WO2003092846A2 - Dispositifs microfluidiques en plastique permettant des separations bidimensionnelles de molecules biologiques - Google Patents

Dispositifs microfluidiques en plastique permettant des separations bidimensionnelles de molecules biologiques Download PDF

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WO2003092846A2
WO2003092846A2 PCT/US2003/013580 US0313580W WO03092846A2 WO 2003092846 A2 WO2003092846 A2 WO 2003092846A2 US 0313580 W US0313580 W US 0313580W WO 03092846 A2 WO03092846 A2 WO 03092846A2
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dimension
microchannels
microchannel
separation
voltage
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PCT/US2003/013580
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WO2003092846A3 (fr
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Cheng Sheng Lee
Don Devoe
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Cheng Sheng Lee
Don Devoe
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Priority claimed from US10/135,385 external-priority patent/US6929730B2/en
Priority claimed from US10/135,386 external-priority patent/US6974526B2/en
Application filed by Cheng Sheng Lee, Don Devoe filed Critical Cheng Sheng Lee
Priority to AU2003232028A priority Critical patent/AU2003232028A1/en
Publication of WO2003092846A2 publication Critical patent/WO2003092846A2/fr
Publication of WO2003092846A3 publication Critical patent/WO2003092846A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/54—Heating or cooling apparatus; Heat insulating devices using spatial temperature gradients

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • SNP single-nucleotide polymorphism
  • SSCP single-stranded conformation polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • matrix-assisted laser desorption/ionization mass spectrometry 5 'nuclease assay
  • single nucleotide primer extension single nucleotide primer extension
  • 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 rwo-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.
  • 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.
  • TDGS two-dimensional gene scanning
  • a mutation scanning system should not only be accurate but also capable of operating at a high throughput in a cost- effective manner.
  • 2-D DNA gel electrophoresis is relatively cost-effective in comparison with other mutation detection techniques.
  • TDGS suffers from the fact that it is not a high-throughput platform for large-scale 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.
  • 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.
  • 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.
  • microfluidic devices for 2-D DNA gel electrophoresis 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.
  • 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).
  • SDS sodium dodecyl sulfate
  • 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 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 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 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.
  • PAGE devices is a lack of methods to transfer proteins simultaneously from first to second dimensions without significant loss in resolution.
  • protein 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.
  • 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.
  • 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).
  • any separation accomplished in the first dimension is completely retained upon transfer to the second dimension.
  • the separation in the second dimension is performed using a temperature gradient (e.g., a spatial or temporal temperature gradient).
  • 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.
  • a 2-D plastic microfluidic network 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.
  • 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.
  • 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.
  • DNA fragments (or other materials) in the second dimension are resolved by using a temporal or a spatial temperature gradient.
  • 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).
  • LIFD laser-induced fluorescence detection
  • 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.
  • 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.
  • microchannels and reservoirs may be implemented to control intersection voltages and enable advantageous separation techniques.
  • other microchannels e.g., tertiary
  • voltage control microchannels may be implemented to enable advantageous manipulation of samples.
  • 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.
  • a spatial temperature gradient is formed along the length of the one or more second-dimension microchannels.
  • 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 ⁇
  • 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 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.
  • 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).
  • 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.
  • a polymeric membrane sandwiched between the upper and the lower microchannels may serve as a hydrodynamic barrier, enabling the i introduction of two different separation media in the upper and the lower microchannels. 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 to the second dimension.
  • 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.
  • 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. 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.
  • proteins may be covalently labeled with a florescent dye.
  • the labeled proteins may be monitored using a florescent detector attached to the microfluidic system.
  • microchannels fabricated by polydimethylsiloxane (PDMS) substrates may be used which provide low florescence background during detection and enable better signal to background resolution.
  • PDMS polydimethylsiloxane
  • LIFD laser induced florescent detection
  • 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 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.
  • microfluidic 2-D PAGE of the present invention may be advantageous for the 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.
  • 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 oftheinvention.
  • 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 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 fluorescence detection in a second dimension of a microchannel array according to one embodiment of the invention.
  • 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.
  • 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.
  • 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 3b.
  • an array of one or more second-dimension microchannels 4 may each have a first end 4a and a second end 4b.
  • 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.
  • the apparatus may further comprise one or more reservoirs (5, 6, 7, 8) and voltage sources (N13, N14, N15, N16) associated with each of the reservoirs, respectively.
  • a first reservoir 5 may be in fluid communication with a first end 3a of the first microchannel 3
  • a second reservoir 6 may be in fluid communication with a second end 3b of the first microchannel 3.
  • 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.
  • different configurations of microchannels and reservoirs may be used. Not all embodiments may use four reservoirs. More or less may be used.
  • the apparatus may further comprise one or more injection microchannels 30 (as illustrated in FIG. 4), wherein the 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.
  • the apparatus may further comprise a sample injection inlet reservoir 31 intersecting the 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.
  • one or more second- dimension separation inlet reservoirs 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.
  • 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 (N13, N14, N15, N16).
  • one or more of electrodes (9-12) may also be connected to ground potential (e.g., ⁇ 0 Volts).
  • 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.
  • 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 (3 a, 3b) of the first dimension microchannel 3. In such embodiments, no second dimension inlet reservoir may be provided.
  • one or more second- 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 may intersect the first end 11a of the one or more tertiary microchannels 11.
  • the second end l ib of the one or more tertiary microchannels 11 may terminate at one or more points between the first 3 a 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 one or more points between the first and second ends 3a, 3b of the first dimension microchannel 3.
  • the one or more points at which the second ends (l ib) of the tertiary microchannels 11 and second dimension microchannels 4 terminate at the first dimension microchannel 3 may be staggered.
  • 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 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 l ib of the tertiary microchannels 11 terminate at the first dimension microchannel 3 by half the distance between adjacent tertiary microchannels 11.
  • the one or more second dimension separation inlet reservoirs may be omitted.
  • reservoirs may be filled with an electrolyte solution.
  • the electrolyte solution may include a buffer (e.g, an electrophoresis buffer, or a salt solution).
  • the electrolyte solution may contain IX 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.
  • microchannels 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 ⁇ m may have an average width of 30 ⁇ m. However, both depth and width preferably range from 5 to 200 ⁇ m. 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.
  • microchannels 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 N/cm to 1000 N/cm.
  • a high voltage power supply may be attached to a second end of a selected number of the one or more separation electrodes (e.g., electrodes 9-12).
  • microfluidic device of the present invention is used for separating D ⁇ A, peptides, and other biological or chemical composites.
  • a plastic substrate such as poly(methylmethacrylate)
  • PMMA polydimethylsiloxane
  • PDMS polydimethylsiloxane
  • ⁇ on-plastic materials such as glass or silica may also be used to fabricate the 2-D microfluidic apparatus of the present invention.
  • 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. 2 A, 2B, 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.
  • 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.
  • 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 4b of the one or more second- dimension microchannels 4.
  • 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 4b 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.
  • 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.
  • microchannels e.g. 3, 4
  • microchannels with an average depth of 10 ⁇ m may have an average width of 30 ⁇ m.
  • both depth and width preferably range from 5 to 200 ⁇ m.
  • the width mentioned herein is from trapezoidal shaped microchannel cross- sections.
  • microchannel cross-sections may be used, for example rectangular, circular, or semi-circular cross-sections.
  • the microchannels e.g. 3, 4
  • the microchannels 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.
  • a preferred separation media for electrophoresis in microchannels is IX 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.
  • a voltage source (V13, V14, V15, VI 6) 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.
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a relatively high electric field 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 outlet reservoir to ensure efficient transfer of the biomolecules from the first- dimension microchannel into the one or more second dimension microchannels 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.
  • 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.
  • 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.
  • 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.
  • one voltage source 35 and 37
  • 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.
  • a single voltage control microchannel 36 may be combined with a second-dimension outlet reservoir 8.
  • 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.
  • groups of one or more tertiary microchannels 11 may intersect one or more tertiary inlet reservoirs 10.
  • groups of one or more second-dimension microchannels 4 may intersect one or more second-dimension outlet reservoirs 8
  • FIG. 7 depicted in FIG.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (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 resistive elements (42, 43).
  • TGGE temperature-gradient gel electrophoresis
  • 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.
  • 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.
  • bulk wires and or foil may be integrated into the plastic substrate using a hot embossing technique.
  • 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.
  • PDMS polydimethylsiloxane
  • 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.
  • first 1 and second 2 planar substrates made be made from various materials, including, glass or silicon
  • 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.
  • 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.
  • 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.
  • the 2-D plastic microfluidic device separates protein analytes with ultrahigh resolution based on their differences in isoelectric point and size.
  • 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.
  • the material e.g. proteins
  • the material 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.
  • one or more reservoirs 10 at the end of one or more tertiary microchannels 11 may be used for introducing the electrophoresis buffer containing SDS and SYPRO fluorescent dyes during electrokinetic transfer of proteins from first dimension to second dimension.
  • proteins may be covalently labeled with a suitable florescent dye and detected by a suitable florescence detector.
  • a fluorescent dye is 5-carboxy 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.
  • the proteins may be prepared in a suitable solution (e.g., a solution including urea, dithiothreitol, and Tris-HCI with approximate concentrations of 8M, 100 mM and 0.1 M respectively) with a pH preferably ranging from 4 to 10.
  • a suitable solution e.g., a solution including urea, dithiothreitol, and Tris-HCI with approximate concentrations of 8M, 100 mM and 0.1 M respectively
  • 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 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 the fluorescence detection of proteins and they provide low fluorescence background. Other materials may be used.
  • 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 creation of pH gradient in the microchannel.
  • size-dependent separation of SDS-protein complexes in the second dimension is based on their differences in electrophoretic mobility inside a polymer sieving matrix.
  • a pressure filling approach may be used for introducing two different media into the at least one first dimension microchannel and array of second dimension microchannels respectively.
  • Gel solutions may be introduced into microchannel arrays (e.g., 3, 4) by applying pressure in reservoirs.
  • 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 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 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 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 pushing a polymer gel solution.
  • 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.
  • the entire plastic microfluidic network may initially be 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.
  • an external force 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 gel from the second-dimension microchannels.
  • 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 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.
  • the solution containing any carrier ampholytes for the isoelectric focusing may then be introduced via pressure from reservoir 5 for filling the first dimension channel connecting reservoirs 5 and 6.
  • 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.
  • 2-D protein separation 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 (C).
  • D illustrates the assembly of 2-D protein separation platform.
  • the pressure needed for pushing a solution through the membrane with a pore diameter of approximately 0.1 ⁇ m may be at least 50 times higher than that required for introducing an aqueous solution into the microchannels.
  • the solutions may take a lower flow-resistance path.
  • two different electrophoresis media may be separately filled in the upper and lower microchannels.
  • 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.
  • 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 separating DNA, peptides, and other chemical and biological composites.
  • focused proteins in the first dimension are simultaneously transferred to the second dimension by hydrodynamic pressure.
  • the two dimensions should be orthogonal, and any separation accomplished by the first dimension should ideally be retained upon transfer to the second dimension.
  • First dimensional microchannels 3 connecting reservoirs 5 and 6 may be 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 hydrostatic pressure may be therefore in place across the entire isoelectric focusing channel during this step.
  • the transfer process may be similar to dead-end membrane filtration.
  • 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.
  • a polymeric membrane serves as the injection ports for transfer of proteins.
  • the voltage may be turned off and the solution containing catholyte in 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.
  • 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 applying two separate positive potentials at reservoir 6 and 8 using two high voltage power supplies (VI 4, VI 6).
  • 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 into the second separation dimension.
  • 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.
  • 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.
  • a positive electric potential may then be applied briefly at reservoir 8 for rapid electrokinetic injection and filling of SDS and dye 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.
  • the proteins focused in the first dimension may be incubated with newly introduced SDS and dye for approximately 5-10 minutes.
  • 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 reservoir 10 while applying a positive potential at reservoir 8 and letting reservoirs 5 and 6 float.
  • 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 transferred into the second dimension (e.g., 4) electrokinetically.
  • 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.
  • electrokinetic transfer method 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.
  • 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.
  • 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.
  • 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 piano 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 ⁇ m 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 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 sequence dependent separations with real-time fluorescence detection and imaging.
  • 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.
  • 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.
  • 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.
  • 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 vertically in a narrow line across the array.
  • 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.
  • CCD charged-coupled device
  • the CCD sensor is comprised of 26 ⁇ m 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 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.
  • microfluidic 2-D PAGE may be integrated with mass spectrometry employing matrix-assisted laser desorption/ionization (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 system offers large-scale analysis of proteins and "differential display” proteomics for comparisons of protein expression under various environmental and physiological conditions.

Abstract

Dans un mode de réalisation, l'invention concerne un dispositif microfluidique destiné à réaliser des séparations biomoléculaires bidimensionnelles. Selon un aspect de l'invention, après une séparation dans la première dimension à l'intérieur d'un microcanal, la matière échantillon est transférée électrocinétiquement et simultanément vers un réseau de microcanaux dans la seconde dimension (par ex., par modification des potentiels électriques au niveau des réservoirs reliés aux microcanaux). De préférence, toute séparation réalisée dans la première dimension est entièrement conservée après le transfert vers la seconde dimension. Selon un autre aspect de l'invention, la séparation dans la seconde dimension est réalisée au moyen d'un gradient de température (tel qu'un gradient de température spatial ou temporel). Selon un mode de réalisation de l'invention, la matière biomoléculaire comprend de l'ADN, la séparation dans la première dimension est une séparation fondée sur la taille, et la séparation dans la seconde dimension est une séparation fondée sur la séquence. Selon un autre mode de réalisation, le dispositif bidimensionnel microfluidique comprend un premier et un second substrat planaire incluant au moins un microcanal dans la première dimension se prolongeant dans un premier sens ainsi qu'un réseau de microcanaux dans la seconde dimension se prolongeant dans un second sens de préférence perpendiculaire à la première dimension. Les extrémités de certains au moins des microcanaux sont en communication fluidique avec une pluralité de réservoirs. Les substrats peuvent également comprendre une pluralité de microcanaux et de réservoirs. Ces réservoirs sont en communication électrique avec une pluralité d'électrodes et de sources d'alimentation en tension. Ce dispositif permet des séparations bidimensionnelles de protéines et d'autres biomolécules. Selon un autre aspect de l'invention, une séparation fondée sur le point isoélectrique est possible dans une première dimension, une séparation fondée sur la taille étant possible dans une seconde dimension.
PCT/US2003/013580 2002-05-01 2003-05-01 Dispositifs microfluidiques en plastique permettant des separations bidimensionnelles de molecules biologiques WO2003092846A2 (fr)

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WO2005089910A1 (fr) * 2004-03-17 2005-09-29 Ciphergen Biosystems, Inc. Filtre a plusieurs compartiments et procede de filtrage au moyen de ce filtre
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