WO2008058204A1 - Dna purification in a multi-stage, multi-phase microchip - Google Patents

Abstract

The present invention allows for improved efficiency in microfluidic-based nucleic acid purification by using a multi-stage microfluidic device, where each stage uses a different phase to specifically remove contaminants, with the last stage being a nucleic acid binding phase to selectively capture the nucleic acid in a sample. In the contaminant removal stage(s), the sample is cleansed of contaminant(s) that can adversely affect the nucleic acid binding of the last stage of in subsequent reaction involving the purified DNA, such as polymerase chain reaction (PCR).

Description

DNA PURIFICATION IN A MULTI-STAGE. MULTI-PHASE MICROCHIP

The present invention claims priority from U.S. Provisional Application Serial No. 60/857,276, filed November 7, 2006, which is incorporated herein by reference. BACKGROUND OF THE INVENTION

DNA purification and pre-concentration is a requirement for most genetic analysis applications, primarily due to the complex nature of biological samples. Whole blood is a particularly complex mixture of nucleic acids, proteins, lipids, metabolites, and inorganic ions, and some of these species, e.g., hemoglobin and heparin, are known to inhibit PCR amplification of DNA (1), a core methodology for almost all genetic analyses. Typically, 1 μL of a lysed human blood sample contains 35 ng of DNA amidst -150 μg of protein. Purification of this DNA is most often performed using solid phase extraction (SPE), which has largely replaced liquid extraction methods for purifying DNA from biological samples. However, the proteins in biological samples, particularly many of those in whole blood, bind avidly to the solid phase, thus limiting the DNA binding capacity, and requiring a wash step to effectively remove protein PCR inhibitors from the solid phase.

The development of solid phase extraction for DNA purification, has also allowed for miniaturization of the DNA extraction process to minimize sample handling, reduce contamination, and expedite analysis time (2,3). Silica phases are the most often utilized solid supports for miniaturized DNA SPE (4), but the proclivity of proteins to bind to the silica surfaces necessitates a wash step. Tian et al (5) reported DNA purification from white blood cells and whole blood on silica beads in a bed volume of only 0.5 μL, but noted decreased capacity and lower extraction efficiencies in comparison with the extraction of pre-purified genomic DNA. This indicated that competition from proteins for binding sites was problematic and, as a result, only 0,2 μL of whole blood could be purified on the device. Breadmore et al, (6) used a tetraethoxyortho silicate (TEOS)-based sol-gel to immobilize the silica beads in a rnicrodevice, but could also show effective extraction of DNA from only 0.2 μL of whole blood. Wu et al. (7) utilized a tetramethyl orthosilicate (TMOS) based sol-gel phase using poly- (ethylene glycol) (PEG) as the porogen to provide a large silica surface area, but again, only 0.2 μL of whole blood was loaded, with 60% of DNA recovered in -12 μL of elution buffer. A microdevice coupling on-chip DNA purification and amplification was shown to successfully amplify extracted DNA from a slightly larger volume of blood (0.6 μL), but the capacity and extraction efficiency were not measured (3). The integrated device illustrated a problem with the wash step, in that residual isopropanol from the wash step contaminated the early eluted fractions (which coincidentally contained much of the DNA) and, subsequently, inhibited the amplification. Thus, it is clear that the predominate presence of proteins in any complex biological sample (including blood) complicates the extraction of DNA decreases the binding capacity of the solid phase for DNA, and necessitates the use of a wash step with a reagent (isopropanol) that is a inhibitor of the subsequent PCR-based DNA amplification.

To minimize protein binding and avoid the use of chao tropic/organic reagents in the DNA purification process, alternative solid phases have been developed. Witek et al. (8) reported the preconcentration of DNA using a photo activated polycarbonate (PPC) microfluidic device with a capacity of 150 ± 30 ng of DNA from E. coli whole cell lysate. An extensive wash step was required, but the device could be dried before the DNA was eluted to remove any inhibition due to the ethanol in the wash buffer. Larger elution volumes were required, however, and this work did not address DNA purification from human whole blood. Nakagawa etal. (9) demonstrated the use of an amine-coated microchip to extract DNA from whole blood. This method avoided the use of PCR- inhibiting reagents, such as isopropanol, but a large elution volume (>15 μL) and poor efficiency (27-40%) were reported as only 10 ng of DNA were recovered from the -44 ng of DNA typically present in 1.25 μL of blood. The problematic nature of protein binding in DNA extraction methodologies is, perhaps, best encaspsulated by the work of Cao et al. (10) who used a novel chitosan-coated multichannel microchip to demonstrate DNA purification in a total aqueous system (i.e., avoiding the use of organic reagents). High extraction efficiency (75%) in small elution volumes and increased blood capacity (up to 1.5 μL) were observed due to low protein binding to the chitosan phase. While volume capacity of 1.5 μL of blood represents a 7-fold improvement, larger capacity still is required for many clinical analyses. Approaches to improving the capacity of extraction phases for chaotrope-based

DNA purification from blood will likely exploit one of two paths ~ increase the surface area of the phase or decrease the protein bonding to potential DNA binding sites. Increasing extraction phase surface area can be achieved by packing smaller diameter beads (e.g., 5 μm), resulting in high back pressure, or by etching pillars in the channels during fabrication (1 1), which increases fabrication cost. Larger functional surface areas for DNA binding can also be met by monolithic materials (12). With the advantages of large surface area, controllable pore size and high mass transfer from the porous structure, monoliths has been recently used for DNA isolation (13,14) or preconcentration (15), A commercially-available large volume monolithic matrix for nucleic acid purification, the anion-exchange based CM® monolithic disk (BIA Separations, Slovenia), has been reported to have greatly increased binding capacity for plasmid and genomic DNA (14), however, the DNA is eluted under high salt and high pH conditions; both of which are problematic for the subsequent PCR (16,17). Our group recently reported on the use of a 3-(trimethoxysilyl)propyl methacrylate (TMSPM) monolith grafted with tetramethyl orthosilicate (TMOS) in a capillary format for DNA purification (15), showing significantly increased DNA binding capacities with purified genomic DNA over other silica phases. The liquid precursor, and UV-induced photopolymerization (18), allowing accurate placement (19) and reproducible fabrication of the monolith, make this method ideal for transfer to a microfluidic device. Use of this phase with blood, however, was limited due to proclivity of proteins for binding to the matrix, thus occupying potential DNA binding sites.

SUMMARY OF THE INVENTION

The present invention allows for improved efficiency in microfluidic-based nucleic acid purification by using a multi-stage microfluidic device, where each stage uses a different phase to specifically remove contaminants, with the last stage being a nucleic acid binding phase to selectively capture the nucleic acid in a sample. The last stage involves the use of a column to reversibly capture the nucleic acids, preferably a grafted, UV photo-polymerized silica-based monolithic micro-column described in WO 2006/093865, which is incorporated herein by reference, or a matrix entrapped chitosan column disclosed in WO 2006/088907, which is incorporated herein by reference. Prior to nucleic acid capture in the last stage, the sample, in at least one contaminant removal stage, is preferably cleansed of contaminant(s) that can adversely affect the nucleic acid binding of the last stage of in subsequent reaction involving the purified DNA, such as polymerase chain reaction (PCR). Those contaminants may include most any component of a cell lysate or reagents used the process of lysing the cell. They may be proteins, peptides, lipids, carbohydrates, small molecules, etc. For example, prior to the last

(nucleic acid capture) stage the sample may pass through a micro-column containing Cl 8 packing to selectively remove proteins, but allowing the nucleic acids to pass therethrough. The nucleic acids are then reversible captured in the grafted, UV photo- polymerized silica-based monolithic micro-column. The bound nucleic acids can subsequently be eluted from the column using a minimal volume of a low ionic strength buffer, which may further allows direct PCR analysis of the extracted nucleic acid without further sample cleaning steps. The present inventors have discovered that by removing the contaminants prior to nucleic acid purification, it possible to improve nucleic acid purification efficiency by increasing capacity for nucleic acid extraction in the last stage.

The apparatus of the present invention relates to a microfluidic device having at least two fluidly linked stages, with the down stream most (last) stage including at least one micro-column for binding nucleic acids. The micro-column is preferably a grafted, UV photo-polymerized silica-based monolithic column for reversibly adsorbing nucleic acids; or a matrix immobilized chitosan column. The upstream (contaminant removal) stage(s) including at least one micro-column for removing contaminants that can adversely affect the nucleic acid binding in the last stage.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic of a simple embodiment of the present invention.

Figure 2 is a schematic of an embodiment of the present invention which contains more than one contaminant removal stage. Figure 3 is a schematic of an embodiment of the present invention where more than one column in parallel is used in the contaminant removal stage.

Figure 4 shows typical breakthrough curves obtained from purified genomic DNA loading on monolithic microdevices derivatized with TMOS for varying times (25, 45, and 75 min). Sample (18 ng DNA/μL in 6 M Gu-HCl, pH 5.8) was loaded at 4 μL/min and fractions collected every 10 μL. Inset: straight channel microchip utilized for monolith optimization; channel dimensions (top width x depth * bottom width): 570x185x200 μm; monolithic phase length: 4 mm. Inset table shows chip and monolith binding capacities for each derivatization time on different chips (n =3).

Figure 5 shows a DNA breakthrough curve obtained on a monolithic column with human whole blood as the sample. Channel and load conditions as in Figure 4; TMOS derivatization time: 45 min.

Figure 6 shows the optimization of the pre-column channel design for reverse phase protein capture from lysed human whole blood sample. Reverse phase length: 10 mm; Flow rate: 4 μL/min. Figure 7 shows the Effect of Triton X-100 concentration on protein binding to the

Cl 8 reverse phase pre-column. Inset: parallel 4 chamber microchip design utilized for Triton X-100 experiments; Chamber dimensions (top width x depth x bottom width): 2770x385x2000 μm; Packed bed length: 10 mm; Flow rate: 5 μL/min. Fractions were collected every 5 μL.

Figure 8 shows an integrated protein capture/DNA extraction in two-stage, dual- phase microdevice for DNA extraction from whole blood. A) Cl 8 beads and monolith in channels; B) dye-filled device (same as the device of Figure 8A) showing protein capture section (Cl 8 section) and DNA extraction section (monolithic section) in different color shadings. Reverse phase dimensions as in Figure 7, monolith dimensions as in Figure 4.

Figure 9 show the elution of DNA and proteins from the Cl 8 phase. The flow rate was 5 μL/min and fractions were collected every 5 μL. A) Continuous loading of a whole blood lysis solution; B) loading of a lysis solution containing 10 μL of blood. Reverse phase chamber dimensions as in Figure 7.

Figure 10 shows the extraction profiles showing device-to-device reproducibility for DNA extractions from whole blood using the two-stage, dual-phase microdevice. Left insets show the well packed Cl 8 phase device which provides reproducible results and the poorly packed device which significantly decreases the extraction capacity of the monolith. Right inset shows PCR profiles from amplification of a 139bp gelsolin fragment directly from the eluted DNA with no wash step, DNA was eluted in 10 mM Tris, 1 mM EDTA, pH 8.0 at 4 μL/min with fractions collected every 1 μL.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below:

As used herein, the term "purify" and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term "purify" does not necessarily indicate that complete purity of the particular molecule will be achieved during the process, but rather an concentration of the particular molecule wilt be improved when compared to the starting material. A "highly purified" compound as used herein refers to a compound that is greater than 90% pure.

As used herein a "chaotropic agent" is an agent that is capable of disrupting the hydrogen bonding between water and nucleic acid and includes, but is not limited, to urea, guanidine hydrochloride, guanidine thiocyanate, and potassium iodine.

As used herein, the term "derivatization" and like terms refer to the process of bonding the molecules of a suitable silica-based monomer reagent to the surface of a silica-based monolithic column. The term "grafted" refers to the state of the silica-based monolithic column after derivatization.

As used herein, the term "immobilization" and like terms refer to the attachment or entrapment, either chemically or otherwise, of material to another entity (e.g., a solid support) in a manner that restricts the movement of the material.

As used herein a "micro-column" is a micro-channel or portion thereof having a matrix comprising pores of a preferred size of about 0.1-15 μm in diameter, more preferably about 1-10 μm, most preferably about 4-6 μm; or having material immobilized on the wall of the micro-channel. The matrix and the material are designed to selectively sequester selected compounds. The micro-column preferably has an average cross sectional dimension of about 5 mm to about 100 μm , more preferably about 1 mm to about 7,000 μm , and most preferably 0.3 mm to about 30,000 μm . As used herein a "micro-channel" is a passageway (in any form, including a closed channel, a capillary, a trench, groove or the like) formed on or in a microfiuidic substrate (a chip, bed, wafer, laminate, or the like) having at least one region with a cross sectional dimension of about 5 mm² to about 100 μm², preferably about 1 mm² to about 7,000 μm , and more preferably 0.3 mm to about 30,000 μm . A "microfiuidic device" is an apparatus or component of an apparatus that includes at least one micro-channel.

As used herein, the term "sol-gel¹¹ refers to preparations composed of porous metal oxide glass structures. The phrase "monolithic column" and like phrases refer to a solid bonded network of silica prepared by a hydrolysis - condensation polymerization reaction of suitable monomers. The materials used to produce the monolithic column can include, but are not limited to, aluminates, aluminosilicates, titanates, ormosils (organically modified silanes), and other metal oxides.

"Fluidly connected" or variations thereof, as used herein, refers to a condition wherein two domains are connected to each other such that fluid can pass from one domain to another.

The present invention is directed to a microfiuidic device for efficiently purifying nucleic acids. Referring to Figure 1, in a simple embodiment of the present invention, the microfiuidic device 10 includes an inlet port 12 that is fluidly connected to a contaminant removal stage 14 that contains a packing material to remove any contaminants that may deleteriously affect the binding of nucleic acids to the grafted, UV photo-polymerized silica-based monolithic micro-column of the nucleic acid capture (last) stage 16. The inlet port 12, contaminant removal stage 14, nucleic acid capture stage 16, and outlet port 18 are fluid connected with micro-channels 20. Although Figure 1 illustrates only one contaminant removal stage, in certain embodiments, more than one stage may be used to remove multiple contaminants. For example, a stage may contain a column to remove proteins, while another stage may contain a column to remove lipids. A multi-stage contaminant removal microfluidic device is illustrated in Figure 2, which has a first contaminant removal stage 14a and a second contaminant removal stage 14b.

Importantly, however, the contaminant removal stage(s) preferably does not capture or bind the nucleic acids, thus, allowing in the nucleic acid to pass through to the last (nucleic acid capture) stage. This ensures that most, if not all, of the nucleic acids in the original sample is available for capture in the nucleic acid capture stage 16, thereby allowing full (or close thereto) recovery of the nucleic acids.

Depending on the contaminants in the sample, one skilled in the art would be equipped to determine the number of stages required to selectively remove the contaminants without undue experimentation. The contaminant(s) can adversely affect the nucleic acid purification, for example by competing with the nucleic acid binding in the column used during the nucleic acid capture stage such as the case with proteins, or by inhibiting PCR involving the purified nucleic acids such as the case with indigo dyes. The contaminants may be, but are not limited to, proteins, peptides, lipids, oils, organic acids, carbohydrates, small molecules, amino acids, protein hydrolysates, etc. To remove proteins or peptides, the following non- limiting list of columns are appropriate: C30, C22_> Cl 8, C12, C8, C5, C4, C2, styrene, styrene/divinylbenzene co-polymer(S-DVB), aminopropyl phase (ph), benzene sulfonic acid phase, polyamide phase, quaternary amine, and cyanopropyl bonded phase (CN). To remove oils or lipids, the following non- limiting list of columns are appropriate: Cl 8, cyanopropyl bonded phase (CN), diol or dihydroxy phase, aminopropyl phase (ph), ethyl en ediamine-N-propyl phase, and silver ion phase. To remove carbohydrates, organic acids, or dyes, the non-limiting list of following columns are appropriate: lectins, Cl 8, C8, sulfonated styrene/divinylbenzene, polyhydroxymethacrylate, polymethylmethacrylate, polystyrene, polyvinyl alcohol, quaternary amine phase, benzene sulfonic acid phase, polyamide phase, cyanopropyl bonded phase (CN), aminopropyl phase (ph), and phenyl. To remove small molecules, the following non-limiting list of columns are appropriate; aminopropyl phase (ph), benzene sulfonic acid phase, polyamide phase, quaternary amine, benzene sulfonic acid phase, styrene, cyanopropyl bonded phase (CN). To remove amino acids or protein hydrolysates, the following non-limiting list of columns are appropriate: silica, Cl 8, Cl 2, C8, C5, C4, C2, phenyl-hexyl, cyanopropyl bonded phase (CN), aminopropyl phase (ph), polystyrene/divinylbenzene (PS/DVB) copolymer phase, polyamide phase, and quaternary amine phase. Instead of targeting particular classes of compounds, the column can selectively binds certain moieties on the selected compounds. For example, immobilized metal affinity columns (IMAC) can be used to bind phosphate groups.

In an embodiment of the present invention, each stage may contain more than one column in parallel or in series to efficiently remove the contaminants. Figure 3 shows an embodiment where the contamination removal stage includes more than one identical column 14 in parallel to effect improved efficiency in removal of the contaminant. Several contaminant removal stages may be placed in series where each stage may contain one or more identical columns placed either in series or in parallel. If more than one column are used in each stage, it is preferred that they are placed in parallel to effect high throughput processing of the sample, In certain embodiments, the nucleic acid capture stage (16) may also contain more than one columns in parallel or series.

In operation, a sample is placed in the inlet port 12, and then flowed through the contaminant removal stage(s) 14 where the contaminant(s) is captured by the packing material inside the column, while allowing the nucleic acid to pass therethrough to the nucleic acid capture stage 16. At the nucleic acid capture stage 16, the nucleic acids are bounded to an absorption material, preferably a grafted, UV photo -polymerized silica- based monolithic column or a matrix immobilize chitosan column. That binding, however, is reversible and the nucleic acid may be eluted from the monolith using an appropriate buffer. The eluted nucleic acid may be used for subsequent amplification and/or analysis. As such, although the Figures 1 -3 show an outlet port 18 , this outlet port 18 may be substituted with a PCR domain or an analysis domain as discussed in detailed in WO/2007/047336, which is incorporated herein by reference.

The preferred grafted, UV photo-polymerized silica-based monolithic column used in the nucleic acid capture stage is prepared in a similar method as described in WO 2006/093865, which is incorporated herein by reference. This material is a silica based monolith polymer whose surface has been derivatized (grafted) with a silica based monomer. To prepare the grafted, UV photo-polymerized silica-based monolithic column of the present invention, a silica-based monomer solution is prepared by is comprised of TMSPM and is present in the polymerization mixture in a concentration of 15 to 30%, more preferably about 19 to 27%, and most preferably 23 to 25%. The photoinitiator, can be, but not limited to, benzophenone, dimethoxyacetophenone, xanthone, thioxanthone, and mixtures thereof with a commercially available mixture. The porogenic solvent functions to foster the development of a suitable pore structure in the monolithic column during the polymerization process. The porogenic solvent can be, but is not limited to, water, organic solvents, and mixtures thereof. Organic porogenic solvents can be, but not limited to, hydrocarbons, alcohols, ketones, aldehydes, organic acid esters, soluble polymer solutions, and mixtures thereof. In a preferred embodiment, the solution is comprised of toluene, as the preferred porogenic solvent, in a concentration of about 70 to 85%, more preferably about 73 to 81%, and most preferably 75 to 77%.

The polymerization mixture is then introduced into the channel (column) in the microfiuidc device. In one embodiment, the channel is conditioned prior to addition of the polymerization mixture. Conditioning of the channel comprises the steps of rinsing the interior surface of the channel with a basic solution (for example, 1 M NaOH), water, an acidic solution (for example, 0.1 M HCl), and an alcohol solution; flushing the interior surface of the channel with a silica-based flushing solution; and allowing the channel to dry. In a preferred embodiment, the silica-based flushing solution is comprised of TMSPM in a concentration of about 5 to 50%, more preferably about 20 to 40%, and most preferably 30 to 35%.

In situ polymerization of the polymerization mixture within the channel is then initiated to form a polymerized monolithic column within the channel. In one

15 embodiment, polymerization is initiated by exposing the polymerization mixture within the channel to a UV light source, such as, for example, a Model RSMlOOW 100 W broad UV wavelength lamp. In a further embodiment, the length of the monolithic column formed in the channel through polymerization is controlled by selective exposure of sections of the polymerization mixture within the channel to UV light. To achieve selective exposure, an extra UV-resistant mask with selective section of window in the area of the desired monolith was placed below the channel and other portions of the channel are protected with a UV-resistant material, such as, for example, UV-block mask, a black cloth, a black-tape covered plate piece during UV exposure to prevent polymerization at these particular locations in the channel.

Once the photo-polymerized silica-based monolithic column has been formed in the channel, the silica monomer may be grafted onto the surface of the monolithic column by flushing the column with a silica-based monomer reagent. In a preferred embodiment, the silica-based monomer reagent is comprised of tetramethylorthosilicate (TMOS), which has enhanced hydrolysis properties under acid-catalyzed conditions and bonds to the surface of the monolithic column at a faster rate, thereby reducing the total fabrication time for the monolithic column. In a further embodiment, TMOS is preferably present in the silica-based monomer reagent in a concentration of about 85%. The reagent is vortexed at room temperature for one minute and then pressure flushed through the column at room temperature. In a further embodiment, the reagent is flushed through the column for 45 minutes. The hydrolyzed TMOS molecules are the bound to the column. In yet another embodiment, TMOS is bound to the column through condensation with unreacted siloxane/silanol groups that are present on the surface of a photo-polymerized

16 monolithic column prepared using TMSPM, allowing the formation of a continuous network of silicon dioxide and increasing the number of silica binding sites on the surface of the column as a result.

The resulting grafted, UV photo-polymerized silica-based monolithic column possesses enhanced capacity and efficiency for extraction of nucleic acids. Binding of nucleic acids to this matrix utilizes the same mechanism as all silica based phases for nucleic acid extraction, with the presence of the chaotropic salt causing the nucleic acids to adsorb on the silica surface. Optimal chaotropic concentration, pH range, and temperature will vary with the phase utilized and can be determined by one skilled in the art through routine experimentation. Release from the surface occurs into a low ionic strength aqueous buffer following removal of the chaotropic agent.

The other preferred nucleic acid binding phase is the matrix entrapped chitosan disclosed in WO 2006/088907, which is disclosed herein by reference. This column requires the formation of chitosan that is immobilized to a matrix, preferably of another polymer, more preferably of a sol-gel. Immobilized, as used herein, means that the chitosan may be physically contained in the matrix or may be chemically linked to the matrix material. Physical containment of the chitosan means that the chitosan is physically trapped within the matrix without being chemically bonded to the matrix material. On the other hand, the chitosan may also be chemically bonded to the matrix material through ionic, covalent, or other chemical bonds, chitosan as a copolymer with another polymer, thereby being entrapped in a matrix. The copolymerization may contain various crosslinking to form a solid or a gel.

17 In a preferred embodiment, the chitosan and the matrix material are copolymerized to form a copolymer, preferably a chitosan/sol-gel composition, In this embodiment, the sol-gel are preferably formed from silanes, such as aldehyde triethoxysilanes, aminopropyl triethoxysilanes, 3-glycidyloxypropyl trimethoxysilane (GPTMS), most preferably GPTMS. The sol-gel is formed either under the acidic condition pH from 0.1 to 6), most preferably between 2-5, or under the basic condition pH from 8 to 12, most preferably between 8 to 10. The addition of 0.1% to 50 %methanol or ethanol is preferably accelerates the form the chitosan/sol-gel copolymer. The reaction temperature is from 10 centigrade to 90 centigrade, preferably at 30 centigrade. The reaction time of forming of chitosan/sol-gel copolymer is from 1 min to 64 hours, depending on the pH value, reaction temperature, and concentration of methanol and ethanol. A preferred chitosan/sol gel composition may be made with chitosan and GPTMS are polymerized to form a cross-link copolymer (chitosan/sol gel). The copolymer may be used alone or coated onto a bead The matrix entrapped chitosan can be used to purify nucleic acids from a sample using mild conditions that do not affect, even temporarily, the chemical integrity of the nucleic acid. The method comprises contacting the sample with the matrix entrapped chitosan phase which is able to bind the nucleic acids at a first pH, and extracting the nucleic acid from the solid phase by using an elution solvent at a second pH. In the binding step, the matrix entrapped chitosan phase selectively binds the nucleic acid and retained it thereon. The binding pH is preferably about 3-6, more preferably about 4-5, and most preferably about 5. The elution pH is preferably greater than about 8, more preferably about 8-10, and most preferably about 9. Preferably, the elution step is carried

18 out in the substantial absence of NaOH, preferably also the substantial absence of other alkali metal hydroxides, more preferably the substantial absence of strong mineral bases. Substantial absence means that the concentration is less than 25 mM, preferably less than 20 mM, more preferably less than 15 mM or 10 mM. Preferably, the multi-stage, multi-phase system of the present invention is used to purify nucleic acid in a micro-total analysis system (μ-TAS). There are many formats, materials, and size scales for constructing μ-TAS. Common μ-TAS devices are disclosed in U.S. Patent Nos. 6,692,700 to Handique et al.; 6,919,046 to O'Connor et al.; 6,551 ,841 to Wilding et al.; 6,630,353 to Parce et al.; 6,620,625 to WoIk et al.; and 6,517,234 to Kopf-Sill et al.; the disclosures of which are incorporated herein by reference. Typically, a μ-TAS device is made up of two or more substrates that are bonded together. Microscale components for processing fluids are disposed on a surface of one or more of the substrates. These microscale components include, but are not limited to, reaction chambers, electrophoresis modules, microchannels, fluid reservoirs, detectors, valves, or mixers. When the substrates are bonded together, the microscale components are enclosed and sandwiched between the substrates. A major advantage of using a μ-TAS resides in the fact that the monolithic column can be used a part of an integrated system for analysis.

Various aspects of embodiments of the present method and system may be implemented with the following methods and systems disclosed in WO 2003/104774, WO 2006/069305, WO 2006/088907, and WO/2007/047336, which are incorporated herein by reference.

19 Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following example is given to illustrate the present invention. It should be understood that the invention is not to be limited to the specific conditions or details described in this example.

EXAMPLE

Materials and Methods Materials and reagents

3-(Trimethoxysilyl)propyl methacrylate (TMSPM, minimum 98%), tetramethyl orthosilicate (TMOS, 98%), λ-phage DNA and PCR reagents were obtained from Sigma- Aldrich (Milwaukee, WI). Methanol (99.9%), hexane (99.9%), toluene (99.9%), 2- propanol (HPLC grade), guanidine hydrochloride (GuHCl, electrophoresis grade), ethanol (95%), and Tris(hydroxymethyl)aminomethane (Tris) were purchased from

Fisher Scientific (Fairlawn, NJ). EDTA was obtained from American Research Products (Solon, OH). Photoinitiator Irgacure 1800 was generously donated by Ciba (Tarrytown, NY). Octadecyl (C18) silica beads (13-17 μm particle size, 300 A wide pores, carbon content 11%) were obtained from J.T. Baker (Phillipsburg, NJ). Picogreen® dsDNA assay kit was obtained from Molecular Probes (Eugene, OR) for DNA quantification and BCATM protein assay kit was obtained from Pierce (Rockford, IL) for protein quantification. Polyetherether-ketone (PEEK) tubing of 360 μm o.d., ferrules, rings and nut assemblies for chip-based microchip connections were purchased from Upchurch

20 Scientific (Oak Harbor, WA). All solutions were prepared with nanopure water Barnstead/Thermolyne, Dubuque, IA).

Microchip fabrication All glass microchips were fabricated as described previously (22) through standard photolithography, wet etching, and thermal bonding (640 °C, 8 h). Borofloat glass slides (127 mm x 127 mmx 1.1 mm) were purchased from Telic (Valencia, CA), pre-coated with chrome and positive photoresist (AZl 500 resist, 5300 A). Channels were etched using 100% hydrofluoric acid (HF), and differentia! depths were achieved by etching in a stepwise fashion using HF-resistant dicing tape (Semiconductor Equipment Corporation, Moorpark, CA). Multiple chip designs were investigated to optimize the various SPE steps: monolith DNA extraction, protein capture and two-stage DNA extraction. The monolith DNA extraction design contained channels (20 mm long, 200 μm initial width) which were etched 185 μm deep. The channels for the protein capture pre-column were etched 200 or 385 μm deep, and contained a 30 μm deep weir to retain the beads. The two-stage DNA extraction device utilized the monolith DNA extraction and protein capture columns, which were connected by a 10 mm long, 20 μrn initial width channel etched 200 μm deep. Reservoirs were drilled into a coverplate using triple ripple diamond tipped bits of 1.1-mm or 1 ,8-mm diameter (Crystalite Corporation, Lewis Center, OH).

Preparation of the photo-polymerized monolith on a microchip

The microchip channel was treated with 1 M NaOH for 20 min, ddH2θ for 10 min

21 and 1.2 M HCl for another 20 min, then rinsed with 95% ethanol and dried in an 80°C oven for 1 h. The channel was then modified using 30% (v/v) TMSPM solution (in 95% ethanol), adjusted to pH 5 with acetic acid, for 12 h in the dark (15). The treated channel was subsequently rinsed with 95% ethanol and dried in the 80 ⁰C oven for 1 h before use. The monomer solution preparation was similar to the one described previously (15). A precursor solution consisting of 85% (v/v) TMSPM and 15% (v/v) 0.1 M HCl was stirred at room temperature in the dark for 1 min to afford a clear solution. The monomer solution was prepared with 24% (v/v) precursor solution, 76% (v/v) toluene and 5% (w/v) photo-initiator Irgacure 1800 and stirred in the dark at room temperature for 30 sec. The treated channels were then filled with the monomer solution and the reservoirs covered with a plastic sheet to prevent solvent evaporation. The microchip was sandwiched between tape covered glass slides with a 1 mm x 4 mm window in the tape on the top glass providing the exposure area; the microchip was exposed to UV light for 5 min using a 100 W broad UV wavelength lamp (Model RSMlOOW, Regent Lighting Corp. Burlington, NC) to initiate polymerization. After polymerization, the microchip was mounted in a plexiglas holder and ethanol was flushed through the channel using PEEK® tubing and a syringe pump (KD Scientific, Holliston, MA) to remove excess monomer reagent and visualize the monolith.

Once the monolith was formed, the surface was derivatized by treatment with 85% TMOS solution, as optimized in our previous work (15). Briefly, a mixture of

TMOS and 0.1 M HCl (85%/ 15% v/v) was vortexed at room temperature for 1 min. The TMOS solution was then flushed through the monolith at room temperature at a flow rate of 4 μL/min (various derivatization times were investigated). The monolith was then

22 washed for 15 min with 95% ethanol to remove the excess TMOS reagent at 4 μL/min,

DNA extraction on the monolith

To investigate DNA extraction on the monolith, a pre-purified human genomic DNA sample (18 ng/μL) was prepared in equilibration buffer (6 M Gu-HCl, pH 5.8). Before sample loading, the SPE monolithic column was rinsed with equilibration buffer for 10 min at a flow rate of 4 μL /min. The DNA sample was then deiivered onto the column using a syringe at a constant flow rate (4 μL/min). For measurement of DNA capacity, a fraction was collected every 10 μL and the amount of DNA present in each fraction was quantified using a modified Picogreen^® assay (Molecular Probes, Eugene, OR). One μL of each fraction was diluted into 49 μL TE solution and mixed with another 50 μL 0.5% Picogreen solution in a 96-well PCR plate then incubated for 10 min at room temperature, protected from light. After incubation, sample fluorescence was measured using a Bio-Rad IQTM 5 real-time PCR instrument (Hercules, CA, USA). A standard calibration curve was used to quantify the extracted DNA. For DNA extraction, 15 ng (load sample concentration: 1.5 ng/μL), 50, or 100 ng (load sample concentration: 5 ng/μL) of genomic DNA were loaded onto the monolith, a wash step was subsequently performed using 20 μL 80% (v/v) 2-propanol in water to remove unbound DNA and proteins/contaminants. DNA was then eluted off the monolith with 20 μL TE buffer at a 4 μL/min flow rate, and the total amount of DNA eluted was determined using the Picrogreen® assay.

The blood capacity of the monolith was determined using human whole blood,

23 which was chemically-treated prior to extraction to release the DNA from the white blood cells. For different chip capacity investigations, proteinase K (10 μL of a 10 mg/mL solution prepared in ddH2θ) was added to either 8 μL or 32 μL of whole blood; the solution was then diluted to 400 μL (0.02 or 0.08 μL blood/μL lysate) in cell lysis buffer (6 M Gu-HCl, 0.1-1 % v/v Triton, pH 5,8) and vortexed for 1 min before incubation in a water bath at 56⁰C for 10 min. While directly loading the 0.02 μL blood/μL lysate sample onto the monolithic phase, fractions were collected every 5 μL and the DNA present in each fraction was quantified using the modified Picrogreen® assay.

Protein capacity measurement of the reverse phase pre-column The reverse phase was used before the monolith as a pre-column to capture proteins present in human blood. To determine the protein capacity of the reverse phase pre-column, a lysed blood sample was loaded. The Cl 8 pre-column was prepared by packing the channels with 13-17 μm octadecyl (C 18) silica beads to a length of 10 mm then equilibrating the phase for 10 min at a flow rate of 4 μL/min. For the pre-column optimization experiments, the 0.08 μL blood/μL lysate was loaded at a flow rate of 5 μL/min. To determine the Triton X-100 effect on protein binding to the reverse phase, sample was loaded at 5 μL/min and up to 40 fractions (5 μL each) were collected during the loading step. The protein and DNA present in each fraction were quantified using a modified BCA® protein assay and the modified Pico green® assay, respectively. For the modified BCA® protein assay, 1 μL of each aliquot was diluted 10-fold in ddtkO prior to mixing the samples with the BCA working reagent to avoid reagent precipitation due to

24 the high Gu-HCl concentration in the samples. Working reagent (80 μL) was then added and the samples incubated at 37⁰C for 30 min before absorbance was measured at 562 nm using a spectrophotometer (Shimadzu UV-1650PC, Columbia, MD). A standard calibration curve was generated for protein quantification,

DNA extraction on the integrated protein capture/DNA extraction microchip For DNA extraction on the integrated microchip, the 4 mm long DNA capture matrix was prepared in the monolithic channel and the surface was derivatized using TMOS solution, which was allowed to flow only in the monolithic channel. The precolumn section was then packed with Cl 8 beads followed by equilibration buffer loading for 10 min at a flow rate of 4 μL /min to equilibrate both phases. Before DNA extraction from blood samples, the monolith was pre-conditioned by running a mock extraction, loading 100 ng of λ-phage DNA, following by eluting with 20 μL elution buffer (10 mM Tris, 1 mM EDTA, pH 8). DNA extraction from a blood sample (0.08 μL blood/μL lysate) was performed in 4 steps. In the first step, 120 μL of blood sample was loaded onto the pre-column at 5 μL /min, and the waste was collected before the DNA monolith.

At this point, the reservoir between the columns was closed and another 10 μL of blood sample was loaded onto the pre-column at 4 μL /min, with the column effluent now passing through the monolith. The loading of blood sample was then stopped and equilibration buffer was loaded to move the DNA recovered from the Cl 8 phase to the monolithic phase. The amount of protein and DNA present in each fraction collected from the monolith during the loading step was determined. The final step was elution of

25 the DNA, by passing elution buffer through the monolith only at 4 μL/min. Twelve fractions of 1 μL were collected, with 0.5 μL of each fraction used for DNA quantification and the remaining 0.5 μL used for PCR amplification of a 139-bp gelsolin fragment. Thermocycling conditions were as follows: 94°C for 30 sec, 6O⁰C for 30 sec and 72⁰C for 30 sec (30 cycles) with a 3 min pre-incubation at 95⁰C and a final extension of 1 min at 72°C. PCR product were analyzed using microchip gel electrophoresis performed in an Agilent Bioanalyzer 2100 (Palo Alto, CA) using the commercially available DNA 1000 kit.

Results and Discussions

The development of a UV-initiated monolith presented the possibility for generating more reproducible DNA purification microdevices, as a result of the ability to accurately control the characteristics and the placement of the immobilized phase within the microchannel architecture. However, the performance of this monolith for DNA extraction in a microfluidic platform had to be investigated to determine the capacity, efficiency and the possible application of this method to real biological crude samples, such as whole blood.

Binding Capacity and DNA Extraction Efficiency of Monolith A DNA extraction method that provides reasonable DNA binding capacity is essential for most clinical applications involving genetic analysis of nucleic acids from complex samples, e.g., whole blood. Since we had shown that the DNA binding site density of a derivatized TMSPM monolith surface was closely related to the density of

26 the derivatized TMOS layer (15), the effect of TMOS derivatization time on the monolith DNA binding capacity was investigated. To determine the DNA binding capacity of the monoliths established in the microdevice shown in the inset of Figure 4, a human genomic DNA (hgDNA) sample (18 ng/μL) was continuously loaded through each monolith and fractions (10 μL) were collected from the channel outlet to generate breakthrough curves. Figure 4 shows the DNA breakthrough curves obtained with a 4 mm long monolith in a straight channel chip (570 μm wide, 185 μm deep) using three different derivatization times - 25, 45 and 75 min. The capacity of each device was determined from the first derivative of the breakthrough curve, while the binding capacity of the monolith itself was calculated based on the volume of the monolith in the device. The shortest monolith derivatization time investigated (25 min) was observed to not only generate the lowest DNA binding chip capacity (3.9 ± 0.6 μg/μL monolith, n = 3), but was also associated with a higher standard deviation on different chips compared to 45 and 75 min derivatization times - this was possibly to the result of an incomplete condensation reaction. The 45 min derivatization time showed a slightly higher binding capacity with less variation between monoliths. The longest derivatization time of 75 min provided a monolith with a binding capacity of 6.0 ± 0.4 μg/μL monolith (n = 3), which is superior to any other microfluidic DNA extraction phase reported to date. It is worthy of note that the curve for the 75 min derivatization time had a significantly shallower profile, which is consistent with a previous report on the CIM® monolithic disk (14), and was attributed to the longer relaxation time needed for large DNA molecules to adapt their shape when passing through the narrow channels of the monolith. Decreased pore size, owing to the TMOS derivatized layer, was also indicated by the substantially

27 increased back pressure after the 75 min derivatization. Generated under these conditions, the monolith was shown to be much less robust and more physically unstable under the high running pressure required; thus, a derivatization time of 45 min, which offered lower back pressure but sufficient binding capacity, was selected for further experiments.

Having defined the reaction conditions for creating the monolith within the microdevice, the extraction efficiency of the device was determined by loading samples containing various masses of DNA (15, 50 and 100 ng). The first extraction on any new monolith suffered from low extraction efficiency, which was consistent with previously report results from Wu et al. (7) on monolithic TMOS-based sol-gels, probably owing to the surface pre-condition and irreversible binding of DNA to sites in dead-end flow paths. Overall, about 30-40 ng of DNA were consistently lost during the initial extractions on each newly formed monolith. Better extraction efficiencies were achieved after the chip was pre-conditioned. Table 1 shows the efficiency and reproducibility of consecutive hgDNA extractions (n = 3) on five different monolithic devices after the surface preconditioning.

28 * Chip dimension (top width xdepth xbottom width) : 570 x 185 x 200 μm. Monolithic phase length: 4 mm. Phase volume: 0.4 μL. TMOS derivatization time: 45 min. Loading gDNA: 1.5 or 5 ng/ μL. Flow rate: 4 μL/min, All DNA samples are dissolved in equilibration buffer ( 6 M Gu-HCl₅ pH 5.8).

^b Extraction efficiency.

For the 15 ng DNA samples, 12.44 ± 0.48 ng could be recovered, corresponding to an extraction efficiency of 83 ± 3%, with device-to-device performance less than 5% RSD. For larger amounts of DNA, the overall average efficiency for microchip extraction was roughly 87% (n = 9), with a run-to-run (intra-chip) RSD of less than 4% (n -- 3); inter-chip (chip-to-chip) extraction efficiency was roughly 85% with a RSD of less than 3% (n = 5). All extracted DNA was observed to be eluted in just 3 μL of solution (data not shown), a substantial decrease in elution volume relative to the most silica-based microdevices. The performance of the monolith was further investigated with the extraction of

DNA from, perhaps, the most challenging of biological samples - human whole blood. To determine the DNA binding capacity, a lysed whole blood sample was loaded, and the fractions collected (as they passed through a non pre-conditioned monolith) were quantified for DNA to generate the breakthrough curve shown in Figure 5. Breakthrough occurred after 0.8 μL of blood was loaded and plateaued at ~1 μL. This indicated that, despite the large capacity observed with pre-purified hgDNA (Figure 4), a DNA binding capacity of less than 35 ng was achieved. The decreased DNA capacity with whole blood most likely involved loss of functional sites for DNA binding to the large mass of proteins (and other lysate components) present in the lysed blood. The extraction efficiency for the blood sample was -35-40% and probably involved some of

29 the proteins blocking of the dead end sites in the monolith. It became clear that, in order to maximally exploit the extraction capabilities of the derivatized TMSPM monolith, interference from protein binding to the monolith had to be reduced or eliminated. Consequently, a method for on-chip removal of proteins from blood was investigated for eventual implementation upstream of the monolith.

Protein Capture Using a Reverse Phase Pre-column

Considering that most blood proteins have reasonable hydro phobicity, the use of an octadecyl (Cl 8) reverse phase pre-column was investigated as a phase that would effectively capture of the proteins with minimal binding of the DNA. Although Cl 8 phases have been applied to the microdevice capture of microorganisms (20) or for preconcentrating trace-level trypsin digests for proteomics applications (21), the high concentration of proteins present in the blood sample challenges the capacity of the reverse phase. Six different microchannel designs having either a single or parallel chambers, and different widths and depths (Figure 6-inset) were investigated for protein capture from blood prior to DNA binding capacity on the subsequent monolith. A lysed whole blood sample was loaded directly through a commercially-available Cl 8 silica bead phase and fractions collected at the outlet were quantified for protein to obtain a breakthrough curve. The high ionic strength buffer used during the DNA extraction loading step (6 M Gu-HCl) provided an environment favorable for protein binding by increasing the exposure of the hydrophobic portions of the proteins. Unlike the DNA breakthrough curves displayed in Figures 4 and 5, the protein breakthrough curves

30 showed a 'wave', or 'roll-up' character - a unique feature observed in frontal affinity chromatography (23),

This 'wave' occurs when the sample contains proteins that compete for the same binding site but have different affinities. As a stronger binding protein reaches the site at which a weaker binding protein has bound, it re-establishes a local equilibrium, displacing the weaker protein. The weaker binding proteins in the sample are thus concentrated at the front and breakthrough in a 'roll-up' curve, which then returns to a plateau value. As can be seen in Figure 6, a nearly 4-fold increase in the chamber volume from design A to B brought only a 2.5-fold increase in the protein capture capacity. To compare the performance of different depths and widths but with a similar total chamber volume, design C was etched to 385 μm deep - this was the maximum depth achievable without creating chips that often cracked under pressure from flow. This design showed a higher capacity than design B but the same capacity as design D, a parallel chamber design. A small increase in capacity was also observed with both designs D and E where only the width of the chambers was increased. The parallel chambers in design F, interestingly, allowed for a proportional increase in blood protein capacity with the total volume of the chamber, possibly due to the slower flow rate through each chamber promoting better interaction of the protein with the reverse phase. The number of parallel chambers could be increased for higher performance, however, the challenge of reproducibly packing beads into all of the chambers increases accordingly. Thus, design F with four parallel chambers was chosen for further experimentation, but with a etch depth that was increased to 385 μm to further enhance the protein binding capacity.

31 With capacity issues addressed, it was imperative to optimize the capture and release kinetic of the DNA with the phase. Triton X-100 (Triton), a well-established agent effective for the lysis of red and white blood cells also aids in the solubilization of cellular membrane proteins, and promotes the dissociation of proteins from nucleic acids - as such, it is a core component of the DNA extraction buffer. However, the lipophilic nature of Triton imparts an inherent affinity for the Cl 8 phase, potentially interfering with protein binding. To elucidate the effect of Triton on the binding of proteins to the Cl 8 phase, the 4-paralIel Cl 8 chamber design (F) was used (inset Figure 7) which had a void volume that was determined to be 15 μL. Figure 7 shows the protein binding breakthrough curves with Triton (concentrations spanning from 0.1 to 1.0%) present in either the bed equilibration and lysis buffers (Figure.7 -left side profiles) or the lysis buffer only (Figure 7-right side profiles). The x-axis is expressed as the volume of blood loaded (μL) excluding the void volume. It should be noted that 7 ± 2% of the proteins were not captured by the Cl 8 phase and were detected in the eluent prior to reaching the breakthrough point; these unretained proteins are assumed to be very hydrophilic. As shown in Figure 7, the presence of Triton in both the lysis and equilibration buffers affected the capacity of the Cl 8 column for protein capture from blood samples. With 1 % Triton in the lysis buffer and the Triton removed from the bed equilibration buffer, the protein binding capacity increased from less than 2 μL of blood to almost 7 μL of blood. In the absence of Triton in the bed equilibration buffer, a decreasing Triton concentration (from 1.0% to 0.1%) provided a significant increase in the amount of proteins which could be captured from the sample. With 0.1% Triton in the lysis buffer,

32 protein breakthrough began at 8.4 μL of blood, with a calculated binding capacity of 9.6 μL. While 0.1% Triton provided optimal protein capture on the C18 phase, this same Triton concentration would have to be effective for sufficiently lysing the white blood cells to release the DNA for capture on the subsequent monolith. Exposing 1 μL of whole blood to 0.1% Triton yielded 35.35 ± 7.24 ng of DNA (n = 14) and 146.05 ± 2.75 μg of protein (n = 7) which did not differ significantly from lysis with 1% Triton (35.22 ± 5.76 ng DNA/ μL blood (n = 18) and 152.73 ± 1.38 μg protein/μL blood (n = 4). These values suggested that 0.1 % Triton was sufficient for comprehensive lysis of the white cells but not the red cells. This was supported by the observation that a red color remained in the pre-column following sample loading when the concentration Triton in the lysis buffer was less than 0.5%. The retained red cells could not be removed by washing with 80% (v/v) 2-propanol and required repacking of the Cl 8 column prior to every extraction. It should also be noted that the observed 'roll-up' effect of the curves decreased with the lower Triton concentrations, suggesting a decreased competition between the released proteins.

A Two-Stage, Dual-phase Microchip Containing Both a C 18 and TMOS Monolith Phase

The two phases (Cl 8 and TMOS monolith) were combined on a single microdevice, with the Cl 8 reverse phase pre-column used for protein capture (stage 1) in series with the monolithic column for DNA extraction (stage 2) (Figure 8). Figure 8A shows the microchip channels filled with the Cl 8 and monolith phases; and Figure 8B shows the channels filled with dyes for better visualization of the microchannel

33 architecture. In order for the DNA to reach the TMOS monolith phase for capture, it had

to traverse the Cl 8 phase -thus, binding of DNA to the Cl 8 phase under the chaotropic conditions associated with the lysis buffer had to be investigated. Figure 9A shows the

protein breakthrough curve (solid squares) from Figure 7, but with an overlay of the DNA breakthrough curve (open triangles) generated under the same conditions. The DNA exited the C18 reverse phase at almost the same time that the proteins broke through, and showed a similar 'roll-up' effect. This suggested that the Cl 8 phase acts as a pre-concentration step for DNA, as it binds to the reverse phase but is then replaced by stronger binding proteins. This is advantageous as it allows the majority of the loaded DNA to be recovered from the Cl 8 column before the bulk of the proteins. It was postulated that, if the load buffer was switched from the sample (lysed blood) to the 6 M Gu-HCl equilibration buffer after a given volume of sample was loaded, the proteins would remain bound while the DNA moved on to the monolith. The majority of protein could then be separated from the DNA, eliminating the competition between the protein and DNA for the binding sites on the monolith. Figure 9B shows the breakthrough profiles for DNA and proteins as lysis solution containing 10 μL of blood was loaded onto the Cl 8 phase, and the loaded sample changed to equilibration buffer. With this approach, significant selectivity could be achieved as shown in Table 2. The net result was that 70% ± 6% of the whole blood proteins could be captured in the stage 1 Cl 8 phase, while allowing >97% of the DNA traversed stage 1 to the stage 2 TMOS monolith for DNA capture.

Table 2. Protein and DNA capture on the Cl 8 and monolith phases

Phases Protein/DNA Amount

34

DNA Extraction Using Two-stage, Dual Phase Microchip

As mentioned previously, successful and reproducible extractions required each newly prepared TMOS coated monolith to be pre-conditioned to block dead-end binding sites with a large mass (100 ng) of λ-phage DNA. Any non-human source of DNA presumably could suffice (e.g., salmon sperm DNA) with the only prerequisite being that it does not interfere with subsequent PCR amplifications of the human genomic DNA target(s). It has also been noted that some hydrophilic proteins pass directly through the Cl 8 column without binding, and these might interact with the monolith and occupy binding sites prior to the DNA. Consequently, the monolithic column was, temporarily disconnected from the pre-column when sample was loaded (by opening the reservoir between the two stages) - reconnection occurred prior to switching from loading lysed blood sample to equilibration buffer. Fractions collected at the outlet of the TMOSderivatized monolith were quantified for proteins and DNA, and these are reported in Table 2. From the difference in protein mass detected before and after the monolith, it was determined that tens of micrograms of proteins still bound to the monolith, resulting

35 in about 30% of the DNA recovered from stage 1 passing through the monolith unbound. After the DNA was bound to the monolith, it was eluted using a low ionic strength TE buffer, delivered through reservoir 2 (Figure 9A), allowing it to pass only through the monolith. Figure 10 illustrates the results from extraction of DNA from a 10 μL of blood sample. The 240 ± 2 ng DNA recovered from the 10 μL of blood, loaded in a 125 μL total volume lysis solution, represented an extraction efficiency of 69 ± 1% with the two- stage device. This compares favorably with the 50-65% extraction efficiency for whole blood observed with other chip-based solid phases, but represents recovery of significantly more DNA than other microchip solid phases. Fractions 2 and 3 contained more than 50% of the extracted DNA (130.1 ± 2.0 ng) in only 2 μL of eluent with 88.4 ± 1 1 % (211.3 ± 1.5 ng) eluting in just 5 μL; this represents a >20-fold increase in the DNA concentration -a characteristic of significant importance for downstream processing. With respect to the reproducible nature of the two -stage process, the upper three elution profiles of Figure 10 were obtained from three different two-stage microchips when the Cl 8 bed was correctly packed, as determined by visual inspection (upper inset photo). The three curves provided essentially the same profile with similar fraction-tofraction quantitative recovery of DNA. The lower profile (lower inset photo) show that poor bed packing (again, determined by visual inspection) decreases the extraction reproducibility. Future work will address this packing issue. The power of the two-stage DNA extraction system is illustrated in the Figure 10 inset data which show the results from amplification of a 139-bp fragment of the human gelsolin gene present in the eluted DNA fractions. The protein product of this gene plays an important role in the 'gel' to 'sol'

36 transformation in cell motility, through the severing and capping of actin filaments, thereby regulating filament length - these filaments are involved in cell structure, motility, apoptosis, and cancer (24, 25). The electrophero grams generated by microchipbased electrophoresis of the PCR products generated by amplification of the first six fractions {0.5 μL of each) eluted from the two-stage system during a whole blood extraction. Successful amplification of the gelsolin fragment from the extracted DNA represents unequivocal proof that PCR inhibitors (including proteins, such as hemoglobin) have been successfully removed using the Cl 8 pre-column.

Note that we not only see the clear-cut amplification of the 139-bp gelsolin fragment but, with the exception of fraction 1 , all fractions contained PCR-amplifiable DNA. This stems from an important benefit of the two-stage extraction system, specifically that the isopropanol wash step, usually required to remove proteins from the solid phase for a successful PCR amplification, is not need here. This simplified the process and accelerates the purification process. While there is still some carryover of residual guanidine, a known PCR inhibitor, in the first fraction, this is minor compared to the carryover of isopropanol which can significantly up to half of the eluted fractions containing DNA. The issue of residual guanidine can be addressed in future work by decreasing the monolith channel dead volume.

Conclusions

The need for high DNA binding capacity is of utmost importance with many clinical applications that rely on whole blood as a source of genomic DNA. Examples include the detection of endogenous nucleic acid sequences associated with rare cells

37 (disease markers in cancer cells) or the detection of infectious agents (exogenous DNA associated with bacteria) - both requiring the purification of DNA from samples exceeding the nanoliter volume scale. Previous microdevices exploiting chaotropic methods for DNA isolation have only demonstrated the processing of human whole blood samples in the 0.1-1.5 μL range with DNA extraction efficiency reaching to 50-60%. Inherent in most of these microchip methods is a protein wash that serves to assure optimal extraction efficiency but increases the number of process steps involved in the purification process.

This work reported here demonstrates a high capacity, high extraction efficiency, surface-derivatized monolith for DNA extraction, the reproducible fabrication of which was shown by high run-to-run and device-to-device reproducibility for genomic DNA extractions. The novel two-stage, dual phase microdevice design is key the ability to handle up to 10 μL of whole blood, which is facilitated by the capture proteins by the inline Cl 8 phase - this represents an increase of at least 50-fold compared to that from most other DNA purification microdevices. Successful amplification of a fragment of a cancer marker gene, gelsolin, following purification of the DNA from the human whole blood illustrated the effectiveness of the method. The ability to achieve this without a protein wash step accelerates the analytical process, reduces the number of analytical steps and eliminates any potential sample contamination that may occur from switching syringes or tubing. With the majority of the extracted DNA released from the monolith in as little as 2 μL, this system is ideal for the concentration and purification of DNA from whole blood for downstream applications on integrated microfluidic devices.

38 Although certain presently preferred embodiments of the invention have been specifically described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the various embodiments shown and described herein may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of law.

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43

Claims

What is claimed is:

1. A microfluidic apparatus comprising a contaminant removal stage and a nucleic acid purification stage down stream of the contaminant removal stage.

2. The microfluidic apparatus of claim 1, wherein the contaminant is selected from the group consisting of proteins, peptides, oils, lipids, carbohydrates, organic acids, small molecules, amino acids, dyes and protein hydrolysates.

3. The microfluidic apparatus of claim 1 , wherein the contaminant removal stage contains a column selected from the group consisting of C30, C22, C18, C12, C8, C5, C4, C2, styrene, styrene/divinylbenzene co-polymer(S-DVB), aminopropyl phase (ph), benzene sulfonic acid phase, polyamide phase, quaternary amine, and cyanopropyl bonded phase (CN), when the contaminant is of proteins or peptides.

4. The microfluidic apparatus of claim 1 , wherein the contaminant removal stage contains a column selected from the group consisting of Cl 8, cyanopropyl bonded phase (CN), diol or dihydroxy phase, aminopropyl phase (ph), ethylenediamine-N-propyl phase, and silver ion phase, when the contaminant is oils or lipids.

5. The microfluidic apparatus of claim 1, wherein the contaminant removal stage contains a column selected from the group consisting of lectins, Cl 8, C8, sulfonated styrene/divinylbenzene, polyhydroxymethacrylate, polymethylmethacrylate, polystyrene, polyvinyl alcohol, quaternary amine phase, benzene sulfonic acid phase, polyamide phase,

44 cyanopropyl bonded phase (CN), aminopropyl phase (ph), and phenyl, when the contaminant is carbohydrates, organic acids, or dyes.

6. The microfluidic apparatus of claim 1 , wherein the contaminant removal stage contains a column selected from the group consisting of aminopropyl phase (ph), benzene sulfonic acid phase, polyamide phase, quaternary amine, benzene sulfonic acid phase, styrene, cyanopropyl bonded phase (CN) when the contaminant is small molecules.

7. The microfluidic apparatus of claim 1, wherein the contaminant removal stage contains a column selected from the group consisting of C18, C12, C8, C5, C4, C2, phenyl -hexyl, cyanopropyl bonded phase (CN), aminopropyl phase (ph), polystyrene/divinylbenzene (PS/DVB) copolymer phase, polyamide phase, and quaternary amine phase, when the contaminant is amino acids or protein hydrolysates.

8. The microfluidic apparatus of claim 1 , wherein the nucleic acid purification stage contains a column of grafted, UV photo-polymerized silica-based monolith or matrix entrapped chitosan.

9. The microfluidic apparatus of claim 8, wherein the grafted, UV photo- polymerized silica-based monolith is formed from 3-(Trimethoxysilyl)propyI methacrylate (TMSPM) monolith whose surfaces are grafted to tetramethylorthosilicate (TMOS).

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10. The microfluidic apparatus of claim 1 , wherein the contaminant removal stage contains a plurality of columns in series.

11. A method for purifying a nucleic acid comprising the steps of a. flowing a sample through a contaminant removal stage where the contaminant in the sample is selectively sequestered; b. flowing the remaining sample through a nucleic acid purification stage where the nucleic acid is selectively and reversibly bound; and c. eluting the nucleic acid from the nucleic acid purification stage.

12. The method of claim 11 , wherein the contaminant is selected from the group consisting of proteins, peptides, oils, lipids, carbohydrates, organic acids, small molecules, amino acids, dyes, and protein hydrolysates,

13. The method of claim 11 , wherein the contaminant removal stage contains a column selected from the group consisting of C30, C22, C 18, C 12, C8, C5, C4, C2, styrene, styrene/divinylbenzene co-polymer(S-DVB), aminopropyl phase (ph), benzene sulfonic acid phase, polyamide phase, quaternary amine, and cyanopropyl bonded phase (CN), when the contaminant is of proteins or peptides.

14. The method of claim 11, wherein the contaminant removal stage contains a column selected from the group consisting of C 18, cyanopropyl bonded phase (CN), diol

46 or dihydroxy phase, aminopropyl phase (ph), ethyl enediamine-N-propyl phase, and silver ion phase, when the contaminant is oils or lipids.

15. The method of claim 11 , wherein the contaminant removal stage contains a column selected from the group consisting of lectins, Cl 8, C8, sulfonated styrene/divinylbenzene, polyhydroxymethacrylate, polymethylmethacrylate, polystyrene, polyvinyl alcohol, quaternary amine phase, benzene sulfonic acid phase, polyamide phase, cyanopropyl bonded phase (CN), aminopropyl phase (ph), and phenyl, when the contaminant is carbohydrates, organic acids, or dyes.

16. The method of claim 11 , wherein the contaminant removal stage contains a column selected from the group consisting of aminopropyl phase (ph), benzene sulfonic acid phase, polyamide phase, quaternary amine, benzene sulfonic acid phase, styrene, cyanopropyl bonded phase (CN) when the contaminant is small molecules.

17. The method of claim 11 , wherein the contaminant removal stage contains a column selected from the group consisting of Cl 8, C12, C8, C5, QA, 02, phenyl-hexyl, cyanopropyl bonded phase (CN), aminopropyl phase (ph), polystyrene/divinylbenzene (PS/DVB) copolymer phase, polyamide phase, and quaternary amine phase, when the contaminant is amino acids or protein hydrolysates.

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18. The method of claim 11 , wherein the nucleic acid purification stage contains a column of grafted, UV photo-polymerized silica-based monolith or matrix entrapped chitosan.

19. The method of claim 18, wherein the grafted, UV photo-polymerized silica-based monolith is formed from 3-(Trimethoxysilyl)propyl methacrylate (TMSPM) monolith whose surfaces are grafted to tetramethylorthosilicate (TMOS).

20. The method of claim 11, wherein the contaminant removal stage contains a plurality of columns in series.

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