FOR HIGH CONDUCTANCE AND HIGH VOLTAGE DIELECTROPHORESIS (DEP)

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This work was supported by NCI Cancer Center Grant CCTP4MH awarded by the National Institutes of Health (NIH). The Government of the United States of America may have certain rights in this invention.

TECHNICAL FIELD

[0002] The present application relates generally to dielectrophoresis methods and devices, and more particularly, to pore-based high voltage electrokinetic devices for performing

dielectrophoresis.

BACKGROUND

[0003] Much of biomolecular research and clinical diagnostic efforts involve the separation and identification of a variety of rare cells, bacteria, virus, and biomarkers (e.g., exosomes, DNA, RNA, antibodies, other proteins, etc.) in complex samples such as blood, plasma, serum, saliva, and urine. A variety of dielectrophoresis (DEP) devices are used for separating out cells from blood or other high-conductance samples, or separating nanoparticulates from other size particles in either blood or other high-conductance samples and buffers. DEP devices can provide good separation output from samples, but such separation techniques usually require dilution of the samples to low-conductance conditions. Additional sample preparation operations may be required to obtain reliable results from operation of such DEP devices. The requirements for sample preparation can delay the results, by prolonging the time from presentation of a sample to output of results, as well as significantly increase the cost of the assay procedure. Improvements such as more rapid sample testing, analysis, and diagnostics could be achieved with reduced time for DEP sample preparation. Such improvements would be beneficial for biomolecular research and clinical diagnostic efforts.

SUMMARY

[0004] Disclosed herein are novel concepts, designs, and features for high-voltage DEP devices that provide single and multiple "pore based" AC/DC electrokinetic devices and improved features and methods for carrying out dielectrophoresis (DEP) under high conductance conditions. These new electrokinetic devices and methods allow rapid isolation, separation, concentration, detection and analysis of cells, bacteria, virus, cell free circulating (cfc) DNA, cfc- RNA, and other cellular nanoparticulates (exosomes, mitochondria, vacuoles, cell membrane fragments etc.), and also allow drug delivery and delivery of other nanoparticles to be carried out under high conductance conditions directly from blood, plasma, serum, urine, and other clinical, biological, and environmental samples. That is, a sample comprising biological materials such as blood, plasma, serum, urine, and other clinical, biological, and environmental samples can remain substantially unaltered from an in vivo condition, such as the condition in which the sample exists when it is collected for analysis, and the constituent materials of which the sample is comprised can be separated.

[0005] The devices, systems, methods and techniques described herein can substantially eliminate or greatly reduce sample preparation time and allow for "seamless sample-to-answer" analysis and/or diagnostics to be rapidly carried out. This can include, but is not limited to, both "pre" and "post" DEP separation analysis by general and/or specific fluorescent stains (DNA, RNA, nuclei, membranes, cellular organelles, exosomes (cellular nanoparticulates), proteins, enzymes and antibodies; analysis of cells, bacteria, virus, nuclei, high molecular weight (hmw) or cell free circulating (cfc) DNA, cfc-RNA and low molecular weight (lmw) DNA by fluorescent stains and/or fluorescent probe hybridization (FISH, etc.); post analysis of cells, bacteria, virus, nuclei, DNA and RNA by polymerase chain reaction (PCR), DNA/RNA sequencing and other genotyping techniques; and pre and post analysis of cells, bacteria, virus, antibodies, enzymes and proteins by well know to the art immunoassay techniques and as well as other protein and enzyme detection methods and techniques. In a properly configured device constructed in accordance with this disclosure, all of the separation, concentration and analysis operations can be carried out in the same chambered compartment (in-situ) in which the DEP separation occurs. Alternatively, the highly concentrated and differentially separated analytes and materials (DNA, RNA cellular nanoparticulates, etc) collected in the device can be transferred to a separate container (PCR tube, sample tube, etc.) for subsequent analysis outside of the chambered compartment. The above examples do not exclude other types of detection techniques from being used with the device, techniques that might include radio-isotopes, colorometric, chemiluminescence, electrochemical, or other methods of biosensing for DNA, RNA, proteins, enzymes, antibodies, biomolecules, cells, bacteria and virus. Finally, the devices of this invention can be scaled in size from micro-devices, which would process very small sample volumes (a few microliters), to very large flow through macro-devices, which could handle large sample (100's of milliliters). It should be noted that while the DEP devices and methods described herein may be used with high voltages for DEP separation under high conductance conditions, such DEP devices and methods in accordance with various other embodiments, may be utilized at lower voltages and/or under lower conductance conditions.

[0006] Some of the background for the techniques described herein may be better understood with reference to the description in PCT International Publication WO 2009/146143 entitled "Ex-Vivo Multi-Dimensional System for the Separation and Isolation of Cells, Vesicles, Nanoparticles and Biomarkers" (UCSD filing 2007-205-1).

[0007] Other related documents for background information on DEP techniques generally include: (1) Krishnan R, Dehlinger DA, Gemmen GJ, Mifflin RL, Esener S and Heller MJ, "Interaction of nanoparticles at the DEP microelectrode interface under high conductance conditions", Electrochemical Communications, Vol. 11, No. 8, 1661-1666, 2009; (2) Krishnan R and Heller MJ, "An AC electrokinetic method for the enhanced detection of DNA

nanoparticles", J. Biophotonics, Vol. 2, No. 4, pp. 253-261, 2009; and (3) Krishnan R, Sullivan BD, Mifflin RL, Esener SC, and Heller MJ, "Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions," Electrophoresis, Vol. 29, No. 9, pp. 1765-1774, May 2008.

[0008] The techniques disclosed herein provide improvements to resolve the issues for biomolecular research and clinical diagnostic efforts described above. Other types of DEP devices that have been used to separate out cells from either blood or other high conductance samples, or nanoparticulates from other size particles in either blood or other high conductance buffers, require that the samples be diluted to low-conductance conditions. These conventional DEP devices do not have the performance characteristics, robustness, and usefulness to carry out the separation applications in the manner described herein. Such conventional devices cannot provide for "seamless sample to answer" or point of care diagnostics as described herein, wherein the sample can remain substantially unaltered from an in vivo condition comprising the condition in which the sample exists when it is collected for analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] For a more complete understanding of example embodiments, reference is now made to the following descriptions taken in connection with the accompanying drawings in which:

[0010] Figure 1 illustrates a first step in sample-to-answer diagnostics wherein a blood sample is provided in accordance with various embodiments of the present invention;

[0011] Figure 2 first step in sample-to-answer diagnostics wherein a DEP field is applied to the blood sample of Figure 1 in accordance with various embodiments of the present invention;

[0012] Figure 3 illustrates a third step in sample-to-answer diagnostics wherein a fluidic wash is applied to the blood sample of Figure 2 in accordance with various embodiments of the present invention;

[0013] Figure 4 illustrates a fourth step in sample-to-answer diagnostics wherein a fluorescent stain and wash are added to the blood sample of Figure 1 and particulates material in accordance with various embodiments of the present invention;

[0014] Figure 5 illustrates a fifth step in sample-to-answer diagnostics wherein fluorescent DNA/RNA is detected and quantitated in accordance with various embodiments of the present invention;

[0015] Figure 6 illustrates a sixth step in sample-to-answer diagnostics wherein the blood sample of Figure 1 is separated from various elements in accordance with various embodiments of the present invention; [0016] Figure 7 illustrates a seventh step in sample-to-answer diagnostics wherein in- situ post fluorescent staining, fluorescent immunoassay, FISH, and PCR procedures may be used in accordance with various embodiments of the present invention to carry out

cell/bacteria/virus/CNP/antibody complex analysis; [0017] Figure 8 illustrates an eighth step in sample-to-answer diagnostics wherein final detection of cells, bacteria, virus, CNPs, and antibody complexes is carried out in accordance with various embodiments of the present invention;

[0018] Figures 9 A and 9B show side and top views, respectively, of an exemplary multi- chambered multi-pore electrokinetic device;

[0019] Figure 10 illustrates an exemplary single-pore, two-chamber high voltage

dielectrophoresis device configured in accordance with one embodiment of the present invention;

[0020] Figure 11 illustrates experimental results from utilizing the high voltage

dielectrophoresis device of Figure 10;

[0021] Figure 12 illustrates an exemplary pipette tip high voltage dielectrophoresis device configured in accordance with another embodiment of the present invention;

[0022] Figure 13 illustrates experimental results from utilizing the pipette tip high voltage dielectrophoresis device of Figure 12; and [0023] Figure 14 illustrates top and perspective views of differing exemplary electrode and pore geometries and configurations in accordance with various embodiments of the present invention.

DETAILED DESCRIPTION

[0024] The separation, concentration and detection of high molecular weight DNA (hmw- DNA), cell free circulating (cfc-DNA), lower molecular weight (lmw) DNA, and nanoparticles from whole blood using the AC/DC electrokinetic dielectrophoretic (DEP) devices and methods described herein have been demonstrated. Both fluorescent nanoparticles and fluorescent- stained hmw DNA in undiluted whole blood samples were separated, highly concentrated and held in DEP high field regions and then detected after the blood cells were removed by a fiuidic wash. In buffy coat blood, with reduced cell numbers, nanoparticles concentrated into the DEP high field regions while the blood cells concentrated into the DEP low field regions. A fiuidic wash then selectively removed the cells while the nanoparticles remained trapped. More importantly, the inventors have now also demonstrated that unlabeled hmw DNA can also be isolated into the high field regions, and then stained with a fluorescent dye for subsequent detection, demonstrating an intrinsic DEP advantage of separating unlabeled analytes. Overall, in accordance with the description herein, DEP techniques can now be developed as a

"seamless" sample-to-answer tool that can be used with complex biological samples (blood, plasma, etc.) for a variety of research and diagnostic applications.

[0025] Details of the methods and devices of the inventors are provided in the following paragraphs and illustrations. The methods and devices described herein can provide more rapid output from DEP techniques that are characterized as simplified sample-to-answer diagnostics.

[0026] An illustration of the concepts for the DEP separation of DNA/Nanoparticles from blood and other high conductance samples in accordance with this disclosure is provided in Figures 1 through 14 below.

[0027] Figure 1 shows a first step in sample-to-answer diagnostics where DEP techniques are used to carry out separation of high molecular weight DNA in whole blood. In Figure 1 , a blood sample with cell free-circulating hmw DNA/RNA is applied (i.e., is provided) to the DEP device 100. The DEP electrodes 110 are represented by the circular "plate" areas on the substrate surface 120 below the illustrated blood sample particulates.

[0028] Figure 2 shows a second step in sample-to-answer diagnostics where DEP is used to carry out separation of high molecular weight DNA in whole blood, wherein the DEP field is applied to the blood sample 130. [0029] Figure 3 shows a third step in sample-to-answer diagnostics where DEP is used to carry out separation of high molecular weight DNA in whole blood, in which a fluidic wash 140 is applied to the blood sample to remove cells.

[0030] Figure 4 shows the fourth step in sample-to-answer diagnostics where DEP is used to carry out separation of high molecular weight DNA in whole blood, in which fluorescent DNA/RNA stain and wash 150 are added to the blood sample and particulates material.

[0031] Figure 5 shows a fifth step in sample-to-answer diagnostics where DEP is used to carry out separation of high molecular weight DNA in whole blood, wherein the device will detect and quantitate the fluorescent DNA/RNA. [0032] Figure 6 shows the next operation where DEP is used to carry out separation of bacteria, virus, cellular nanoparticulates or CNP's (which can include cellular membrane, nuclei, vacuoles, endoplasmic reticulum, mitochondria, etc.), antibody complexes and other biomarkers from whole blood. [0033] Figure 7 shows the post fluorescent staining, fluorescent immunoassay, FISH, and PCR procedures, which can be used in-situ (in the same compartment of the device) to carry out analysis of cells, bacteria, virus, CNPs and antibody complexes.

[0034] Figure 8 shows the final detection of cells, bacteria, virus, CNPs and antibody complexes by the device described herein. [0035] Figures 9A and 9B show a basic design for a three chambered multi-pore electrokinetic DEP device 900 in which the electrodes 910 are placed into separate chambers 920 and positive DEP regions and negative DEP regions are created within an inner sample chamber 930 by passage of the AC DEP field through pore or hole structures 940. These devices are described in the PCT International Publication WO 2009/146143 entitled "Ex-Vivo Multi-Dimensional System for the Separation and Isolation of Cells, Vesicles, Nano-particles and Biomarkers" (UCSD filing 2007-205-1).

[0036] Figure 10 shows a two-chambered and single-pore electrokinetic DEP device which can be constructed in accordance with this disclosure, and used for the rapid separation of cells, bacteria, virus, exosomes, cfc-DNA /RNA, nanoparticles, antibodies, proteins from blood and other high conductance biological samples.

[0037] Figure 11 shows actual experimental results for using a two-chamber single-pore device constructed in accordance with this disclosure for separating 10 micron beads and 40 nm red fluorescent nanoparticles.

[0038] Figure 12 shows a DEP pipette tip device, which represents another type of AC/DC device which can be constructed in accordance with this disclosure. In these devices, the pore or hole structure is provided by a pipette tip or a capillary tube.

[0039] Figure 13 shows PCR results from cfc-DNA extracted from CLL cancer patient blood samples without sample preparation such as required by conventional techniques. A pipette tip device such as illustrated in Figure 12 was used to achieve the results. [0040] Figure 14 shows various electrode, hole/pore and pipette/capillary tip configurations and geometries (other than circular) contemplated in accordance with this disclosure which can produce better DEP high field regions for improved concentration of analytes. For example, Figure 14 illustrates a capillary tip device 1400, where an elliptical pore 1410 is formed at the tip of the capillary tip device, around which is an electrode 1420. Figure 14 also illustrates a variety of different electrode and pore geometries/configurations (1430, 1432, 1434, 1436, 1438, and 1440), any of which are contemplated in accordance with this disclosure. These geometries include, but are not limited to, elliptical, clover leaf, square, rectangular, triangular, etc.

geometries. Such structures may also be three dimensional. [0041] In general, for the devices of this invention and the previous invention a large variety design geometries can be used to form desired positive DEP (high field) regions and the negative DEP (low field) regions for separating, concentrating and trapping the desired cells,

nanoparticles, and biomarkers within specific regions of the sample chamber. The earlier chambered pore/hole devices are shown in Figures 9 A and 9B, and the new devices of this invention are shown in Figure 10 and Figure 12. Device pore/hole structures can have diameters from 100 nanometers to 10 millimeters, more preferably from about 1 micron to 5 millimeters, and most preferably from 10 microns to 1 millimeter. The pore structure platform or substrate material can be fabricated from glass, silicon, ceramic materials (alumina, etc.), plastic, rubber, PDMS or combinations of these and other materials. Materials such as glass, silicon and ceramics are sometimes preferable because of their high heat conductivity properties that allow removal of Joule heating and associated bubbling, which can sometime occur around the pore/hole structure under high voltage and/or high conductance conditions. The pore/hole structures may be covered with a membrane, or filled with sieving gels, hydrogels or filtering materials which can control, confine or prevent cells, nanoparticles or other entities from diffusing or being transported into the inner chambers. However, the AC/DC electric fields, solute molecules, buffer and other small molecules can pass through the chambers. Pore/hole structures can be filled with a porous gel sieving materials such as but not limited to agarose, polyacrylamide, or other hydrogel materials or synthetic micro/nano/molecular size sieving materials. Incorporation of sieving gel or hydrogel materials of defined pore size can provide better separation of the desired analyte within the gel material itself. For example, the separation of higher molecular weight cfc-DNA from lower molecular weight apoptotic DNA can be achieved within the agarose or polyacrylamide gel. Pore/hole structures may also be covered with a porous membrane or filter material including but not limited to paper, cellulose or nylon membranes. Pore over-layers or fillings can have thicknesses from about 1 micron to about 20 millimeters, or more preferably form about 10 microns to 5 millimeters, or most preferably from 20 microns to 1 millimeter. [0042] By segregating the electrode structures (platinum, palladium, gold, carbon, etc.) into separate chambers, these unique DEP devices as described herein substantially eliminate the electrode-associated electrochemistry effects including, bubbling, heating and chaotic fluidic movement from influencing the analyte separations that are occurring in the inner/sample chamber during the higher voltage AC DEP and DC electrophoretic processes. The inner/sample chamber and outer/lower chamber electrodes can be designed in a variety of geometries including circular (as shown in Figure 10) and parallel tracks around the hole/pore structure. It is further contemplated within this disclosure to use other non-circular configurations and geometries for the inner sample chamber electrode(s) and the hole/pore structure(s) which can produce better DEP high field regions for improved concentration of analytes. Some non- limiting examples of these non-circular structures are shown in Figure 14. Additionally, it should be noted that the arrangement of the non-circular inner sample chamber electrode structure relative to the non-circular hole/pore structure can also be used to produce more optimal DEP high field regions for concentrating analytes.

[0043] In addition to the AC voltage level, the geometry of the electrode(s) and their distance to the pore structure can determine the strength of the DEP high field region at the pore/hole; as well as the strength and geometry (shape) of the DEP low field trapping regions. The inner/sample chamber electrode structure(s) can be placed from about 10 microns to 10 centimeters form the pore structure(s), more preferably from about 100 microns to 10 millimeters from the pore structure(s), or most preferably from about 500 microns to 2 millimeters from the pore structure. The outer/lower chamber electrode structure(s) can be placed from about 10 microns to 10 centimeters form the underside of the pore structure(s), more preferably from about 100 microns to 10 millimeters from the pore structure(s), or most preferably from about 500 microns to 2 millimeters from the pore structure. The disclosed chambered pore/hole devices can be operated at both very low as well as very high AC voltages (1 volt to 10,000 volts pk-pk), but most preferably in a range from about 10 volts to 500 volts pk-pk. The disclosed devices can be operated at AC frequencies that range from 1 kHz to 100 MHz. According to well know classical DEP work, high resolution separation of cells, bacteria, virus and nanoparticles can be achieved when DEP is carried out at specific AC frequencies in the above frequency ranges.

[0044] One of the major advantages of the disclosed devices is that they can be operated under very low conductance conditions as well as very high conductance conditions (1 μΞ/ι to 10 S/m). The ability of the devices to be operated under the high conductance conditions means that, if desired, they can be used directly with un-diluted high conductance (0.5 S/m to 1.5 S/m) clinical, biological and buffered samples, some of which include, but are not limited to, blood, buffy coat blood, serum, plasma, urine and saliva. In addition to AC DEP processes, the disclosed devices can also be used to carry out DC electrophoretic transport in the sample chamber and electrophoretic separations within the pore gel sieving materials. The devices can be operated at DC voltages which range from 1 volt to 2000 volts. Combinations of AC dielectrophoretic and DC electrophoretic field application (simultaneous, parallel, series, pulsing, positive or negative DC off-sets) provides major advantages for separating, concentrating and trapping desired analytes (bacteria, virus, exosomes, cfc-DNA, cfc-R A, nanoparticles, etc.) during fluidic movement or washing; for overall higher efficiency separations of the desired analytes; and for better resolution, concentration and trapping of the desired analytes when in very complex high conductance biological samples, i.e., blood, plasma, serum, etc. Overall, the disclosed devices and systems can be operated in the AC frequency range of from 1000 Hz to 100 MHz, at AC voltages from 1 volt to 5000 volts pk-pk, at DC voltages from 1 volt to 2000 volts, at fluidic flow rates of from 1 microliters per minute to 10 milliliter per minute and in temperature ranges from 1° C to 100° C. While both AC and DC can be run through the same set of electrodes, it is also within the scope of the this invention to incorporate separate sets of electrodes in the device which allow AC DEP and DC electrophoretic processes to be run in parallel with different AC and DC field geometries. [0045] Figure 10 shows a relatively simple high- voltage single-pore two-chamber DEP device 1000 connected to an AC/DC power supply 1005 that can be constructed from very simple plastic, glass, silicon, ceramic, paper or nylon membrane, sponge, porous gel (agarose or polyacrylamide), and other materials. Figure 10 shows the device 1000 being used for the DEP separation of nanoparticles (cfc-DNA) from micron-size particles (cells) 1010, where the smaller, centrally- located nanoparticles indicated by the arrow 1020 are concentrated around the high field region, which occurs at the edge of the pore structure 1030 of a porous material 1035 (e.g., a small hole in a glass or plastic base 1040 atop a membrane 1045); and the micron-size larger particles 1010 are shown farther from the central pore 1030, in the low field regions radiating out to one or more platinum ring electrodes 1050 (in an inner sample chamber 1060). The DEP high- field region occurs around the edges of the pore structure 1030 because this is where the DEP field between an outer buffer chamber 1070 and the inner sample chamber 1060 is most constricted.

[0046] Figure 10 represents just one basic version of these new devices of which a variety of forms are envisioned by this invention. These include but are not limited to multiplexed electrode and chambered devices; scaled micro-devices to macro-devices to handle sample volumes from a few microliters to hundreds of milliliters; all ranges of sample preparation devices that allow reconfigurable AC and DC electric field patterns to be created; devices that combine DC electrophoretic and fluidic processes; sample preparation devices; exosomes/cfc- DNA/cfc-R A from apoptotic/lmw-DNA separation devices; sample preparation/diagnostic devices that include high sensitivity detection and analysis components; sample preparation to DNA/RNA sequencing systems; pathogen isolation to genotyping/sequencing systems; lab-on- chip devices; point of care (POC) systems; seamless sample to answer systems with in-situ detection and analysis; and other clinical diagnostic components, devices, systems or versions.

[0047] It is also in the scope of this invention to: (1) incorporate more than one pore/hole structure, and/or other types of structures including but not limited to slits or lane structures; (2) design pore or hole structures/geometries and inner chamber electrode structures/geometries so that the improved DEP high field (analyte concentration) regions occur at certain regions of the pore structure (some of which are illustrated in, e.g., Figure 14); (3) fill pore structures with agarose, polyacrylamide or other sieving materials or hydrogels and combinations which allow higher resolution separation of the desired analytes to occur within the gel sieving material itself; (4) use a DC (electrophoretic) duty cycles along with AC DEP to better collect

(concentrate) analytes in a preferred region of the pore or inner/sample chamber structure; (5) use appropriate levels of positive DC bias (voltage) to further resolve or separate desired analytes (hmw/cfc-DNA, cfc-RNA) within gel sieving material; (6) use negative DC bias (voltage ) to electrophorese (expel) the purified analyte (hmw/cfc-DNA, cfc-RNA) back into the sample chamber; (7) use combinations of positive and negative DC bias to carry out cell, bacteria, virus or organelle (nuclei, mitochondria) lysis by concentrating denaturants (SDS, etc.) onto the collected cells, lysing the cells and then re-concentrating the DNA or RNA while other materials are washed away; (8) separate the sample chamber electrode into a third chamber connected via a conductance channel (e.g., membrane, gel, etc.) back to the sample chamber; (9) incorporate input and output flow channels for both introducing reagents and sample materials, and then extracting and/or collecting the analytes (e.g., cells, bacteria, virus, exosomes, cfc-DNA, cfc- RNA, nanoparticles, antibodies, etc.); (10) provide linear flow devices with parallel electrode structures and pores which move blood cells to low field regions while virus, exosomes, cfc- DNA, cfc-R A and nanoparticles are trapped or collected in the high field pore structures; (11) use appropriate AC frequencies (well known in the art of DEP) which allow separation of bacteria from blood, via bacteria in high field region and blood cells into low field regions; (12) use appropriate AC frequencies (well known in the art of DEP) which allow separation of various cell types (blood cells, cancer cells, stem cells) to be carried out by trapping/collecting in DEP low field regions; (13) use high voltage AC frequency generators and DC power supplies and computer control systems and programs to regulate the application of AC and DC voltages to the devices; (14) provide associated controlled fluidic pumping for sample/reagent delivery; for in-situ staining; for in-situ PCR; for in-situ immunoassay; for in-situ isolation, preparation, fragmentation and labeling of DNA for sequencing; and for final analyte collection; (15) provide associated optical, fluorescent, electronic components for process monitoring and for in-situ detection and analysis (including PCR, staining, or immunoassay) of analytes in the device chamber. Overall, it is in the scope of this invention to provide all associated components and other devices, which will allow the devices of the invention to be used for applications that range from sample preparation to complete seamless sample-to-answer diagnostics.

[0048] Figure 11 shows actual experimental results for using a two-chamber single-pore device constructed in accordance with this disclosure. The DEP separation of 10 micron beads and 40 nm red fluorescent nanoparticles in lxTBE buffer on a two-chamber single-pore (-800 μιη diameter) device at 10 kHz and high AC voltage of 160 volts pk-pk. The left side frame of

Figure 11 shows 10 micron beads moving to low field regions, and the right side frame of Figure 11 shows red fluorescent nanobeads concentrating in the high field regions at the edge of the pore (the dashed circle in the center of each illustration frame).

[0049] Figure 12 shows a DEP pipette tip device 1200 configured for use with a pipette (e.g., a plastic pipette 1205, which represents another general type of high voltage AC/DC device

(powered with, e.g., an AC/DC power supply 1210) that can be constructed in accordance with this disclosure. The pipette tip component 1220 has a pore/hole structure 1230, a hydrogel filling 1240 in a lower section 1245, a buffer reservoir 1250 above the hydrogel 1240 and an electrode 1260. A lower sample chamber 1270 provides a reservoir for the sample and has, e.g., a platinum ring-an electrode 1275. Figure 12 shows the DEP separation of nanoparticles from blood cells (1280), where the smaller fluorescent 40nm nanoparticles 1290 (cfc-DNA) are concentrated around the DEP high field region, which generally occurs at the edge of the pipette tip hole structure 1230; and micron-size blood cells are concentrated in the low field region radiating out to the platinum ring electrode 1275 (in the sample chamber 1270). The DEP high field region occurs around the edges of the pipette tip hole structure 1230 because this is where the DEP field between the sample separation chamber 1270 and inner area (inside the pipette 1205) is most constricted.

[0050] In addition to pipette tip devices, closely related capillary tube devices can also be designed and constructed as disclosed in this invention. Both types of devices and lower sample chambers can be constructed from a variety of the commonly used plastic pipette tips, glass pipettes, Pasteur pipettes, plastic/class capillary/microcapillary tubes, plastic tubing along with glass/plastic slides, platinum or gold electrodes, agarose gel, polyacrylamide or other porous gel sieving materials, membranes, PDMS, plastic, rubber or glass construction materials.

Alternatively, plastic/glass pipette tip and plastic/glass capillary tube devices can be specially fabricated with precision holes; with specially designed tip structures to create unique DEP high field geometries and better analyte collection/trapping regions; and with printed, painted or sputtered electrodes in-side and/or out-side on the pipette or capillary tube structure. Separate lower/sample chambers can be easily constructed on glass slides, within common laboratory plastic or glass sample tubes, fabricated with PDMS technology or common laboratory microtiter plates can be used.

[0051] In general, the pipette tip or capillary device can have hole diameters, which range from 100 nanometers to 1 centimeter, more preferably from 50 microns to 5 millimeters, and most preferably from 100 microns to 1 millimeter. Again, as with the chambered pore/hole devices, by segregating the electrode structures (platinum, palladium, gold, carbon, etc.) into separate chambers, these unique DEP devices as described herein substantially eliminate the electrode associated electrochemistry effects including, bubbling, heating and chaotic fluidic movement from influencing the analyte separations that are occurring in the lower sample chamber during the high voltage AC DEP and DC electrophoretic processes. With regard to these devices, the electrode structure within the pipette tip or capillary tube can be as simple as a platinum, palladium or gold wire, and the electrode in the lower/sample chamber can be circular, as is shown in Figure 12. In addition to the AC voltage, the geometry of the lower chamber electrode, and the distance of both electrodes to the pore structure can determine the strength of the DEP high field region at the pore; as well as the strength and geometry (shape) of the low field trapping regions. Again, it is within the scope of this invention to use other non-circular configurations and geometries for the inner sample chamber electrode and the pipette/capillary tip structure which can produce better DEP high field regions for improved concentration of analytes. The geometry of these structures can also be three dimensional, and the arrangement of the non-circular inner sample chamber electrode structure relative to the pipette/capillary tip structure can also be used to produce more optimal DEP high field regions for concentrating analytes. Examples of such non-circular structures are shown in Figure 14.

[0052] With regard to the pipette tip or capillary tube component, depending on thickness of membrane covering over the pore or the amount of sieving gel in the pipette tip or capillary tube, the electrode structure can be placed from about 10 microns to 20 centimeters form the pore structure, more preferably from about 100 microns to 5 millimeters from the pore structure, or most preferably from about 500 microns to 2 millimeters from the pore structure. If separate AC and DC electrodes are used in the pipette tip or capillary tube, the AC electrode can be placed down through the sieving gel material much nearer to the pore, and the DC electrode placed further up above the gel filling in the buffer chamber. The lower/sample chamber electrode structure can be placed from about 10 microns to 10 centimeters from the pore structure, more preferably from about 100 microns to 10 millimeters from the pore structure, or most preferably from about 500 microns to 2 millimeters from the pore structure. The distance of the pipette tip or capillary tube hole to the bottom of the lower/sample chamber can influence the strength and the trapping efficiency of the high field region and the strength and trapping efficiency of the low field regions.

[0053] The disclosed pipette tip and capillary tube devices can be operated at both very low as well as very high AC voltages (1 volt to 10,000 volts pk-pk), but most preferably in a range from about 10 volts to 500 volts pk-pk. The disclosed devices can be operated at AC frequencies that range from 1 kHz to 100 MHz, and in temperature ranges from 1 °C to 100 °C. According to well know classical DEP work, high resolution separation of cells, bacteria, virus and

nanoparticles can be achieved when DEP is carried out at specific AC frequencies in the above frequency ranges. Scaled micro-devices to macro-devices can be designed to handle sample volumes from a few microliters to several milliliters.

[0054] As with the other pore/hole devices, one of the major advantages of the pipette tip/capillary tube devices is that they can be operated under very low conductance conditions as well as very high conductance conditions (1 μΞ/ι to 10 S/m). The ability of the devices to be operated under the high conductance conditions means that, if desired, they can be used directly with un-diluted high conductance (0.5 S/m to 1.5 S/m) clinical, biological and buffered samples, some of which include but are not limited to blood, serum, plasma, urine and saliva. In addition to AC DEP processes, the disclosed devices can also be used to carry out DC electrophoretic transport in the sample chamber and high resolution electrophoretic separations within the gel sieving materials inside the pipette tip or capillary tube structure. The devices can be operated at DC voltages which range from 1 volt to 2000 volts. Combinations of AC dielectrophoretic and DC electrophoretic field application (simultaneous, parallel, series, pulsing, positive or negative DC off-sets) provides major advantages for separating, concentrating and trapping desired analytes (bacteria, virus, exosomes, cfc-DNA, cfc-R A, nanoparticles, etc.) during fluidic movement or washing; for overall higher efficiency separations of the desired analytes; and for better resolution, concentration and trapping of the desired analytes when in very complex high conductance biological samples, i.e., blood, plasma, serum, etc. While both AC and DC can be run through the same set of electrodes, it is also within the scope of the this invention to incorporate separate sets of electrodes in the device which allow AC DEP and DC

electrophoretic processes to be run in parallel with different AC and DC field geometries. Figure 12 represents just one basic version of these new devices of which a variety of forms are envisioned, as numerous types and sizes of pipette tips and capillary tubes are readily available.

[0055] The pipette tip and capillary tube devices of this invention have two other important advantages. The first is that the pipette tip or capillary tube can be filled with a larger volume (length) of hydrogel sieving material (agarose, polyacrylamide, etc.) which allows a higher resolution separation of analytes to occur when using DC electrophoresis. By way of example, when a positive DC bias (voltage) is applied, lower molecular weight DNA and small proteins will move quickly from the pore/hole through the hydrogel and up into the buffer chamber. Larger macromolecules, high molecular weight DNA and intermediate size nanoparticles will move at a slower rate through the hydrogel. Very high molecular weight DNA, cfc-DNA and large nanoparticles will move very slowly and remain nearer the pore. Thus, different components in the sample which are initially concentrated around the pore via the AC DEP process, or are in very high concentration in the sample (proteins), can be resolved by DC electrophoresis within the hydrogel. After resolving the analytes within the hydrogel, application of a negative DC bias (voltage) allows the analytes to electrophoresed (expelled) in reverse order and collected in sample tubes for analysis. Just one of many important applications would be the separation from whole blood samples of very small amounts of disease related high molecular weight cfc-DNA and cfc-RNA from larger amounts of low molecular weight apoptotic DNA and blood proteins. Separation parameters and selection of appropriate agarose gel and

polyacrylamide gel concentrations are well known in the art of DC electrophoresis. The final advantage of pipette tip and capillary tube devices is that they can be combined into multiple tip arrangements for automated sample preparation, and used with conventional type microtiter plates (96, 384, 1536 wells) as sample wells. For such multiple tip automated systems, both electrodes can be incorporated into the tip device, or electrodes can be incorporate into modified microtiter plates .

[0056] Figure 13 shows PCR results from cfc-DNA extracted from CLL cancer patient blood samples without sample preparation such as required by conventional techniques. A pipette tip device such as illustrated in Figure 12 was used to isolate cfc-DNA from about 25 μΐ of CLL cancer patient whole blood samples, by applying 200 volts pk-pk at about 5-10 kHz AC for 5 minutes. The pipette tip was removed from the blood sample and the collected cfc-DNA was deposited into a PCR tube (with PCR buffer) and PCR was then carried out. The results in Figure 13 show the PCR detection of patient-specific VH-L immunoglobulin rearrangements, as indicated by the circled relatively bright bands across the middle of the image. With this configuration, blood-to-PCR can be carried out in about five minutes from sample presentation. [0057] The present invention has been described above in terms of presently preferred embodiments so that an understanding of the present invention can be conveyed. There are, however, many configurations and permutations of the devices, system, and separation mechanisms not specifically described herein but with which the present invention is applicable. The present invention should therefore not be seen as limited to the particular embodiments described herein, but rather, it should be understood that the present invention has wide applicability with respect to biological separation systems generally. All modifications, variations, or equivalent arrangements and implementations that are within the scope of the attached claims should therefore be considered within the scope of the invention.