Abstract:
One embodiment of the invention relates to a microfluidic apparatus for performing two dimensional biomolecular separations. According to one aspect of the invention, after 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 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.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority from U.S. Provisional Patent Application Ser. No. 60/287,801, filed May 1, 2001, which is incorporated herein by reference in its entirety. 
    
    
     The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant Numbers: R43CA092819, and R43GM062738, awarded by the National Institutes of Health, and Grant Number DAAH 01-02 -C-R136, awarded by the Defense Advanced Research Projects Agency. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to a system and method for using a microfluidic apparatus for performing two dimensional separations of biomolecular materials. 
     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 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 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 filly 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. 
     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 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 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 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 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 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 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 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 several large human disease genes, including CFTR (cystic fibrosis transmembrane regulator gene), RB1 (retinoblastoma tumor suppressor gene), MLH1 (MutL protein 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 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 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 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 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 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 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 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 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 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. 
     These and other drawbacks exist with known systems and methods. 
     SUMMARY OF THE INVENTION 
     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 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 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. 
     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 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 (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 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. 
     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 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 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 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 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 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 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 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 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 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). 
     These and other objects, features, and advantages of the invention will be apparent through the detailed description of the preferred embodiments and the drawings 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 
         FIG. 1  is a schematic of a microfluidic apparatus according to one embodiment of the invention. 
         FIG. 2A  is a side view of a microfluidic apparatus according to one embodiment of the invention. 
         FIG. 2B  is a front sectional view of a microfluidic apparatus according to one embodiment of the invention. 
         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 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 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 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. 
         FIG. 11  is a schematic of a laser-induced fluorescence detection setup for line-based fluorescence detection in a second dimension of a microchannel array according to one embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION 
     According to an embodiment of the invention illustrated in  FIG. 1 , a microfluidic 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. 
     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  3   a  and a second end  3   b . Similarly, an array of one or more second-dimension microchannels  4  may each have a first end  4   a  and a second end  4   b.    
     According to one embodiment the first end  4   a  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. 
     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 (V 13 , V 14 , V 15 , V 16 ) associated with each of the reservoirs, respectively. For example, a first reservoir  5  may be in fluid communication with a first end  3   a  of the first microchannel  3 , and a second reservoir  6  may be in fluid communication with a second end  3   b  of the first microchannel  3 . Additionally, a third reservoir  7  may be in fluid communication with a first end  4   a  of each of the second dimension microchannels  4 , and a fourth reservoir  8  may be in fluid communication with a second end  4   b  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 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 injection microchannels have a first end  30   a  and a second end  30   b , and wherein the one or more injection microchannels  30  intersect the first-dimension microchannel  3  near the first end  3   a  of the first-dimension microchannel  3 . According to another embodiment, the apparatus may further comprise a sample injection inlet reservoir  31  intersecting the first end  30   a  of the injection microchannel  30 , a sample injection outlet reservoir  32  intersecting the second end  30   b  of the injection microchannel  30 , a first-dimension separation inlet reservoir  61  intersecting the first end  3   a  of a first-dimension microchannel  3  and a first-dimension separation outlet reservoir  62  intersecting a second end  3   b  of a first-dimension microchannel  3 . As shown in  FIG. 1 , one or more second-dimension separation inlet reservoirs (e.g. reservoir  7 ) may intersect a first end  4   a  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  4   b  of the one or more second-dimension microchannels  4 . 
     According to one embodiment of the invention, the one or more reservoirs ( 5 - 8 ,  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 (V 13 , V 14 , V 15 , V 16 ). Likewise, one or 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 ( 3   a ,  3   b ) 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 ( 4   a ,  4   b ) of the second dimension microchannels  4 . Other configurations may be used. For example, in one embodiment, the second ends  4   b  of the one or more second dimension microchannels  4  may terminate at one or more points between the first and second ends ( 3   a ,  3   b ) 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-dimension separation outlet reservoirs  8  may intersect the second end  4   b  of the one or more second-dimension microchannels  4 , and one or more tertiary inlet reservoirs  10  may intersect the first end  11  a of the one or more tertiary microchannels  11 . The second end  11   b  of the one or more tertiary microchannels  11  may terminate at one or more points between the first  3   a  and second  3   b  ends of the first dimension microchannel  3 , and the first ends  4   a  of the one or more second dimension microchannels  4  may terminate at one or more points between the first and second ends  3   a ,  3   b  of the first dimension microchannel  3 . In this embodiment, the one or more points at which the second ends ( 4   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 microchannels  4 , and the one or more points at which the first ends  4   a  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  11   b  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 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 1×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA). The 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 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. 
     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 ,  2 B, one or more heating elements  17  may be bonded between (or otherwise integrated with) the first  1  and second  2  planar substrates. 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 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 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 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 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° C. at the intersection between the first dimension microchannel  3  and the one or more second-dimension microchannel  4 , to about 70-90° C. at the second end  4   b  of the one or more second-dimension microchannels  4 . Alternatively, the spatial temperature gradient may vary from about 70-90° 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  4   b  of the one or more second-dimension microchannels  4 . The spatial temperature 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. 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. 
     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 μm may have an average width of 30 μm. However, both depth and width preferably range from 5 to 200 μm. 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 lengths may be used. Some embodiments may have other microchannel dimensions for various 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 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 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 1×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, other biomolecules and chemical composites. 
     According to one embodiment of the invention, a voltage source (V 13 , V 14 , V 15 , V 16 ) 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 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 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 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  30   a  of the injection microchannels  30  towards the second end  30   b  of the injection microchannel  30 . A high voltage may be applied to an electrode (not 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  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 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 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 of the biomolecules may occur through the application of an electric field along the 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 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 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 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 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 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 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-dimension microchannel  3  into the one or more second dimension microchannels  4 . 
     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 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 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 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 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 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  11 , and one or more voltage control microchannels  36  may intersect the one or more second-dimension microchannels  4 . 
     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   
     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 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 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 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 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 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  (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 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 (V 44 , V 45 , V 46 , V 47 ) are applied at each end of the one or more resistive elements ( 42 ,  43 ), thereby creating a voltage drop along the length of the 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 (V 44 , V 45 , V 46 , V 47 ) and the resistance of the one or more resistive elements ( 42 ,  43 ). 
     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 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 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. 
     A preferred method of electrode integration may be realized by depositing evaporated and/or sputtered platinum films on a polycarbonate plastic substrate, followed 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 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 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 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 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 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 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 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 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. 
     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-channel monitoring of DNA fragments near the end of the second-dimension microchannel array. As shown in  FIG. 11 , 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 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 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 μm pixels positioned in a 1024×128 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 532 nm rejection band filter (OD&gt;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 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 preparation for nucleic acid extraction (from blood, tissue, etc.), purification/isolation, amplification, digestion, and tagging. 
     In some embodiments, the electrokinetic transfer method may be performed to transfer proteins, peptides, and other chemical or biological composites from one dimension to another dimension of a gel electrophoresis device. As used herein, electrokinetic transfer includes a method or a system which transfer materials from a channel and/or chamber containing structure in one dimension, to similar structures in other dimensions, through the application of electric fields to the materials, thereby causing the transfer of the materials. 
     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 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.