AU2003232028A8 - Plastic microfluidics enabling two-dimensional separations of biological molecules - Google Patents

Description

WO 03/092846 PCT/US03/13580 PLASTIC MICROFLUIDICS ENABLING TWO-DIMENSIONAL SEPARATIONS OF BIOLOGICAL MOLECULES 5 FIELD OF THE INVENTION The invention relates to a microfluidic apparatus, system and method for performing two-dimensional (2-D) separations of biomolecular materials including DNA and protein separations. 10 BACKGROUND OF THE INVENTION A major goal of the Human Genome Project is to provide researchers with an optimal infrastructure for finding and characterizing new genes. The availability of 15 genetic and physical maps of the human genome may greatly accelerate the identification of human genes, including disease genes, and allow subsequent characterization of these genes. Once the genome maps and consensus sequences are obtained, the ability to assess individual variation may open the way to gene discovery and gene diagnosis. Such gene discovery programs may lead to new insights into the organization and 20 functioning of the human genome and its role in the etiology of disease, providing new and highly accurate diagnostic and prognostic tests. Ultimately, the availability of fully characterized genes encoding a variety of functions may provide the raw materials for novel gene therapies and rational drug discovery/design. Other benefits may be recognized. 25 Rapid and accurate identification of DNA sequence heterogeneity has been recognized as being of major importance in disease management. Comprehensive testing for gene mutational differences can provide diagnostic and prognostic information, which, in the context of integrated relational databases, could offer the opportunity for individualized, more effective health care. Practical examples include current attempts to 30 initiate pre-symptomatic testing programs by looking for mutations in genes predisposing to common diseases such as breast and colon cancer. A recent estimate for single-nucleotide polymorphism (SNP) due to single-base substitution in the genome approximates 1 SNP/1000 bp. Other types of SNP involve WO 03/092846 PCT/US03/13580 insertion and deletion and are found to occur at -1/12 kb. Thus far, nucleotide sequencing remains the gold standard for accurate detection and identification of mutational differences. However, large-scale DNA sequencing to detect mutations is not efficient because of the low frequency of SNP. Furthermore, the high costs involved in 5 sequencing have prompted the development of a large number of potentially more cost effective, alternative, pre-screening techniques. These include single-stranded conformation polymorphism (SSCP) and SSCP-derived methods, chemical or enzymatic mismatch cleavage, denaturing gradient gel electrophoresis (DGGE), matrix-assisted laser desorption/ionization mass spectrometry, 5'nuclease assay, single nucleotide primer 10 extension, and chip-based oligonucleotide arrays, among others. Two-dimensional (2-D) gel electrophoresis is a commonly used technique for separating proteins based on molecular weight and isoelectric point. This technique is also used for separating DNA molecules based on size and base-pair sequence for detecting mutations or SNPs. The 2-D format for DNA separation increases the number 15 of target fragments that can be analyzed simultaneously. 2-D DNA gel electrophoresis has been used to two-dimensionally resolve the entire E. coli genome and detect differences. DNA fragments can be resolved in two dimensions based on their differences in size and sequence. Sequence-dependent separation is typically achieved in the second dimension using DGGE. Apart from 20 nucleotide sequencing, DGGE is the only known method which offers virtually 100% theoretical sensitivity for mutation detection. Provided the sequence of the fragment of interest is known, DGGE can be simulated on the basis of the melting theory using a computer algorithm. By attaching a GC-rich fragment to one of the PCR (Polymerase Chain Reaction) primers, the target fragment can be designed so that it will always be the 25 lowest melting domain, providing absolute sensitivity to all kinds of mutations. It is known to combine 2-D DNA gel electrophoresis with extensive PCR multiplexing to produce a high resolution system known as a two-dimensional gene scanning (TDGS) system. TDGS systems can be used for detecting mutational variants in multiple genes in parallel. The resolving power of TDGS has been demonstrated for 30 several large human disease genes, including CFTR (cystic fibrosis transmembrane regulator gene), RB1 (retinoblastoma tumor suppressor gene), MLH1 (MutL protein 2 WO 03/092846 PCT/US03/13580 homolog 1), TP53 (p53 tumor suppressor gene), BRCA1 (breast and ovarian cancer susceptibility gene 1), and TSC1 (tuberous sclerosis complex gene 1), as well as for a part of the mitochondrial genome. To be suitable for true large-scale analysis, including for example, analysis of 5 essentially all human genes in population-based studies, a mutation scanning system should not only be accurate but also capable of operating at a high throughput in a cost effective manner. At present, 2-D DNA gel electrophoresis is relatively cost-effective in comparison with other mutation detection techniques. However, TDGS suffers from the fact that it is not a high-throughput platform for large-scale DNA analysis. Despite the 10 selectivity and sensitivity of conventional 2-D DNA analysis, this technique as practiced today is a collection of manually intensive and time-consuming tasks, prone to irreproducibility and poor quantitative accuracy. Microfluidic systems generally are known and are convenient for performing high-throughput bioassays and bioanalyses. One problem with existing systems is the 15 materials and fabrication procedures used in existing commercial microfluidic devices. Currently, the majority of devices are made from glass or silicon. These materials are often chosen, not because of their suitability for the applications at hand, but rather because the technology is readily transferable from established procedures. A limitation with glass or silicon-based microfluidic devices is the high cost of fabrication and the 20 brittleness of the material. Separations by DGGE are based on the fact that the electrophoretic mobility of a partially melted DNA molecule is greatly reduced compared to an unmelted molecule. When a mixture of molecules, differing by single base changes, is separated by electrophoresis under partially denaturing conditions, they display different states of 25 equilibrium between the unmelted DNA fragment and the partially melted form. The fraction of time spent by the DNA molecules in the slower, partially melted form varies among specific sequences. Less stable species move more slowly than the more stable ones in an electric field, resulting in efficient separation. The generation of a temperature gradient in a capillary via ohmic heat produced 30 by a voltage ramp over time is known, as is the use of DGGE in capillary electrophoresis. While these results have some favorable aspects, constructing the gradients is not quite 3 WO 03/092846 PCT/US03/13580 straightforward, particularly for the development of multiple-capillary arrays. Others have demonstrated a 96-capillary array electrophoresis system for screening SNP by surrounding the capillaries with thermal conductive paste and controlling the temporal temperature gradient through the use of an external heating plate. Various drawbacks 5 exist with these approaches. Another problem with microfluidic devices for 2-D DNA gel electrophoresis is the lack of convenient, effective methodology to transfer DNA molecules from a first dimension to a second dimension after separation of molecules in the first dimension. Microfluidic devices for 2-D DNA gel electrophoresis also suffers from the lack of a 10 convenient method or device for high throughput and high resolution second dimension separation. Current approaches using DGGE or other currently available gel based methods for this sequence-dependent separation in microfluidic devices have limitations in handling for high throughput purposes. In another aspect, existing protein analysis technology is largely based upon two 15 dimensional polyacrylamide gel electrophoresis (2-D PAGE), which has undeniably assumed a major role and is central to much of what is now described as "proteomics." Typically, proteins are separated by charge in a first dimension, based on isoelectric focusing in a pH gradient medium, and by size in a second dimension, based on molecular weight in a polyacrylamide gel containing sodium dodecyl sulfate (SDS). 20 When proteins are radiolabeled, or stained, their positions in the gel are detected by autoradiography, or densitometry, respectively. Despite the selectivity of 2-D PAGE, existing techniques are a collection of manually intensive procedures and time-consuming tasks prone to irreproducibility and poor quantitative accuracy. Thus, automated, high resolution, rapid, reproducible, and 25 ultrasensitive 2-D separation techniques would be advantageous for large-scale analysis of proteins. Microfluidic platforms offer fast, accurate, and low cost electrokinetic systems for high-throughput 2-D PAGE. One drawback of existing systems is a lack of methodology to detect protein separations in microchannels. Performance of the 30 isoelectric focusing and the size based separation can be monitored by detecting the proteins in microchannels. A robust detection system of proteins in microchannels, is not 4 WO 03/092846 PCT/US03/13580 only important for identification of proteins, but also important for quantification of proteins, with accuracy and resolution. Another drawback of the application of existing microfluidic techniques to 2-D PAGE devices is a lack of methods to introduce different separation media into different 5 dimensions in the same unit. Performing both charge and size based separations in one miniaturized 2-D PAGE device is desirable for high-throughput purpose. Another drawback of the application bf existing microfluidic techniques to 2-D PAGE devices is a lack of methods to transfer proteins simultaneously from first to second dimensions without significant loss in resolution. In existing methods, protein 10 analytes are continuously sampled in the first dimension and transferred to the second dimension. To date, sufficient resolution has not been achieved using existing methods. These and other drawbacks exist. SUMMARY OF THE INVENTION 15 One object of the invention is to overcome these and other drawbacks in existing systems and methods. One embodiment of the invention relates to a microfluidic apparatus for performing 2-D biomolecular separations. According to one aspect of the invention, after 20 a first dimension separation in a first microchannel, the sample material is electrokinetically and simultaneously transferred to an array of microchannels in the second dimension (e.g., by changing the electric potentials at the reservoirs connected to the microchannels). Preferably any separation accomplished in the first dimension is completely retained upon transfer to the second dimension. According to another aspect 25 of the invention, the separation in the second dimension is performed using a temperature gradient (e.g., a spatial or temporal temperature gradient). According to one embodiment of the invention, the biomolecular material comprises DNA and the first dimension separation is a sized-based separation and the second dimension separation is a sequence based separation. 30 According to another aspect of the invention, to automate and increase the throughput of 2-D DNA gel electrophoresis, a 2-D plastic microfluidic network is provided for rapidly and accurately resolving DNA fragments based on their differences 5 WO 03/092846 PCT/US03/13580 in size and sequence. The first dimension size-based separation may be performed in a known manner. Instead of continuously sampling DNA analytes eluted from the first size-separation dimension, one aspect of the invention relates to electrokinetically and simultaneously transferring the size-separated DNA fragments from the first dimension 5 (e.g., a microchannel extending from left to right and connecting first and second reservoirs) to a microchannel array between third (and in some embodiments) and fourth reservoirs for performing a sequence-dependent separation. Preferably, the electrokinetic transfer occurs simultaneously in each of the second dimension microchannels. Increased throughput can be achieved by rapid size-based separations (e.g., in the range of 0-200 10 seconds) followed by simultaneous transfer of size-separated DNA fragments together with parallel sequence-dependent separations in the second dimension. This simultaneous transfer approach also significantly simplifies the procedures compared to those involved in continuous sampling and separation of the eluants from the first dimension. 15 According to another aspect of the invention, instead of using denaturing reagents such as urea and formamide, DNA fragments (or other materials) in the second dimension are resolved by using a temporal or a spatial temperature gradient. Since the "melting" of DNA fragments is a function of base sequence with GC-rich regions being more stable than AT-rich regions, sequence differences between fragments may be 20 revealed as migration differences. Thus, the invention provides an automated, cost effective, high throughput, rapid, and reproducible 2-D microfluidic gene scanner. Ultrasensitive measurements of these DNA fragments may then be achieved with an integrated optical detection system (e.g., by using laser-induced fluorescence detection (LIFD) with the addition of intercalating dyes such as ethidium bromide and thiazole 25 orange in the electrophoresis buffer). This 2-D DNA separation platform can perform effectively with even minute DNA samples and enables automation and true system integration of size and sequence-dependent separations with real time fluorescence detection and imaging. According to one embodiment, the second dimension transfer and the second 30 dimension separation may occur by applying an electric field along the length of the one or more second-dimension microchannels while applying a temperature gradient, thereby 6 WO 03/092846 PCT/US03/13580 denaturing the biomolecules and further separating the biomolecules based on their migration time through the gel contained therein. According to some embodiments of the invention, various combinations and configurations of microchannels and reservoirs may be implemented to control 5 intersection voltages and enable advantageous separation techniques. For example, in addition to first and second dimension microchannels, other microchannels (e.g., tertiary) may be implemented to enable advantageous separation techniques. Likewise, voltage control microchannels may be implemented to enable advantageous manipulation of samples. In addition, other reservoirs, grouping of microchannels (e.g., parallel groups 10 feeding into respective reservoirs, multiple groups feeding into single, common microchannels, etc.) resistive elements and other configurations may enable advantageous sample separation and manipulation. According to one embodiment a spatial temperature gradient is formed along the length of the one or more second-dimension microchannels. According to another 15 embodiment, a temporal gradient is used. The temporal or spatial temperature gradient may be created using a variety of techniques including internal and external heat sources. One aspect of the invention relates to 2-D microfluidic networks formed in plastic substrates (e.g., using template imprinting technologies) and integration of this technology with the computerized design of PCR primers that generate a large number of 20 DGGE-optimized target fragments in one single reaction, i.e. a PCR multiplex. The combination of the high throughput and cost-effective 2-D microfluidic gene scanner with the principle of the PCR multiplex may enable an extensive parallel gene scanner for mutation detection in large human disease genes, for exploring human genetic variability in population-based studies, and for other purposes. This may facilitate genome typing of 25 human individuals, comprehensive mutation analysis, and other advantages.\ Direct detection of all possible DNA variations at high accuracy in a cost effective manner will allow for the identification of all possible variants of the multiple genes determining disease susceptibility, disease progression, and response to therapy (pharmacogenomics). 30 7 WO 03/092846 PCT/US03/13580 Another advantage of the invention is that the microfluidic 2-D device may comprise first and second planar substrates which include at least a first dimension microchannel extending in a first direction and an array of second dimension microchannels extending in a second direction, preferably, orthogonal to the first 5 dimension. The ends of at least some of the microchannels are in fluid communication with a plurality of reservoirs. The substrates may further comprise a number of microchannels and reservoirs. The reservoirs are in electrical communication with a plurality of electrodes and voltage power sources. The device enables two dimensional separations of proteins and other biomolecules. According to another aspect of the 10 invention, an isoelectric point based separation is enabled in a first dimension, and a size based separation in a second dimension. A further advantage of the invention is that it enables introduction of two different media in different microchannels of the same 2-D microfluidic device (e.g., a media for isoelectric point based separation, and a media for size based separation). In one 15 embodiment, a pressure filling technique may be used to introduce the two different media. Electroosmotic or other electrokinetic pumping may also be used to introduce the two different media. In some embodiments, a polymeric membrane sandwiched between the upper and the lower microchannels may serve as a hydrodynamic barrier, enabling the introduction of two different separation media in the upper and the lower microchannels. 20 Other filling approaches may be used. Another advantage of the invention is that it enables simultaneous transfer of proteins from first dimension microchannels to second dimension microchannels (e.g., by changing the electric potentials at the reservoirs connected to the microchannels). Any separation accomplished in the first dimension may be completely retained upon transfer 25 to the second dimension. In some embodiments, the transfer of material (e.g., proteins) from the first to the second dimension may be achieved by hydrodynamic pressure at the reservoirs connecting first dimension microchannels. In other embodiments, isoelectric focused proteins in the first dimension may be electrokinetically injected into the second dimension, by altering the electric potentials at the reservoirs connecting microchannels. 30 This simultaneous transfer approach also significantly simplifies the procedures 8 WO 03/092846 PCT/US03/13580 compared to those involved in continuous sampling and separation of the eluants from the first dimension. Another advantage of the invention is that it enables high resolution detection of proteins in microchannels. In one embodiment, proteins may be covalently labeled with 5 a florescent dye. During first and second dimension separations, the labeled proteins may be monitored using a florescent detector attached to the microfluidic system. In some embodiments, microchannels fabricated by polydimethylsiloxane (PDMS) substrates may be used which provide low florescence background during detection and enable better signal to background resolution. According to another embodiment of the invention, 10 laser induced florescent detection (LIFD) may be employed for the detection of SDS protein complexes using non-covalent, environment-sensitive, fluorescent probes. Another advantage of the invention is that it enables integration of 2-D microfluidic networks formed in plastic substrates (e.g., using template imprinting, injection molding, laser machining, or a combination of these technologies) with LIFD 15 and mass spectrometry detection for automation, high throughput, reproducibility, robustness, and ultrahigh resolution. These capabilities are advantageous for large-scale proteome analysis and for "differential display" of protein expressions under various physiological conditions. The microfluidic 2-D PAGE of the present invention may be advantageous for the 20 study of organisms having fully sequenced genomes, and may identify proteins (and their modifications in many cases) as well as provide quantitative measurements of expression levels. Other uses will be apparent. These and other features and advantages of the invention will be more fully appreciated from the detailed description of the preferred embodiments and the drawings 25 attached hereto. It is also to be understood that both the foregoing general description and the following detailed description are exemplary and not restrictive of the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS 30 FIG. 1 is a schematic of a microfluidic apparatus according to one embodiment of the invention. 9 WO 03/092846 PCT/US03/13580 FIG. 2A is a side view of a microfluidic apparatus according to one embodiment of theinvention. FIG. 2B is a front sectional view of a microfluidic apparatus according to one embodiment of the invention. 5 FIG. 3 illustrates electrokinetic transfer of DNA from first dimension to second dimension according to one embodiment of the invention. FIG. 4 is a schematic of a microfluidic apparatus with tertiary microchannels according to one embodiment of the invention. FIG. 5 is a schematic of a microfluidic apparatus with voltage control 10 microchannels according to one embodiment of the invention. FIG. 6 is a schematic of a microfluidic apparatus comprising a voltage control microchannel combined with second-dimension outlet reservoir according to one embodiment of the invention. FIG. 7 is a schematic of a microfluidic apparatus showing voltage control 15 microchannels intersecting other microchannels according to one embodiment of the invention. FIG. 8 is a schematic of a microfluidic apparatus showing grouping of tertiary or second-dimension microchannels according to one embodiment of the invention. FIG. 9 is a schematic of a microfluidic apparatus showing groups of tertiary or 20 second-dimension microchannels merging into single common microchannels according to one embodiment of the invention. FIG. 10 is a schematic of a microfluidic apparatus showing electrically resistive elements intersecting tertiary or second-dimension microchannels according to one embodiment of the invention. 25 FIG. 11 is a schematic of microfluidic 2-D PAGE device lacking second dimensional inlet reservoir according to one embodiment of the invention. FIG. 12 is a schematic microfluidic 2-D PAGE with a polymeric membrane strip for introduction of two different media according to one embodiment of the invention. FIG. 13 is a schematic of a laser-induced fluorescence detection setup for line-based 30 fluorescence detection in a second dimension of a microchannel array according to one embodiment of the invention. 10 WO 03/092846 PCT/US03/13580 DETAILED DESCRIPTION OF THE INVENTION According to an embodiment of the invention illustrated in FIG. 1, a microfluidic 5 2-D gel electrophoresis apparatus is provided. Microfluidic 2-D gel electrophoresis apparatus may comprise a first planar substrate 1 containing one or more first-dimension microchannels 3 for first dimension separation, as well as a second planar substrate 2 (bonded to first planar substrate 1) to provide enclosure for one or more second dimension microchannels 4 for second dimension separation. 10 According to one embodiment, the first-dimension microchannel 3 may extend in a first direction, while an array of one or more second-dimension microchannels 4 may extend from, or intersect with, the first-dimension microchannel 3 in a second direction. Preferably the second direction is orthogonal to the first direction. The first-dimension microchannel 3 may have a first end 3a and a second end 3b. Similarly, an array of one 15 or more second-dimension microchannels 4 may each have a first end 4a and a second end 4b. According to one embodiment the first end 4a of the one or more second dimension microchannels 4 may intersect the first-dimension microchannel 3 at various locations along the length of the first dimension microchannel. 20 According to one embodiment, as illustrated in FIG. 1, the apparatus may further comprise one or more reservoirs (5, 6, 7, 8) and voltage sources (V13, V14, V15, V16) associated with each of the reservoirs, respectively. For example, a first reservoir 5 may be in fluid communication with a first end 3a of the first microchannel 3, and a second reservoir 6 may be in fluid communication with a second end 3b of the first microchannel 25 3. Additionally, a third reservoir 7 may be in fluid communication with a first end 4a of each of the second dimension microchannels 4, and a fourth reservoir 8 may be in fluid communication with a second end 4b of the second dimension microchannels 4. In other embodiments, some of which are described herein, different configurations of microchannels and reservoirs may be used. Not all embodiments may use four 30 reservoirs. More or less may be used. According to one embodiment of the invention, the apparatus may further comprise one or more injection microchannels 30 (as illustrated in FIG. 4), wherein the 11 WO 03/092846 PCT/US03/13580 injection microchannels have a first end 30a and a second end 30b, and wherein the one or more injection microchannels 30 intersect the first-dimension microchannel 3 near the first end 3a of the first-dimension microchannel 3. According to another embodiment, the apparatus may further comprise a sample injection inlet reservoir 31 intersecting the 5 first end 30a of the injection microchannel 30, a sample injection outlet reservoir 32 intersecting the second end 30b of the injection microchannel 30, a first-dimension separation inlet reservoir 61 intersecting the first end 3a of a first-dimension microchannel 3 and a first-dimension separation outlet reservoir 62 intersecting a second end 3b of a first-dimension microchannel 3. As shown in FIG. 1, one or more second 10 dimension separation inlet reservoirs (e.g. reservoir 7) may intersect a first end 4a of the one or more second-dimension microchannels 4, and one or more second-dimension separation outlet reservoirs (e.g., reservoir 8) may intersect a second end 4b of the one or more second-dimension microchannels 4. According to one embodiment of the invention, the one or more reservoirs (5-8, 15 61, 62) may be formed in the first 1 or second 2 substrate, and a plurality of separation electrodes (9, 10, 11, 12) may be provided. A first end (indicated schematically) of separation electrodes (9-12) may be located in communication with the reservoirs 5-8, respectively. A second end (indicated schematically) of the separation electrodes 9-12 may be attached to one or more voltage sources (V13, V14, V15, V16). Likewise, one or 20 more of electrodes (9-12) may also be connected to ground potential (e.g., -0 Volts). As illustrated in FIG. 1, the device may comprise one or more inlet reservoirs (e.g. reservoir 5) and outlet reservoirs (e.g. reservoir 6) at the ends (3a, 3b) of the first microchannel 3, and one or more inlet reservoirs (e.g. reservoir 7) and one or more outlet reservoirs (e.g. reservoir 8) at the ends (4a, 4b) of the second dimension microchannels 4. 25 Other configurations may be used. For example, in one embodiment, the second ends 4b of the one or more second dimension microchannels 4 may terminate at one or more points between the first and second ends (3a, 3b) of the first dimension microchannel 3. In such embodiments, no second dimension inlet reservoir may be provided. In another embodiment, shown, for example in FIG. 4, one or more second 30 dimension separation outlet reservoirs 8 may intersect the second end 4b of the one or more second-dimension microchannels 4, and one or more tertiary inlet reservoirs 10 12 WO 03/092846 PCT/US03/13580 may intersect the first end 11 a of the one or more tertiary microchannels 11. The second end 1 lb of the one or more tertiary microchannels 11 may terminate at one or more points between the first 3a and second 3b ends of the first dimension microchannel 3, and the first ends 4a of the one or more second dimension microchannels 4 may terminate at 5 one or more points between the first and second ends 3a, 3b of the first dimension microchannel 3. In this embodiment, the one or more points at which the second ends (11 b) of the tertiary microchannels 11 and second dimension microchannels 4 terminate at the first dimension microchannel 3 may be staggered. Preferably, the number of tertiary microchannels 11 is equal to one more than the number of secondary 10 microchannels 4, and the one or more points at which the first ends 4a of the second dimension microchannels 4 terminate at the first dimension microchannel 3 are staggered from the one or more points at which the second ends 1 lb of the tertiary microchannels 11 terminate at the first dimension microchannel 3 by half the distance between adjacent tertiary microchannels 11. In this embodiment, the one or more second dimension 15 separation inlet reservoirs may be omitted. According to one embodiment, reservoirs (e.g., reservoirs 5, 6, 7, 8) may be filled with an electrolyte solution. The electrolyte solution may include a buffer (e.g, an electrophoresis buffer, or a salt solution). In some embodiments, the electrolyte solution may contain 1X TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA). The 20 electrolyte solution may also have a pH over a broad range of pH values, with a preferred pH ranging between 6 and 10, or more preferably with a pH of about 8-9. In some embodiments, microchannels (e.g., 3, 4) may have depth to width ratio of approximately 1:3. Other ratios and dimensions may be used. For example, microchannels with an average depth of 10 pn may have an average width of 30 Im. 25 However, both depth and width preferably range from 5 to 200 pn. For illustrative purpose, the width mentioned herein is from trapezoidal shaped microchannel cross sections. Other shapes for microchannel cross-sections may be used, for example rectangular, circular, or semi-circular cross-sections. The microchannels (e.g. 3, 4) can be any suitable length. A preferred length ranges from about 1 to about 10 cm. Other 30 lengths may be used. Some embodiments may have other microchannel dimensions for various applications. 13 WO 03/092846 PCT/US03/13580 Spacing between the intersections determines the size of the sample plug being introduced into the second dimension microchannel array 4. The extent of resolution loss during the transfer step is mainly dependent upon the spacing and focused protein bandwidth achieved from isoelectric focusing in the first dimension microchannel 3. 5 According to one embodiment, microchannels (e.g., 3, 4) may be filled with any suitable carrier ampholyte solution for first dimension separation and any gel solution preferably with SDS for second dimension separation of proteins. A preferred voltage for separation of proteins range from 100 V/cm to 1000 V/cm. A high voltage power supply may be attached to a second end of a selected number of the one or more separation 10 electrodes (e.g., electrodes 9-12). Due to the extremely large surface area to volume ratio of microchannels (e.g., 3, 4) for efficient heat dissipation, the application of high electric voltage enables rapid and excellent separation of proteins in the microfluidic network. In some embodiments, microfluidic device of the present invention is used for separating DNA, peptides, and other biological or chemical composites. 15 In some embodiments, a plastic substrate such as poly(methylmethacrylate) (PMMA) or polycarbonate may be used for fabrication of the microfluidic 2-D apparatus. In one embodiment, a polydimethylsiloxane (PDMS) layer combined with a rigid substrate is used for fabrication of the 2-D microfluidic apparatus. Non-plastic materials such as glass or silica may also be used to fabricate the 2-D microfluidic apparatus of the 20 present invention. In one embodiment, the grounding and separation electrodes may be formed from any suitable thin film metal deposited and patterned onto the first 1 and second 2 planar substrate. Additionally, the temporal or spatial temperature gradient may be created using a variety of techniques including internal and external heat sources. 25 According to one embodiment of the invention, one or more heating elements 17 may be affixed to an exposed outer surface of the first 1 or second 2 planar substrate for controlling the temperature of the substrates. According to another embodiment of the invention, as illustrated in FIGS. 2A, 2B, one or more heating elements 17 may be bonded between (or otherwise integrated with) the first 1 and second 2 planar substrates. 30 A nonconducting dielectric film 18 may also be placed between the heating elements 17 and the second planar substrate 2 containing one or more microchannels. The one or 14 WO 03/092846 PCT/US03/13580 more heating elements 17 may be shaped to provide a desired temperature distribution across the planar substrate (1, 2) when current is passed through the one or more heating elements 17. In some embodiments, the temperature gradient may comprise a temporal temperature gradient, wherein the one or more heating elements 17 may induce a constant 5 spatial temperature across the entire length and width of the one or more second dimension microchannels 4, and wherein the constant spatial temperature is varied with time. In other embodiments, a linear spatial temperature profile may be imposed along the length of the one or more second-dimension microchannels 4. Resistive heating of the one or more heating elements 17 may be used to produce 10 the desired temperature gradient. The heating elements may be made from any suitable material. Platinum may, for example, be used as a preferred heating element 17 material for imposing temperature gradient along microchannels. By using platinum heating elements 17, the local temperature may be monitored by measuring changes in resistance. Platinum may be replaced with other less expensive electrode materials with acceptable 15 temperature coefficients of resistance including, for example, thin film gold, metal foil, conductive polymer(s), conductive ink, electrically-conductive wire, or other materials. Other temperature control structures and techniques may be used. The spatial temperature gradient may vary from about 20-25 0 C at the intersection between the first dimension microchannel 3 and the one or more second-dimension 20 microchannel 4, to about 70-90 0 C at the second end 4b of the one or more second dimension microchannels 4. Alternatively, the spatial temperature gradient may vary from about 70-90 0 C at the intersection between the first dimension microchannel 3 and the one or more second-dimension microchannel 4, to about 20-25 "C at the second end 4b of the one or more second-dimension microchannels 4. The spatial temperature 25 gradient may be replaced by a temporal temperature gradient where the one or more heating elements 17 induces a constant spatial temperature across the entire length and width of the one or more second-dimension microchannel 4 and the constant spatial temperature is varied with time. The constant spatial temperature may be varied from an initial temperature of about 20-25 "C to a final temperature of about 70-90 "C. 30 Alternatively, the constant spatial temperature may be varied from an initial temperature of about 70-90 "C to a final temperature of about 20-25 "C. 15 WO 03/092846 PCT/US03/13580 In some embodiments, microchannels (e.g. 3, 4) may have depth to width ratio of approximately 1:3. Other ratios and dimensions may be used. For example, microchannels with an average depth of 10 pm may have an average width of 30 pm. However, both depth and width preferably range from 5 to 200 pm. For illustrative 5 purpose, the width mentioned herein is from trapezoidal shaped microchannel cross sections. Other shapes for microchannel cross-sections may be used, for example rectangular, circular, or semi-circular cross-sections. The microchannels (e.g. 3, 4) can be any suitable length. A preferred length ranges from about 1 to about 10 cm. Other lengths may be used. Some embodiments may have other microchannel dimensions for various 10 applications. The number of microchannels (e.g. 3, 4, 11) and the spacing therebetween, may be application dependent. The spacing between the second dimension microchannels 4 in the array may determine the size of the sample plug being introduced from the first to the second dimensions. The extent of resolution loss during the transfer step is in part 15 dependent upon the spacing and the DNA bandwidth achieved from size-based separation in the first dimension. Minimal resolution loss may be achieved as there may be no mixing during the electrokinetic transfer of DNA fragments. The number second dimension of microchannels in the array may also range from 10 to 1000, or more. Separation efficiency and resolution of DNA fragments may be dependent upon 20 the size-sieving polymer characteristics and the applied electric potential. According to one aspect of the invention, a preferred separation media for electrophoresis in microchannels (e.g. 3, 4) is 1X TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) containing 2% poly(ethylene oxide) (PEO). It should be noted that microchannels (e.g. 3, 4) may be filled with any other polymeric media for separating DNA, protein, 25 other biomolecules and chemical composites. According to one embodiment of the invention, a voltage source (V13, V14, V15, V16) may be attached to a second end (indicated schematically) of a selected number of the one or more separation electrodes (indicated schematically). Due to the extremely large surface area to volume ratio of microchannels for efficient heat dissipation, the 30 application of an electric field may enable rapid and excellent separation of DNA fragments in a microfluidic network. A preferred electric field for separating DNA 16 WO 03/092846 PCT/US03/13580 fragments in the present invention range from 100-1000 V/cm, however, other electric field strengths may be used. Various methods of operation may be implemented consistent with the objectives of the invention. According to one embodiment, as illustrated in FIG. 4, a method of 5 operation of the invention may include performing two-dimensional gel electrophoresis of biomolecules by applying a suitable electric field along the length of an injection microchannel 30. A sample stream containing the biomolecules of interest may be injected from the first end 30a of the injection microchannels 30 towards the second end 30b of the injection microchannel 30. A high voltage may be applied to an electrode (not 10 shown) disposed within the injection outlet reservoir 32, while a grounding voltage may be applied to an electrode (not shown) disposed within the injection inlet reservoir 31. All other reservoirs may be disconnected from any voltage source. This arrangement may cause the sample stream to cross through a portion of the first-dimension microchannel 3. By removing the high electric field within the injection microchannel 30 15 and applying a high electric field along the length of the first-dimension microchannel 3, biomolecules within the sample stream that crosses through the first-dimension microchannel 3 may be separated within the first-dimension microchannel 3 according to their migration time through the gel contained therein. This may result in separation of the biomolecules based on their size. By applying a high voltage to an electrode (not 20 shown) disposed within the first-dimension outlet reservoir (e.g., 6, 62), and by grounding an electrode (not shown) disposed within the first-dimension inlet reservoir (e.g., 5, 61) and disconnecting all other reservoirs from any voltage source, the separated sample stream may pass by the one or more second-dimension microchannels 4 intersecting with the first-dimension microchannels 3. The first-dimension separation 25 may be performed within the first-dimension microchannel 3 before transferring the separated sample stream past the one or more second-dimension microchannels 4 intersecting with the first-dimension microchannels 3, or first-dimension separation may be performed during this transfer process. According to an embodiment of the invention, further separation and denaturing 30 of the biomolecules may occur through the application of an electric field along the 17 WO 03/092846 PCT/US03/13580 length of the one or more second-dimension microchannels 4, while simultaneously applying a temperature gradient. According to one embodiment, a spatial temperature gradient may be formed along the length of the one or more second-dimension microchannels 4. A voltage may 5 be applied to an electrode (not shown) disposed within the second-dimension outlet reservoir 8, and a grounding voltage may be applied to the electrode disposed within the second-dimension inlet reservoir 7. Each of the remaining reservoirs may be disconnected from any voltage source. According to one embodiment of the invention, as illustrated in FIG. 3, a 10 relatively low voltage may be applied to the first-dimension outlet reservoir 6, while a grounding voltage may be applied to the first-dimension inlet reservoir 5. The one or more second-dimension inlet reservoirs 7 may be disconnected from any voltage source. Pursuant to this arrangement, when a relatively high electric field is applied along the length of the one or more second-dimension separation microchannels 4, a small electric 15 field may be simultaneously generated along the length of the first-dimension microchannel 3, thereby causing biomolecules to be drawn slightly towards the first dimension outlet reservoir to ensure efficient transfer of the biomolecules from the first dimension microchannel into the one or more second dimension microchannels 4. According to one embodiment of the invention, as illustrated in FIG. 4, a 20 grounding voltage may be applied to the one or more tertiary reservoirs 10, while a high voltage may be applied to the one or more second-dimension outlet reservoirs 8. All other reservoirs may be disconnected from any voltage source. Pursuant to this arrangement, a high electric field is applied along the length of the one or more second dimension separation microchannels 4, with said electric field passing from the one or 25 more tertiary microchannels 11 through the one or more regions of the first-dimension microchannel 3 between adjacent tertiary 11 and second-dimension microchannels 4, and into the one or more second-dimension microchannels 4, thereby causing biomolecules within the first-dimension microchannel 3 to be drawn into the one or more second dimension microchannels 4 to ensure efficient transfer of the biomolecules from the first 30 dimension microchannel 3 into the one or more second dimension microchannels 4. 18 WO 03/092846 PCT/US03/13580 According to another aspect of the invention, one or more intersection control voltages may be applied to the one or more second-dimension separation outlet reservoirs 8 or tertiary inlet reservoirs 10, as illustrated in FIG. 4, and the one or more second-dimension separation inlet reservoirs 7 (see FIG. 1). This may control the 5 electric field lines at the intersection of the one or more first-dimension separation microchannels 3 and the one or more second-dimension separation microchannels 4 in such a manner that the distribution of biomolecules undergoing separation during the first-dimension separation step are not substantially affected by the intersections. According to an embodiment, as depicted in FIG. 5, the one or more intersection 10 control voltages may be applied using a plurality of voltage sources, wherein one voltage source ( 35 and 37) may be applied to the one or more inlet reservoirs 35 of the one or more voltage control microchannels 36, and a second voltage source may be connected to the one or more outlet reservoirs 37 of the one or more voltage control microchannels 36 to generate a potential gradient along fluid within the one or more voltage control 15 microchannels 36. The geometry of the one or more voltage control microchannels 36 may be selected such that the intersection control voltage at the one or more intersections of the voltage control microchannels 36 and the second-dimension microchannels 4 and/or tertiary microchannels 11 is set by the voltages applied at the voltage control reservoirs (not shown in the figure). Further, the one or more intersection control 20 voltages may be chosen such that the voltage within the one or more second-dimension microchannels 4 and/or tertiary microchannels 11 near the intersection of the one or more first-dimension separation microchannels 3 and the one or more second-dimension separation microchannels 4 (connected to the reservoir at which the intersection control voltage is applied) is slightly different than the voltage within the intersection itself. In 25 this embodiment, the one or more tertiary inlet reservoirs 10 are omitted. According to another aspect of the invention, depicted in FIG. 6, a single voltage control microchannel 36 may be combined with a second-dimension outlet reservoir 8. According to another aspect of the invention, depicted in FIG. 7, one or more voltage control microchannels 36 may intersect the one or more tertiary microchannels 30 11, and one or more voltage control microchannels 36 may intersect the one or more second-dimension microchannels 4. 19 WO 03/092846 PCT/US03/13580 According to another aspect of the invention, depicted in FIG. 8, groups of one or more tertiary microchannels 11 may intersect one or more tertiary inlet reservoirs 10. Similarly, groups of one or more second-dimension microchannels 4 may intersect one or more second-dimension outlet reservoirs 8 5 According to another aspect of the invention, depicted in FIG. 9, groups of one or more tertiary microchannels 11 may merge into a single common tertiary microchannel 52, which intersects the one or more tertiary inlet reservoirs 10. Similarly, groups of one or more second-dimension microchannels 4 may merge into a single common second dimension microchannel 51, which intersects the one or more second-dimension outlet 10 reservoirs 8. According to one embodiment, the one or more intersection control voltages may be applied using a plurality of voltage sources, wherein one voltage source may be connected to the first end of a first resistive element, and a second voltage source may be connected to the second end of the first resistive element to generate a potential gradient 15 along the first resistive element. The resistive element may placed in electrical contact with the one or more second-dimension separation inlet reservoirs such that the intersection control voltage in each reservoir is set by the voltage of the first resistive element at the point of electrical contact. Further, the one or more intersection control voltages may be chosen such that the voltage near the intersection of the one or more 20 first-dimension separation microchannels 3 and the one or more second-dimension separation microchannels 4 (connected to the reservoir at which the intersection control voltage is applied) is slightly different than the voltage within the intersection itself. A third voltage source may be connected to the first end of a second resistive element, and a fourth voltage source may be connected to the second end of the second 25 resistive element to generate a potential gradient along the second resistive element. The second resistive element may then be placed in electrical contact with the one or more second-dimension separation inlet reservoirs, such that the intersection control voltage in each reservoir is set by the voltage of the second resistive element at the point of electrical contact. The one or more intersection control voltages may be chosen such that 30 the voltage near the intersection of the one or more first-dimension separation microchannels 3 and the one or more second-dimension separation microchannels 4 20 WO 03/092846 PCT/US03/13580 (connected to the reservoir at which the intersection control voltage is applied) is slightly lower than the voltage within the intersection itself. According to another aspect of the invention, depicted in FIG 10, one or more electrically-resistive elements (42, 43) such as a thin-film metal, wire, conductive 5 polymer, or similar material may intersect the one or more tertiary microchannels 11 and the one or more second-dimension microchannels 4, with the one or more resistive elements (42, 43) in electrical contact with the fluid within the microchannels. One or more voltage sources (V44, V45, V46, V47) are applied at each end of the one or more resistive elements (42, 43), thereby creating a voltage drop along the length of the 10 resistive elements (42, 43). Since the one or more resistive elements (42, 43) are in electrical contact with the fluid at the points of intersection with the microchannels, the local voltage at each point in the microchannel may be controlled in this manner, with the voltages defined by the one or more voltage sources (V44, V45, V46, V47) and the resistance of the one or more resistive elements (42, 43). 15 In at least some embodiments of the invention, temperature-gradient gel electrophoresis (TGGE) may be used instead of DGGE. In TGGE, instead of a denaturing gradient along the gel, a spatial or temporal temperature gradient is used to perform the same function. Since the "melting" of DNA fragments is a function of base sequence with GC-rich regions being more stable than AT-rich regions, sequence 20 differences between fragments will be revealed as migration differences. Ultrasensitive measurements of these DNA fragments may be performed by using LIFD with the addition of intercalating dyes such as ethidium bromide and thiazole orange in the electrophoresis buffer. Other optical techniques may be used. According to one embodiment of the present invention, a method to integrate 25 electrodes into plastic substrates for imposing temperature gradient is provided. Integrating the electrodes directly into the microfluidic device may significantly reduce the overall size and cost of the device. In addition, by heating the fluidic channels directly, the thermal mass associated with external heating elements may be eliminated, resulting in faster thermal time constants, and more rapid, overall separation speeds. 30 A preferred method of electrode integration may be realized by depositing evaporated and/or sputtered platinum films on a polycarbonate plastic substrate, followed 21 WO 03/092846 PCT/US03/13580 by a lamination of a thin plastic layer atop the metallized plastic to prevent direct contact between the thermal electrodes and separation samples. In some embodiments, bulk wires and/or foil may be integrated into the plastic substrate using a hot embossing technique. In one embodiment of the invention, the 5 electrodes may be isolated from the separation channels preferably by a thin polydimethylsiloxane (PDMS) or by any laminated plastic layer, to prevent modification of microchannel surface chemistry. According to one embodiment of the invention, performance of the fabricated microchannel devices with integrated temperature-control electrodes is assessed by 10 coating the topside of the channels with commercially-available microencapsulated thermochromic liquid crystals, which change colors with variations in temperature. The one or more separation electrodes may include a thin film metal deposited and patterned onto first or second planar substrate. While the first 1 and second 2 planar substrates made be made from various 15 materials, including, glass or silicon, various advantages may be obtained from the use of plastic, e.g., polycarbonate plastic. One of the advantages of the use of plastic substrates in the present invention is that it may not suffer from the adverse effects of sample leakage at channel junctions caused by diffusion and unwanted electro-osmotic flows. Sample leakage at channel junctions has been one of the problems in microfluidic 20 devices. These leakages are primarily caused by the combined effects of sample diffusion and undesired electroosmotic flows. Plastic substrates used in the present invention are relatively hydrophobic and exhibit smaller electroosmotic flow than silica and others due to their lack of significant surface charge. It should be noted that microfluidic 2-D electrophoresis device can also be made up of glass, silicon or any other combination of 25 dissimilar materials including glass, PDMS, plastic, and silicon. PDMS may have some particular advantages. PDMS is optically transparent at the wavelengths required for the fluorescence detection of DNA fragments. The low background fluorescence associated with PDMS may offer a better substrate than many other plastic materials for fluorescence detection. In addition, the PDMS substrate 30 containing the microfluidic network is oxidized in an oxygen plasma. The plasma introduces silanol groups (Si-OH) at the expense of methyl groups (Si-CH 3 ). These 22 WO 03/092846 PCT/US03/13580 silanol groups may then condense with appropriate groups (OH, COOH, ketone) on another surface when the two PDMS layers are brought into conformal contact. Oxidized PDMS also seals irreversibly to other materials, including glass, Si, SiO 2 , quartz, silicon nitride, polyethylene, polystyrene, and glassy carbon. Oxidation of the PDMS has the 5 additional advantage that it renders the surface hydrophilic because of the presence of silanol groups. These negatively charged channels have greater resistance to adsorption of hydrophobic and negatively charged analytes (i.e. DNA fragments) than unmodified PDMS. According to one aspect of the invention, the 2-D plastic microfluidic device 10 separates protein analytes with ultrahigh resolution based on their differences in isoelectric point and size. As illustrated in FIG. 1, one or more microchannels 3 extending from left to right in the figure and connecting one or more reservoirs 5 and 6 may be employed for performing a non-native isoelectric focusing separation in a first dimension. Once the first dimension separation is complete, the material (e.g. proteins) 15 may be transferred into a microchannel array connecting one or more reservoirs 8 for performing a parallel and high throughput size-dependent separation. The transfer to the second dimension may occur virtually simultaneously in each microchannel of the second dimension array of microchannels 4. In some embodiments, one or more reservoirs 10 (as illustrated in FIG. 4) at the end of one or more tertiary microchannels 11 may be used 20 for introducing the electrophoresis buffer containing SDS and SYPRO fluorescent dyes during electrokinetic transfer of proteins from first dimension to second dimension. To monitor the performance of isoelectric focusing in first dimension microchannels 3, proteins may be covalently labeled with a suitable florescent dye and detected by a suitable florescence detector. An example of a fluorescent dye is 5-carboxy 25 fluorescein succinimidyl ester which may be used to label the proteins. Other dyes or labeling techniques may be used. An example of a detector is a Zeiss fluorescence microscope. Other detectors and detection techniques may be used. Because of the large number of amino groups on protein molecules, very small amounts of fluorescein dye is needed. These labeled proteins may be denatured and reduced at approximate 30 concentration of 1 mg/ml for each protein. The proteins may be prepared in a suitable solution (e.g., a solution including urea, dithiothreitol, and Tris-HCl with approximate 23 WO 03/092846 PCT/US03/13580 concentrations of 8M, 100 mM and 0.1 M respectively) with a pH preferably ranging from 4 to 10. The protein solution may be kept under a nitrogen atmosphere for about four hours in room temperature. The denatured and reduced proteins may be desalted using a column preferably by PD-10 column. Eluted proteins may be dried preferably by 5 vacuum and stored at a preferred temperature of about -20 *C. These proteins may be reconstituted in the solution containing carrier ampholytes (approximately 2% pharmalyte 3-10) and urea (between 1 and 3 M) for performing non-native isoelectric focusing. Plastic microchannels made out of PDMS substrate are advantageous for isoelectric focusing because they are optically transparent at the wavelengths required for 10 the fluorescence detection of proteins and they provide low fluorescence background. Other materials may be used. According to one aspect of the invention, two different media are introduced in a 2-D microfluidic network for performing two dimensional separations of proteins. Isoelectric focusing in the first dimension involves the use of carrier ampholytes for the 15 creation of pH gradient in the microchannel. However, size-dependent separation of SDS-protein complexes in the second dimension is based on their differences in electrophoretic mobility inside a polymer sieving matrix. In one embodiment, a pressure filling approach may be used for introducing two different media into the at least one first dimension microchannel and array of second 20 dimension microchannels respectively. Gel solutions may be introduced into microchannel arrays (e.g., 3, 4) by applying pressure in reservoirs. As illustrated in FIG. 11, reservoir 8 and the connecting microchannel array 4 may be filled with a polymer gel solution. Various polymers, including polyacrylamide, polyethylene oxide, and branched dextran, can be employed for preparing the gel solution. The gel solution may be 25 introduced into the array by applying pressure at reservoir 8. The flow velocity may be controlled by the pressure gradient, the viscosity of the solution, and the channel dimensions. The filling process may be monitored by adding a preferred fluorescent dye, ethidium bromide (excitation: 514 nm; emission: 604 nm), into the gel solution and using a preferred fluorescent detector, Zeiss fluorescence microscope, equipped with a 30 computer controlled moving stage. The filling may be stopped as soon as the gel solution reaches the intersections of the microchannel array and the channel connecting reservoirs 24 WO 03/092846 PCT/US03/13580 5 and 6. The solution containing carrier ampholytes for isoelectric focusing separation may then be introduced via pressure from reservoir 5 for filling the channel connecting reservoirs 5 and 6. Because of the low viscosity in the ampholyte solution, the pressure needed for introducing the ampholyte solution is much lower than that required for 5 pushing a polymer gel solution. Thus, a 2-D plastic microfluidic network may be filled with two different separation media using this two-step pressure filling approach. It should be noted that any suitable fluorescent dye may be added to gel and any suitable detector may be used to monitor the filling process. In another embodiment, the entire plastic microfluidic network may initially be 10 filled with a polymer gel solution by pressure. The pressure level required for filling the microfluidic network depends upon the cross-sectional dimensions and lengths of the microchannels, in addition to the viscosity of the gel solution. In the case of embodiments where the bonding strength between the top and bottom layers of the device is not sufficient to hold the device together during high-pressure filling, an external force 15 may be applied to hold the layers together during the filling process. The filling of the microfluidic network is followed by removal of the polymer gel in the microchannels connecting reservoirs 5 and 6 as illustrated in FIG. 11. Removal of gel in the first dimension microchannel may be achieved by applying a pressure to reservoir 5 while leaving reservoir 6 open, and closing all other reservoirs in the device to prevent loss of 20 gel from the second-dimension microchannels. In another embodiment, removal of the gel is achieved using an electroosmotic process. The electroosmotic removal process may be induced by applying a positive electric voltage ranging from 1 to 10 kV at reservoirs 5 containing a preferred salt, NaOH, solution. The speed and completeness of electroosmotic removal approach may be monitored by adding preferred dye ethidium 25 bromide into the gel solution and using a preferred fluorescence detector Zeiss fluorescence microscope equipped with a computer controlled moving stage. The removal speed is expected to increase as the section of microchannel containing NaOH instead of polymer gel solution increases. As soon as the removal process is complete, the solution containing any carrier ampholytes for the isoelectric focusing may then be 30 introduced via pressure from reservoir 5 for filling the first dimension channel connecting reservoirs 5 and 6. 25 WO 03/092846 PCT/US03/13580 In yet another embodiment, a preferred polymeric membrane, polyvinylidene fluoride (PVDF), sandwiched between the upper and lower microchannels may serve as a hydrodynamic barrier, providing the initial filling of two different separation media in the upper and lower microchannels. As illustrated in FIG. 12, 2-D protein separation 5 platform may comprise the upper substrate (A) containing two or more reservoirs (5 and 6) for isoelectric focusing in the upper channel 3 and two or more reservoirs (7 and 8) for size based separation in the lower channel array 4, one or more microchannels 3 for performing isoelectric focusing separation, a polymeric membrane strip (B), and the lower substrate containing a microchannel array 4 for performing size-based separation 10 (C). In FIG. 12, D illustrates the assembly of 2-D protein separation platform. According to one aspect of the present invention, the pressure needed for pushing a solution through the membrane with a pore diameter of approximately 0.1 ptm may be at least 50 times higher than that required for introducing an aqueous solution into the microchannels. Furthermore, the solutions may take a lower flow-resistance path. Thus, 15 two different electrophoresis media may be separately filled in the upper and lower microchannels. According to another aspect of the present invention, the membrane (B) at the intersections between the upper and the lower microchannels may also serve as injection ports when pressure applied at both ends of the upper microchannel for transferring proteins from the first to the second dimension. In analogy to a dead-end 20 microfiltration process, the focused protein zones in the upper channel may simultaneously permeate through the exposed membrane areas at the nearby intersections. The methods of the present invention for introducing two different media in the same microfluidic device may also be used for other media in two dimensions for 25 separating DNA, peptides, and other chemical and biological composites. According one aspect of the present invention, focused proteins in the first dimension are simultaneously transferred to the second dimension by hydrodynamic pressure. To fulfill the requirements of a comprehensive 2-D separation system, the two dimensions should be orthogonal, and any separation accomplished by the first dimension 30 should ideally be retained upon transfer to the second dimension. First dimensional microchannels 3 connecting reservoirs 5 and 6 (as illustrated in FIG. 11) may be 26 WO 03/092846 PCT/US03/13580 disconnected from the high voltage power supply as soon as the focusing is complete in the first dimension. Equal pressure may then be applied at reservoirs 5 and 6 to drive the solution containing focused protein analytes into the parallel microchannel array. Since the flow resistance in the gel filled microchannel array is extremely high, a constant 5 hydrostatic pressure may be therefore in place across the entire isoelectric focusing channel during this step. Thus, the transfer process may be similar to dead-end membrane filtration. In some embodiments, a highly viscous polymer gel solution at the intersections between the first and second separation dimensions may serve as the individual injection ports for quantitative transfer of focused protein bands. In other 10 embodiments, a polymeric membrane serves as the injection ports for transfer of proteins. In another aspect of the present invention, focused proteins in the first dimension (e.g., 3) may be electrokinetically transferred to the second dimension (e.g. 4) using end reservoirs (e.g., 5, 6). As soon as the focusing is complete in the first separation dimension, the voltage may be turned off and the solution containing catholyte in 15 reservoir 6 (as illustrated FIG. 11) may be replaced with a Tris buffer containing SDS and a florescent dye, preferably, SYPRO orange. A positive electric potential may then be reapplied briefly at reservoir 5 for rapid electrokinetic injection and filling of SDS and dye within the first separation dimension (e.g., 3), followed by the incubation of focused proteins with SDS and dye for approximately 5-10 minutes. Since the focused proteins 20 from the first separation dimension (e.g., 3) (non-native isoelectric focusing) may be already denatured and reduced, the rapid formation of SDS-protein complexes may not only prepare protein analytes for size based separation in the second dimension (e.g., 4), but may also establish the foundation for performing electrokinetic protein transfer. The SDS-protein complexes may be electrokinetically injected by grounding reservoir 5 while 25 applying two separate positive potentials at reservoir 6 and 8 using two high voltage power supplies (V14, V16). The smaller positive potential at reservoir 6 may be employed to continuously drive the proteins in the first dimension toward the channel junctions. As soon as the proteins reach the channel junctions, the larger positive potential at reservoir 8 may commence the electrokinetic injection of focused proteins 30 into the second separation dimension. 27 WO 03/092846 PCT/US03/13580 In yet another aspect of the present invention, focused proteins in the first dimension may be electrokinetically transferred to the second dimension using tertiary reservoirs 10 as illustrated in FIG. 4. This method may involve the use of a set of tertiary microchannels 11 to introduce SDS and a florescent dye, preferably, SYPRO orange. In 5 this method, as soon as the focusing is complete in the first separation dimension (e.g., 3), the voltage may be turned off and the solution in the one or more reservoirs labeled 10 (as illustrated in FIG. 4) may be replaced with a Tris buffer containing SDS and dye. With the potentials at reservoirs 5 and 6 floating, a positive electric potential may then be applied briefly at reservoir 8 for rapid electrokinetic injection and filling of SDS and dye 10 from the tertiary microchannels 11 leading from reservoir 10, through the portion of the first-dimension separation channels 3 between the tertiary microchannels 11, and partially into the second dimension separation channels 4. Once this portion of the microchannel network may be filled, the proteins focused in the first dimension may be incubated with newly introduced SDS and dye for approximately 5-10 minutes. Since the 15 focused proteins from the first separation dimension (non-native isoelectric focusing) may be already denatured and reduced, the rapid formation of SDS-protein complexes may not only prepare protein analytes for the size-based separation in second dimension, but may also establish the foundation for performing electrokinetic protein transfer. After incubation, the SDS-protein complexes may be electrokinetically injected by grounding 20 reservoir 10 while applying a positive potential at reservoir 8 and letting reservoirs 5 and 6 float. Because each tertiary microchannel 11 may intersect the first-dimension microchannel in between two adjacent second-dimension microchannels 4, the applied field may force the majority of SDS-protein complexes that lie within the first dimension microchannel 3 between the extent of the second-dimension microchannels 4 to be 25 transferred into the second dimension (e.g., 4) electrokinetically. In some embodiments, electrokinetic transfer method may be performed to transfer DNA, peptides, and other chemical or biological composites from one dimension to another dimension of the gel electrophoresis system. As used herein, "electrokinetic transfer method" includes a method or a system which transfer materials from a channel 30 and/or chamber containing structure in one dimension to similar structures in other 28 WO 03/092846 PCT/US03/13580 dimensions, through the application of electric fields to the materials, thereby causing the transfer of the materials. One of the objects of the present invention is integration of the 2D microfluidics platform with an ultrasensitive (e.g. LIFD) system for the simultaneous and multi 5 channel monitoring of DNA fragments near the end of the second-dimension microchannel array. As shown in FIG. 13, excitation of the intercalated ethidium bromide is performed by the argon ion laser 21 (e.g. tuned to 514 nm). One molecule of ethidium bromide, present in the electrophoresis buffer, intercalates at every 4-5 base pairs of double-stranded DNA. Upon intercalation, the quantum yield of ethidium 10 bromide increases 20-30 fold while its fluorescence emission blue-shifts from 604 nm to 590 nm. The output beam from the laser is diverged, collimated to span the entire second dimension microchannel array, and focused vertically in a narrow line across the array. For example, in one embodiment, this is achieved by directing the laser beam (e.g., with a mirror 22) to a (diverging) 2.5 cm focal length plano-concave cylindrical lens 23 in series 15 with a (collimating) 10 cm focal length plano-convex cylindrical lens 24 and a (focusing) 5.0 cm focal length plano convex cylindrical lens 25, respectively. The fluorescence in each channel of the array is independently monitored using a charged-coupled device (CCD) camera 27 with a 50 mm macro Nikon camera lens. The CCD sensor is comprised of 26 gm pixels positioned in a 1024 x 128 array. The system is arranged so that a single 20 column of pixels on the sensor is designated to measure the fluorescence intensity emitted from each individual channel over time. A 532 nm rejection band filter (OD > 6) is used in series with a 595 nm bandpass emission filter to eliminate laser scatter. The 2-D DNA separation platform of the present invention may require only minute DNA samples, and may enable automation and true system integration of size and 25 sequence dependent separations with real-time fluorescence detection and imaging. In some embodiments, microfluidic 2-D DNA gel device of the present invention may be integrated with PCR based multicolor detection system that will allow multiplexing mutation detections for multiple genes by using different dye-labeled primers in a known manner. The techniques in this system may require automated sample 30 preparation for nucleic acid extraction (from blood, tissue, etc.), purification/isolation, amplification, digestion, and tagging. 29 WO 03/092846 PCT/US03/13580 One of the advantages of the present invention is integration of a optical source device and an image capturing device for monitoring the detection of SDS-protein complexes near the end of second dimension microchannel array using non-covalent, environment-sensitive, fluorescent probes. 5 For example, florescent probes such as SYPRO orange (excitation: 470 nm, emission: 570 nm) or SYPRO red (excitation: 550 nm, emission: 630 nm) may be employed for pre-electrophoretic staining of proteins. As shown in FIG. 13, an argon ion laser 21 may be used for excitation. The output beam from the laser is diverged, collimated to span the entire second dimension microchannel array, and focused 10 vertically in a narrow line across the array. This is achieved using a (diverging) 2.5 cm focal length plano-concave cylindrical lens 23 in series with a (collimating) 10 cm focal length plano-convex cylindrical lens 24 and a (focusing) 5.0 cm focal length plano convex cylindrical lens 25, respectively. The fluorescence in each channel of the array is independently monitored using a charged-coupled device (CCD) camera 27 with a 50 15 mm macro Nikon camera lens. The CCD sensor is comprised of 26 pm pixels positioned in a 1024 x 128 array. The system is arranged so that a single column of pixels on the sensor is designated to measure the fluorescence intensity emitted from each individual channel over time. A holographic notch filter may be located in front of the image capturing device to filter out any laser scattering. It should be noted that any suitable 20 detecting devices and image capturing devices known to one skilled in the art can be used with microfluidic 2-D PAGE system for monitoring the protein separations in the microchannel array. According to one aspect of the present invention, microfluidic 2-D PAGE may be integrated with mass spectrometry employing matrix-assisted laser desorption/ionization 25 (MALDI) and electrospray ionization (ESI) for integrating protein separation, quantification, identification, and sequencing processes. Electrospray ionization may be integrated into the microfluidic network by extending the second-dimension microchannels to the outer edge of the device to form electrospray tips, or by combining traditional silica capillary electrospray tips into the microfluidic system. The integrated 30 system offers large-scale analysis of proteins and "differential display" proteomics for 30 WO 03/092846 PCT/US03/13580 comparisons of protein expression under various environmental and physiological conditions. Other embodiments, uses and advantages of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the 5 invention disclosed herein. The specification should be considered exemplary only, and the scope of the invention is accordingly intended to be limited only by the following claims. 31

Claims (151)

1. A microfluidic apparatus for performing two-dimensional biomolecular separations, 5 the apparatus comprising: at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; 10 means for transferring the separated sample to the microchannels of the array of second dimension microchannels; means for performing a second separation in the second dimension microchannels, where the second separation is performed by applying a temperature gradient. 15

2. The apparatus of claim 1 wherein the temperature gradient comprises a spatial temperature gradient.

3. The apparatus of claim 1 wherein the temperature gradient comprises a temporal temperature gradient. 20

4. The apparatus of claim 1 further comprising internal heating means for producing the temperature gradient.

5. The apparatus of claim 1 further comprising external heating means for producing 25 the temperature gradient.

6. The apparatus of claim 4 wherein the internal heating means comprises electrodes embedded within the apparatus. 32 WO 03/092846 PCT/US03/13580

7. The apparatus of claim 1 wherein one or more heating elements is affixed to an exposed outer surface of a planar substrate, and by which the temperature of the substrate may be controlled. 5

8. The apparatus of claim 1 wherein one or more heating elements is bonded between a first and second planar substrates, and wherein the one or more heating elements is shaped to provide a desired temperature distribution across the first and second planar substrates when current is passed through the one or more heating elements. 10

9. The apparatus of claim 8 wherein the one or more heating elements comprises thin film gold.

10. The apparatus of claim 8 wherein the one or more heating elements comprise metal foil. 15

11. The apparatus of claim 8 wherein the one or more heating elements comprise conductive polymer.

12. The apparatus of claim 8 wherein the one or more heating elements comprise 20 conductive ink.

13. The apparatus of claim 8 wherein the one or more heating elements comprise an electrically-conductive wire. 25

14. The apparatus of claim 1 wherein a nonconducting dielectric film is located between a heating element and a second planar substrate containing one or more second dimension microchannels.

15. The apparatus of claim 1 further comprising one or more separation electrodes 30 wherein the one or more separation electrodes comprise a thin film metal deposited and patterned onto a planar substrate. 33 WO 03/092846 PCT/US03/13580

16. The apparatus of claim 1 further comprising one or more separation electrodes wherein the one or more separation electrodes comprise electrically-conductive wires positioned between a first and second planar substrates. 5

17. The apparatus of claim 1 wherein the biomolecular separation is performed on a biomolecular material and the biomolecular material comprises DNA, and wherein a first dimension separation is a size-based separation and a second dimension separation is a sequence-based separation. 10

18. The apparatus of claim 17 wherein the first dimension separation is substantially retained upon transfer to the second dimension.

19. The apparatus of claim 17 wherein the first-dimension separation medium 15 comprises a gel solution.

20. The apparatus of claim 17 wherein the second-dimension separation medium comprises a gel solution. 20

21. The apparatus of claim 19 wherein the gel solution is a sieving matrix selected from the group consisting of: cross-linked polyacrylamide, linear polyacrylamide, polydimethylamide, N-acrylamoniethoxyethanol, hydroxyethylcellulose [HEC], poly(ethylene glycol), poly(ethylene oxide) [PEO], poly(vinylpyrrolidone) [PVP], or nonionic polymeric surfactants (n-alkyl polyoxyethylene ethers). 25

22. The apparatus of claim 20 wherein the gel solution is a sieving matrix selected from the group consisting of: cross-linked polyacrylamide, linear polyacrylamide, polydimethylamide, N-acrylamoniethoxyethanol, hydroxyethylcellulose [HEC], poly(ethylene glycol), poly(ethylene oxide) [PEO], poly(vinylpyrrolidone) [PVP], or 30 nonionic polymeric surfactants (n-alkyl polyoxyethylene ethers). 34 WO 03/092846 PCT/US03/13580

23. The apparatus of claim 1 further comprising a detector placed near a second end of the array of second-dimension microchannels for monitoring the separated biomolecules.

24. The apparatus of claim 1 further comprising measurement means for monitoring 5 DNA fragments resolved from the second separation dimension.

25. The apparatus of claim 1 further comprising an integrated optical detection system.

26. The apparatus of claim 1 further comprising an integrated laser-induced 10 fluorescence detection system.

27. The apparatus of claim 1 further comprising an integrated laser-induced fluorescence detection system capable of simultaneously monitoring each second dimension microchannels in the array of second-dimension microchannels. 15

28. The apparatus of claim 1 wherein first ends of the second-dimension microchannels terminate at the at least one first-dimension microchannel at one or more points between the first and second ends of the at least one first-dimension microchannel, and wherein an outlet reservoir is in fluid communication with the second ends of the second-dimension 20 microchannels.

29. The apparatus of claim 1 wherein the second-dimension microchannels have first and second ends and the at least one first dimension microchannel intersects the second dimension microchannels at a position somewhere between the first and second ends of 25 the second-dimension microchannels.

30. The apparatus of claim 29 wherein an inlet reservoir is in fluid communication with the first end of the second dimension microchannels and an outlet reservoir is in fluid communication with the second end of the second dimension microchannels. 30 35 WO 03/092846 PCT/US03/13580

31. The apparatus of claim 29 wherein the first ends of the second-dimension microchannels terminate at the at least one first-dimension microchannel and further comprising an array of tertiary microchannels, wherein a second end of the tertiary microchannels terminate at the at least one first-dimension microchannel. 5

32. The apparatus of claim 31 wherein the points at which the second-dimension microchannels intersect with the at least one first-dimension microchannel are staggered with respect to the points at which the tertiary microchannels intersect with the at least one first-dimension microchannel. 10

33. The apparatus of claim 31 wherein an outlet reservoir is in fluid communication with the second end of the second dimension microchannels and one or more inlet reservoirs are in fluid communication with the first end of the tertiary microchannels. 15

34. The apparatus of claim 1 further comprising first and second planar substrates and wherein the first and second planar substrates comprise glass.

35. The apparatus of claim 1 further comprising first and second planar substrates and wherein the first and second planar substrates comprise plastic. 20

36. The apparatus of claim 1 further comprising first and second planar substrates and wherein the first and second planar substrates comprise polycarbonate plastic.

37. The apparatus of claim 1 further comprising first and second planar substrates and 25 wherein the first and second planar substrates comprise a combination of dissimilar materials.

38. The apparatus of claim 1 wherein the at least one first dimension microchannel and the second dimension microchannels have an inner width of between about 5 pm and 30 about 200 pm. 36 WO 03/092846 PCT/US03/13580

39. The apparatus of claim 1 wherein the at least one first dimension microchannel and the second dimension microchannels have an average inner width of between about 5 pim and about 80 pm. 5

40. The apparatus of claim 1 wherein the at least one first dimension microchannel and the second dimension microchannels have an average inner width of between about 5 pm and about 20 pm.

41. The apparatus of claim 1 wherein the at least one first dimension microchannel and 10 the second dimension microchannels possess different average widths.

42. The apparatus of claim 1 wherein the at least one first-dimension microchannel has an average width substantially smaller than the second-dimension microchannels. 15

43. The apparatus of claim 1 wherein the second-dimension microchannels have an average width substantially smaller than the at least one first-dimension microchannel.

44. The apparatus of claim 1 wherein the at least one first dimension microchannel and the second dimension microchannels have an inner depth of between about 5 gm and 20 about 200 pm.

45. The apparatus of claim 1 wherein the at least one first dimension microchannel and the second dimension microchannels have an average inner depth of between about 5 im and about 80 pm. 25

46. The apparatus of claim 1 wherein the at least one first dimension microchannel and the second dimension microchannels have an average inner depth of between about 5 pim and about 20 pm. 30

47. The apparatus of claim 1 wherein the at least one first-dimension microchannel is between about 1 cm and about 50 cm long. 37 WO 03/092846 PCT/US03/13580

48. The apparatus of claim 1 wherein the at least one first-dimension microchannel is between about 1 cm and about 4 cm long. 5

49. The apparatus of claim 1 wherein the second-dimension microchannels are between about 1 cm and about 50 cm long.

50. The apparatus of claim I wherein the second-dimension microchannels are between about 1 cm and about 4 cm long. 10

51. The apparatus of claim 1 further comprising an electric field and wherein the electric field along the at least one first-dimension microchannels is between about 100 V/cm and about 1000 V/cm. 15

52. The apparatus of claim 1 further comprising an electric field and wherein the electric field along the second-dimension microchannels is between about 100 V/cm and about 1000 V/cm.

53. A method for performing two-dimensional biomolecular separations, the method 20 comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; 25 transferring the separated sample to the array of second dimension microchannels; and performing a second separation in the second dimension microchannels, where the second separation is performed by applying a temperature gradient.

54. The method of claim 53 wherein the temperature gradient is applied using one or 30 more heating elements affixed to the external surface of a first or a second planar substrate. 38 WO 03/092846 PCT/US03/13580

55. The method of claim 53 wherein the temperature gradient is applied using one or more heating elements enclosed between a first and second planar substrate, wherein resistive heating of the one or more heating elements produces the temperature gradient. 5

56. The method of claim 53 wherein the temperature gradient varies from about 23 degrees Celcius at the intersection between the at least one first-dimension microchannel and the second-dimension microchannel, to about 90 degrees Celcius at a second end of the second-dimension microchannels. 10

57. The method of claim 53 wherein the temperature gradient varies from about 23 degrees Celcius at the intersection between the at least one first-dimension microchannel and the second-dimension microchannels, to about 70 degrees Celcius at a second end of the second-dimension microchannels. 15

58. The method of claim 53 wherein the temperature gradient varies from about 90 degrees Celcius at the intersection between the at least one first-dimension microchannel and the second-dimension microchannel, to about 23 degrees Celcius at a second end of the second-dimension microchannels. 20

59. The method of claim 53 wherein the temperature gradient varies from about 70 degrees Celcius at the intersection between the at least one first-dimension microchannel and the second-dimension microchannel, to about 23 degrees Celcius at a second end of the second-dimension microchannels. 25

60. The method of claim 53 wherein the temperature gradient is a temporal temperature gradient, wherein; b) one or more heating elements induce a constant spatial temperature across a length and width of the second-dimension microchannels; and 30 b) the constant spatial temperature is varied with time; 39 WO 03/092846 PCT/US03/13580

61. The method of claim 60 wherein the constant spatial temperature is varied from an initial temperature of about 23 degrees Celcius to a final temperature of about 90 degrees Celcius. 5

62. The method of claim 60 wherein the constant spatial temperature is varied from an initial temperature of about 23 degrees Celcius to a final temperature of about 70 degrees Celcius.

63. The method of claim 60 wherein the constant spatial temperature is varied from an 10 initial temperature of about 90 degrees Celcius to a final temperature of about 23 degrees Celcius.

64. The method of claim 60 wherein the constant spatial temperature is varied from an initial temperature of about 70 degrees Celcius to a final temperature of about 23 degrees 15 Celcius.

65. The method of claim 53 wherein the biomolecular separations are performed on biomolecules and wherein the biomolecules are DNA molecules. 20

66. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension 25 microchannel to produce a separated sample; means for electrokinetically transferring the separated sample simultaneously to the second dimension microchannels; and means for performing a second separation in the second dimension microchannels, where the second separation is performed by applying a temperature gradient. 30 40 WO 03/092846 PCT/US03/13580

67. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: at least one first dimension microchannel for performing a first biomolecular separation; 5 an array of one or more second dimension microchannels for performing a second separation; an array of one or more tertiary microchannels; one or more electrodes that intersect the one or more second dimension microchannels and the one or more tertiary microchannels; 10 one or more voltage sources operatively connected to the one or more electrodes to control the voltage at the points of intersection with the microchannels.

68. A method for performing two-dimensional biomolecular separations, the method 15 comprising the steps of: providing a first dimension microchannel; providing an array of second dimension microchannels; performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and 20 electrokinetically transferring the separated sample to the second dimension microchannels.

69. A method for performing two-dimensional biomolecular separations, the method comprising the steps of: 25 providing at least one first dimension microchannel; providing an array of second dimension microchannels; performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and simultaneously transferring the separated sample to the second dimension microchannels. 30 41 WO 03/092846 PCT/US03/13580

70. A method for performing two-dimensional biomolecular separations, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; 5 performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and electrokinetically transferring the separated sample simultaneously to the second dimension microchannels. 10

71. A method for performing two-dimensional biomolecular separations, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; performing a first biomolecular separation in the first dimension microchannel to 15 produce a separated sample; and electrokinetically transferring the separated sample simultaneously to the second dimension microchannels; and performing a second separation in the second dimension microchannels, where the second separation is performed by applying a temperature gradient. 20

72. A method for performing two-dimensional biomolecular separations, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; 25 providing at least one voltage-control microchannel; performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and applying a voltage gradient in the voltage-control microchannels to individually define the voltage within the second-dimension microchannels near the intersections of 30 the first-dimension microchannel and second-dimension microchannels to be nearly equal 42 WO 03/092846 PCT/US03/13580 to the voltage within the first-dimension microchannel near the intersections of the first dimension microchannel and second-dimension microchannels; and electrokinetically transferring the separated sample simultaneously to the second dimension microchannels; and 5 performing a second separation in the second dimension microchannels, where the second separation is performed by applying a temperature gradient.

73. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: 10 a) at least one first-dimension microchannel for receiving a first-dimension separation medium, wherein the at least one first dimension channel has a first end and a second end and extends in a first direction; b) an array of one or more second-dimension microchannels for receiving a second-dimension separation medium, wherein the microchannels of the array of one or 15 more second-dimension microchannels each have a first end and a second end, extend in a second direction orthogonal to the first direction and intersect with the at least one first dimension microchannel; c) a first reservoir in fluid communication with the at least one first dimension microchannel, 20 d) at least a first electrode, having a first end and a second end, the first end being in electrical communication with the first reservoir, g) at least one voltage source in electrical communication with the second end of the first electrode; h) at least a second reservoir in fluid communication with microchannels of the 25 array of second dimension microchannels; i) at least a second electrode, having a first end and a second end, the first end being in electrical communication with the second reservoir; and j) at least one voltage source in electrical communication with the second end of the second electrode. 30 43 WO 03/092846 PCT/US03/13580

74. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: a) a first planar substrate containing one or more microchannels; b) a second planar substrate bonded to the first planar substrate to provide 5 enclosure of the one or more microchannels; c) a first-dimension microchannel containing a first-dimension separation medium, wherein the channel has a first end and a second end; d) an array of one or more second-dimension microchannels containing a second dimension separation medium, wherein the microchannels have a first end and a second 10 end, and wherein the one or more second-dimension microchannels intersect the first dimension microchannel; e) one or more injection microchannels, wherein the microchannels have a first end and a second end, and wherein the one or more injection microchannels intersect the first-dimension microchannel near the first end of the first-dimension microchannel; 15 f) one or more reservoirs formed in the first or second substrate having disposed therein an electrolyte solution and a first end of one or more separation electrodes, and wherein the reservoirs are located at the end of the one or more microchannels; g) one or more high voltage power supplies attached to a second end of a selected number of the one or more separation electrodes; and 20 h) one or more grounding electrodes attached to the second end of a selected number of the one or more separation electrodes

75. The apparatus of claim 74 wherein the one or more reservoirs include: a) a sample injection inlet reservoir intersecting the first end of the injection 25 microchannel; b) a sample injection outlet reservoir intersecting the second end of the injection microchannel; c) a first-dimension separation inlet reservoir intersecting the first end of the first dimension microchannel; 30 d) a first-dimension separation outlet reservoir intersecting the second end of the first-dimension microchannel; 44 WO 03/092846 PCT/US03/13580 e) one or more second-dimension separation inlet reservoirs intersecting the first end of the one or more second-dimension microchannels; and f) one or more second-dimension separation outlet reservoirs intersecting the second end of the one or more second-dimension microchannels. 5

76. A method of performing two-dimensional gel electrophoresis of biomolecules, comprising: a) applying a high electric field along the length of the injection microchannel, thereby injecting a sample stream containing the biomolecules of interest from the first 10 end of the injection microchannels towards the second end of the injection microchannel, wherein; 1) a high voltage is applied to the electrode disposed within the injection outlet reservoir; 2) a grounding voltage is applied to the electrode disposed within the 15 injection inlet reservoir; 3) all other reservoirs are disconnected from any voltage source; 4) the sample stream crosses through a portion of the first-dimension microchannel. b) applying a high electric field along the length of the first-dimension 20 microchannel, thereby separating the biomolecules based on their migration time through the gel contained therein and resulting in separation of the biomolecules based on their size, wherein; 1) a high voltage is applied to the electrode disposed within the first dimension outlet reservoir; 25 2) a grounding voltage is applied to the electrode disposed within the first dimension inlet reservoir; 3) all other reservoirs are disconnected from any voltage source; 4) the separated sample stream passes by the one or more second dimension microchannels intersecting with the first-dimension microchannels. 30 c) applying a high electric field along the length of the one or more second dimension microchannels while applying a temperature gradient, thereby denaturing the 45 WO 03/092846 PCT/US03/13580 biomolecules and further separating the biomolecules based on their migration time through the gel contained therein, wherein; 1) a spatial temperature gradient is formed along the length of the one or more second-dimension microchannels; 5 2) a high voltage is applied to the electrode disposed within the second dimension outlet reservoir; 3) a grounding voltage is applied to the electrode disposed within the second-dimension inlet reservoir; 4) all other reservoirs are disconnected from any voltage source. 10

77. The method of claim 76 wherein a low voltage is applied to the first-dimension outlet reservoir, with a grounding voltage applied to the one or more first-dimension inlet reservoirs, and the second-dimension inlet reservoir is disconnected from any voltage source, during application of the high electric field along the length of the one or more 15 second-dimension separation microchannels, thereby generating a small electric field along the length of the first-dimension microchannel and causing biomolecules to be drawn slightly towards the first-dimension outlet reservoir to ensure efficient transfer of the biomolecules from the first-dimension microchannel into the one or more second dimension microchannels. 20

78. The method of claim 76 wherein one or more intersection control voltages are applied to the one or more second-dimension separation inlet reservoirs and the one or more second-dimension separation outlet reservoirs to control the electric field lines at the intersection of the one or more first-dimension separation microchannels and the one 25 or more second-dimension separation microchannels in such a manner that the distribution of biomolecules undergoing separation during the first-dimension separation step are not substantially affected by the intersections.

79. The method of claim 76 wherein one or more intersection control voltages are 30 applied to the one or more voltage-control microchannel inlet reservoirs and the one or more voltage-control microchannel outlet reservoirs to control the electric field lines at 46 WO 03/092846 PCT/US03/13580 the intersection of the one or more first-dimension separation microchannels and the one or more second-dimension separation microchannels in such a manner that the distribution of biomolecules undergoing separation during the first-dimension separation step are not substantially affected by the intersections. 5

80. The method of claim 76 wherein the one or more intersection control voltages are applied using a plurality of voltage sources, wherein; a) one voltage source is connected to the first end of a first resistive element; b) a second voltage source is connected to the second end of the first resistive 10 element to generate a potential gradient along the first resistive element; c) the resistive element is placed in electrical contact with the one or more second-dimension separation inlet microchannels such that the intersection control voltage at each point of electrical contact is set by the voltage of the first resistive element at the point of electrical contact; 15 d) the one or more intersection control voltages are chosen such that the voltage near the intersection of the one or more first-dimension separation microchannels and the one or more second-dimension separation microchannels at which the intersection control voltage is applied is slightly different than the voltage within the intersection itself. 20 e) A third voltage source is connected to the first end of a second resistive element; f) a fourth voltage source is connected to the second end of the second resistive element to generate a potential gradient along the second resistive element; g) the resistive element is placed in electrical contact with the one or more 25 second-dimension separation inlet reservoirs such that the intersection control voltage in each reservoir is set by the voltage of the second resistive element at the point of electrical contact; h) the one or more intersection control voltages are chosen such that the voltage near the intersection of the one or more first-dimension separation 30 microchannels and the one or more second-dimension separation microchannels connected to the reservoir at which the intersection control 47 WO 03/092846 PCT/US03/13580 voltage is applied is slightly different than the voltage within the intersection itself

81. The method of claim 76 wherein the one or more intersection control voltages are 5 applied using a plurality of voltage sources, wherein; a) one voltage source is connected to the inlet reservoir of a first voltage-control microchannel; b) a second voltage source is connected to the outlet reservoir of a first voltage control microchannel to generate a potential gradient along the first voltage 10 control microchannel; c) the first voltage-control microchannel intersects the one or more second dimension separation microchannels such that the intersection control voltage in each second-dimension separation microchannel is set by the voltage of the inlet reservoir and outlet reservoir of the first voltage-control microchannel; 15 d) the one or more intersection control voltages are chosen such that the voltage near the intersection of the one or more first-dimension separation microchannels and the one or more second-dimension separation microchannels is slightly different than the voltage within the intersection itself. 20 e) A third voltage source is connected to the inlet reservoir of a second voltage control microchannel; f) a fourth voltage source is connected to the outlet reservoir of a second voltage-control microchannel to generate a potential gradient along the second voltage-control microchannel; 25 g) the second voltage-control microchannel intersects the one or more tertiary microchannels such that the intersection control voltage in each tertiary microchannel is set by the voltage of the inlet reservoir and outlet reservoir of the second voltage-control microchannel; h) the one or more intersection control voltages are chosen such that the voltage 30 near the intersection of the one or more first-dimension separation 48 WO 03/092846 PCT/US03/13580 microchannels and the one or more tertiary microchannels is slightly different than the voltage within the intersection itself.

82. A microfluidic apparatus for performing two-dimensional biomolecular separations, 5 the apparatus comprising: at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and 10 means for electrokinetically transferring the separated sample to the second dimension microchannels.

83. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: 15 at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and means for simultaneously transferring the separated sample to the second dimension 20 microchannels.

84. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: at least one first dimension microchannel; 25 an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and means for electrokinetically transferring the separated sample simultaneously to the second dimension microchannels 49 WO 03/092846 PCT/US03/13580

85. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first separation in the first dimension microchannel to produce a separated sample; means for transferring the separated sample to the second dimension microchannels; and means for introducing a first media in the first dimension microchannel and a second media into the array of second dimension microchannels.

86. The apparatus of claim 85 wherein the first media is a media for enabling separation based on isoelectric focusing.

87. The apparatus of claim 85 wherein the second media is a media for enabling size based separation.

88. The apparatus of claim 85 wherein the means for introducing comprises pressure filling means.

89. The apparatus of claim 85 wherein the means for introducing comprises means for filling each of the first dimension microchannel and second dimension microchannels with a second media, electroosmotically removing the second media from one of the first dimension microchannel or the second dimension microchannels and introducing a first media therein.

90. The apparatus of claim 85 further comprising a hydrodynamic barrier between the first dimension microchannel and second dimension microchannels to enable each to be filled with a different media.

91. The apparatus of claim 85, wherein the biomolecular separation is to be performed on a biomolecular material and the biomolecular material comprises protein, the first 50 WO 03/092846 PCT/US03/13580 dimension separation is a isoelectric point-based separation and the second dimension separation is a size-based separation.

92. The apparatus of claim 85, wherein the first dimension separation is substantially retained upon transfer to the second dimension.

93. The apparatus of claim 85, wherein the first media comprises a media for isoelectric point based separation.

94. The apparatus of claim 85, wherein the second media comprises a media for size based separation.

95. The apparatus of claim 85, wherein the first media comprises a media for isoelectric focusing in the first dimension, comprising carrier ampholytes for the creation of pH gradient in the first microchannel.

96. The apparatus of claim 85, wherein the second media comprises a media for size based separation of SDS-protein complexes in the second dimension based on their differences in electrophoretic mobility inside a polymer sieving matrix.

97. The apparatus of claim 85, wherein the second media is a sieving matrix selected from the group consisting of: cross-linked polyacrylamide, linear polyacrylamide, polydimethylamide, N acrylamoniethoxyethanol, hydroxyethylcellulose [HEC], poly(ethylene glycol), poly(ethylene oxide) [PEO], poly(vinylpyrrolidone) [PVP], or nonionic polymeric surfactants (n-alkyl polyoxyethylene ethers).

98. The apparatus of claim 85, further comprising a detector placed near a second end of the array of second-dimension separation microchannels for differential display of protein expressions. 51 WO 03/092846 PCT/US03/13580

99. The apparatus of claim 85, further comprising a detector capable of monitoring substantially all of the full length and breadth of the array of second-dimension separation microchannels for differential display of protein expressions.

100. The apparatus of claim 85, further comprising a detector to monitor the performance of isoelectric focusing in the at least one first dimension microchannel, wherein proteins may be covalently labeled with a suitable florescent dye and detected by a suitable florescence detector.

101. The apparatus of claim 85, further comprising a detector to monitor the performance of isoelectric focusing in first dimension microchannels, wherein proteins in a biomolecular sample may be covalently labeled with a suitable florescent dye and detected by a suitable florescence detector and the microchannels are formed from a substrate optically transparent at the wavelengths used for the fluorescence detection.

102. The apparatus of claim 85 further comprising an integrated optical detection system.

103. The apparatus of claim 85 further comprising an integrated laser-induced fluorescence detection system.

104. The apparatus of claim 85, wherein first ends of the second dimension microchannels in the array of second-dimension microchannels terminate at the at least one first-dimension microchannel at one or more points between first and second ends of the at least one first-dimension microchannel, and wherein at least one outlet reservoir is in fluid communication with second ends of the second-dimension microchannels.

105. The apparatus of claim 85, wherein the second-dimension microchannels have first and second ends and the at least one first dimension microchannel intersects the second dimension microchannels at a position somewhere between the first and second ends of the second-dimension microchannels. 52 WO 03/092846 PCT/US03/13580

106. The apparatus of claim 85, wherein an inlet reservoir is in fluid communication with the first end of the second dimension microchannels and an outlet reservoir is in fluid communication with the second end of the second dimension microchannels.

107. The apparatus of claim 85, wherein the first ends of the second-dimension microchannels terminate at the at least one first-dimension microchannel and further comprising an array of tertiary microchannels, wherein a second end of the tertiary microchannels terminate at the at least one first-dimension microchannel.

108. The apparatus of claim 107, wherein points at which the second-dimension microchannels intersect with the at least one first-dimension microchannel are staggered with respect to points at which the tertiary microchannels intersect with the at least one first-dimension microchannel.

109. The apparatus of claim 107, wherein one or more outlet reservoirs are in fluid communication with second ends of the second dimension microchannels and one or more inlet reservoirs are in fluid communication with first ends of the tertiary microchannels.

110. The apparatus of claim 107, wherein groups of the array of tertiary microchannels merge into a first end of a smaller number of one or more merged tertiary microchannels, and wherein'bne or more outlet reservoirs are in fluid communication with the second end of the second-dimension microchannels and one or more inlet reservoirs are in fluid communication with a second end of the merged tertiary microchannels.

111. The apparatus of claim 110, wherein a length of the merged tertiary microchannels is significantly larger than a length of a voltage-control microchannel, thereby increasing an electrical resistivity of the merged tertiary microchannels compared with an electrical resistivity of the voltage-control microchannels when filled with a similar conductive solution. 53 WO 03/092846 PCT/US03/13580

112. The apparatus of claim 107, wherein groups of the array of second-dimension microchannels merge into a first end of a smaller number of one or more second dimension microchannels, and wherein one or more outlet reservoirs are in fluid communication with a second end of the merged second-dimension microchannels and one or more inlet reservoirs are in fluid communication with the second end of the tertiary microchannels.

113. The apparatus of claim 112, wherein a length of the merged second-dimension microchannels is substantially larger than a length of a voltage-control microchannel, thereby increasing an electrical resistivity of the merged second-dimension microchannels compared with an electrical resistivity of the voltage-control microchannels when filled with a similar conductive solution.

114. The apparatus of claim 85 further comprising a voltage control microchannel, wherein a cross-sectional area of the voltage control microchannels is similar to a cross sectional area of the at least one first-dimension microchannel, thereby creating a similar electrical resistivity in each channel when filled with a similar conductive solution.

115. The apparatus of claim 107 further comprising a voltage control microchannel, wherein a cross-sectional area of the voltage control microchannel is substantially smaller than a cross-sectional area of the second-dimension microchannels and tertiary microchannels, thereby increasing an electrical resistivity of the second-dimension and tertiary microchannels compared with an electrical resistivity of the voltage-control microchannel when filled with a similar conductive solution.

116. The apparatus of claim 85 further comprising first and second planar substrates wherein the first and second planar substrates comprise glass.

117. The apparatus of claim 85 further comprising first and second planar substrates wherein the first and second planar substrates comprise plastic. 54 WO 03/092846 PCT/US03/13580

118. The apparatus of claim 85 further comprising first and second planar substrates wherein the first and second planar substrates comprise polycarbonate plastic.

119. The apparatus of claim 85 further comprising first and second planar substrates wherein the first and second planar substrates comprise a combination of dissimilar materials including glass, polydimethylsiloxane (PDMS), plastic, or silicon.

120. The apparatus of claim 85, wherein the at least one first dimension microchannel and the second dimension microchannels have an inner width of between about 5 ptm and about 200 pm.

121. The apparatus of claim 85, wherein at least one first dimension microchannel and the second dimension microchannels have an average inner width of between about 5 pm and about 80 pm.

122. The apparatus of claim 85, wherein the at least one first dimension microchannel and the second dimension microchannels have an average inner width of between about 5 pm and about 20 pm.

123. The apparatus of claim 85, wherein the at least one first dimension microchannel and the second dimension microchannels possess different average widths.

124. The apparatus of claim 85, wherein the at least one first-dimension microchannel has an average width substantially smaller than the second-dimension microchannels.

125. The apparatus of claim 85, wherein the second-dimension microchannels have an average width substantially smaller than the at least one first-dimension microchannel. 55 WO 03/092846 PCT/US03/13580

126. The apparatus of claim 85, wherein the at least one first dimension microchannel and the second dimension microchannels have an inner depth of between about 5 pm and about 200 pim.

127. The apparatus of claim 85, wherein the at least one first dimension microchannel and the second dimension microchannels have an average inner depth of between about 5 pim and about 80 jim.

128. The apparatus of claim 85, wherein the at least one first dimension microchannel and the second dimension microchannels have an average inner depth of between about 5 gm and about 20 pm.

129. The apparatus of claim 85, wherein the at least one first dimension microchannel and the second dimension microchannels possess different average inner depths.

130. The apparatus of claim 85, wherein the at least one first-dimension microchannel has an average inner depth substantially smaller than the average inner depths of the second-dimension microchannels.

131. The apparatus of claim 85, wherein the second-dimension microchannels have average inner depths substantially smaller than the average inner depth of the at least one first-dimension microchannel.

132. The apparatus of claim 85, wherein the at least one first-dimension microchannel is between about 1 cm and about 50 cm long.

133. The apparatus of claim 85, wherein the at least one first-dimension microchannel is between about 1 cm and about 4 cm long.

134. The apparatus of claim 85, wherein the second-dimension microchannels are between about 1 cm and about 50 cm long. 56 WO 03/092846 PCT/US03/13580

135. The apparatus of claim 85, wherein the second-dimension microchannels are between about 1 cm and about 4 cm long.

136. A microfluidic apparatus for performing two-dimensional protein separations, the apparatus comprising: at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and means for simultaneously transferring the separated sample to the second dimension microchannels.

137. A microfluidic apparatus for performing two-dimensional protein separations, the apparatus comprising: at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; and means for electrokinetically transferring the separated sample simultaneously to the second dimension microchannels.

138. A microfluidic apparatus for performing two-dimensional biomolecular separations, the apparatus comprising: at least one first dimension microchannel; an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; means for transferring the separated sample simultaneously to the second dimension microchannels; and means for performing a second separation in the second dimension microchannels; and 57 WO 03/092846 PCT/US03/13580 means for introducing a first media in the first dimension microchannel and a second media into the array of second dimension microchannels.

139. A microfluidic apparatus for performing two-dimensional protein separations, the apparatus comprising: at least one first dimension microchannel for performing a first protein separation; an array of one or more second dimension microchannels for performing a second separation; an array of one or more tertiary microchannels; one or more electrodes that intersect the one or more second dimension microchannels and the one or more tertiary microchannels; one or more voltage sources operatively connected to the one or more electrodes to control the voltage at the points of intersection with the microchannels.

140. A microfluidic apparatus for performing two-dimensional protein separations, the apparatus comprising: at least one first dimension microchannel for performing a first protein separation; an array of one or more second dimension microchannels for performing a second separation; an array of one or more tertiary microchannels; one or more voltage-control microchannels that intersect the one or more second dimension microchannels and the one or more tertiary microchannels; one or more voltage sources operatively connected to the one or more voltage control microchannels to control the voltage at the points of intersection with the second dimension and tertiary microchannels.

141. A method for performing two-dimensional biomolecular separations with a microfluidic apparatus, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; 58 WO 03/092846 PCT/US03/13580 performing a first separation in the first dimension microchannel to produce a separated sample; transferring the separated sample to the second dimension microchannels; and introducing a first media in the first dimension microchannel and a second media into the array of second dimension microchannels.

142. The method of claim 141, wherein the first media is a media for enabling separation based on isoelectric focusing.

143. The method of claim 141, wherein the second media is a media for enabling size based separation.

144. The method of claim 141, wherein the step of introducing comprises pressure filling the second-dimension microchannels with a second media without filling the first dimension microchannel, followed by pressure filling the first-dimension microchannel with a first media.

145. The method of claim 141, wherein the step of introducing comprises filling each of the first dimension microchannel and second dimension microchannels with a second media, electroosmotically removing the second media from one of the first dimension microchannel or the second dimension microchannels and introducing a first media therein.

146. The method of claim 141 further comprising the step of providing a hydrodynamic barrier between the first dimension microchannel and second dimension microchannels to enable each to be filled with a different media.

147. A method for performing two-dimensional protein separation with a microfluidic apparatus, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; 59 WO 03/092846 PCT/US03/13580 performing a first protein separation in the first dimension microchannel to produce a separated sample; and simultaneously transferring the separated sample to the second dimension microchannels.

148. A method for performing two-dimensional protein separation with a microfluidic apparatus, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; performing a first protein separation in the first dimension microchannel to produce a separated sample; and electrokinetically transferring the separated sample simultaneously to the second dimension microchannels.

149. A method for performing two-dimensional biomolecular separations with a microfluidic apparatus, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; means for performing a first biomolecular separation in the first dimension microchannel to produce a separated sample; means for transferring the separated sample simultaneously to the second dimension microchannels; performing a second separation in the second dimension microchannels; and introducing a first media in the first dimension microchannel and a second media into the array of second dimension microchannels.

150. A method for performing two-dimensional biomolecular separations with a microfluidic apparatus, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; providing an array of one or more tertiary microchannels; 60 WO 03/092846 PCT/US03/13580 providing one or more electrodes that intersect the one or more second dimension microchannels and the one or more tertiary microchannels; operating one or more voltage sources operatively connected to the one or more electrodes to control the voltage at the points of intersection with the microchannels.

151. A method for performing two-dimensional biomolecular separations with a microfluidic apparatus, the method comprising the steps of: providing at least one first dimension microchannel; providing an array of second dimension microchannels; providing an array of one or more tertiary microchannels; providing one or more voltage-control microchannels that intersect the one or more second dimension microchannels and the one or more tertiary microchannels; operating one or more voltage sources operatively connected to the one or more voltage-control microchannels to control the voltage at the points of intersection with the microchannels. 61