitable first substrate 101. A dielectric layer 103 A is applied to cover electrodes El and E1D to electrically separate El and E1D electrodes from each other, and at the same time electrically separate El and E1D electrodes (belonging to the first electrode layer) from E2 and E2D electrodes (belonging to the second electrode layer). Another thin lower layer 103B of hydrophobic insulation is applied to cover and thereby electrically isolate control electrode arrays E2 and E2D. Upper plate 104 comprises control electrodes embedded in or formed on a suitable up substrate 105. Preferably, a thin upper layer 107 of hydrophobic insulation is also applied to upper plate 105 to cover and electrically isolate electrodes LI, L2 and L3.

Standard IC or LCD manufacturing processes can be applied to the making of DMC devices suitable to be used in biochemical analyses, for example, techniques for forming a layer may include (but not limited to) deposition such as plasma enhanced chemical vapor deposition (PECVD), sputtering, or spinning coating, etc.; techniques for removing a layer may include (but not limited to) etching such as wet etching, plasma etching, etc.; and patterning techniques include (but not limited to)UV lithography, electron beam lithography, etc.

As one kind of digital micro f uidic device, device 100 can include other micro fluidic components and/or microelectronic components. For example, said device can include resistive heating areas, microchannels, micropumps, pressure sensors, optical waveguides, and/or biosensing or chemosensing elements which are connected to an MOS (Metal Oxide Semiconductor) circuitry.

As a preferred implementation, micro fluidic device 100 can include electrode selector. The said electrode selector is connected to electrodes in the first electrode layer and the second electrode layer on the first substrate, and electrodes in the third electrode layer on the second substrate, and can be used select electrodes and supply voltages to the selected electrodes.

As another preferred implementation, device 100 can include at least a temperature control element to control the temperature of some portion(s) of the device. The temperature control element, such as a Peltier, can be set separated from device 100, but making contact with at least a portion of said device; or it can be integrated on to device 100, for example, a thin film resistive heater to be bonded to the outer surface of said device; and device 100 can include both integrated temperature control element(s) and separated ones. The said temperature control element(s) can control the temperature of the portion(s) of the device to any specified value(s) from 0 to 100 degrees Celsius. Furthermore, device 100 can include liquid inlet(s) and outlet(s) which connect the space of said device which can contain liquid.

Figures 2A to 2E illustrate the establishment of dielectrophoretic potential cages and the manipulation of them. L is an electrode in the upper substrate. ED represents an array of narrow electrodes on the bottom substrate of device 100 (i.e., the narrow electrodes E1D or E2D on bottom plate 102). The dotted lines on the dielectrophoretic potential cages represent equal electrical potential points (or equal electric field) generated by the electrodes. The circled "n" with the dotted lines indicates the cage is a negative dielectrophoresis (nDEP) cage. Any particle that experiences sufficient nDEP forces, e.g., the circled "n" in the Figures 2A to 2E, will be attracted to and trapped inside the cages. A cage may trap one or more particles, thus permitting them either to levitate steadily or to move within with the said cage, or both. Typically, the "cage" traps and keeps particles against gravity, Brownian motion, etc.

Figures 2B to 2D demonstrate the move of an nDEP by varying the voltage patterns on electrodes ED. By manipulating the voltages on the individually addressable electrodes ED, the positions of the nDEP cages can be shifted. The effect is that the trapped particles will follow the movement of the nDEP cages. Using this principle, the separation of different types of particles can be achieved if DEP conditions (applied field strengths and/or frequencies) can be found so that only one type of particles experiences nDEP force (and moves with the nDEP cages) while the others experience null DEP force and so remain stationary. It should be mentioned that "+" and "-" signs do not represent positive or negative voltages. They only indicate that the voltages of two electrodes with same signs, "+" or are in phase, i.e., 0 degree phase difference; if one electrode is marked with "+", and the other with the voltages on these two electrodes have opposite phase, i.e., 180 degrees phase difference. It should also be pointed out that in practice the voltage phase difference between two electrodes can be values between -180 degrees to 180 degrees. As a matter of fact, for smoother particle control purposes, the phase difference between two adjacent electrodes may be kept at less than 180 degrees.

Figures 3 A to 3E illustrate the motion of particles in an aqueous solution under the influence of DEP. 300 shows a portion of device 100 which is capable of generate the dielectrophoretic force to manipulate the particles contained within said device. For illustration purposes, ED is an array of evenly spaced elongated electrodes. By applying AC voltages to the electrodes ED and electrode L in upper substrate, potential cages within the spaces between L and ED are generated.

The potential cages have elongated shape as the narrow electrodes ED in the bottom substrate have elongated shapes. By shifting the voltage distribution pattern on ED pattern to the right, the three dimensional dielectrophoretic potential cages CI, C2 and C3 are moved to the right. From Figure 3 A to Figure 3B, cages CI, C2 and C3 are all moved to the right by one unit of the electrode spacing. So, all the particles contained in the said cages are also move to the right by one unit from Figure 3A to Figure 3B. From Figure 3B to Figure 3C, cages CI, C2 and C3, along with the particles contained within, are all moved to the right by another unit of the electrode spacing. In Figure 3C, cage CI reaches the right most driving electrode, and cage C4 is just formed. From Figure 3C to Figure 3D, cages C2, C3 and C4, along with the particles contained within, are all moved to the right by one unit of driving electrode spacing. Particles previously contained in cage CI stays in the right most driving electrode of ED. The electric configuration of Figure 3 A and 3D are the same. By repeating steps from Figure 3A to 3C, all the particles of interests will eventually be moved to the location of the rightmost electrode which is involved in the dielectrophoretic effects.

Figures 4A through 4L illustrate one embodiment of using a DMF device 100 to redistribute particles. In Figure 4 A, voltages of the same phase are applied to the first narrow electrodes EID of the bottom substrate of device 100, and zero (or very small) voltages are applied to the second electrodes E2 in the bottom substrate and electrode L in top substrate; without being bound to theory, due to the electrowetting effect generated by electrodes EID, droplet D, which is above E2 in space (see Figure 1A and 1C for their related positions), spreads along electrodes EID. Due to the space alternation of where electrodes EID and E2 overlap and do not overlap, the potion of those electrodes E2 located right above EID has shielding effect, which stops the further spread of droplet D along electrodes E2. This makes droplet D to form a shape of a rectangle (approximately), which is similar to the shape where electrodes EID and E2 do not overlap. Since droplet D is changed to square shape from it natural round shape, it is easier to operate the particles contained in square shaped droplet D.

From the discussion above, it can be seen that a droplet takes a flattened round shape. It is not as effective to separate different kind of particles utilizing dielectrophoretic effect for the particles in a droplet of flattened round shape, which makes it less convenient to measure or further handle of said particles. To make it easier to utilize the dielectrophoretic effect, there exist methods which form the droplet to a square or other desired shapes by utilizing the surface of a top plate and the indentation of a bottom plate or inner surface(s) to control the size and shape of the droplet. In the microfluidic device of present invention, the bottom plate are designed to have the first electrode layer which contains the first narrow electrodes and the second electrode layer; furthermore, there are regions where some of the electrodes in the second electrode layer overlap with at least some of the narrow electrodes in the first electrode layer and regions where such overlap does not occur, and these overlapping and non-overlapping regions alternate in space. Due to the electrowetting effect and non-overlapping region(s), the shape and size of a droplet can be easily controlled, which makes it more effective to operate the particles in the droplet utilizing the dielectrophoretic effect.

Figure 4B is a zoomed in version of Figure 4A with only part of the droplet related electrodes EID shown. Figures 4C to 4K are step-wise illustrations of the changes of the voltage distribution on EID - by repeating the shifting of the voltage distribution to the right, particles marked as type 1 are moved to the right by one unit (the spacing of EID electrodes) at a time, and finally all of them are moved to the rightmost electrode of EID which overlaps with the liquid droplet. It should be pointed out that, from Figure 4B to Figure 4K, the voltage of the electrode in the upper plate and the voltages of the narrow electrodes marked "-" have the same phase, and the frequency of all the voltages on all said electrodes meets the following condition: when AC voltages of said frequency are applied to the said narrow electrodes and the electrode in the upper plate, the dielectrophoretic cage can only trap type 1 particles, and not any particles of other types.

Following the same or similar approaches as illustrated in Figures 4B to 4K, by applying voltages of a different desired frequency to the narrow electrodes and the electrode in the upper plate, type 2 particles can be singled out and operated too. Figure 4L shows that all type 2 particles have been moved to the leftmost electrode of EID which overlaps with the droplet. Figure 4L shows that type 1 and type 2 particles have been redistributed. The concentration of type 1 particles on the right side of the droplet and the concentration of type 2 particles on the left side of the droplet are both much higher than their corresponding concentrations before the redistributions occur. This makes it much easier to measure the said particles, and hence improves the corresponding detection sensitivities dramatically.

Figures 5A through 5E demonstrate the further separation of two different types of particles "1" and "2" into two isolated droplets after the two particles are redistributed as indicated in Figure 4L. It should be mentioned that process shown in Figures 5A to 5E should take place before significant diffusion happens after the redistributed populations "1" and "2". In Figure 5A, the similar DC or low frequency voltages are applied to electrodes EID and El, and the droplet takes a relatively rectangular shape. In Figures 5B and 5C, voltages V2 and V4 are high enough so that the droplet starts to split due to electro wetting. V3 is connected to ground or very small voltage. Figure 5D shows the original droplet D is split into two separate daughter droplets Dl and D2. Now the majority of population "1" particles are in droplet Dl, while most of "2" particles are in droplet D2.

For more effectively utilizing dielectrophoresis, the same set of electrodes can be designed to have different geometry at different locations on the devices to make it possible to effectively manipulate particles of difference sizes, shapes and types. Figures 6A to 6E are essentially the same as Figures 5A to 5E respectively, except all the control electrodes (not just portions of) on the bottom plate are shown to give an overall illustration.

Aside from varying the frequency of the electric field, other parameters can also be adjusted, including the electrical conductivity and/or the real permittivity of the medium and/or its pH value.

It should be mentioned that, when in a medium (aqueous solution), particles experience buoyancy force besides the dielectrophoretic force. This should be taken into account when designing particle manipulation experiments especially when trying to operate (such as to trap) particles.

When a particle moves in a medium, it also experiences viscous forces. As shown in Eqn 1, the strength of the dielectrophoretic force is proportional to the particle size, but the strength if the viscous force is proportional to its surface area. Without being bound to the theory, the result is that it takes longer for smaller particles to move from one location to another than larger particles. So, to properly concentrate particles in a particular region, it is necessary to design wait times in order to account for slower particle dynamics.

Additionally or alternatively, other forces may be used to enhance the movement of the particles. These may include, but not limited to, hydrodynamic, ultrasonic, electrophoretic or optical forces.

A wide variety of particles, biological or non-biological, can be manipulated utilizing this invention, employing suitable electrode arrays and other parameters. For example, the shape, width and the spacing of the electrodes, the gap between the top and bottom plates, the frequency of the electric field, etc., could be altered for different bio-particles such as DNA, proteins, prions, viruses, cells, etc., or chemically activated particles such as coated latex beads.

The fields of use of the invention include the characterization of the particles suspended in an aqueous medium or other fluid utilizing dielectrophoretic effect. This is required for the inspection of liquefied food products, biological fluids such as whole blood, plasma, or other body fluids (such as saliva and urine), or of liquids sampled during a chemical production process, etc. In these cases, a rapid means would be provided for checking the presence, viability and homogeneity of the particles of interest.

The invention could also be employed for the analysis of fluids containing several particle types. Examples include biological fluids such as urine, where the relative composition of Gram- positive and Gram-negative bacteria could be ascertained by obtaining dielectrophoretic measurements over a range of conductivity and pH values, for example, to identify the presence of a dominant infective organism.

Due to their high-affinity and specificity, immunoassays are among the most widely used and sensitive techniques for the detection and quantitation of analytes such as viruses, peptides, polynucleotides, proteins (such as antibodies, toxins, cytokines), and other small molecules. In a clinical lab, immunoassays are currently used to test for cardiac markers, tumor markers, hormones, drugs, infectious agents, and immune response, etc., and with new tests being added continuously. Among the various immunoassay formats, heterogeneous immunoassays are the most common due to their higher sensitivity. In heterogeneous immunoassays, there are typically three steps: 1) capture - the type of reaction involves the forming of labeled antigen-antibody complex. 2) separation - the process of separating the bound antigen-antibody complex from free antigen. 3) detection - the measurement of signal from the bound antigen-antibody complex.

In heterogeneous immunoassays, the antibody-antigen complexes are typically immobilized on a solid surface (a well plate or magnetic microbeads) and unbound molecules from the sample are washed away. With this invention, the separation of bound and unbound molecules can be done by dielectrophoresis on DMF device, without the need to immobilize analytes to a solid surface. This, hence, reduces the complexity and the cost of the overall system.

AFP (Alpha-fetoprotein) is a glycoprotein with a molecular weight of approximately 70,000 Daltons. It is normally produced during fetal and neonatal development by the liver, yolksac, and in small concentrations by the gastrointestinal tract. After birth, serum AFP concentrations decrease rapidly, and, after the second year of life, only trace amounts normally exist.

Elevation of serum AFP to abnormally high values occurs in several malignant diseases, most notably nonseminomatous testicular cancer and primary hepatocellular carcinoma. In the case of nonseminomatous testicular cancer, a direct relationship has been observed between the incidence of elevated AFP levels and the stage of disease. Elevated AFP levels have been observed in patients diagnosed with seminoma. Elevated serum AFP concentrations also have been measured in patients with other noncancerous diseases, including neonatal hyperbilirubinemia, ataxia telangiectasia, hereditary tyrosinemia, acute viral hepatitis, chronic active hepatitis, and cirrhosis, etc.

Figure 7 shows an example of using a DMF device according to current invention to carry out a fluorescence (or chemiluminescence, absorbance, etc.) immunoassay to measure the concentration of AFP in patient serum. S701, a DMF device is loaded with patient serum sample and detection reagent, which contains primary capture antibodies, blocking proteins, and reporter secondary antibodies for detecting specified antigens. Reporter molecules are signal-generating molecules such as fluorescent molecules, chemiluminescent molecules, enzymes, quantum dots, biotins, etc. S702, similar to the way a droplet is split into two daughter droplets illustrated in Figures 5A to 5E, two droplets are dispensed from the serum sample and the reagent respectively, transported, merged, mixed and incubated, by applying DC or low frequency AC voltages to the corresponding electrodes in the device based on the locations of the serum sample and the reagent. S703, after the formation of capture antibody-antigen-reporter antibody complex, by applying DC or low frequency AC voltages to the corresponding electrodes, move the combined droplet to a location where the droplet overlaps with the first narrow electrodes so that dielectrophoresis can be carried out. S704, similar to the way used in Figure 4a, apply voltages of the same phase to the corresponding first narrow electrodes, which makes the combined droplet form a rectangle shape; similar to the ways illustrated in Figures 4B to 4K, apply AC voltages of a specified frequency to the first narrow electrodes and the electrode in upper substrate, which results in the generated dielectrophoretic potential cages capable of trapping and moving the said complexes in the droplet; afterwards, apply AC voltages of a different frequency to the first narrow electrodes and the electrode in upper substrate so that molecules not involved in the formation of the complexes can be trapped and moved, which results in the separation of the bound and unbound molecules in the combined droplet. S705, measure the optical signal, such as fluorescence, or chemiluminecence, or absorbance, etc., from the region where the bound complexes reside. S706, move the waste droplet to a storage location in the device.

If desired, after the separation of the bound and unbound complexes are complete in S704, the droplet can be split into two daughter droplets with bound and unbound complexes separated into each of the two droplets, as illustrated in Figure 5A to 5E. Then optical detection, such as fluorescence, absorbance, or chemiluminecence, etc., can be carried out on the daughter droplet with bound complexes.

It should be pointed here and will not be further described that, as is known to technical people in this field, it makes the sample or droplet move by applying proper voltages to the corresponding electrodes.

Figure 7 is an example of AFP being measured by running an immunoassay on a DMF device. Similar methods can be used to detect many other analytes such as bacteria, viruses, cells, etc.

Thanks to the particle manipulation capabilities presented in this invention, immobilizing the antibody-antigen complexes using things such as a well-plates or microbeads is no longer needed, as separation can be achieved on the DMF device simply by controlling the electrodes. This adds huge benefit - robust measurement, cost effective measurement, easy-to-use system, etc.

So far, the samples being analyzed on a microfluidic device are generally pretreated (sample preparation) before being loaded to the device. Sample preparation is an important step in most analytical techniques, because the measurement techniques are not always responsive to the analyte in its in-situ form, or the results are distorted by interfering species. Traditionally, the term sample preparation refers to the concentration of an analyte, the exchange of solvent, or the removal of interfering substances before analysis. In biochemical analysis, sample preparation is typically highly labor intensive and time consuming with multiple steps required such as to collect DNA, RNA or proteins from raw samples (such as whole blood, saliva, urine, sweat, spinal fluid, and stool, etc. ).

In general, sample preparation can be separated into two major steps: 1) cell or tissue lysis - to break open cells without denaturing or degrading sensitive macromolecules inside, such as DNAs or proteins, and 2) extraction or separation - to extract analyte(s) of interest from lysed cells. In a microfluidic system, cell lysis can be categorized into four major groups.

a. Mechanical lysis - to use cellular contact forces to crush or burst the cells.

b. Thermal lysis - to use high temperature to disrupt the cell membranes. c. Chemical lysis - to use a chemical buffer or enzyme to break down the cell membranes.

d. Electrical lysis - to induce cell membrane porosity with a low-strength electric field, or to break cells with a stronger field.

Without being bound to theory, with current invention, cell lysis can be done easily thermally, chemically, or electrically on a DMF device from this invention. Using dielectrophoresis, extraction or separation can be achieved on the device too. In other words, this invention makes a DMF device a truly integrated one - capable of sample preparation, measurement, and analysis.

Figure 8 shows an example of extracting DNA sample from a whole blood and analyzing it using a DMF device in this invention. S801, a DMF device is loaded with the patient whole blood sample and the reagent (including DNA primers, DNA polymerases, dNTPs, etc.) for running realtime PCR. S802, based on electrowetting effect by applying voltages to the corresponding electrodes in the device, dispense one or more sample droplets and move it (them) to a region (regions) on device for thermal treatment. S803, break the cells by raising the temperature of sample droplet(s) to around 100 degrees Celsius for a brief period of time (for example, 30 seconds). S804, move the sample droplet to a location where it overlaps with the first narrow electrodes by applying voltages to the corresponding electrodes; isolate the DNA(s) of interest by applying voltages to the first narrow electrodes and the electrode(s) in the upper substrate. S805, split the sample droplet into two daughter droplets, with the DNA(s) of interest in one of them, using electrowetting effect by applying voltages to the corresponding electrodes. S806, based on electrowetting effect by applying voltages to the corresponding electrodes in the device, dispense one or more reagent droplet(s), transport and merge it (them) with the DNA droplet(s). S807, carry out real-time PCR measurement(s) on the combined droplet(s). S808, move the measured droplet(s) to a waste storage location on the device.

Figure 8 shows only one of the many possible applications for carrying out biochemical analyses by simply loading a DMF device of this invention with raw material and the corresponding reagents. The DMF device provides all kinds of functions such as extracting analytes from the raw material, carrying out detection on the analytes, and running analyses, etc. Examples include, but not limited to, blood chemistry measurements, such as blood gases, glucose, electrolytes, urea, etc., in whole blood; the measurement of Trichomonas vaginalis in urine for bladder cancer diagnostics; the measurement of sweat electrolytes in sweat for cystic fibrosis diagnostics; and the measurement of interleukin 1-beta (IL-Ιβ) and interleukin 8 (IL-8), etc., in saliva, to detect oral squamous cell carcinoma; etc.

Many types of cells or macromolecules have demonstrated different physiology as a function of its environment, such as conductivity, viscosity, tonicity, pH, etc. As a sensitive technique, dielectrophoresis provides a means to detect subtle changes in cell physiology. For example, the dielectrophoretic cross-over frequency method has been applied to the detection of human red blood cells' responses to toxicants such as paraquat, styrene oxide, N-nitroso-N-methylurea and puromycin (Biochim Biophy Acta, Vol 1564, P 449, 2002).

Figure 9 is an example of studying cell physiology by measuring its dielectric behavior over a certain frequency range using a DMF device of this invention. S901, a DMF device is loaded with liquid samples which contain cells of interest. The liquid samples can be different solutions containing cells of the same type or different types of cells. S902, using electrowetting effect by applying voltages to the corresponding electrodes on the device, dispense sample droplets and move them to locations where they overlap with the first narrow electrodes or the second narrow electrodes on DMF device. S903, perform dielectrophoresis on the droplets by applying voltages to the narrow electrodes and the electrode(s) in the upper substrate, and measure the speeds and directions of the cells using optical (or electrical) detection. S904, move the measured droplet(s) to a waste storage location on the device. S905, using the data collected in S903, analyze the dielectric characteristics of the cells as a function of the electrical field frequency. S906, provide information regarding the cells' physiology based on the data analyses.

As it can be seen, this invention offers a methodology useful for clinical studies, point-of-care testing, and many other fields. This includes the development of automated sample preparation (such as cell separation, cell lysis, molecular extraction and purification, concentration, mixing with reagents, and maybe amplification, etc.), measurement, and analysis. Several advantages associated with this invention can be easily seen from the above mentioned examples. In addition to the advantages inherited from DMF in general, the present invention adds more advantages including the following.

a. The droplet shape can be controlled utilizing the electrowetting effect and overlapping arrangement of the two electrode layers in the bottom plate, as opposed to relying on the indentation or inner surfaces formed by the top and bottom plates. This reduced the device manufacturing complexity effectively.

b. A device in this invention includes both the narrow electrodes for generating the dielectrophoretic effect and the subset electrodes that work with said narrow electrodes to generate electrowetting effect. This makes it convenient for the users.

c. These devices are more integrated as sample preparation can be perform in the device, as opposed to carried out using other methods before the sample is loaded to the device. So, measurements can be done on raw materials directly.

d. Detection sensitivity is enhanced as particles suspended in liquid can be redistributed or concentrated.

e. As different particles can be separated by controlling the electrodes in the device, magnetic beads and external magnetic devices are no longer needed for particle manipulations for the detection of particles interested. This simplifies the processes and lowers the costs of using the devices.

f. The capacity of redistributing or separating particles increases the flexibility and multiplicity of the detections.

g. Many biochemical analysis steps can be integrated and automated in the device, such as sampling, sample preparation, transport, mixing, dilution, concentration, separation, incubation, reaction, detection, waste storage, etc.

h. Multiple analytes can be measured concurrently.

i. Many different types of analyses can be performed simultaneously.

j. The mixing of a sample and a reagent can be expedited using electrophoresis or dielectrophoresis on the device.

k. Calibration and sample measurement can be performed simultaneously. Calibration droplets can be generated and measured simultaneously along with the sample droplets. Calibration does not require cessation of sample measurement.

It should be mentioned that temperature of the DMF device (a portion or the whole device) can be controlled to reduce the side effect of Joule heating.

Although not discussed in detail, when utilizing dielectrophoretic effects, it should also be mentioned that amplitudes of the voltages applied to the electrodes can also be adjusted (besides frequency), which is often done in practice, for the effectiveness and efficiency of particle manipulations.

From the discussions above, it can be seen that this invention presents a true point-of-care testing microfluidic device and operation method. With this invention, cell lysis and target isolation/separation can be part of the device functions. A DMF device in this invention possesses a rather complete set of functions including sample preparation, measurement, analysis, and diagnostics, etc. Combining with the internet and cloud computing, this invention provides a good foundation of building a healthcare system including patient diagnostics, on-line medical consulting, remote patient-doctor interactions, etc.

It should be mentioned that the above described examples and the above mentioned advantages are by no means exhaustive. The flexible nature of this invention can be utilized for many applications and does have a lot of advantages comparing other technologies such as single layered EWOD or channel-based microfluidics.

All printed patents and publications referred to in this application are hereby incorporated herein in their entirely.

While the preferred embodiments of the invention have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

The above described embodiments are only used to illustrate the principles and their effects of this invention, and are not used to limit the scope of the invention. For people who are familiar with this technical field, various modifications and changes can be made without violating the spirit and scope of the invention. So, all modifications and changes without departing from the spirit and technical guidelines by anyone with common knowledge in this technical field are still covered by the current invention.