Patent Abstract:
The present invention relates to methods and systems for microfluidic DNA sample preparation. More specifically, embodiments of the present invention relate to methods and systems for the isolation of DNA from patient samples on a microfluidic device and use of the DNA for downstream processing, such as performing amplification reactions and thermal melt analysis on the microfluidic device.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/081,967, filed on Jul. 18, 2008, which is incorporated herein by reference in its entirety. This application is related to U.S. patent application Ser. No. 12/505,195, filed on Jul. 17, 2009, entitled “METHODS AND SYSTEMS FOR DNA ISOLATION ON A MICROFLUIDIC DEVICE,” and naming Michele R. Stone as the inventor, which application is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND 
     1. Field of the Invention 
     The present invention relates to methods and systems for microfluidic DNA sample preparation. More specifically, embodiments of the present invention relate to methods and systems for the isolation of DNA from patient samples on a microfluidic device and use of the DNA for downstream processing, such as performing amplification reactions and thermal melt analysis on the microfluidic device. 
     2. Description of Related Art 
     The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is perhaps the most well-known of a number of different amplification techniques. 
     PCR is one of the more sensitive methods for nucleic acid analysis. However, many substances in clinical samples, including blood, can affect PCR and can result in substantial error in the PCR results. Thus, DNA isolation and purification are critical to methods for DNA analysis. Conventional DNA preparation requires large volume samples and requires a long process time. Microfluidic technology makes it possible to use much less sample and less time for DNA sample preparation. Solid phase extraction methods have been applied in DNA sample preparation. DNA is selectively extracted on the solid phase while other substances in the sample are washed out of the extraction column. For instance, Breadmore et al. ( Anal Chem  75(8): 1880-1886, 2003) reported on a microchip-based DNA purification method using silica beads packed into glass microchips and immobilized within a sol-gel. Alternatively, DNA isolation can be achieved by nuclei size sieving. 
     Since DNA only exists in the nuclei of cells, DNA samples can be prepared by selectively isolating nuclei from the sample. Traditional nuclei isolation is slow and has low efficiency. Generally, nuclei isolation is performed by selective lysis of cellular membranes while keeping the nuclei intact. Nuclei are then isolated by centrifuge, sediment or sieving. Dignani et al. ( Nucl Acids Res  11: 1475-1489, 1983) reported isolation of nuclei from samples by centrifugation. U.S. Pat. No. 5,447,864 discloses a method of isolating nuclei using a DNA mesh. U.S. Pat. No. 6,852,851 discloses a method of isolating nuclei in a microfabricated apparatus that contains a plurality of radially dispersed micro-channels. U.S. Pat. No. 6,992,181 describes the use of a CD device for the purification of DNA or cell nuclei. This method requires moving parts and centrifugal force to isolate DNA and or cell nuclei, using a barrier in the channel to impede flow of DNA and nuclei. Palaniappan et al. ( Anal Chem  76:6247-6253, 2004) reported a continuous flow microfluidic device for rapid erythrocyte lysis. VanDelinder et al. ( Anal Chem  78:3765-3771, 2006) reported a separation of plasma from whole human blood in a continuous cross-flow in a molded microfluidic device. To increase mixing of lysis buffer with blood sample in microfluidic channel, Palaniappan et al. ( Anal Chem  78:5453-5461, 2006) reported a microfluidic channel with the channel floors that are patterned with double herringbone microridges. VanDelinder et al. ( Anal Chem  79:2023-2030, 2007) describe a perfusion in microfluidic cross-flow for particles and cells. Particles flow in the main channel while a perfusion flows through the side channels to exchange the medium of suspension. 
     There are several problems with current technology of purifying DNA by isolating nuclei from cells. First, the conventional approach is slow. Usually, the conventional approach takes hours to finish from cell lysis to the nuclei isolation. For example, the purification process described in U.S. Pat. No. 6,852,851 is carried out in a plurality of micro channels with a mesh built into the micro channel. However, because the size of micro channels is limited, the process can treat only limited sample sizes from 100 nl to 1 μl. Another problem is with the method of releasing DNA and/or nuclei from membrane. For example, DNAse is used in U.S. Pat. No. 5,447,864 to release nuclei from membrane. However, the addition of DNAse will fail the down stream process. In U.S. Pat. No. 5,447,864, sodium dodecyl sulfate solution or proteinase K is used to disrupt the nuclear envelope in order to release DNA. However, these lysis reagents will also seriously inhibit the downstream process. The conventional nuclear lysis method is to use high concentration sodium chloride (0.5 M) to disrupt the nuclear membrane. However, the high concentration sodium chloride will also inhibit the downstream process. 
     In addition, the current technologies require specific buffers for DNA binding and washing, most of which are not compatible with down stream applications such as PCR. These technologies also have a wide range of efficiencies in the overall quantity of DNA that is purified. This can be a significant problem when samples are to be used in microfluidics. The multiple reagents that are typically required for DNA purification would demand that moving parts, such as valves, be constructed into a microfluidic device for the introduction of multiple reagents in a solid phase extraction. In a microfluidic system, solid phase extraction or the use of multiple reagents is complicated and can lead to system failures. 
     Although the various methods exist to capture nuclei for use in down stream application or to separate specific cells from a sample population, none of these methods describes a single device that is capable of extracting cell nuclei and isolating the nucleic acid contained in the cell nucleic that is suitable for microfluidic processing and down stream processes such as amplification reactions and detection analysis. Thus, there is a need to develop microfluidic systems and methods for DNA isolation. 
     SUMMARY OF THE INVENTION 
     The present invention relates to methods and systems for microfluidic DNA sample preparation. More specifically, embodiments of the present invention relate to methods and systems for the isolation of DNA from patient samples on a microfluidic device and use of the DNA for downstream processing, such as performing amplification reactions and thermal melt analysis on the microfluidic device. 
     In one aspect, the present invention provides a method of purifying DNA from a sample (e.g., a patient sample or other sample) in a microfluidic device. According to this aspect, the method comprises: (a) mixing the sample and a lysis buffer in a mixing region of a microfluidic device; (b) selectively lysing the cellular membranes of cells in the sample without lysing the nuclear membranes of cells in a cell lysing region of the microfluidic device to produce intact nuclei from the cells; (c) trapping the intact nuclei from the sample on a membrane in a cell trapping region of the microfluidic device while allowing other components of the sample to flow through the membrane and into a waste collection region of the microfluidic device; (d) lysing the intact nuclei trapped on the membrane; (e) releasing the DNA from the lysed nuclei; and (f) collecting the released DNA in a DNA collection region of the microfluidic device. 
     In some embodiments, the sample is a patient sample which could be, for example, a blood sample, a urine sample, a saliva sample, a sputum sample, a cerebrospinal fluid sample, a body fluid sample or a tissue sample which contain white blood cells. In other embodiments, the patient sample comprises white blood cells. In additional embodiments, the patient sample is first enriched for white blood cells prior to the selective lysis of the cellular membrane. In some embodiments, the enrichment of white blood cells is performed by filtration. In additional embodiments, the enrichment of white blood cells is performed using antibodies. In some embodiments, the antibodies are coupled to a solid phase, such as beads, magnetic beads, particles, polymeric beads, chromatographic resin, filter paper, a membrane or a hydrogel. 
     In some embodiments, the selective lysis is performed by contacting the patient sample, either whole or after white blood cell enrichment, with a buffer (referred to herein as a lysis buffer or nuclei isolation buffer) that selectively permeabilizes cellular membranes while leaving the nuclei of the cells intact. Nuclei isolation buffers that have these characteristics are well known to the skilled artisan. Products that include nuclei isolation buffers for selectively lysing cellular membranes are commercially available. Suitable commercial products that include such buffers, include, but are not limited to, Nuclei EZ Prep Nuclei Isolation Kit (NUC-101) (Sigma, St. Louis, Mo., USA), Nuclear/Cytosol Fractionation Kit (K266-100) (BioVision Research Products, Mountain View, Calif., USA), NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockville, Ill., USA), Nuclear Extraction Kit (Imgenex, Corp., San Diego, Calif., USA), Nuclear Extract Kit (Active Motif, Carlsbad, Calif., USA), and Qproteome Nuclear Protein Kit (Qiagen, Valencia, Calif., USA). See also, U.S. Pat. Nos. 5,447,864, 6,852,851 and 7,262,283. It is known that the type of nuclei in question may determine which nuclei isolation buffer will be required. See, U.S. Pat. No. 5,447,864 for a discussion of factors that can be optimized for preparing a suitable selective lysis buffer for different cell types. 
     In one embodiment, the lysis buffer is a hypotonic buffer. For example, a commercial hypotonic lysis buffer can be purchased from Sigma Aldrich, Nuclei EZ lysis buffer (N 3408). A kit is also available from Sigma Aldrich, Nuclei EZ Prep Nuclei Isolation Kit (Nuc-101). A common recipe for a 10× hypotonic solution is, 100 mM HEPES, pH 7.9, with 15 mM MgCl 2  and 100 mM KCl. In another embodiment, the lysis buffer is a hypotonic buffer that comprises a detergent. Suitable detergents include, but are not limited to ionic detergents, such as lithium lauryl sulfate, sodium deoxycholate, and Chaps, or non-ionic detergents, such as Triton X-100, Tween 20, Np-40, and IGEPAL CA-630. In another embodiment, the lysis buffer is an isotonic buffer. For example, Sigma Aldrich offers a kit, CelLytic Nuclear Extraction kit, which contains an isotonic lysis buffer. A common recipe for a 5× isotonic lysis buffer is, 50 mM Tris HCl, pH 7.5, with 10 mM MgCl 2 , 15 mM CaCl 2 , and 1.5M Sucrose. In an additional embodiment, the buffer is an isotonic buffer that comprises a detergent which may be an ionic detergent or a non-ionic detergent. In other embodiments, the selective lysis is performed using a hypotonic lysis buffer that contains a weak detergent. In further embodiments, the patient sample and the hypotonic lysis buffer are mixed in a 1:1 ratio. In additional embodiments, the selective lysis of the cellular membranes totally lyses red blood cells. 
     In some embodiments, the steps of lysing the intact nuclei trapped on the membrane and releasing the DNA from the lysed nuclei comprise flowing an elution buffer over the intact nuclei trapped on the membrane. 
     In some embodiments, the elution buffer is a buffer in which the DNA is compatible. In other embodiments, elution buffer comprises a Tris buffer, KCl and a zwitterion. In one embodiment, the zwitterion is betaine, trimethylamine-N-oxide, trimethylamine hydrochloride or trimethylamine bromide. In other embodiments, the elution buffer is an amplification reaction buffer that may contain the non-assay specific amplification reagents. In additional embodiments, the amplification reaction buffer is a PCR buffer that may contain the non-assay specific PCR reagents. In further embodiments, the elution buffer contains a dye that binds to DNA. In additional embodiments, the dye is useful for quantifying the amount of DNA in the channel. In additional embodiments, the nuclei are lysed by heat to release the DNA from the nuclei. In some embodiments, the nuclei are subjected to heat prior to an amplification reaction. In other embodiments, the nuclei are subjected to heat during the amplification reaction and the nuclei lysis region is the initial region of microfluidic device in which the amplification reaction is conducted. 
     In some embodiments, the intact nuclei trapped on the membrane are lysed by applying heat to the trapped nuclei. In one embodiment, the trapped nuclei are heated for approximately 1 to 10 minutes at a temperature in the range of approximately 35° C. to 95° C. In another embodiment, the trapped nuclei are heated for approximately 7 minutes at a temperature of approximately 50° C. In a further embodiment, the DNA released from the lysed nuclei flows to the DNA collection region of the microfluidic device by flowing an elution buffer over the DNA. In some embodiments, the elution buffer is as described herein. 
     In another aspect, the present invention provides a microfluidic device for purifying DNA from a patient sample. In accordance with this aspect, the microfluidic device comprises a sample port and lysis buffer port in fluid communication with a mixing region of the microfluidic device. The mixing region is configured to permit mixing of a patient sample from the sample port and lysis buffer from the lysis buffer port. The microfluidic device also comprises a cell lysis region in fluid communication with the mixing region. The cell lysis region is configured to permit the lysis buffer to selectively lyse cellular membranes of cells in the patient sample without lysing nuclear membranes of the cells to produce intact nuclei from the cells in the patient sample. The microfluidic device further comprises a nuclei trapping region wherein intact nuclei from the patient sample are trapped on a membrane while other components of the patient sample flow through the membrane and into a waste collection region of the microfluidic device. The nuclei trapping region is in fluid communication with the cell lysis region. The microfluidic device also comprises a nuclei lysis region in which the nuclear membranes of the intact nuclei are lysed to release the DNA. The microfluidic device further comprises a DNA collection region in the microfluidic device wherein DNA released from the trapped intact nuclei is collected. 
     In some embodiments, the microfluidic device further comprises an elution buffer port in fluid communication with the nuclei trapping region and the nuclei lysing region. The elution buffer from the elution buffer port can be controlled to flow through the nuclei trapping region and the nuclei lysing region to lyse the nuclear membranes of the trapped intact nuclei to release the DNA. In one embodiment, the elution buffer is one in which the DNA is compatible. In other embodiments, elution buffer comprises a Tris buffer, KCl and a zwitterion. In one embodiment, the zwitterion is betaine, trimethylamine-N-oxide, trimethylamine hydrochloride or trimethylamine bromide. In other embodiments, the elution buffer is an amplification reaction buffer that may contain the non-assay specific amplification reagents. In additional embodiments, the amplification reaction buffer is a PCR buffer that may contain the non-assay specific PCR reagents. 
     In some embodiments, the microfluidic device further comprises a heat source which is configured to provide heat to the intact nuclei in the nuclei lysis region sufficient to lyse the nuclear membranes thereby releasing the DNA. In one embodiment, the heat source is controlled to heat the nuclei for approximately 1 to 10 minutes at a temperature in the range of approximately 35° C. to 95° C. In another embodiment, the heat source is controlled to heat the nuclei for approximately 7 minutes at a temperature of approximately 50° C. 
     In some embodiments, the DNA released from the lysed nuclei flows to the DNA collection region of the microfluidic device by flowing an elution buffer over the DNA. In other embodiments, the membrane is made of silicon, glass, polymers, polyester, polycarbonate or nitrocellulose. In additional embodiments, the membrane has a round or rectangular shape. In one embodiment, the membrane has a pore size from approximately 500 nm to 10 μm. In another embodiment, the membrane has a pore size from approximately 0.5 μm to 10 μm. 
     In some embodiments, the microfluidic device comprises multiple layers. In one embodiment, the microfluidic device further comprises: (1) a first layer comprising the lysis buffer port, the patient sample port, an elution buffer port, a purified DNA collection port and a waste port; (2) a second layer comprising a network of microchannels that transports the lysis buffer solution and the patient sample to the mixing region of the microfluidic device; (3) a third layer comprising a network of microchannels; (4) the membrane located between the second and third layers. In some embodiments, the patient sample and the lysis buffer solution mix in the mixing region and flow in the microchannels to the cell lysis region, and wherein the patient sample and the lysis buffer solution flow from the cell lysis region to the membrane in the nuclei trapping region, and wherein the other components of the patient sample flow through the membrane and into a microchannel in the third layer and to the waste collection region of the microfluidic device, and wherein the DNA released from the nuclei flows through the membrane and into a microchannel located in the third layer and to the DNA collection region. 
     In other embodiments, the microfluidic device further comprises: (1) a first layer comprising the lysis buffer port, the patient sample port, an elution buffer port, a purified DNA collection port and a waste port; (2) a second layer comprising a network of microchannels that transports the lysis buffer solution and the patient sample to the mixing region of the microfluidic device; (3) a third layer comprising a hole through which fluid flows from the microchannels in the second layer and onto the membrane; (4) a fourth layer comprising a hole through which fluid flows from the membrane and into microchannels located in a fifth layer, therein the fifth layer further comprising the waste collection region and the DNA collection region. 
     In another aspect, the present invention provides another microfluidic device for purifying DNA from a patient sample. In accordance with this aspect, the microfluidic device comprises a cell lysis region configured such that a lysis buffer is permitted to mix with the patient sample resulting in the selective lysing of cellular membranes of cells in the patient sample without lysing nuclear membranes of the cells to produce intact nuclei from the cells in the patient sample. The microfluidic device also comprises a cross-flow filtration region in which intact nuclei are separated from other components of the patient sample by a filter. The filter has a pore size such that the intact nuclei do not pass through the filter and the other components of the patient sample pass through the filter and are carried away by a cross-flow buffer that is controlled to flow through the cross-flow filtration region. The microfluidic device further comprises an interface channel in fluid communication with said cross-flow filtration region through which purified nuclei flow for downstream analysis. 
     In some embodiments, the cross-flow filtration region comprises a microfluidic separation channel in fluid communication with the cell lysis region and configured to receive the intact nuclei and the other components of the patient sample from the cell lysis region. The filter is constructed in the microfluidic channel. The cross-flow filtration region also comprises a cross-flow buffer port configured to permit the cross-flow buffer to flow across the microfluidic separation channel and through the filter. The cross-flow buffer, as it flows across the separation channel, facilitates removal of the other contents of the patient sample from the separation channel through the filter. The intact nuclei flow through the separation channel. 
     In some embodiments, the flow of one of the lysed patient sample and the cross-flow buffer is driven by a pressure differential and the flow of the other of the lysed patient sample and cross-flow buffer is driven by an electrophoretic voltage potential. In one embodiment, the pore size of the filter is between approximately 2 μm to 10 μm. In another embodiment, the pore size of the filter is approximately 5 μm. In one embodiment, the filter is a membrane. In another embodiment, the filter is an array of pillars that forms as size exclusion barrier. 
     In some embodiments, the cross-flow filtration region is configured to separate the intact nuclei, bacteria and viruses from the lysed patient sample. In one embodiment, the cross-flow filtration region comprises a first filter to separate the intact nuclei, a second filter to separate bacteria and a third filter to separate viruses. In another embodiment, the first filter is located closest to a cross-flow buffer port, the second filter located next closest to the cross-flow buffer port, and the third filter located furthest from the cross-flow buffer port. In a one embodiment, the pore size of the first filter is between approximately 2 μm to 10 μm, the pore size of the second filter is between approximately 0.2 μm to 2 μm, and the pore size of the third filter is between approximately 10 nm to 400 nm. In another embodiment, wherein the pore size of the first filter is approximately 8 μm, the pore size of the second filter is approximately 0.4 μm, and the pore size of the third filter is approximately 100 nm. 
     In some embodiments, the microfluidic device further comprises more than one cross-flow filtration region in which each cross-flow filtration region receives a portion of the lysed patient sample from the cell lysis region, and each cross-flow filtration region is in fluid communication with one or more interface channels. In one embodiment, the filter of one cross-flow filtration system has a pore size that is different from the pore size of one other cross-flow filtration system. 
     In some embodiments the microfluidic device further comprises a nuclei concentration region in which the intact nuclei from the cross-flow filtration region are concentrated. In one embodiment, the nuclei concentration region comprises a concentration channel having a sample input section, a sample outlet section and a wall portion configured to prevent intact nuclei from flowing through said wall portion and to allow the other contents of the patient sample to flow through said wall portion. In one embodiment, the wall portion is a filter. In another embodiment, the filter comprises a set of pillars placed along the concentration channel. 
     In some embodiments, the patient sample is as described herein. In other embodiments, the patient sample comprises white blood cells. In further embodiments, the downstream analysis comprises an amplification reaction in which nucleic acid is amplified and/or a detection procedure for determining the presence or absence of an amplified product. 
     In another aspect, the present invention provides a microfluidic system for purifying DNA from a patient sample. In accordance with this aspect, the microfluidic system comprises a microfluidic device. The system also comprises a cell lysis region in the microfluidic device configured such that a lysis buffer is permitted to mix with the patient sample resulting in the selective lysing of cellular membranes of cells in the patient sample without lysing nuclear membranes of the cells to produce intact nuclei from the cells in the patient sample. The system further comprises a cross-flow filtration region in the microfluidic device in which intact nuclei are separated from other components of the patient sample by a filter. The filter has a pore size such that the intact nuclei do not pass through the filter and the other components of the patient sample pass through the filter and are carried away by a cross-flow buffer that is controlled to flow through the cross-flow filtration region. The system also comprises a nuclei lysis region in the microfluidic device in which the nuclear membranes of the intact nuclei are lysed to release the DNA. The nuclei lysis region is in fluid communication with the cross-flow filtration region. The system also comprises an amplification reaction region in the microfluidic device in which the nucleic acid is amplified and a detection region in the microfluidic device for determining the presence or absence of an amplified product. 
     In some embodiments, the nuclei lysis region is part of the amplification reaction region. In other embodiments, the regions are in separate microfluidic devices. In one embodiment, the cell lysis region, the cross-flow filtration region, and the nucleic lysis region are in one microfluidic device and the amplification region and the detection region are in a second microfluidic device. 
     In another aspect, the present invention provides a method for purifying DNA from a patient sample in a microfluidic device. In accordance with this aspect, the method comprises mixing a patient sample containing cells and a lysis buffer in a mixing region of said microfluidic device. The lysis buffer selectively lyses cellular membranes without lysing nuclear membranes. The method also comprises selectively lysing in a cell lysing region of the microfluidic device the cellular membranes of the cells in the patient sample without lysing the nuclear membranes of the cells to produce intact nuclei from the cells. The method further comprises separating the intact nuclei from the patient sample in a cross-flow filtration region of said microfluidic device. The cross-flow filtration region comprises a filter having a pore size such that the intact nuclei do not pass through the filter and the other components of the patient sample pass through the filter and are carried away by a cross-flow buffer that is controlled to flow through the cross-flow filtration region. The method also comprises flowing purified nuclei through an interface channel in fluid communication with said cross-flow filtration region for downstream analysis. 
     In some embodiments, the method further comprises driving the flow of one of the lysed patient sample and the cross-flow buffer by a pressure differential and driving the flow of the other of the lysed patient sample and the cross-flow buffer by an electrophoretic voltage potential. In other embodiments, the method further comprises separating the intact nuclei, bacteria and viruses from the lysed patient sample in said cross-flow filtration region in which each of the intact nuclei, bacteria and viruses are released into separate channels with the cross-flow buffer. In another embodiment, the method further comprises separating the intact nuclei, bacteria and viruses from the lysed patient sample in said cross-flow filtration region by a series of filters each having a different pore size. In another embodiment, the method further comprises concentrating the intact nuclei prior to sending the intact nuclei for downstream analysis. In some embodiments, the method further comprises separating the intact nuclei from the lysed patient sample utilizing more than one cross-flow filtration region, each receiving a portion of the lysed patient sample. In some embodiments the patient sample is as described herein. In other embodiments, purifying DNA from cells in a patient sample comprises purifying DNA from white blood cells in the patient sample. 
     In another aspect, the present invention provides a method of determining the presence or absence of a nucleic acid in a patient sample. In accordance with this aspect, the method comprises mixing a patient sample containing cells and a lysis buffer in a mixing region of said microfluidic device. The lysis buffer selectively lyses cellular membranes without lysing nuclear membranes. The method also comprises selectively lysing in a cell lysing region of the microfluidic device the cellular membranes of the cells in the patient sample without lysing the nuclear membranes of the cells to produce intact nuclei from the cells. The method further comprises separating the intact nuclei from the patient sample in a cross-flow filtration region of the microfluidic device. The cross-flow filtration region comprises a filter having a pore size such that the intact nuclei do not pass through the filter and the other components of the patient sample pass through the filter and are carried away by a cross-flow buffer that is controlled to flow through the cross-flow filtration region. The method also comprises lysing the nuclei to release the nucleic acid in the microfluidic device. The method further comprises amplifying the nucleic acid in the microfluidic device; and determining the presence or absence of an amplified product. The presence of the amplified product indicates the presence of the nucleic acid in the patient sample. 
     In some embodiments, the patient sample is as described herein. In other embodiments the patient sample contains white blood cells. In additional embodiments, the method further comprises enriching the patient sample for white blood cells prior to the selective lysis of the cellular membranes. The enrichment for white blood cells can be performed as described herein. In further embodiments, the mixing of the patient sample and lysis buffer, selectively lysing, separating intact nuclei and lysing the nuclei are performed in one microfluidic device and the amplification and detection are performed in a second microfluidic device. In other embodiments, the mixing of the patient sample and lysis buffer, selectively lysing and separating intact nuclei are performed in one microfluidic device and the lysing the nuclei, amplification and detection are performed in a second microfluidic device. 
     In another aspect, the present invention provides another microfluidic system for isolating DNA from cells in a patient sample. In accordance with this aspect, the microfluidic system comprises a lysis buffer storage device for storing a lysis buffer in which the lysis buffer selectively lyses cellular membranes without lysing nuclear membranes. The system also comprises an elution buffer storage device for storing an elution buffer. The system further comprises a sample card having multiple chambers for receiving the patient sample. Each chamber in the sample card comprises an inlet, a filter and an outlet. The system also comprises a flow control system for controlling flow of the lysis buffer and the elution buffer to each chamber of the sample card. The flow control system controls the flow of lysis buffer into each chamber of the sample card such that the lysis buffer selectively lyses cellular membranes to release nuclei and cell debris causing the cell debris to flow through the filter into a waste receptacle positionable beneath the sample card and without lysing nuclear membranes of nuclei in the patient sample which become trapped on the filter. The system further comprises a temperature control system for heating the filter in the sample card sufficient to lyse nuclei trapped on said filter and release DNA. The system also comprises an interface chip comprising multiple DNA sample wells and DNA sample outlets. The interface chip is positionable beneath the sample card and is configured to receive the DNA released from the lysed nuclei trapped on said filter. The system further comprises a main controller in communication with the temperature control system, and the flow control system. 
     In some embodiments, the temperature control system comprises a heating source and a heat sensor. In other embodiments, the flow control system comprises a pump and a solution delivery chip, wherein the solution delivery chip comprises multiple channels for delivering lysis buffer and elution buffer to each chamber of the sample card. In further embodiments, the flow control system further comprises a pressure control system. The pressure control system comprises an air source, a pressure sensor for controlling the delivery of the elution buffer and the lysis buffer to each chamber of the sample card. In some embodiments, the multiple chambers of the sample card are in fluid communication with one another. In other embodiments, the sample card is disposable. In some embodiments, the sample card is configured to contain multiple different patient samples. In other embodiments, the sample card is configured to contain one patient sample in multiple chambers. 
     In another aspect, the present invention provides a microfluidic system for determining the presence or absence of a nucleic acid in a patient sample. In accordance with this aspect, the microfluidic system comprises a microfluidic device comprising a sample preparation region, an amplification reaction region and a detection region. The sample preparation region comprises a lysis buffer storage device for storing a lysis buffer in which the lysis buffer selectively lyses cellular membranes without lysing nuclear membranes. The sample preparation region also comprises an elution buffer storage device for storing an elution buffer. The sample preparation region further comprises a sample card having multiple chambers for receiving the patient sample. Each chamber comprises an inlet, a filter and an outlet. The sample card is removably insertable into said sample preparation region of said microfluidic device. The sample preparation region also comprises a flow control system for controlling flow of the lysis buffer and the elution buffer to each chamber of said sample card. The flow control system controlling the flow of lysis buffer into each chamber of the sample card such that the lysis buffer selectively lyses cellular membranes to release nuclei and cell debris causing the cell debris to flow through the filter into a waste receptacle positionable beneath the sample card and without lysing nuclear membranes of nuclei in the patient sample which become trapped on the filter. The sample preparation region further comprises a temperature control system for heating the filter in the sample card sufficient to lyse nuclei trapped on said filter and release DNA. The sample preparation region also comprises an interface chip comprising multiple DNA sample wells and DNA sample outlets, wherein said interface chip is positionable beneath the sample card and is configured to receive the DNA released from the lysed nuclei trapped on said filter. The microfluidic system further comprises a main controller in communication with the temperature control system, the flow control system, and the microfluidic chip. In one embodiment, the main controller controls the flow of DNA from the interface chip to the amplification region and/or the detection region of the microfluidic chip. 
     In some embodiments, the temperature control system comprises a heating source and a heat sensor. In other embodiments, the flow control system comprises a pump and a solution delivery chip in which the solution delivery chip comprises multiple channels for delivering lysis buffer and elution buffer to each chamber of the sample card. In some embodiments, the multiple chambers of the sample card are in fluid communication. In other embodiments, the flow control system further comprises a pressure control system, wherein the pressure control system comprises an air source, a pressure sensor for controlling the delivery of the elution buffer and the lysis buffer to each chamber of the sample card. In some embodiments, the multiple chambers of the sample card are in fluid communication in which a patient sample in one chamber can be driven into other chambers. In other embodiments, the sample card is disposable. 
     In another aspect, the present invention provides a method for isolating DNA from cells in a patient sample. In accordance with this aspect, the method comprises providing a microfluidic system comprising (i) a sample card having multiple chambers for receiving the patient sample, wherein each chamber comprises an inlet, a filter and an outlet, said sample card being removably insertable into said microfluidic system, (ii) a flow control system for controlling flow of a lysis buffer and an elution buffer to each chamber of the sample card, (iii) a temperature control system for heating the filter in the sample card; (iv) a waste receptical positionable beneath the sample card, and (v) an interface chip comprising multiple DNA sample wells and DNA sample outlets, wherein said interface chip is positionable beneath the sample card. 
     The method also comprises loading the patient sample into the chambers of the sample card. The method further comprises inserting the sample card into the microfluidic system. The method also comprises delivering lysis buffer to the chamber of the sample card and selectively lysing cellular membranes of the patient sample without lysing nuclear membranes of nuclei, producing a solution comprising lysis buffer, intact nuclei and cellular debris. The method further comprises controlling the flow control system to drive the lysis buffer and the cellular debris through the filter and into the waste receptacle, thereby trapping the intact nuclei on the filter. The method also comprises controlling the temperature control system to heat the filter causing the intact nuclei trapped on the filter to lyse, thereby releasing DNA. The method further comprises delivering an elution buffer to the chambers of the sample card. The method also comprises controlling the flow control system to drive the elution buffer and the DNA to the DNA sample wells in the interface chip. In some embodiments, the lysis buffer is repeatedly delivered to the chambers of the sample card to clean the filters. 
     The above and other embodiments of the present invention are described below with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements. 
         FIG. 1  is a functional block diagram of a DNA preparation and analysis system. 
         FIG. 2  shows a schematic illustration of a microfluidic DNA sample preparation device in accordance with an embodiment of the invention. 
         FIG. 3A  illustrates a multi-layered microfluidic sample preparation device in accordance with an embodiment of the invention. 
         FIG. 3B  is a longitudinal cross-sectional view of a multi-layered microfluidic sample preparation device in accordance with an embodiment of the invention. 
         FIG. 3C  is a transverse cross-sectional view of a multi-layered microfluidic sample preparation device in accordance with an embodiment of the invention. 
         FIG. 4  is an exploded view of a multi-layered microfluidic sample preparation device in accordance with an embodiment of the invention. 
         FIG. 5  illustrates a top view of layer  1  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 6  is a longitudinal cross-sectional view of layer  1  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 7  illustrates a top view of layer  2  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 8  illustrates a top view of layer  3  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 9  is a longitudinal cross-sectional view of layer  3  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 10  illustrates a top view of layer  4  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 11  is a longitudinal cross-sectional view of layer  4  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 12  illustrates a top view of layer  5  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 13  is a longitudinal cross-sectional view of layer  5  of the microfluidic sample preparation device of  FIG. 4 . 
         FIG. 14  is a flow chart illustrating a process according to an embodiment of the invention. 
         FIGS. 15A and 15B  show trapping of nucleic by the membrane.  FIG. 15A  shows the membrane before trapping the nuclei.  FIG. 15B  shows the membrane after trapping the nuclei which are dyed with a fluorescence dye. 
         FIG. 16  is a graph showing the results of an experiment. 
         FIG. 17  shows a schematic illustration of a cross-flow microfluidic device for sample preparation in accordance with other embodiments of the present invention. 
         FIG. 18  shows a schematic illustration of a cross-flow filter in accordance with an embodiment of the present invention. 
         FIG. 19  shows a schematic illustration of a cross-flow filter in accordance with other embodiments of the present invention. 
         FIG. 20  shows a schematic illustration of a cross-flow microfluidic device for sample preparation in accordance with further embodiments of the present invention. 
         FIG. 21  is a transverse cross-sectional view of the cross-flow microfluidic device shown in  FIG. 20 . 
         FIG. 22  illustrates a sample concentrator in accordance with embodiments of the present invention. 
         FIG. 23  illustrates a system for sample preparation in accordance with other embodiments of the present invention. 
         FIG. 24  is a flow chart illustrating a process for sample preparation according to an embodiment of the invention. 
         FIG. 25  is a flow chart illustrating a process for determining the presence or absence of a nucleic acid in a sample according to an embodiment of the invention. 
         FIG. 26  shows a schematic illustration of a microfluidic device for sample preparation in accordance with other embodiments of the present invention. 
         FIG. 27  illustrates a sample card in accordance with an embodiment of the invention. 
         FIG. 28  illustrates a sample card in accordance with another embodiment of the invention. 
         FIG. 29  illustrates a flow control system in accordance with an embodiment of the invention. 
         FIG. 30  illustrates a solution delivery chip in accordance with an embodiment of the invention. 
         FIG. 31  illustrates a pressure control chip in accordance with an embodiment of the invention. 
         FIG. 32  is a flow chart illustrating a process for sample preparation according to an embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       FIG. 1  illustrates a microfluidic DNA analysis system  100  according to some embodiments of the invention. As illustrated in  FIG. 1 , system  100  includes a DNA sample preparation sub-system  102  (a.k.a., “the sample preparation unit”), a DNA amplification, analysis and detection subsystem  104 , and a main control system  101 . The present application is primarily directed to the sample preparation unit  102  and earlier filed applications describe various embodiments of subsystem  104  (see e.g., U.S. Pat. Pub. Nos. 2008/0003588, 2008/0130971, 2008/0176230, and 2009/0053726, all of which are incorporated herein in their entirety by this reference). 
       FIG. 2  shows a schematic illustration of a component  200  of sample preparation unit  102  in accordance with an embodiment of the invention. More specifically,  FIG. 2  shows a schematic illustration of a microfluidic DNA sample preparation device  200 . As illustrated in  FIG. 2 , device  200  comprises a chip  201 , a well  202  formed in chip  201  for storing a lysis buffer, and one or more microfluidic channels  206  formed in chip  201  that fluidly connect well  202  to a mixing region  208  formed in chip  201 , thereby providing a path for the lysis buffer in well  202  to travel to the mixing region  208 . Device  200  also includes a sample well  204  formed in chip  201  for storing a sample to analyzed (e.g., a blood sample). Like well  202 , well  204  is connected in fluid communication with mixing region  208  via one or more channels  205  formed in chip  201 . 
     Mixing region  208  is configured to permit a sample from well  204  and lysis buffer from well  202  to mix. Mixing region  208 , which my simply be a small channel, is connected in fluid communication with a filter  210  (e.g., a permeable membrane or other filter) disposed in chip  201  via a microfluidic channel  209  formed in chip  201 . In some embodiments, filter  210  is made of any combination of one or more of the following: silicon, glass, polymers, polyester, polycarbonate, and nitrocellulose. Filter  210  may have a round shape, rectangular shape, or other shape. In some embodiments, filter  210  comprises a number of pores and the pore sizes may range from 500 nanometers (nm) to 10 micrometers (um). For example, in some embodiments, the pore sizes range from approximately 0.5 um to 10 um.  FIGS. 15   a  and  15   b  illustrate an exemplary embodiment of filter  210 . 
     Channel  209  is configured to function as a cell lysis region. That is, channel  209  is configured to permit the lysis buffer from well  202  to selectively lyse cellular membranes of cells in the patient sample from well  204  without lysing nuclear membranes of the cells to produce intact nuclei from the cells in the patient sample. For example, in the cell lysis region red blood cells may be disrupted before reaching the filter  210  while white blood cells are partially lysed such that nuclei are intact when the mixture reaches filter  210 . 
     As further shown in  FIG. 2 , a waste well  212 , a DNA collection well  214 , and an elution buffer well  216  are also formed in chip  201 . Additionally, each of the wells  212 ,  214  and  216  are connected in fluid communication with filter  210  via microfluidic channels  213 ,  215  and  217 , respectively. In some embodiments, during use, well  216  stores an elution buffer which can be, for example, a Tris buffer, KCl and/or a zwitterion. Main control system  101 , as illustrated in  FIG. 1 , can cause the elution buffer to flow into filter  210 . 
     During use of chip  200 , filter  210  forms a nuclei trapping region wherein the intact nuclei from the sample are trapped by filter  210  while other components of the patient sample flow through filter  210  and into waste collection region  212 . Filter  210  also functions as a nuclei lysis region in which the nuclear membranes of the intact nuclei are lysed to release the DNA. The released DNA is forced to flow to DNA collection region  214 . 
     As further shown in  FIG. 2 , device  200  may include a heat source  250 . Heat source  250  may be formed in or on chip  201  or may be structurally separate from chip  201 . Heat source  250  may be an electrical heater (i.e., a device that converts electrical energy into heat) or other type of heat producer. Heat source  250  may be controlled by a temperature controller  252 , which may be a module of main controller  101  or a separate component that is in communication with main controller  101 . Heat source  250  is controlled, configured and arranged to transfer heat to filter  210  at desired times. For example, when intact nuclei are trapped by filter  210 , heat source  250  may be controlled to cause the transfer of heat to filter  210 , which heat is preferably sufficient to lyse or facilitate the lysing of the nuclear membranes of the nuclei, thereby releasing the DNA contained by the nuclear membranes. 
     In some embodiments, chip  201  is a multilayer chip. That is, chip  201  may comprise two or more boards. Referring now to  FIGS. 3A-3C , a multilayered embodiment of device  200  is illustrated. In the non-limiting embodiment illustrated in  FIG. 3A , device  200  includes six layers. However, fewer or more layers also could be used.  FIG. 3B  shows a longitudinal cross-sectional view of device  200  in accordance with the embodiment shown in  FIG. 3A , and  FIG. 3C  shows a transverse cross-sectional view of device  200  in accordance with the embodiment shown in  FIG. 3A . 
       FIG. 4  illustrates an exploded view of the embodiment of device  200  shown in  FIG. 3A . In the first layer (or top layer)  401 , a top view of which is shown in  FIG. 5 , wells  202 ,  204  and  216  are formed for containing the lysis buffer, sample and elution buffer, respectively. Also formed in layer  401  is a through hole  402  in fluid communication with DNA collector well  214  and a through hole  404  in fluid communication with waste well  212 . In one non-limiting embodiment, layer  401  is preferably approximately five (5) millimeters thick and is made from Poly(methyl methacrylate) (PMMA). Other thicknesses and materials also may be used for this layer in additional embodiments. 
     As further shown in  FIG. 5  and  FIG. 6 , which is a longitudinal cross sectional view of layer  401 , well  202  has a port  405  in its bottom surface so that fluid can flow from well into channels  206 . Likewise, well  204  has a port  406  in its bottom surface so that fluid can flow from well  204  into channel  205 . Thus, ports  405  and  406  are in fluid communication with mixing region  208 . Similarly, well  216  has port  407  in its bottom surface so that fluid can flow from well  216  into channel  217 . As shown in  FIG. 4 , channels  205 ,  206  and  217  are formed in the second layer  411  of device  200 . 
       FIG. 7  illustrates a top view of second layer  411 . As shown in  FIG. 7 , formed in second layer  411  are a mixing region  208 , channel  209 , through holes  413 ,  414  and  415 , and closed bottom wells  416 ,  417 , and  418 . Channel  209  fluidly connects mixing region  208  with hole  413  so that a lysis buffer and sample mixture, which may be formed in mixing region  208 , can flow into hole  413  and down to the third layer  421  of device  200 . Likewise, channel  217  connects well  418 , which is positioned directly beneath hole  407  of elution buffer well  216 , with hole  413  so that elution buffer can flow from well  216  into hole  413  and down to the third layer of device  200 . Through hole  414  is positioned beneath through hole  404  so that fluid may flow through hole  414  into hole  404 , and through hole  415  is positioned beneath through hole  402  so that fluid may flow through hole  415  into hole  402 . In one non-limiting embodiment, the second layer  411  of device  200  may comprise a 150 micrometer thick cyclic olefin copolymer (COC) film. Other thicknesses and materials also may be used for this layer in additional embodiments. 
       FIG. 8  illustrates a top view of third layer  421  which includes through holes  422 ,  423  and  424 . Through hole  422  is positioned beneath through hole  414  so that fluid may flow through hole  422  into hole  414 , through hole  423  is positioned beneath through hole  413 , so that fluid may flow through hole  413  into hole  423 , and through hole  424  is positioned beneath through hole  415  so that fluid may flow through hole  423  into hole  415 .  FIG. 9  shows a longitudinal cross-sectional view of layer  421 . In one non-limiting example, the third layer  421  of device  200  may comprise a 1 millimeter thick PMMA board. Other thicknesses and materials also may be used for this layer in additional embodiments. 
       FIG. 10  illustrates a top view of fourth layer  431  which includes through holes  432 ,  433  and  434 . Through hole  432  is positioned beneath through hole  422  so that fluid may flow through hole  432  into hole  422 , through hole  433  is positioned beneath through hole  423 , so that fluid may flow through hole  423  into hole  433 , and through hole  434  is positioned beneath through hole  424  so that fluid may flow through hole  434  into hole  424 .  FIG. 11  shows a longitudinal cross-sectional view of layer  421 . In one non-limiting embodiment, the fourth layer  431  of device  200  may comprise a 1 millimeter thick PMMA board. Other thicknesses and materials also may be used for this layer in additional embodiments. 
     As shown in  FIG. 4 , filter  210  is sandwiched between the third and fourth layers of device  200 . In some embodiments, filter  210  may be made of any combination of one or more of silicon, glass, polymers, polyester, polycarbonate, and nitrocellulose, as described above. In one non-limiting embodiment, filter  210  is approximately 9 mm by 9 mm and has a thickness of approximately 10 μm. The filter may have other thicknesses and dimensions in additional embodiments. 
       FIG. 12  illustrates a top view of fifth layer  441 . As shown in that figure, closed bottom wells  212 ,  443  and  214  are formed on the top surface of layer  441 . Also, microfluidic channel  215  is formed on the top surface of layer  441  as well as channel  213 , which connects well  443  with waste collection well  212 . Closed bottom wells  212 ,  443 , and  214  are positioned beneath through holes  432 ,  433 ,  434 , respectively.  FIG. 13  shows a longitudinal cross-sectional view of layer  441 . On one non-limiting embodiment, the fifth layer  441  of device  200  may comprise a 150 micrometer thick COC film. Other thicknesses and materials also may be used for this layer in additional embodiments. 
     As illustrated in  FIG. 4 , the sixth layer of device  200  is a base layer  451 . In one non-limiting embodiment, layer  451  may comprise a 1 millimeter thick board made of PMMA. Other thicknesses and materials also may be used for this layer in additional embodiments. In some embodiments, the third, fourth and sixth layers are removed, thereby creating a three layer device. 
     Referring now to  FIG. 14 , a flow chart illustrating a process  1400  for preparing DNA for analysis using device  200  is shown. Process  1400  may begin in step  1402 , where a sample (e.g., a sample of blood containing white blood cells) is introduced into sample well  204 . In step  1404 , a lysis buffer is introduced into lysis buffer well  202 . In step  1405 , the lysis buffer is forced to flow out of well  202  through port  405  and channel  206  into mixing region  208 . At or about the same time, the sample is forced to flow out of sample well  204  through port  406  and channel  205  into mixing region  208 . In step  1406 , the lysis buffer and sample mix in the mixing region  208  and the mixture is forced to flow to filter  210  via channel  209 . In embodiments where the sample contains white blood cells, the sample may be enriched for white blood cells prior to introducing the sample into mixing region  208 . 
     While the lysis buffer/patient sample mixture is in channel  209  and travelling towards filter  210 , the lysis buffer selectively lyses the cellular membranes of cells in the patient sample without lysing the nuclear membranes of cells to produce intact nuclei from the cells, as reflected in step  1407 . When the mixture reaches filter  210 , the mixture preferably contains released intact nuclei from the patient sample. In step  1408 , the intact nuclei are trapped by filter  210  while the waste (i.e., other components of the sample and lysis buffer) passes through filter  210  and is forced to travel via channel  213  to waste collection well  212 . 
     In step  1410 , the intact nuclei trapped on filter  210  are lysed, thereby releasing DNA from the lysed nuclei. In some embodiments, the step of lysing the intact nuclei comprises causing an elution buffer in well  216  to flow to filter  210  via channel  217  and/or heating the trapped nuclei using heater  250 . In some embodiments, the elution buffer comprises a Tris buffer, KCl and/or a zwitterion. In some embodiments, the elution buffer is an amplification reaction buffer. In embodiments where heat is used to lyse the trapped nuclei, the trapped nuclei may be heated at a temperature in the range of approximately 35 degrees centigrade to 95 degrees centigrade for approximately 1 to 10 minutes. For example, in one embodiment, the trapped nuclei may be heated at a temperature in the range of approximately 50 degrees centigrade for approximately 7 minutes. 
     In step  1412 , after lysing the intact, trapped nuclei, the DNA released from the nuclei is collected in the DNA collection well  214 . For example, the released DNA flows out of filter  210  and to well  214  via channel  215 . In some embodiments, the released DNA flows to well  214  by flowing an elution buffer from well  216  such that the elution buffer flows out of port  407  and into channel  217 , then through channel  217  to and through the filter  210  where the released DNA mixes with the elution buffer, and then flows through channel  215  into well  214 . Once in well  214 , the mixture containing the released DNA and elution buffer can be removed from chip  201  via through holes  402 ,  415 ,  424 , and  434 , all of which are in fluid communication with well  214 . 
     While not shown, it is well known in the art that device  200  may be coupled to a flow control system (e.g., a system that comprises one or more pumps) for causing the various buffers, samples and mixtures to flow as described above. Additionally, device  200  may be coupled with a microfluidic platform. The DNA purified by device  200  may be directly delivered by a pump to a well in the microfluidic platform, and further mix with other PCR components. 
       FIGS. 15A and 15B  show trapping of nucleic acid by the membrane in accordance with an embodiment of the present invention. Specifically,  FIG. 15A  shows fluorescence emitted from membrane prior to the membrane trapping dye stained nuclei, and  FIG. 15B  fluorescence emitted from membrane after the membrane has trapped dye stained nuclei. 
     Referring now to  FIG. 16 , a graph is provided showing results achieved from using an above-described method. In particular, DNA purification was tested using 9 patient blood samples. After obtaining purified DNA using this method, one fraction of purified DNA sample was quantified by Pico green method (fluorescence based method for measuring total DNA concentration). Another fraction of purified DNA sample was quantified by real time-PCR. The results show that real time-PCR result is comparable to Pico green assay, indicating that no significant amount of inhibits exist in the purified DNA sample. 
     The table below provides representative dimensions for many of the above described components of device  200 . These dimensions are illustrative and not intended to be limiting in any way. 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Component 
                 Dimensions 
               
               
                   
                   
               
             
             
               
                   
                 chip 201 
                 length: 42 mm; width: 28 mm; height: 8.3 mm 
               
               
                   
                 channel 206 
                 length: 28 mm; width: 150 μm; depth: 150 μm 
               
               
                   
                 channel 205 
                 length: 5.4 mm; width: 100 μm; depth: 150 μm 
               
               
                   
                 channel 209 
                 length: 100 mm; width: 200 μm; depth: 150 μm 
               
               
                   
                 channel 213 
                 length: 7.75 mm; width: 400 μm; depth: 150 μm 
               
               
                   
                 channel 215 
                 length: 42 mm; width: 100 μm; depth: 150 μm 
               
               
                   
                 channel 217 
                 length: 36 mm; width: 100 μm; depth: 150 μm 
               
               
                   
                   
               
             
          
         
       
     
     Referring now to  FIG. 17 , a schematic illustration is provided of various components of sample a preparation sub-system  102  according to other embodiments of the present invention. As shown in  FIG. 17 , system  102  may include a sample well  1702  for containing a sample (e.g., a sample of blood containing white and red blood cells), a lysis buffer well  1704  for containing a lysis buffer, and a channel  1706  in fluid communication with wells  1702  and  1704  via channels  1703  and  1705 , respectively. Channel  1706  may function as a cell lysis region. That is, channel  1706  may be configured such that the lysis buffer from well  1702  is permitted to mix with the sample from sample well  1704  resulting in the selective lysing of cellular membranes of cells in the sample without lysing nuclear membranes of the cells to produce intact nuclei from the cells in the sample. 
     As further shown in  FIG. 17 , system  102  may include a cross-flow filtration region  1708  in fluid communication with channel  1706 . In some embodiments, in region  1708  intact nuclei (or white cells or other target components) are separated from other components of the sample (e.g., proteins and other PCR inhibitors) by one or more cross-flow filters  1710 , each having a pore size such that the target components (e.g., intact nuclei) are prevented from being carried away by a cross-flow buffer that is controlled to flow through the cross-flow filtration region, but other components of the patient sample flow through filters and are carried away by the cross-flow buffer. In the non-limiting embodiment shown in  FIG. 17 , cross-flow filtration region  1708  includes four cross flow filters  1710  that are connected in parallel. In other embodiments, region  1708  may have one, two, three or five or more filters  1710 . Additionally, in some embodiments, some filters  1710  may be arranged in series. Moreover, each filter  1710  may have a different average pore size. 
     System  102  may also include a concentrator region  1712 , which is in fluid communication with cross flow filtration region  1708 , in which the intact nuclei from the cross-flow filtration region are concentrated. An interface region  1714  may be in fluid communication with concentrator  1712 . As will be explained herein, purified intact nuclei preferably exit concentrator  1712  and enter interface region  1714 , which includes one or more interface channels through which purified nuclei flow for downstream processing and analysis. 
       FIG. 18  further illustrates an embodiment of cross-flow filter  1710 . As shown in  FIG. 18 , cross-flow filter  1710  includes a microfluidic separation channel  1802 , which, as shown in  FIG. 17 , is in fluid communication with the cell lysis region  1706  and concentrator  1712 . Channel  1802  is configured such that fluid entering channel  1802  from channel  1706  can flow through channel  1802  such that the fluid will reach and enter the concentration region  1712 . A filter  1810  and a filter  1812  are disposed in a middle portion of channel  1802 . Filters  1810  and  1812  are arranged to form a separation chamber  1814 . 
     In operation, while the lysis buffer/sample mixture is flowing through channel  1802  (i.e., from the input end  1832  to the output end  1834 ), a cross-flow fluid is introduced into the portion of channel  1802  having the filters  1810  and  1812  via a cross-flow buffer input port  1804 . The cross-flow fluid exits this portion of the channel  1802  via a cross-flow buffer output port  1806 . Advantageously, there is a differential (e.g., a pressure differential or voltage differential, such as an electrophoretic voltage potential, or a gravitational field) between ports  1804  and  1806  that causes the cross-flow fluid that enters channel  1802  via port  1804  to flow first through filter  1810 , then through separation chamber  1814 , then through filter  1812 , and finally out of channel  1802  via exit port  1806 . There is also a force (e.g., pressure, electric, gravity) that causes fluid entering channel  1802  to flow from end  1832  to end  1834 . As the cross-flow buffer flows across separation chamber  1814  (as illustrated by the dashed lines), the cross-flow buffer together with filters  1810  and  1812  facilitate the separation of the intact nuclei from the other components of the mixture that flows into channel  1802  from cell lysis region  1706 . More specifically, the pore size of the filters  1812  and  1810  are such that the intact nuclei do not pass through filter  1812 , but are driven toward the concentrator region  1712  by the flow of fluid from end  1832  to end  1834 , whereas other, smaller components of the sample are driven through filter  1812  and driven towards exit port  1806  via the cross-flow of the cross-flow buffer. In this manner, intact nuclei (or other target material) can be efficiently separated from the other components of the sample. Preferably, one type of force (e.g., air pressure) is used to cause fluid entering channel  1802  to flow from end  1832  to end  1834 , while a different type of force (e.g. an electrical field, gravity) is used to cause the cross-flow fluid to flow from  1804  to  1806 . 
     In some embodiments, the size of the pores of filters  1810  and  1812  is between approximately 1 um and 15 um. For example, the size of the pores of filter  1812  may be about 5 um. In some embodiments, filters  1810  and  1812  may consists of or include a membrane and/or an array of pillars. 
     Referring now to  FIG. 19 , a cross-flow filter  1710  according to another embodiment is illustrated. As shown in  FIG. 19 , cross-flow filter  1710  may include a microfluidic separation channel  1902 . In an exemplary embodiment, filters  1910 ,  1912 ,  1916 , and  1918  are disposed in a middle portion of channel  1902 . Filters  1910 ,  1912 ,  1916 , and  1918  are arranged to form separation chambers  1914   a ,  1914   b , and  1914   c.    
     In operation, while the lysis buffer/sample mixture is flowing through channel  1902  (e.g., from the input end  1932  towards an output end  1934   a ), a cross-flow fluid is introduced into the portion of channel  1902  having the filters via a cross-flow buffer input port  1904 . The cross-flow fluid exits the portion of channel  1902  having the filters via a cross-flow buffer output port  1906 . Advantageously, there is a differential (e.g., a pressure differential or voltage differential, such as an electrophoretic voltage potential) between ports  1904  and  1906  that causes the cross-flow fluid that enters channel  1902  via port  1904  to flow first through filter  1910 , then through separation chamber  1914   a , then through filter  1912 , then through separation chamber  1914   b , then through filter  1916 , then through separation chamber  1914   c , then through filter  1918 , and finally out of channel  1902  via exit port  1906 . There is also a differential (e.g., pressure or electric) that causes fluid entering separation chambers  1914   a ,  1914   b , and  1914   c  to flow towards ends  1934   a ,  1934   b , and  1934   c , respectively. As the cross-flow buffer flows across a separation chamber  1914   a , the cross-flow buffer together with the filters that form the separation chamber  1914   a  facilitate the separation of desired components (e.g., intact nuclei, bacteria, viruses) from the other components of the mixture that flows into the separation chamber. 
     More specifically, for example, the pore size of the filters  1912  and  1910  are such that the intact nuclei do not pass through filter  1912 , but are driven toward end  1934  by a differential, whereas other, smaller components of the sample (e.g., bacteria, viruses or waste material) are driven through filter  1912  and into separation chamber  1914   b  by the flow of the cross-flow buffer. For example, the average pore size of filter  1912  may be between approximately 1 um and 15 um. In one embodiment, the average pore size is about 8 um. 
     The pore size of the filter  1916  may be such that bacteria does not pass through filter  1916 , but are driven toward end  1934   b  by a differential, whereas other, smaller components of the sample (e.g., viruses) are driven through filter  1916  into separation chamber  1914   c  by the flow of the cross-flow fluid. For example, the average pore size of filter  1916  may be between approximately 0.2 um and 2 um. In one embodiment, the average pore size is about 0.4 um. The pore size of the filter  1918  may be such that viruses do not pass through filter  1918 , but are driven toward end  1934   c  by a differential, whereas other, smaller components of the sample are driven through filter  1918  and driven towards exit port  1906  via the cross-flow of the cross-flow buffer. For example, the average pore size of filter  1918  may be between approximately 10 nm 400 nm. In one embodiment, the average pore size is about 100 nm. Port  1961  may be used to create a pressure differential between port  1961  and end  1934   b  so that a fluid in chamber  1914   b  will flow towards end  1934   b . Likewise, Port  1962  may be used to create a pressure differential between port  1962  and end  1934   c  so that a fluid in chamber  1914   c  will flow towards end  1934   c.    
     In the above manner, intact nuclei, bacteria and viruses may be separated from the sample collected in separate target collection ports using the cross flow filter  1710  as illustrated in  FIG. 19 , in accordance with one embodiment. 
     Referring now to  FIG. 20 , a layered embodiment of filter  1710  in illustrated. As shown in  FIG. 20 , a filter  1710  may include three layers: a top layer  2002 , a middle layer  2004 , and a bottom layer  2006 . A filter  2008  may be formed in top layer  2002 , one or more separation channels  2010  may be formed in middle layer  2004 , and a filter  2012  may be formed in bottom layer  2006 . At least a portion of the separation channel  2010  extends from the top surface of layer  2004  to the bottom surface of layer  2004 , as shown in  FIG. 21 , which shows a cross sectional view of this embodiment of filter  1710 . As shown in  FIG. 21 , layer  2002  is positioned on top of layer  2004  such that filter  2008  is on top of channel  2010 , thereby forming a top, porous wall of channel  2010 . Likewise, as shown in  FIG. 21 , layer  2006  is positioned beneath layer  2004  such that filter  2012  is underneath channel  2010 , thereby forming a bottom, porous wall of channel  2010 . 
     In operation, while the lysis buffer/sample mixture is flowing through channels  2010  (i.e., from the input end  2006  to the output end  2014 ), a cross-flow fluid is introduced into the portion of channel  2010  having the filters  2008  and  2012  via a cross-flow buffer input port (not shown). The cross-flow fluid exits this portion of the channel  2010  via a cross-flow buffer output port (also not shown). As with previous embodiments, there is a differential (e.g., a pressure differential or voltage differential, such as an electrophoretic voltage potential, or a gravitational field) between the cross-flow buffer input and output ports that causes the cross-flow fluid that enters channel  2010  to flow first through filter  2008 , then through the separation chamber, then through filter  2012 , and finally out of channel via the exit port. There is also a force (e.g., pressure, electric, gravity) that causes fluid entering channel  2010  to flow from end  2006  to end  2014 . As with previous embodiments, as the cross-flow buffer flows across separation chamber, the cross-flow buffer together with filters  2008  and  2012  facilitate the separation of the intact nuclei from the other components of the mixture that flows into channel  2010  from, for example, the cell lysis region  1706 . 
     Referring back to  FIG. 17 , as described above, system  102  may include a concentrator  1712 , as further illustrated in  FIG. 22 . As shown in  FIG. 22 , a concentrator  1712 , in accordance with one exemplary embodiment, includes a generally triangular shaped channel  2208  (i.e., a channel wherein the width of the channel increases as one moves from an input end to an output end). At the input end of channel  2208  there is an inlet  2202  providing a means for a fluid (e.g., the purified sample collected in cross flow filtration region  1708 , which also contain some waste components from the blood sample and lysis buffer) to enter into channel  2208 . At the opposite end of channel  2208  (i.e., at the output end) there are two waste outlets ( 2204   a  and  2204   b ), each of which provides a means for additional waste to exit channel  2208 , and a sample outlet  2206  that provides a means for the desired concentrated fluid to exit channel  2208 . As further shown in  FIG. 22 , two filters ( 2210   a  and  2210   b ) are disposed in channel  2208  and together form a separation chamber  2212  in channel  2208 . Separation chamber  2212  includes an fluid entry point  2214  that is positioned downstream from inlet  2202  and a fluid exit point  2216  that is adjacent the output end of channel  2208  and that is in fluid communication with outlet  2206 , but not in fluid communication with any of the waste outlets  2204 . 
     Referring back to  FIG. 17 , it can be seen that after the intact nuclei exit filtration region  1708 , the intact nuclei, as well as any waste matter not removed by filtration region  1708 , will flow into the channel  2208  of concentrator  1712 . Referring back now to  FIG. 22 , when the intact nuclei and any waste material enter channel  2208 , the mixture will be forced to flow into separation chamber  2212  by, for example, a pressure differential (or other force) between the input end and the output end of channel  2208 . When the mixture is in chamber  2212 , some of the mixture will flow through filter  2210   a  towards waste outlet  2204   a , some will flow through filter  2210   b  towards waste outlet  2204   b , and the rest will flow the entire length of chamber  2212  and into channel  2223  and eventually to outlet  2206 . For example, the pressure in chamber  2212  may be higher than the pressure at outlets  2204   a ,  2204   b  and  2206 , thereby forcing some of the mixture to flow to the outlets. Advantageously, the filters  2210  are configured such that the intact nuclei in the mixture are not able to pass through or enter the filter, but any waste material is able to flow through the filter. Accordingly, the mixture that leaves chamber  2212  will have a higher concentration of intact nuclei than the mixture that entered chamber  2212 . 
     As illustrated in  FIG. 17 , outlet  2206  of concentrator  1712  is in fluid communication with an inlet of an interface channel of interface region  1714 . Accordingly, in some embodiments, as described above, a mixture containing a concentrated amount of intact nuclei may flow into the interface channels of interface region  1714  for further testing and analysis. 
     The interface region  1714  in accordance with one embodiment is further illustrated in  FIG. 23 . As shown in  FIG. 23 , interface region  1714  may contain a number of microfluidic channels  2304 , such as, for example, 8 microfluidic channels. In some embodiments, the intact nuclei that exit concentrator  1712  are forced to flow through channels  2304  as is know in the art. As is also known in the art, as the intact nuclei flow, DNA from the intact nuclei may be released by lysing the nuclei. The DNA released from the intact nuclei may be amplified as they traverse channels  2304  using, for example, a PCR technique. In such an embodiment, a temperature control system  2306  controls the temperature of the DNA flowing though channels  2304  to create the PCR reaction. Thus, a portion of channels  2304  may be considered an amplification region. A camera  2302  may be positioned relative to channels  2304  to record fluorescent emissions from channels  2304  and thereby detect amplification of the DNA. Systems and methods for amplifying DNA and detecting the amplification of the DNA are described in the above-referenced patents. 
     In some embodiments, a concentrator is not used and the purified intact nuclei from the cross flow filtration region  1708  are caused to flow directly into the interface region  1714 . 
     Referring now to  FIG. 24 , a flow chart is provided which illustrates a process  2400  according to an embodiment of the invention for using the system shown in  FIG. 17 . Process  2400  may begin in step  2402 , wherein fluid from well  1702  (e.g., a blood sample) and a lysis buffer from well  1704  are mixed. That is, the blood sample and lysis buff are forced to flow out of wells  1702  and  1704 , respectively, and into channel  1706 , where the sample and lysis buffer mix. In some embodiments the fluids may be forced out of wells  1702  and  1704  by creating a pressure differential or an electrophoretic voltage. 
     In step  2404 , while the mixture is flowing along channel  1706 , the lysis buffer selectively lyses, in cell lysing region  1706 , the cellular membranes of the cells in the sample without lysing the nuclear membranes of the cells to produce intact nuclei from the cells. 
     In step  2406 , the intact nuclei is separated from the sample in a cross-flow filtration region  1708 , which includes one or more cross-flow filters  1710  that has a pore size such that the intact nuclei are not permitted do not pass through the filter and the other components of the patient sample pass through the filter and are carried away by a cross-flow buffer that is controlled to flow through the cross-flow filtration region. In some embodiments, the cross-flow buffer is driven through the cross-flow filtration region by a pressure differential or by an electrophoretic voltage. In step  2408 , which is optional, the intact nuclei are concentrated by concentrator  1712 . In step  2410 , the concentrated nuclei are forced to flow through an interface channel  1714  for downstream analysis. 
     In some embodiments, the cross-flow region may have multiple filters to filter out not only intact nuclei from the sample, but also bacteria and viruses. For example, the cross-flow filtration region may have three filters, one for separating nuclei from the sample, one for separation bacteria from the sample, and one for separating viruses from the sample, as described above in connection with  FIG. 19 . In other embodiments, the cross-flow filter  1710  is configured such that the purified sample from output end  1834  is recirculated back into the input end  1832  for additional passes through separation chamber  1814  for further purification. In one embodiment, this recirculation is accomplished by providing a channel connecting output end  1834  with input end  1832  to permit fluid flow there between, and controllably driving fluid from the output end into the input end by, for example, pressure differential. 
     Referring now to  FIG. 25 , a flow chart is provided illustrating a process  2500  of determining the presence or absence of a nucleic acid in a patient sample according to an embodiment of the invention. Process  2500  may begin in step  2502 , where a patient sample containing cells is mixed with a lysis buffer in a mixing region of a microfluidic device (e.g., region  1706 ), wherein the lysis buffer selectively lyses cellular membranes without lysing nuclear membranes. Next, in step  2504 , the lysis buffer selectively lysis in the mixing region  1706  (a.k.a., “cell lysing region”) the cellular membranes of the cells in the patient sample without lysing the nuclear membranes of the cells to produce intact nuclei from the cells. In step  2506 , the intact nuclei are separated from the patient sample in cross-flow filtration region  1708  as described above. In step  2508 , the nuclei are lysed to release nucleic acid. In step  2510 , the nucleic acid is amplified (e.g., amplified using PCR). In step  2512 , the presence or absence of an amplified product is determined, wherein the presence of the amplified product indicates the presence of the nucleic acid in the patient sample. In some embodiments, the patient sample is first enriched for white blood cells prior to the selective lysis of the cellular membranes. In some embodiments, steps  2502 - 2508  are performed one microfluidic device and steps  2510 - 2512  are performed in a different microfluidic device. In other embodiments, steps  2502 - 2506  are performed one microfluidic device and steps  2508 - 2512  are performed in a different microfluidic device. 
     Referring now to  FIG. 26 , subsystem  102  is illustrated in according with another embodiment. As shown in  FIG. 26 , the sample preparation subsystem  102  includes a sample card  2600  that has one or more chambers  2601  that are configured to hold one or more patient samples. Each of the one or more chambers  2601  comprises an inlet  2602 , a filter  2603 , and an outlet  2604 . The sample card  2600  is removably insertable into the sample preparation subsystem  102 , which allows the sample card  2600  to be loaded with the one or more patient samples, and then inserted into the sample preparation subsystem  102 . Each inlet  2602  further comprises one or more channels which may or may not be in fluid communication with each other.  FIG. 27  further illustrates one embodiment of the sample card  2600  containing one or more chambers  2601 . As shown in  FIG. 27 , each chamber  2601  comprises an inlet  2602 , a filter  2603 , and an outlet  2604 . The filter pore size may be between 1 to 15 um, and preferably is approximately 5 um. 
     As shown in  FIG. 26 , the sample preparation subsystem  102  includes a flow control system  2605  that controls the flow of a lysis buffer from a lysis buffer storage device  2606  into each chamber  2601  of the sample card  2600  through inlets  2602 . The lysis buffer contained in the lysis buffer storage device  2606  selectively lyses cellular membranes to release nuclei and cell debris. The flow control system  2605  then causes the cell debris and lysis buffer to flow through the filter  2603 , through outlets  2604 , and into a removably insertable waste receptacle  2607 , while leaving the nuclei trapped on the filter  2603 . The waste receptacle  2607  is positionable beneath the outlets  2604  to receive the cell debris and lysis buffer from the chambers  2601 . The lysis buffer does not lyse the nuclei from the patient sample. The flow control system  2605  also controls the flow of an elution buffer from an elution buffer storage device  2608  into each chamber  2601  of the sample card  2600  through the inlet  2602 . 
     As shown in  FIG. 26 , the sample preparation subsystem  102  includes a temperature control system  2609  that controls the temperature of the sample card  2600 . The temperature control system  2609  heats the sample card  2600 , which causes the nuclei trapped on the filter  2603  to lyse, thereby releasing the DNA of the nuclei. In this embodiment, the temperature control system uses a sensor  2610  and a heat source  2611  to controllably and effectively heat the sample card to lyse the intact nuclei. 
       FIG. 26  further illustrates an interface chip  2612  which is removably insertable into the sample preparation subsystem  102 . The interface chip  2612  is positionable beneath the sample card  2600  and is configured to receive the DNA released from the lysed nuclei trapped on the filter  2603 . As shown in  FIG. 26 , the interface chip  2612  comprises one or more DNA sample wells  2613  which are in fluid communication with one or more DNA sample outlets  2614 . When inserted into the subsystem  102 , the DNA sample wells  2613  are each aligned with an outlet  2604  from the sample card  2600 , which enables each DNA sample well  2613  to collect the DNA released by the lysed nuclei as the DNA exit the sample card  2600  through the outlet  2604 . 
     In this embodiment, the main controller  101  communicates with the temperature control system  2609  and the flow control system  2605 . As those skilled in the art will recognize, many options exist for a main controller  101 , such as, for example, a general purpose computer or a special purpose computer. Other specialized control equipment known in the art could also serve the purpose of the main controller  101 . 
     Referring to  FIG. 28 , a sample card  2600  is illustrated with multiple chambers  2601  connected by fluidic channels  2800 . In this embodiment, connecting chambers  2601  using a fluidic channel  2800  allows different numbers of samples to be tested in varying volumes. For example, in the embodiment shown where all chambers  2601  are in fluidic communication via fluidic channels  2800 , the sample card  2600  would allow one patient sample to be tested in a larger volume because every chamber  2601 , being in fluidic communication, would contain the same sample. Alternatively, if none of the chambers  2601  were in fluidic communication, as shown in  FIG. 27 , the sample card  2600  would allow testing of multiple patient samples simultaneously, in which each sample could be from the same patient or different patients. When none of the chambers  2601  are in fluidic communication, the number of patient samples to be tested would be limited by the number of chambers  2601  on the sample card  2600 . 
     While  FIG. 28  shows all chambers  2601  being connected, other arrangements are contemplated. The number of chambers  2601  connected together on a sample card  2600  could be many different combinations, which would allow for testing of a desired number of patient samples and a desired volume of each patient sample. For example, a sample card A 200  could be configured to connect the chambers  2601  in pairs via fluidic channels  2800 , which would reduce the number of different patient samples by half, while doubling the volume of each patient sample to be tested. The sample card  2600  can be made from various materials which might depend on the testing requirements of each application; the sample card  2600  can be reusable or disposable to reflect the requirements of each testing application. 
       FIG. 29  further illustrates the flow control system  2605  according to one embodiment. As shown in  FIG. 29 , the flow control system  2605  is controlled by the main controller  101  and comprises a pump  2900 , a pressure control system  2901 , a solution delivery chip  2902 , and a pressure control chip  2903 . The pressure control system  2901  comprises an air source and a pressure sensor which allows the flow control system  2605  to control the delivery of the elution buffer and the lysis buffer to the sample card  2600  using pressure to move the solutions. The solution delivery chip  2902  comprises multiple channels for delivering lysis buffer and elution buffer to each chamber  2601  of the sample card  2600 . The pressure control chip  2903  comprises multiple channels for providing pressure to each chamber  2601  of the sample card  2600 . While this embodiment illustrates the use of a solution delivery chip  2902 , a pump  2900 , and a pressure control system  2901  in the flow control system  2605 , the different components can be used in different combinations. For example, the pump  2900  and the solution delivery chip  2902  can be used without the pressure control system  2901 . In another embodiment, the pressure control system  2901  can comprise multiple channels for controlling air pressure in each chamber  2601  of the sample card  2600 . 
     Referring to  FIG. 30 , the solution delivery chip  2902  is illustrated according to one embodiment. As shown in  FIG. 30 , the solution delivery chip comprises multiple channels  3000  in fluidic communication the chambers  2601  of the sample card. The channels  3000  comprise one or more solution inlets  3001  which receive the buffers from the pump  2900 . The channels  3000  further comprise one or more solution outlets  3002  that are in fluid communication with the inlets  2602  of the sample card  2600  which allows the lysis buffer and the elution buffer to be delivered to the chambers  2601  of the sample card  2600  via the solution outlets  3002  of the solution delivery chip  2902 . 
     Referring to  FIG. 31 , the pressure control chip  2903  is illustrated according to one embodiment. As shown in  FIG. 31 , the pressure control chip  2903  comprises multiple pressure channels  3100 , and each pressure channel  3100  is in fluid communication with a pressure inlet  3101  and a pressure outlet  3102 . In this embodiment, each pressure channel is in fluid communication with the same pressure inlet  3101 ; however, other embodiments are contemplated where there may be more than one pressure inlet  3101 . The pressure inlet  3101  is in fluid communication with the pressure control system  2901  in order to deliver pressure to the pressure control chip  2903 . The pressure outlets  3102  are in fluid communication with the inlets  2602  of the sample card  2600 , which allows the pressure control chip  293  to deliver pressure to the chambers  2601  of the sample card  2600 . 
       FIG. 32  is a flowchart illustrating a method  3200  for isolating DNA cells in a patient sample. While the method  3200  is not limited to the system provided in  FIG. 26 , one preferred embodiment of the method can utilize a system similar to that shown in  FIG. 26 , and  FIG. 26  is used for reference purposes to assist in describing the method. As shown in  FIG. 32 , in step  3202  a sample preparation system  102  is provided, wherein the system comprises: (i) a sample card  2600  having multiple chambers  2601 , wherein each chamber  2601  comprises an inlet  2602 , a filter  2603 , and an outlet  2604 , where the sample card  2600  is removably insertable into the sample preparation subsystem  102 ; (ii) a flow control system  2605  for controlling flow of a lysis buffer and an elution buffer to each chamber  2601  of the sample card  2600 ; (iii) a temperature control system  2609  for heating the filter  2603  in the sample card  2600 ; (iv) a removably insertable waste receptacle  2607  which is positionable beneath the sample card  2600 ; and (v) an interface chip  2612  comprising multiple DNA sample wells  2613  and DNA sample outlets  2614 , where the interface chip  2612  is positionable beneath the sample card  2600 . 
     In step  3204 , the patient sample is loaded into the chambers  2601  of the sample card  2600 . In step  3206 , the sample card  2600  containing the patient sample is inserted into the sample preparation system  102 . In step  3208 , the flow control system  2605  delivers a lysis buffer from the lysis buffer storage device  2606  to the chamber  2601  of the sample card  2600  through the inlet  2602 . The lysis buffer selectively lyses the cellular membranes of the patient sample without lysing the nuclear membranes of the nuclei. The reaction produces a solution comprising a lysis buffer, intact nuclei and cellular debris, in the chamber  2601  of the sample card  2600 . In step  3210 , the removably insertable waste receptacle  2607  is positioned below the sample card  2600  and the flow control system  2605  operates to drive the solution through the filter  2603  of the chamber  2601 . The filter  2603  traps the intact nuclei from the solution while the lysis buffer and the cellular debris are driven out of the chamber  2601  through the outlet  2604  by the flow control system  2605 . The lysis buffer and cellular debris are collected when exiting the chamber  2601  through the outlet  2604  in the waste receptacle  2607 . 
     In step  3212 , the temperature control system  2609  operates to heat the filter  2603 , which heats the intact nuclei trapped in the filter  2603 . The heating of the intact nuclei causes the nuclei to lyse, which releases DNA from the nuclei. In step  3214 , the flow control system  2605  operates to deliver the elution buffer from the elution buffer storage device  2608  to the chamber  2601  of the sample card  2600  through the inlet  2602 . In step  3216 , the interface card  2612  is positioned beneath the sample card  2600 , and the flow control system  2605  operates to drive the elution buffer and the DNA through the outlet  2604  of the chamber  2601  while leaving the lysed nuclei on the filter  2603 . The elution buffer and DNA are deposited in the DNA sample well  2613  of the interface chip  2612  after exiting the chamber  2601  though the outlet  2604 . This method thus allows the DNA to be isolated from the patient sample. 
     An optional step following the DNA isolation described above can be to deliver the lysis buffer from the lysis buffer containment device  2606  into the chambers  2601  of the sample card  2600  using the flow control device  2605 , then drive the solution out of the chamber  2601  through the outlet  2604  by operation of the flow control system  2605  in order to clean the filter  2603 . This optional step can be repeated multiple times to provide the desired level of cleanliness of the filter  2603 . 
     While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments. Variations of the embodiments described above may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 
     Additionally, while the processes described above and illustrated in the drawings are shown as a sequence of steps, this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, the order of the steps may be re-arranged, and some steps may be performed in parallel.

Technology Classification (CPC): 2