WO2002044143A9

WO2002044143A9 - Microcapillary arrays for high-throughput screening and separation of differently charged molecules using an electric field

Info

Publication number: WO2002044143A9
Application number: PCT/US2001/044297
Authority: WO
Inventors: David Willoughby; Ashwin Seshia; Paul Swanson; John R Havens
Original assignee: Nanogen Inc
Priority date: 2000-11-28
Filing date: 2001-11-26
Publication date: 2003-11-06
Also published as: AU2002226981A1; WO2002044143A2; WO2002044143A3

Abstract

A system for electrokinetic separation of charged molecules in one or more samples in a microtiter plate comprising a plurality of wells, the apparatus comprising at least one separatory unit comprising: a first and second well in the microtiter plate; a capillary connecting the first and second wells; a detector positioned to detect meolecules passing through the capillary; first and second electrodes positioned in the first and second wells; and a power supply connected to the electrodes.

Description

MICROCAPILLARY ARRAYS FOR HIGH-THROUGHPUT SCREENING

AND SEPARATION OF DIFFERENTLY CHARGED MOLECULES

USING AN ELECTRIC FIELD

FIELD OF THE INVENTION

The field of the present invention relates generally to systems and methods for electro-kinetic analysis of samples containing a molecule of interest using small-scale capillary electrophoresis devices. More particularly the methods and systems disclosed herein may be used to electrophoretically separate molecules of interest in a sample which are differently charged, or which have different electrophoretic mobilities, in a high throughput context. For example, the systems and methods disclosed herein may be used to detect the enzymatic modification of peptide substrates by phosphatases, proteases and kinases.

BACKGROUND OF THE INVENTION

Protein kinases are of particular interest in drug discovery research because they have been shown to be key regulators of many cell functions, including signal transduction (Ullrich and Schlessinger, 1990), transcriptional regulation (Pawson and Bernstein, 1990), cell motility (Miglietta and Nelson, 1988) and cell division (Pines and Hunter, 1990). Protein kinases are enzymes which covalently modify proteins and peptides by the attachment of a phosphate group to one or more sites on the protein. Phosphatases perform the opposite function. Many of the known protein kinases use adenosine triphosphate (ATP) as the phosphate donor, placing the γ-phosphate onto a histidine, tyrosine, serine or threonine residue in the protein. The location of the modification site and the type of residue modified by the kinase are usually specific for each particular kinase.

The added phosphate alters certain structural, thermodynamic and kinetic properties of the phosphorylated protein. Generally, the phosphate adds two negative charges to the protein. This modifies the electrostatic interactions between the protein's constituent amino acids, in turn altering secondary and tertiary protein structure. The phosphate may also form up to three hydrogen bonds or salt bridges with other protein residues, or may otherwise change the conformational equilibrium between different functional states of the protein. These structural changes provide the basis, in a biological system, for altering substrate binding and catalytic activity of the phosphorylated proteins. Phosphorylation and dephosphorylation reactions, under the control of kinases and phosphatases, respectively, can occur rapidly to form stable structures. This makes the phosphorylation system ideal as a regulatory process. Phosphorylation and dephosphorylation reactions may also be part of a cascade of reactions that can amplify a signal that has an extracellular origin, such as hormones and growth factors. Methods for assaying the activity of protein kinases often utilize a synthetic peptide substrate that can be phosphorylated by the kinase protein under study. The most common mechanisms for detecting phosphorylation of the peptide substrates are 1)

Incorporation of P or P phosphate from [ P]γ-ATP into the peptides, purification of the peptides from ATP, and scintillation or Cherenkov counting of the incorporated radionucleotide, 2) Detection of phosphoamino acids with radiolabeled specific antibodies, or 3) Purification of phosphorylated peptides from unphosphorylated peptides by chromatographic or electrophoretic methods, followed by quantification of the purified product.

For example, in one widely used method, a sample containing the kinase of interest is incubated with activators and a substrate in the presence of gamma P-ATP, with an inexpensive substrate, such as histone or casein being used. After a suitable incubation period, the reaction is stopped and an aliquot of the reaction mixture is placed directly onto a filter that binds the substrate. The filter is then washed several times to remove excess radioactivity, and the amount of radiolabeled phosphate incoφorated into the substrate is measured by scintillation counting (Roskoski, 1983).

The use of ³²P in assays, however, poses significant disadvantages. One major problem is that, for sensitive detection, relatively high quantities of P must be used routinely and subsequently disposed. The amount of liquid generated from the washings is not small, and contains P. Due to government restrictions, this waste cannot be disposed of easily. It must be allowed to decay, usually for at least six months, before disposal. Another disadvantage is the hazard posed to personnel working with the isotope. Shielding and special waste containers are inconvenient but necessary for safe handling of the isotope. Further, the lower detection limit of the assay is determined by the level of background phosphorylation and is therefore variable. Although radioisotope methods have been applied in high throughput screening, the high cost and strict safety regulation incurred with the use of radioisotopes in high throughput screening greatly limits their use in drug discovery. For these and other reasons, it would be useful to develop alternative methods and apparatus for high throughput screening that facilitate measuring the kinase dependent phosphorylation of peptides. Further, assays for phosphatases, kinases, and proteases are generally conducted in

384 or 1536-well microtiter plates. These assays make electro kinetic separation more difficult due to the degree of manual manipulation that is required to transfer reaction products from each well to the loading wells of the separation system. Although microtiter well compatible loading and electrophoretic separation systems have been created for use with DNA analysis and sequencing techniques (for example, the system of U.S. Patent No. 5,916,428), these systems are large (owing to their very long electrophoretic separation paths), complex (due to extensive sample loading procedures and specialized detection apparatus), and fairly expensive. For use in a high-throughput screen of a combinatorial library of compounds, a system is needed which is more suitable for rapid, repeated analysis without the need to transfer samples from the microtiter plate format.

SUMMARY OF THE INVENTION

Thus, in one aspect, the present invention provides a system for electro-kinetic separation of molecules of interest in one or more samples in a microtiter plate comprising a plurality of wells, the apparatus comprising at least one separatory unit comprising: a) a first microtiter plate well containing a liquid; b) a second microtiter plate well within the same microtiter plate as the first well, the second well containing a liquid; c) a capillary tube fluid circuit connecting the first and second wells, the capillary tube fluid circuit comprising a detection section in which the passage of a molecule may be detected, the capillary tube fluid circuit also being filled with a liquid; and d) a first and second electrode for applying an electric current across the capillary tube fluid circuit, wherein the first and second electrodes are connected to a power supply, the first and second electrodes being in electrical contact with the first and second wells, respectively. As it is preferred that the system be capable of electro-kinetically acting on several samples at the same time, the systems of the invention preferably comprise a plurality of separatory units. In addition, for ease of handling, it is preferred that the fluid circuits be attached to an armature to hold the fluid circuits in position. Also, means for moving the fluid circuits up and down, in relation to the microtiter plate, and means for aligning the fluid circuits with the microtiter plate wells, are also preferred additions to the basic systems of the invention. In preferred embodiments, the detection section of the fluid circuit is transparent, in order to facilitate the detection of the molecule of interest by light- based means (fluorescence, luminescence, absorption spectroscopy, etc.).

Several alternative embodiments of the invention utilize fluid circuits of diverse construction, including single-piece U-shaped capillary tubes and multi-component fluidic circuits. In a preferred embodiment, the fluid circuit is composed of two substantially vertical components (e.g., fused silica capillary tubes) which are fluidly joined to a substantially horizontal component (e.g. a microchannel formed in a silicon wafer, or in a polymer.) This design has allowed the construction of multiple separatory units on a single microchannel manifold, which also serves as an armature for moving and positioning the separatory units. In another aspect, the current invention also provides a method for simultaneously processing an array of samples containing molecules of differing electrophoretic mobilities. As the methods are especially useful for separating simple mixtures, they are ideal for high throughput screening applications where a substrate is converted to a differently charged molecule by an enzymatic reaction. Samples are loaded directly from multi-well sample plates, and are electrophoresed within the small-scale devices of the invention. The results of electrophoretic separation of the sample may be observed within approximately two minutes. In general, the method for performing electro-kinetic analysis of a sample located in a microtiter plate well, utilizing the above system, comprises the steps of : a) forming at least one separatory unit by placing a first end of a capillary tube fluid circuit and a first electrode into a first microtiter well containing a sample mixture in a liquid, and placing a second end of the capillary tube fluid circuit and a second electrode into a second microtiter well on the same microtiter plate as the first well, wherein the second microtiter well and the capillary tube fluid circuit contain a liquid; b) energizing the electrodes to apply a first electric field across the capillary tube fluid circuit, thereby loading the sample mixture into the capillary tube fluid circuit; c) removing the first and second ends of the capillary tube fluid circuit from the first and second sample wells; d) transferring the first and second ends of the capillary circuit to a third and a fourth well, respectively, wherein the third and fourth wells are both contained within a single microtiter plate, wherein the third and fourth wells are in electrical contact with a third and fourth electrode, respectively, and wherein the third and fourth wells contain a liquid; e) applying a second electric field across the fluidic circuit to effect separation of the sample mixture; and f) detecting the electrophoretic migration of at least one molecule of interest through the detection section of the capillary fluid circuit. Several alternative arrangements of the above methods exist, in which the sample loading (first and second well) and electrophoresis steps (third and fourth wells) take place within the same microtiter plate, or different microtiter plates. Additionally, a single pair of electrodes may be used as the first and second and third and fourth electrodes, or separate electrodes may be used. Liquids in the wells may be the same or different, as long as they support the requirement of electro-kinetic transport of the molecules of interest. In preferred embodiments, the method also includes a separatory unit flushing step, in which the capillary tube fluid circuits are electro-kinetically rinsed with the electrophoresis liquid or buffer in a pair of fifth and sixth wells. Because of the small scale of the individual separatory units used in the methods, they have a great deal of format flexibility, allowing the user to adjust the methods to best fit his particular assay and pre-existing microtiter plate handling and loading equipment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1: A schematic of a simple U-shaped capillary fluid circuit embodiment of the invention. TSIote that the scale of the device is such that the entire electrophoretic separation and detection process may take place within the footprint of a standard microtiter plate. FIGURE 2: A schematic of a multi-separation-unit laminar structure embodiment of the invention, in which the horizontal component of the capillary fluid circuit is made from a silicon wafer with etched microchannels covered by a sheet of Pyrex® glass, and the vertical component of the capillary fluid circuit is made from glass or plastic microcapillaries. FIGURE 3: A chart showing the electropherogram obtained by electrophoresing a sample containing 10% phosphorylated Texas Red labeled Kemptide (TXK) through the device shown in Figure 1. Note that peak area corresponds well to the percentage of Kemptide which is phosphorylated (first peak) as compared to the percentage which is unphosphorylated (second peak). The phosphorylated

Kemptide, which is negatively charged, travels faster through the capillary fluid circuit towards the anode. FIGURE 4: A chart showing the reproducibility of a concentration curve in three separate U-capillary units like the one pictured in Figure 1. Note that the system shows a quantitative response curve for peptide conversion fractions in the desirable 2-10% range, which is useful in kinase and phosphatase assays. FIGURE 5: A chart showing calculated percent conversions in phosphorylated kemptide samples from fluorescence data obtained using the device shown in

Figure 1. FIGURE 6: A chart showing a concentration standard curve generated using the device shown in Figure 1 with known percentage standards. The curve was used to calculate the percent phosphorylation in Figure 5. FIGURE 7: A diagram of the process described in Example 2 for generating a capillary tube fluid circuit using MEMS techniques. FIGURE 8: A diagram of the process described in Example 3 for generating a capillary tube fluid circuit using molded elastomer techniques.

FIGURE 9: A chart showing electropherograms obtained by electrophoresing a series of samples containing phosphorylated or unphosphorylated Texas Red labeled SRC substrate peptide through a MEMS fabricated multi-component fluid circuit device, as described in Example 2.

DEFINITIONS

As used herein, the term "microtiter plate" means, in its broadest sense a plate of material containing a plurality of regularly arrayed sample accepting depressions or voids (wells). Although this definition explicitly includes standard 96, 384, and 1536 well microtiter plate formats in a 8.5 X 11 cm footprint, it also includes non standard arrays of 2 to several thousand wells in footprints from 1cm X 1cm up to 100cm X 100 cm, or any other workable size. Because of their standard use in the biological sciences, standard 8.5 X 11 cm microtiter plates are preferred for use in the systems of the invention. However, "special" microtiter plates with larger reservoir wells are also contemplated for use as the third and fourth well containing plates, and subsequent plates, for supplying running and rinsing buffers in the methods of the invention.

"Capillary" as used herein, denotes a tube having a small interior dimension, so that electro-osmotic forces may be observed in a fluid placed inside the tube when an electric potential is applied across the length of the tube, although embodiments in which EOF forces are not evident are also contemplated within the scope of the invention. In general, capillary tubes preferably have an inner diameter of less than 1000 μm, more preferably less than 700 μm, more preferably less than 500 μm, more preferably less than 200 μm, more preferably less than 100 μm, and most preferably less than about 75 μm. The capillary inner diameter should be large enough, however, to permit the passage of moderately sized molecules and the electrophoresing liquid, and to permit the filling and rising of the fluid circuits with moderate pressure. Thus, the capillary tubes preferably have an interior dimension greater than 100 nm, more preferably greater than 500 n , more preferably greater than 2.0 μm, more preferably greater than 10 μm, and most preferably greater than 25 μm. As used herein, a "substantially vertical" component means that there is a greater vertical element than horizontal element to any angle to the length of the component. Similarly, "substantially horizontal" means that there is a greater horizontal element than vertical element to any angle to the length of the component. Although right angled devices are described and used in the systems and methods. of the invention, and are preferred because of their ease of fabrication, odd- or variably angled devices are also contemplated as obvious variants of the described systems.

In general, where used herein, the term "fluid circuit" is synonymous with the term "capillary tube fluid circuit."

DETAILED DESCRIPTION OF THE INVENTION

The systems and methods described in the present invention permit the rapid analysis of a large number of samples by free solution electro-kinetic flow in a standard microtiter plate-sized footprint. The assembled systems of the invention utilize microcapillary fluid circuits to separate charged molecules in an electrophoresing liquid. In addition to the rapid separation of differently charged molecules of approximately the same size, the systems and methods of the invention may also be used to separate analytes of the same charge with different electrophoretic mobilities in solution. Depending on the net charge of the analytes, under the buffer conditions used for the separation, electro- osmotic flow may be utilized to facilitate the transport of the analytes towards the detection section of the fluid circuit. Unlike several capillary tube electrophoretic devices which have been described, the systems of the invention utilize relatively short electrophoretic path lengths (usually less than 10 cm, more preferable less than 5 cm, and most preferably less than 3.5 cm) in order to efficiently separate simple sample mixtures for analysis within 2-5 minutes. Thus, several complete separatory units may fit simultaneously on a single microtiter plate during operation, and the need for complex handling steps to transfer samples to a larger device is eliminated.

These systems and devices are especially useful in measuring the conversion of labeled substrates by an enzymatic reaction, such as kinase, phosphatase, and protease reactions. For instance, the conversion of a peptide to a phosphorylated peptide by a kinase may be easily measured in the systems of the invention because the phosphorylated product has a - 2 charge as compared to the substrate. Such converted substrates are formed in assays for the inhibitory or stimulatory effects of combinatorial libraries of compounds on kinases, proteases, and phosphatases. Because of the small separatory unit size of the systems of the invention, use of the systems to perform parallel high-throughput analysis of such libraries provides an economical and relatively rapid alternative to conventional capillary electrophoresis devices.

In general, the systems of the invention comprise at least one separatory unit, although systems with a plurality of separatory units are preferred. Each separatory unit consists of a first and second well on the same microtiter plate connected by a capillary tube fluid circuit, and a first and second electrode electrically connected to the first and second well. The capillary tube fluid circuit has a length sufficient to allow electrophoretic separation of molecular analytes, and a cross section small enough to allow the application of high electric fields without excessive power consumption, but large enough to allow filling and cleaning of the circuit with moderate pressure. The capillary tube fluid circuit also comprises a section through which a molecule of interest may be detected (e.g., a transparent section), which may be either specially fabricated or take advantage of an intrinsic property of the capillary tube material. The fluid circuit, and the two wells, contain a liquid which promotes electro-kinetic transport of molecular analytes when an electric field is applied. The liquid may also flow electro-osmotically under the applied electric field. In operation, a sample is contained within the first well. The separatory unit is briefly energized in order to draw an amount of the sample into the capillary tube fluid circuit. The fluid circuit is then moved to a third and fourth well (the new first and second wells of the separatory unit) containing the liquid, which are in contact with a third and fourth electrode, and the system is again energized. Molecules are transported through the fluidic circuit by electro-osmotic flow and/or electrophoretic means, with the molecules of greatest electrophoretic mobility in the direction of the fourth electrode moving at the greatest rate. The separated molecules are detected as they flow through the detection section of the fluid circuit by a detection device, such as fluorometer.

Design of the Capillary Tube Fluidic Circuit for Use in the Systems of the Invention

A central element of the systems of the invention is the capillary tube fluid circuit. The fluidic path formed by the capillary tubes preferably has an inner diameter of less than 700 μm, more preferably less than 500 μm, more preferably less than 200 μm, and most preferably less than about 100 μm, to allow the application of high electric fields and to limit excessive heating during the electrophoresis step. The capillary inner diameter should be large enough, however, to permit the passage of moderately sized molecules and the electrophoresing liquid, and to allow the initial filling of the tube without the use of extraordinary pressure. Thus, the capillary tubes preferably have an interior dimension greater than 100 nm, more preferably greater than 1.0 μm, and most preferably greater than 10 μm.

The capability to promote electro-osmotic flow within the fluid circuit increases the versatility of the system, allowing for the transport of positively charged, negatively charged, and uncharged molecules through the fluidic circuit. However, electro-osmotic flow is not necessary for performing separations in the systems of the invention. In order to facilitate electro-osmotic flow, the internal walls of the capillary tube fluid circuit must have a charged surface. This may be an intrinsic property of the materials comprising the fluid circuit, or may be conferred on the material by chemical modification of their interior surfaces. The capillary tube fluid circuits are preferably comprised of one or more materials including plastic, silicon, ceramics, metals, composite materials, glass, fused silica, or other suitable materials.

The length and dimensions of the capillary tube fluid circuit will depend upon the position and size of the first and second wells of the separatory unit in the microtiter plate. The sample plate may be comprised of glass, plastic or of other suitable non-conductive or insulated material. Standard polystyrene or polycarbonate microtiter plates are suitable for use in the systems. In order to be compatible with standard microtiter plate loaders and handling equipment, microtiter plates with 96, 384, or 1536 wells in an 8.5 X 11 cm footprint are preferred. Thus, for these microtiter plate formats, the ends of the capillary fluid circuit should be about 9.0 mm , 4.5 mm, or 2.25 mm apart, respectively, to connect adjacent wells on the microtiter plate. The capillary fluid circuit should also be high enough (as related to the microtiter plate plane) so that the ends of the fluid circuit may extend down into the wells to make contact with the liquid. Although the capillary tube fluid circuit may connect two wells which are not adjacent to each other (e.g., at opposite sides of the plate), it is preferred that the fluid circuit connect adjacent wells. This decreases the complexity of the system, and longer electro-kinetic separation paths are not necessary in order to separate relatively simple mixtures of molecules with different electrophoretic mobilities. In addition, more separatory units may be packed into the microtiter plate footprint when adjacent wells are used. Thus, it is preferred that the total length of the capillary tube fluid circuit be less than 10 cm, more preferably less than 5 cm, and most preferably less than 3.5 cm.

A single-component fluidic circuit may be formed by bending a capillary tube into a U-shape. The U-shape preferably has a radius substantially equivalent to half of the center-to-center distance between wells of the sample plate. An illustrative fluid circuit may be prepared by bending fused silica tubing into a U-shape, using a propane torch, so that the height of the U is approximately 1.0 cm and the radius is approximately 2.25 mm (for use with a 384 well standard microtiter plate). The internal walls of the fluid circuit are conditioned for electrophoresis by filling the tubes and incubating them for one hour with 1 N NaOH, creating a charged surface. The tubes are then washed twice with purified H₂0. The fluidic circuits are then filled and stored with a buffer comprising lOOmM NaHPO₄ at pH 7.2, 0.005% FC-CAT cationic surfactant, and 0.005% FC-N neutral surfactant (J&W Scientific). For handling, a single fluidic circuit or a plurality of fluidic circuits may then be mounted in a black plastic sheet (in order to minimize background fluorescence during detection of the migrating charged molecule) or printed circuit board (PCB), with the section of the U which is above the plastic sheet or PCB becoming the detection section of the fluid circuit. A system using a simple one-component fluid circuit is illustrated in Figure 1.

Alternately, the fluidic circuits may be comprised of a substantially horizontal component and two substantially vertical components. In the exemplary embodiments, the substantially vertical components are a pair of capillary tubes, and the substantially horizontal component is a microchannel manifold structure. In the preferred multi- component fluid circuits, the capillary tube vertical components are joined by pressure fitting, adhesives, soldering, or other appropriate means, to holes in a multi-layered microchannel manifold structure in order to create complete fluid circuits. However, multi -component circuits comprising joined capillary tubes and the like, are also within the scope of the present invention.

The microchannel manifold comprises from one to several layers of material, including at least one microchannel wafer layer. The microchannel wafer layer may be comprised of plastic, silicon, ceramics, metals, composite materials, glass, fused silica, or other suitable materials. Preferred plastics include polymethyl methacrylate (PMMA) and polydimethylsiloxane (PDMS). PDMS is especially useful because it self-seals with glass and other commonly used materials. The manifold may also include a sealing wafer layer to seal the microchannel structures, which may be comprised of plastic, silicon, ceramics, metals, composite materials, glass, fused silica, or other suitable materials. In order to form a detection section in the fluid circuit, the sealing wafer layer may comprise a transparent material, such as transparent plastic or glass. However, detection systems may utilize fiber optic filaments or other components built into the microchannel layer, or may utilize non-light signals which do not require transparent materials for detection. For instance, electrochemical detection system components my be integrated on a silicon microchannel wafer layer which is sealed with an opaque sealing wafer layer. Preferably, the microchannel wafer layer is comprised of a different material than that of the sealing wafer layer, so that the surface charge on the top wall of the channels differs from that of its sides and bottom. In the embodiments of the systems of the invention, the separated analytes pass around a bend or comer in the fluidic circuit as they transition to either the horizontal channel in the microchannel manifold, or around the top of the U in single component fluid circuits. The bend causes dispersion of the analytes due to the longer path traveled by the analyte molecules on the outer versus the inner surface of the bend. By using different materials to make the microstructure manifold, one may use the charge differential to compensate for the dispersion of analytes, which can cause wide or overlapping bands in the detection section of the fluidic circuit.

In a MEMS approach to forming the microchannel manifolds, the manifold is comprised of a sealing wafer layer, microchannel wafer layer, and a mounting wafer layer, as illustrated in Figure 2. In exemplary manifolds, the sealing wafer layer may be comprised of glass, the microchannel wafer layer of silicon, and the mounting wafer layer of glass or silicon. The manifold may be fabricated and assembled by using any suitable micro-fabrication processes. For example, as illustrate in Figure 7, a mounting wafer is first prepared by using a photoresist to pattern an approximately 525 μm thick silicon wafer for through-holes. A deep reactive ion etch (DRIE) is performed to etch approximately 375-400 μm holes into the silicon wafer to form the through-holes [apertures]. The microchannel wafer layer is prepared by first oxidizing an approximately 300 μm thick substrate and patterning the oxide with an HF etch using a photoresist etch mask to form a through-hole pattern. Through-holes of approximately 25-75 μm are then created by DRIE using thick resist as an etch mask. Another DRIE is then carried out to fabricate the surface microchannels using the patterned oxide as an etch mask. The mounting and microchannel wafer layers are aligned so that the mounting wafer through holes are below the microchannel wafer through holes, in order to form an insertion point for the vertical components of the fluid circuit. The two layers are then fusion bonded and oxidized. The two layers are then anodically bonded to a Pyrex β) glass sealing wafer layer to seal the microchannels. Alternately, the manifold may be fabricated by a Silicon-on-Insulator (SOI) process, in which a single silicon substrate may be used for the microchannel layer. The substrate contains a layer of oxide, which is approximately lμm thick, sandwiched between a first and a second layer of silicon, which are respectively approximately 425μm and approximately 100 μm thick. The substrate may then be machined by processes identical to those described for the two wafer process to form microchannels and approximately 50μm through holes in the 100 μm second layer. Large through holes are machined in the 425 μm first layer. The internal oxide layer is then etched to make a fluid interconnect between. the first and second layers, and the microchannels are capped by anodic bonding to a Pyrex® sealing wafer layer.

The use of the silicon chemistry processes described above require rather involved masking steps and the use of photolithographic equipment. In order to decrease the number of steps necessary to manufacture microchannel manifold, and to simplify . manufacturing, manifolds may also be made from elastomers molded on top of hard photoresist relief structures. Multiple layers of positive SU8 resist are used to create positive-relief U-shaped and post structures for molding microchannel and mounting wafer layers, as described in Example 3. A suitable elastomer, such as PDMS, may then be poured over the mold in a pre-polymerized state, and allowed to polymerize over the relief structures, pressing down over the posts to form through-holes. Alternately, a thermoplastic, such as PMMA, may be hot-molded over the positive-relief mold, provided that the mold is made from a heat-resistant material. The two layers may then be aligned and adhered (if PDMS is used, this can be accomplished simply by cleaning the layers in oxygen plasma and joining them). The microchannels are then sealed by adhering the microchannel wafer layer to a Pyrex® sealing wafer layer. Advantageously, the mounting wafer layer through-holes may be made slightly smaller than the outer diameter of the capillary tube vertical components. This allows the capillary tubes to be friction fit into the mounting wafer, ensuring a tight junction for the fluid circuit.

Once the microchannel manifold is assembled, the substantially vertical components are inserted into the mounting wafer to complete the fluid circuit. Although fused silica capillary tubes are a preferred vertical component material for use.in the invention, other suitable structures and materials may also be used (e.g., plastic tubing.) The vertical components may be joined to the horizontal component's mounting wafer layer by friction fitting, the use of epoxy or other adhesives, or by any suitable means. In a preferred embodiment, the vertical component is a metal-coated fused silica capillary. When a printed circuit board with properly spaced holes is aligned with the mounting wafer layer, the capillary vertical components may be inserted through the circuit board into the microchannel manifold, and then soldered in place to secure the capillary. In this manner, an integrated electrode on the surface of the vertical component of the capillary tube fluid circuit may be fashioned. Using these methods to create composite fluid circuits, several microchannels for individual separatory units may be created in parallel on the same microchannel manifold structure. This structure then may serve not only as the horizontal component of the capillary fluid circuit, but may also serve as an armature for holding and positioning the individual separatory units for use in the systems and methods of the invention. In addition, when metal-coated capillary tubes are used as the vertical components of the fluid circuit, integrated electrodes may be easily formed. Because these methods offer the production of consistent, precisely spaced separatory units which may be easily handled after assembly, they are preferred for forming the capillary fluid circuits for use in the systems and methods of the invention.

Electrode Assembly and Positioning Machinery

The electrode assembly may comprise any electrode configuration that permits the electrodes to be lowered into the sample wells, or the assembly may be integral to the microtiter plates used in the systems. The electrodes may be wires, strips, or other convenient shapes, and may be soldered, deposited, etched, or attached in place with adhesives. Electrodes may be made of any suitable conductive material, including platinum and platinum plated titanium, gold, iridium tin oxide, aluminum, carbon fibers, or conductive polymers. Although non-corroding materials are preferred for use in reusable embodiments of the invention, reactive metals such as aluminum, copper, or steel may be utilized in limited-use devices. In one embodiment, the electrodes are wires which extend parallel to the arms of the capillary tube fluid circuit into the microtiter plate wells. These electrodes may be attached by solder or epoxy to a printed circuit board, may simply be wires which are run along a support structure holding the capillary tube fluid circuit (as in Figure 1), or may be configured in any convenient manner.

In an alternative preferred embodiment, the electrodes are integral with the arms of the capillary tube fluid circuit, such as those formed by coating a capillary tube with metal, as described above. These types of electrodes may be easily soldered to a printed circuit board with holes aligned with the arms of the capillary tube fluid circuit. These electrodes may also be attached by wire bonding or other means to metal leads on the underside of a microfabricated microchannel manifold. In U-shaped single component fluid circuit embodiments, the printed circuit board may also serve as an armature, or handling support, for the fluid circuit. Embodiments utilizing printed circuit boards for maintaining electrical contact between the electrodes and a power source are preferred, as one of ordinary skill in the electrical arts may print circuitry for the individual or tandem control of the electrodes on the board.

Alternatively, in wholly disposable multiple- or single-use devices, the electrodes may be integrally formed within the microtiter plate. For example , the electrodes could be placed within the microtiter plate during polymerization or molding. Or printed circuit board may be sealed to a bottomless well microtiter plate to create electrodes in the bottom of the microtiter plate wells.

In addition to the fluidic circuits and electrodes, the preferred embodiments of the separation systems preferably contain additional components to facilitate the handling and use of the separatory system. In order to properly handle the fluidic circuits, especially in automated embodiments, the system preferably comprises a means for raising, lowering and aligning the capillary first and second lower ends in relation to the sample wells. This may be as simple as having the fluidic circuits and/or electrodes mounted on an armature with a couple of screws or posts to assist in aligning the device with the microtiter plate wells. In partially or wholly automated systems, more complex mechanical or robotic devices may be used, including motorized lifting actuators, optical detection of the microtiter plate for proper alignment, and mechanical means for moving the microtiter plate in relation to the fluid circuits and/or the electrodes.

Additionally, the system may comprise an analyte detection device. Any suitable means for detection may be used in the systems and methods of the invention, including absorption spectroscopy, fluorometry, colorimetry, luminometry, mass spectrometry, electrochemical detection, and radioactivity detection. Fluorometry is preferred for use in the present invention, because of the ease in handling fluorophores, and the commercial availability of fluorescent detection equipment, including excitation lasers and properly filtered cameras. Fluorescently labeled peptides for use in particular kinase, phosphatase, or protease reactions may be made by derivatization with a fluorescent moiety, as has been described in the relevant assay literature. As detection is carried out dynamically, during electrophoresis, each capillary tube fluid circuit may be monitored at its detection section for the passage of the charged molecule of interest by a different detection device. Alternatively, a row or column of separatory units may be rapidly scanned by a device which pivots or is shuttled back and forth over the separatory units. Devices such as those utilized for parallel DNA sequencing applications could be utilized in the present invention.

General Electro-kinetic Separation Methods of the Invention In general operation of the systems of the present invention, the fluidic circuit is first flushed and pre-filled with a suitable electrophoresis liquid, preferably a buffer. Suitable aqueous buffers for use in the electrophoretic methods of the invention include Tris hydrochloride buffers, Tris borate buffers, histidine buffer, sodium or potassium phosphate buffers, and HEPES buffers. Alternatively, an organic or other non-aqueous liquid, such as DMSO, may be used. A particular preferred electrophoresis buffer is 100 M NaHPO₄ at pH 7.2. The liquid used may also contain anionic, cationic, or neutral surfactants to alter the surface charge characteristics of the capillary walls in order to promote electro-osmotic flow towards the cathode or anode. The fluidic circuit is preferably pre-filled by pressure injecting the buffer into the fluidic circuit. In addition, the wells of the microtiter plate used in the method must contain the electrophoretic liquid, or buffer, at a level sufficient to contact the electrodes and the ends of the capillary tube fluid circuit, when the separatory unit is assembled.

To load the sample, one end of the capillary tube fluid circuit is inserted into the sample-containing buffer in a first well, and the second lower capillary end is lowered into the second buffer-containing well. The first and second electrodes are then placed within the wells. In one embodiment of the method, using the device pictured in Figure 1, a DC current with a potential of approximately 500 volts is applied across each fluidic circuit for ten 100 ms pulses, or an approximately three second ramp to 500 volts is applied across the fluidic circuit, to move the sample into the capillary end. The current, voltage and time depend upon such factors as the length and diameter of the capillary tube fluid circuit, buffer composition, and the total electromotive force necessary to move a particular charged substrate or product through the liquid in a reasonable time frame. These factors are well known in the art, and persons skilled in the electrophoretic arts can ascertain, with a minimum of experimentation, the optimal voltage, amperage, and time to use with the systems of the invention in particular applications. In general, useful voltages for sample loading and electro-kinetic separation of samples range from 10V to 1000V per cm of capillary tube fluid circuit, more preferably from 50V to 500V per cm of capillary tube fluid circuit, and most preferably from 100V to 300V per cm of capillary tube fluid circuit. After the sample is loaded, the capillary tube fluid circuit is then removed from the first and second wells, and placed into a third and fourth well containing buffer, which are in contact with a third and fourth electrode. The third and fourth wells may be on the same or a different microtiter plate. In embodiments where the microtiter plate is the same, it is preferred that the third and fourth electrodes be the same as the first and second electrodes, and that they be transferred to the third and fourth wells along with the capillary tube fluid circuit. In an alternative embodiment where the third and fourth wells are on a separate microtiter plate, the third and fourth wells are specialized trough-shaped wells, which each contain a single long electrode. These trough-shaped wells are shared by a plurality of capillary fluid circuits during the electro-kinetic separation of the sample. The electrode may be an integral part of the microtiter plate in these embodiments. In an embodiment utilizing the device Pictured in Figure 1 , the third and fourth wells are on the same microtiter plate, and the first and second electrodes are used as the third and fourth electrodes. A potential of approximately 300-500 volts is applied across each fluidic circuit for 0.25 to 4 minutes to effect the separation of the charged molecules in the sample. Molecular analytes in the sample are separated as they travel up and around the capillary tube fluid circuit towards the anode. Negatively charged molecules, which are attracted to the positively biased anode, migrate more quickly than uncharged or positively charged species. Alternatively, by using different surfactant compositions, the electro-osmotic flow may be changed towards the cathode. In that instance, the positively charged analytes would travel more swiftly towards that cathode than the negatively charged analytes. The separated molecules may be observed by a detection device as they pass through the detection section of the capillary tube fluid circuit, usually the top of the U, or in the microchannel manifold.

Residual sample material remaining in the capillaries can be removed between runs by physically flushing the fluidic circuits with running buffer. Alternately, the fluidic circuits may be flushed by transferring the capillary tube fluid circuit ends to fifth and sixth sample wells containing a fifth and sixth electrode, and applying 300-500 volts across the fluidic circuits for approximately two to three minutes. As above, trough-shaped fifth and sixth wells containing long electrodes are an alternative embodiment of this additional method step. By using this flushing method, nearly continuous running systems for the analysis of large numbers of samples may be constructed. For example, using a 1536 well-format device with 768 capillary tube fluid circuits with integrated electrodes on an actuated armature, 768 samples may be loaded from alternating rows of wells on a microtiter plate, with the other rows containing buffer. The armature may then be raised, and the microtiter plate replaced with a 1536 well plate containing all buffer for electrokinetic separation. After separation and detection of the molecules in the samples, the armature is raised again, and the plate replaced with another all-buffer plate for flushing of the fluid circuits. The capillary tube fluid circuits may be used for approximately 20-100 electrophoresis runs in this fashion, after which the fluidic circuits are preferably subjected to a pressure flushing step. A single fluidic circuit may be run at a time to obtain multiple data points by sequential runs with the same capillary or, alternately, a plurality of fluidic circuits may be run simultaneously. If the electrodes are connected to circuitry which allows their independent control, the fluid circuits may be run at different or similar voltages at the same time, or run in sets. Utilizing these methods, very good sensitivity has been obtained in detecting the enzymatic conversion of substrates (e.g., kinase or phosphatase phosphorylation/dephosphorylation, or protease reactions), as shown by the Kemptide conversion data in Example 1, below. The systems and methods of the invention are able to detect about 10%, more preferably about 1.0%, and most preferably about 0.1% conversion of a labeled substrate in enzymatic reactions. The high sensitivity and convenient format of these systems make them ideal for use in high-throughput drug screening applications.

EXAMPLES The following examples are offered to further illustrate the various aspects of the present invention, and are not meant to limit the invention in any fashion. Based on these examples, and the preceding discussion of the embodiments and uses of the invention, several variations of the invention will become apparent to one of ordinary skill in the art. Such self-evident alterations are also considered to be within the scope of the present invention.

Example 1 : Peptide Separations

Phosphorylated and unphosphorylated versions of Kemptide were prepared synthetically and labeled at the N-terminus with the Texas Red fluorescent dye. The phosphorylated Texas Red- labeled Kemptide has a charge at neutral pH of -1, and the unphosphorylated form had a charge of +1. The peptides were diluted at various ratios to a final total concentration of 20 μm in a sample buffer containing 20 M Tris CL pH 7.5, 5 mM MgC12, 35 mM KHPO₄ pH 7.5, and ImM ATP. The capillary tube fluidic circuits were first flushed and pre-filled with 100 mM NaHPO₄ at pH 7.2, 0.005% FC-CAT cationic surfactant and 0.005% FC-N neutral surfactant (J&W Scientific). In typical operation, each fluidic circuit were used for approximately 20-100 electrophoresis runs after which the fluidic circuits are preferably subjected to a pressure-flushing step. To load the sample, the first and second ends of the fluidic circuits were lowered into a sample mixture in a first sample well, and into an adjacent second sample well containing the buffer. The cathode was then inserted into the first sample well, and the anode inserted into the second well. A DC current of 500 volts was then applied across each fluidic circuit for ten 100 ms pulses, or an approximately three second ramp to 500 volts was applied across the fluidic circuit, in order to pull the sample into the capillary fluid circuit. The sample was then run by removing the first and second lower capillary ends from the first and second sample wells to a third and fourth adjacent sample wells, each containing approximately lOOμl of running buffer. A current of 500 volts was applied across each fluidic circuit for 0.25 to 4 minutes to effect separation of the molecules of the peptide mixture in the samples.

The peptides migrated through the capillary towards the anode and separated into unphosphorylated (slower) and phosphorylated (faster) peaks. A typical electropherogram for a solution containing 18 μm TXK and 2 μ m TXK-PO₄ is shown in Figure 3. The fluorescence in the peak corresponding to phosphorylated kemptide (TXK-PO₄) was divided by the sum of the fluorescence in the TXK-PO₄ peak and the fluorescence in the peak corresponding to unphosphorylated Kemptide (TXK). For three different capillaries the fluorescence ratio of TXK / TXK-PO₄ was reproducible and linearly related to the percentage of phosphorylated peptide relative to total peptide in the sample. The results are graphed in Figure 4. To test direct monitoring of enzymatic phosphorylation of a peptide substrate, 20 um TXK was phosphorylated with 0.1 ng/ μl purified protein kinase A catalytic subunit (Upstate Biotechnology) in a buffer containing 100 mM Tris-Cl pH 7.5, 5 μm cAMP, 13.5 mM MgCl₂, and 90 μm ATP. The reactions were incubated for various times at 30° C, then stopped by heat inactivation at 95° C for 10 minutes. Reaction products were placed in the wells of 384-format microtiter plate and analyzed electrophoretically as described above. Actual percent conversion of TXK to TXK-PO₄ was calculated from TXK / TXK- PO₄ fluorescence ratios using the equation from a standard curve of known standards (curve shown in Figure 6). A graph of the results is shown in Figure 5.

Example 2: Fabrication of Microcapillary Fluid Circuits Using MEMS Approaches.

In order to improve the quality and uniformity of the capillary tube fluid circuits used in the systems of the invention, fluid circuits were manufactured utilizing a MEMS procedure. In these approaches, polyimide-coated fused silica capillaries are bonded to a microchannel manifold structure to generate arrays of U shaped fluidic circuits, wherein the silica capillaries form the vertical arms of the circuit, connected by a horizontal component in the microchannel manifold. The exemplary microchannel manifolds constructed consisted of a Pyrex® glass sealing layer, a silicon microchannel layer, and a glass or silicon mounting wafer with holes for accepting the silica capillary tubes. Several specialized processes were designed to achieve the finished microchannel manifold structure.

One of the processes, using two silicon wafer layers and a Pyrex® wafer layer for the sealing layer, is illustrated in Figure 7. In that process the mounting wafer layer is prepared from a 525 μm thick silicon substrate by applying and patterning a thick photoresist, then etching 375-400 μm through-holes using Deep Reactive Ion Etching (DRIE). The microchannel wafer layer is prepared by first oxidizing a 300 μm thick substrate, then patterning the oxide with a HF etch using a photoresist etch mask. Small 25-75 μm through holes are then created by DRIE using thick resist as an etch mask. Finally, DRIE is used to generate the surface microchannels using the patterned oxide as an etch mask. The mounting and microchannel wafer layers are fusion bonded and oxidized, and then anodically bonded to a Pyrex® glass sealing wafer.

In an alternative Silicon-on-Insulator (SOI) process, a single silicon substrate containing a 1 μm layer of oxide sandwiched between 100 μm and 425 μm silicon layers is machined by DRIE processes identical to those described for the two wafer process. Microchannels and 50 μm through holes are machined in the 100 μm layer, while large through holes are created in the 425 μm silicon layer. The internal oxide layer is then etched to make a fluid interconnect between the layers, and the microchannels are capped by anodic bonding to a Pyrex® sealing wafer layer. In another alternative process, 375-425 μm through holes are generated in a

Pyrex® substrate by water jet ablation to generate the mounting layer. A silicon microchannel wafer layer prepared as described for the two silicon wafer process is then bonded anodically between the Pyrex® mounting wafer layer and a Pyrex® sealing wafer layer in a single step. The last step in each of the assembly processes is the insertion and bonding of commercially available polyimide-coated fused silica microcapillaries into the 375-425 um holes in the mounting wafer layer. The microcapillaries may be held in place by friction fitting, chemical bonding, or by using epoxy or other adhesives.

A device used for testing was constructed from a micromachined microchannel manifold consisting of a silicon microchannel wafer layer bonded between a Pyrex sealing wafer layer and a water jet-machined Pyrex mounting wafer layer. It is interesting to note that the anodically bonded Pyrex® / silicon wafer junctures were water-tight over a surprisingly large area. Water tight junctures of over 25 cm , and even over 100 cm were obtained using this method. Thus, anodically bonded structures such as these may find other uses in mass-microfluidics applications using a large number of repeated structures. Fused silica polyimide-coated 50 μm i.d. microcapillaries were bonded to the assembly, to create an array of 3 -dimensional U-shaped microfluidic circuits. The entire assembly was seated on a PCB fitted with platinum electrodes, which allowed rapid transfer of the array between 384 microtiter trays for sample loading, running, and electro- osmotic capillary flushing. Initial electrophoresis testing on the MEMS device was conducted on SRC kinase peptide substrates modified with a Texas Red fluorophore. Electrophoresis of the peptides was observed in a single fluidic circuit which had 60 μm through holes in the silicon wafer coupled to the microcapillaries, connected by a 4.5 mm long, 70 μm wide, 50 μm deep, trench on the wafer surface. The unphosphorylated (SRC) and phosphorylated (SRC-PO₄) forms of the peptide were diluted separately to 10 μM in 10 mM Tris-Cl pH 8.0 containing 0.01 % Tween 20 detergent. Electrophoresis was conducted at 500 V in 100 mM NaHPO₄ pH 7.2 + 0.01 % of the cationic surfactant FC- CAT. The trench was illuminated and images were acquired on the C5 controller, collecting one frame per second. The results are shown in the chart in Figure 9. Consistent peaks for SRC and SRC-PO₄ were obtained in sequential runs on a single microfluidic circuit.

Example 3: Fabrication of Microcapillary Arrays Using a PDMS Microstrucrure manifold

A microchannel manifold may also be prepared from a single layer of unpatterned glass and two layers of the elastomeric polymer poly(dimethylsiloxane) (PDMS). PDMS microchannels and through holes are prepared by casting the two part pre-polymer against relief structures patterned on silicon wafers in thick SU8 photoresist, as follows: A 50 μm thick layer of SU8 photoresist is spun on a 5" silicon wafer, pre-baked, exposed to UV light through the trenches mask, and post-baked, to define 50 μm wide ridges in the SU8. A 200 μm layer of SU8 is then spun on top of the 50 um layer. The wafer is then pre-baked, exposed through the small through holes mash, and post-baked, to create -50 μm diameter columns in the SU8 coincident with either end of the SU8 ridges. The patterned SU8 is then developed with propylene glycol methyl ether acetate (PGMEA) in order to reveal the relief structure which defined small through holes and connected microchannels. A second 200 μm layer of SU8 photoresist is spun on a 5" silicon wafer, pre-baked, exposed to UV through the large through holes mask, and post- baked, to define 350 μm diameter posts in the SU8. Development in PGMEA generates the large column structures in the SU8.

A 10:1 mixture of PDMS pre-polymer and curing agent is degassed and poured over the SU8 molds. In order to generate through holes in the PDMS layers, the PDMS pre-polymer/ curing agent mixture is compressed on to the SU8 structures so that the tops of the SU8 posts make contact with a sheet of Mylar® film. This assembly is prepared as described in Jo et al., (J. MEMS 9: 76-81 (2000)). The mixture is cured under compression in a 65° C oven.

After curing, the first PDMS layer containing microchannels with connecting through holes is oxidized with oxygen plasma and attached to a Pyrex® wafer such that the microchannel surface of the PDMS contacts the glass surface, creating sealed microchannels between the layers. PDMS is preferred for this structure because the material self-seals with itself, glass, and other common materials, when treated with oxygen plasma. The second piece of PDMS is also oxidized in oxygen plasma, aligned, and attached to the PDMS microchannel layer on the Pyrex®. The two PDMS layers are brought into contact such that the small through holes in the first PDMS layer are centered on the openings of the large through holes in the second PDMS layer.

A printed circuit board (PCB) containing -450 μm diameter holes is positioned above the PDMS side of the PDMS/ Pyrex manifold so that the holes in the PCB are aligned with the large holes on the manifold. A gap remains between the PCB and the PDMS. Fused silica polyimide coated microcapillaries of -365 μm outer diameter (o.d.), ~50 μm inner diameter (i.d.), 13 mm long, are coated on the exterior with platinum by sputtering along the middle 11 mm of their length. Capillaries are inserted through the holes in the PCB and into the holes in the PDMS layers. Because the larger PDMS holes are slightly smaller than the o.d. of the silica capillary, a tight-friction fit forms between the capillaries and the PDMS layer of the manifold. The PCB and capillaries are secured in place by UV curing adhesive. The platinum on the surface of the capillaries is soldered to the leads on the PCB to generate working electrodes. This creates integrated electrodes on the capillary tube fluid circuit, eliminating the need for separate wire electrodes. In addition, the printed circuitry for individual or tandem control of the electrodes may be easily designed and executed on the PCB using traditional techniques.

Alternatively, the a combination of the elastomer molding and MEMS approaches may be used. For instance, a molded PDMS layer may be used for the mounting wafer layer, while an etched silicon wafer is used for the microchannel wafer layer. This embodiment would have the advantage of the precise micro-structure control allowed using MEMS techniques for the microchannel of the capillary fluid circuit combined with the use of smaller elastomeric mounting holes for a tight friction fit with the vertical components of the fluid circuit.

Claims

WE CLAIM:

1. A system for electro-kinetic separation of charged molecules in one or more samples in a microtiter plate comprising a plurality of wells, the apparatus comprising at least one separatory unit comprising: a) a first microtiter plate well containing a liquid; b) a second microtiter plate well within the same microtiter plate as the first well, wherein the second well contains a liquid; c) a capillary tube fluid circuit connecting the first and second wells, the capillary tube fluid circuit comprising a detection section in which the passage of a molecule may be detected, wherein the capillary tube fluid circuit is filled with a liquid; and d) a first and second electrode for applying an electric current across the capillary tube fluid circuit, wherein the first and second electrodes are connected to a power supply, the first and second electrodes being in electrical contact with the first and second wells, respectively.

2. The system of claim 1 , further comprising at least one device for detecting the passage of a molecule of interest through the detection section of the capillary tube fluid circuit.

3. The system of claim 2 wherein the device for detecting of the molecule of interest uses a detection means selected from the group consisting of fluorometry, colorimetry, luminometry, mass spectrometry, absorption spectroscopy, electrochemical detection, and radioactivity detection.

4. The system of claim 2 wherein the molecule of interest is detectably labeled.

5. The system of claim 2 wherein the device for detecting the molecule of interest comprises a fluorescence excitation system positioned to deliver light of a specified wavelength to the molecule of interest through detection section of the capillary tube fluid circuit.

6. The system of claim 2 wherein the device for detecting the passage of a molecule of interest through the detection section of the capillary tube fluid circuit detects light emitted or transmitted through the detection section as the molecule passes through the section.

7. The system of claim 1 wherein the liquids in the first and second well and in the capillary tube fluid circuit are the same.

8. The system of claim 1 wherein the sample is contained within the first microtiter plate well.

9. The system of claim 1 wherein the sample is contained within the capillary tube fluid circuit.

10. The system of claim 1 wherein the capillary tube fluid circuit detection section is transparent to light.

11. The system of claim 1 wherein at least one liquid is an aqueous buffer.

12. The method of claim 11 wherein the aqueous buffer is selected from the group consisting of: Tris hydrochloride buffers, Tris borate buffers, histidine buffer, sodium phosphate buffers, potassium phosphate buffers, and HEPES buffers.

13. The system of claim 1 wherein the internal diameter of the capillary tube fluid circuit is in the range of about 1000 μm to about 100 nm.

14. The system of claim 1 wherein the internal diameter of the capillary tube fluid circuit is in the range of about 500 μm to about 500 nm.

15. The system of claim 1 wherein the internal diameter of the capillary tube fluid circuit is in the range of about 200 μm to about 2.0 μm.

16. The system of claim 1 wherein the internal diameter of the capillary tube fluid circuit is in the range of about 100 μm to about 10 μm.

17. The system of claim 1 wherein the internal diameter of the capillary tube fluid circuit is in the range of about 75 μm to about 25 μm.

18. The system of claim 1 wherein the capillary tube fluid circuit defines a U-shape.

19. The system of claim 18 wherein the capillary tube fluid circuit is comprised of a material selected from the group consisting of glass, silicon, fused silica, silicon oxide, composite materials, and plastic.

20. The system of claim 18 wherein the distance between the first and second arms of the U-shape is approximately equal to the distance between the center of the first and second wells.

21. The system of claim 18 wherein the distance between the first and second arms of the U-shape is approximately 9.0 mm.

22. The system of claim 18 wherein the distance between the first and second arms of the U-shape is approximately 4.5 mm.

23. The system of claim 18 wherein the distance between the first and second arms of the U-shape is approximately 2.25 mm.

24. The system of claim 1 wherein the capillary tube fluid circuit consists of a single . continuous capillary tube.

25. The system of claim 1 wherein the capillary tube fluid circuit is a composite capillary tube structure comprising a plurality of fluidly joined capillary tube components.

26. The system of claim 25 wherein the composite capillary tube structure comprises first and second substantially vertical capillary tube components connected by a substantially horizontal capillary tube component.

27. The system of claim 25 wherein the substantially horizontal capillary tube component comprises the detection section of the capillary tube fluid circuit.

28. The system of claim 27 wherein the detection section is transparent to light.

29. The system of claim 25 wherein the horizontal capillary tube component is a microchannel manifold.

30. The system of claim 29 wherein the microchannel manifold is comprised of a material selected from the group consisting of silicon, glass, ceramics, metals, composite materials, plastic, and combinations thereof.

31. The system of claim 30 wherein the plastic is selected from the group consisting of polymethyl methacrylate and polydimethylsiloxane.

32. The system of claim 30 wherein the microchannel manifold is comprised of a combination of materials including glass and a material selected from the group consisting of silicon and plastic.

33. The system of claim 29 wherein the microchannel manifold comprises a plurality of material layers.

34. The system of claim 33 wherein the wafer comprises a sealing layer and at least one other layer of material which forms the substantially horizontal capillary tube component of the capillary tube fluid circuit.

35. The system of claim 34 wherein the sealing layer comprises a transparent material.

36. The system of claim 35 wherein the transparent sealing layer forms the detection section of the capillary tube fluid circuit.

37. The system of claim 34, wherein the sealing layer is comprised of a material selected from the group consisting of plastic, silicon, ceramics, metals, composite materials and glass.

38. The system of claim 34, wherein the sealing layer and the other layers forming the microchannel manifold comprise different materials.

39. The system of claim 26 wherein the first and second substantially vertical components of the capillary tube fluid circuit are comprised of a material selected from the group consisting of plastic, silicon, ceramics, metals, composite materials, glass and fused silica.

40. The system of claim 39 wherein the first and second substantially vertical components of the capillary tube fluid circuit are comprised of fused silica.

41. The system of claim 39 wherein the first and second substantially vertical components of the capillary tube fluid circuit are comprised of a composite material including a layer of metal.

42. The system of claim 26 wherein the first and second electrodes are integral to the first and second substantially vertical components of the capillary tube fluid circuit.

43. The system of claim 26 wherein the first and second substantially vertical components of the capillary tube fluid circuit are fluidly connected to the substantially horizontal component by a friction fit with the substantially horizontal component.

44. The system of claim 26 wherein the first and second substantially vertical components of the capillary tube fluid circuit are fluidly connected to the substantially horizontal component by soldering.

45. The system of claim 26 wherein the first and second substantially vertical components of the capillary tube fluid circuit are fluidly connected to the substantially horizontal component with an adhesive.

46. The system of claim 1, further comprising a means for raising and lowering the capillary tube fluid circuit orthogonal to the plane of the first and second wells.

47. The system of claim 46 wherein the capillary tube fluid circuit is integrally formed with the means for raising and lowering the capillary tube fluid circuit.

48. The system of claim 46 wherein the means also raises the first and second electrodes.

49. The system of claim 48, wherein the electrodes and capillary tube fluid circuit are integrally formed with the means for raising and lowering the electrodes and capillary tube fluid circuit.

50. The system of claim 1, wherein the system comprises a plurality of separatory units.

51. The system of claim 50, further comprising an armature which holds the plurality of capillary tube fluid circuits each separatory unit in position above the first and second wells of each separatory unit.

52. The system of claim 51, further comprising a means for raising and lowering the armature, with the capillary tube fluid circuits of the separatory units, transverse to the plane of the liquid level in the first and second wells.

53. The system of claim 52, further comprising a means for positioning the armature relative to the first and second wells, so that the ends of the capillary tube fluid circuits of the separatory units are approximately centered in the first and second wells of the separatory units.

54. The system of claim 1 wherein at least one electrode is integrated into the microtiter plate.

55. The system of claim 54 wherein the first electrode is integrated into the microtiter plate.

56. The system of claim 54 wherein the second electrode is integrated into the microtiter plate.

57. The system of claim 1 wherein at least two wells in different separatory units share a common electrode.

58. The system of claim 1 wherein at least two separatory units share a common first well.

59. The system of claim 1 wherein at least two separatory units share a common second well.

60. A method for performing electro-kinetic analysis of a sample located in a microtiter plate well, utilizing the system of claim 1 , the method comprising the steps of : a) forming at least one separatory unit by placing a first end of a capillary tube fluid circuit and a first electrode into a first microtiter well containing a sample mixture in a liquid, and placing a second end of the capillary tube fluid circuit and a second electrode into a second microtiter well on the same microtiter plate as the first well, wherein the second microtiter well contains a liquid and the capillary tube fluid circuit contains a liquid; b) energizing the electrodes to apply a first electric field across the capillary tube fluid circuit, thereby loading the sample mixture into the capillary tube fluid circuit; c) removing the first and second ends of the capillary tube fluid circuit from the first and second sample wells; d) transferring the first and second ends of the capillary circuit to a third and a fourth well, respectively, wherein the third and fourth wells are both contained within a single microtiter plate, wherein the third and fourth wells are in electrical contact with a third and fourth electrode, respectively, and wherein the third and fourth wells contain a liquid; e) applying a second electric field across the fluidic circuit to effect separation of the sample mixture; and f) detecting the electrophoretic migration of at least one molecule of interest through the detection section of the capillary fluid circuit.

61. The method of claim 60 wherein at least one liquid is an aqueous buffer.

62. The method of claim 61 wherein the aqueous buffer is selected from the group consisting of: Tris hydrochloride buffers, Tris borate buffers, histidine buffer, sodium phosphate buffers, potassium phosphate buffers, and HEPES buffers.

63. The method of claim 60 wherein the first and second, and third and fourth electrodes are the same pair of electrodes.

64. The method of claim 60 wherein the first, second, third, and fourth wells are all contained within a single microtiter plate.

65. The method of claim 60 wherein the first and second wells are contained within a different microtiter plate than the third and fourth wells.

66. The method of claim 60 wherein the molecule of interest is detected by detecting the light emitted or transmitted through a transparent section of the capillary tube fluid circuit

67. The method of claim 60 wherein the first and second wells are adjacent to one another.

68. The method of claim 60 wherein the first electric field has a potential of approximately 10 to approximately 1000 volts/cm of capillary tube fluid circuit.

69. The method of claim 60 wherein the first electric field has a potential of approximately 50 to approximately 500 volts/cm of capillary tube fluid circuit.

70. The method of claim 60 wherein the first electric field has a potential of approximately 100 to approximately 300 volts/cm of capillary tube fluid circuit.

71. The method of claim 60 wherein the second electric field has a potential of approximately 10 to approximately 1000 volts/cm of capillary tube fluid circuit.

72. The method of claim 60 wherein the second electric field has a potential of approximately 50 to approximately 500 volts/cm of capillary tube fluid circuit.

73. The method of claim 60 wherein the second electric field has a potential of approximately 100 to approximately 300 volts/cm of capillary tube fluid circuit.

74. The method of claim 71 wherein the electrical current is applied for approximately 0.25 to four minutes.

75. The method of claim 60 wherein the detection in step (f) is by a method selected from the group consisting of fluorometry, luminometry, colorimetry, mass spectrometry, absorption spectroscopy, electrochemical detection, and radioactivity detection.

76. The method of claim 60 further comprising the step of pre-filling the capillary tube fluidic circuit with the liquid by pressure injection prior to step (a).

77. The method of claim 60 comprising the further step of flushing residual sample mixture remaining in the capillary tube fluid circuit subsequent to step (f).

78. The method of claim 77 wherein the flushing step is by pressure injection of the liquid through the capillary tube fluid circuit for a period of time.

79. The method of claim 77 wherein the flushing step is by electro-osmotic flow created by energizing the electrodes for a period of time after the sample has passed through the detection section of the capillary tube fluid circuit.

80. The method of claim 79 further comprising the steps of i) removing the capillary tube fluid circuit from the third and fourth wells; ii) inserting the ends of the capillary tube fluidic circuit into a fifth and sixth well, wherein the fifth and sixth wells contain the fluid, wherein the fifth and sixth wells are contained within a single microtiter plate, and wherein the fifth and sixth wells are in electrical contact with a fifth and sixth electrode, respectively; and iii) energizing the electrodes for a period of time to create electro-osmotic flow of the fluid through the capillary tube fluid circuit.

81. The method of claim 80 wherein the first and second, third and fourth, and fifth and sixth electrodes are the same pair of electrodes.

82. The method of claim 80 wherein the first, second, third, fourth, fifth, and sixth wells are all contained within a single microtiter plate.

83. The method of claim 80 wherein the fifth and sixth wells are contained within a different microtiter plate than the third and fourth or first and second wells.

84. The method of claim 60 wherein at least one electrode is integrated into the microtiter plate.

85. The system of claim 84 wherein the third electrode is integrated into the microtiter plate.

86. The system of claim 84 wherein the fourth electrode is integrated into the microtiter plate.

87. The method of claim 60 wherein at least two wells in different separatory units share a common electrode.

88. The method of claim 60 wherein at least two separatory units share a common third well.

89. The method of claim 60 wherein at least two separatory units share a common fourth well.

90. The method of claim 60 wherein the charged molecule of interest is the product of a substrate reaction wherein the net charge of a substrate is changed in the enzymatic reaction.

91. The method of claim 90 wherein the charged molecule of interest and the substrate both comprise a detectable labeling moiety.

92. The method of claim 91 wherein the labeling moiety is a fluorescent moiety.

93. The method of claim 90 wherein the method is capable of detecting the enzymatic conversion of at least 10% of the substrate.

94. The method of claim 90 wherein the method is capable of detecting the enzymatic conversion of at least 1.0% of the substrate.

95. The method of claim 90 wherein the method is capable of detecting the enzymatic conversion of at least 0.1% of the substrate.