analyte of interest, and sample filters containing a hydrophilic matrix with impregnated chemicals that lyse cell membranes.

Also disclosed is an integrated sample preparation cartridge. The integrated sample preparation cartridge includes a sample preparation device and a cartridge base. The sample preparation device includes a housing defining a passage way between a first opening and a second opening, and a sample filter occupying a section of the passage way, the sample filter includes an adsorbent that specifically binds to an analyte of interest. The cartridge base includes a first port configured to interface with the sample preparation device, a second port configured to interface with a liquid delivery device, and a first channel connecting the first port to the second port.

Also disclosed is a sample purification system. The sample purification system includes a sample preparation device, a cartridge base and a liquid delivery device.

Also disclosed is a method for purifying an analyte from a sample. The method includes passing a sample through a sample preparation device comprising a housing defining a passage way between a first opening and a second opening, and a filter occupying a section of the passage way, wherein the sample filter comprising a material that specifically binds to said analyte and the sample passes through the sample filter while passing through the sample preparation device; and eluting analyte bound to the sample filter with an eluting solution, wherein the sample and the eluting solution enter and exit the housing through the same opening. DESCRIPTION OF THE DRAWINGS

The detailed description will refer to the following drawings, wherein like numerals refer to like elements, and wherein:

Figures 1 A-ID are schematics of various embodiments of a sample preparation device.

Figure 2A-2C are schematics of the three-dimensional view (Figure 2A), the top view (Figure 2B) and the bottom view (Figure 2C) of an embodiment of an integrated sample preparation device.

Figure 3 shows the real-time PCR analysis of Bacillus anthracis nucleic acids purified from a blood sample. Controls include unprocessed Bacillus anthracis suspended in water at a concentration of 10 ⁴ cfu/ml and NTC (no template control).

Figure 4 shows the real-time PCR analysis of Streptococcus pyogenes nucleic acids purified from samples at various Streptococcus pyogenes concentrations. Controls

analyte of interest, and sample filters containing a hydrophilic matrix with impregnated chemicals that lyse cell membranes.

Also disclosed is an integrated sample preparation cartridge. The integrated sample preparation cartridge includes a sample preparation device and a cartridge base. The sample preparation device includes a housing defining a passage way between a first opening and a second opening, and a sample filter occupying a section of the passage way, the sample filter includes an adsorbent that specifically binds to an analyte of interest. The cartridge base includes a first port configured to interface with the sample preparation device, a second port configured to interface with a liquid delivery device, and a first channel connecting the first port to the second port.

Also disclosed is a sample purification system. The sample purification system includes a sample preparation device, a cartridge base and a liquid delivery device.

Also disclosed is a method for purifying an analyte from a sample. The method includes passing a sample through a sample preparation device comprising a housing defining a passage way between a first opening and a second opening, and a filter occupying a section of the passage way, wherein the sample filter comprising a material that specifically binds to said analyte and the sample passes through the sample filter while passing through the sample preparation device; and eluting analyte bound to the sample filter with an eluting solution, wherein the sample and the eluting solution enter and exit the housing through the same opening. DESCRIPTION OF THE DRAWINGS

The detailed description will refer to the following drawings, wherein like numerals refer to like elements, and wherein:

Figures IA-ID are schematics of various embodiments of a sample preparation device.

Figure 2A-2C are schematics of the three-dimensional view (Figure 2A), the top view (Figure 2B) and the bottom view (Figure 2C) of an embodiment of an integrated sample preparation device.

Figure 3 shows the real-time PCR analysis of Bacillus cmthracis nucleic acids purified from a blood sample. Controls include unprocessed Bacillus anthtacis suspended in water at a concentration of 10 ⁴ efu/ml and NTC (no template control).

Figure 4 shows the real-time PCR analysis of Streptococcus pyogenes nucleic acids purified from samples at various Streptococcus pyogenes concentrations. Controls

(1997); Weinert, Science, 277:1450-1451 (1997); Walworth et al., Nature, 363:368- 371 (1993); and Al-Khodairy et a/., Molec. Biol. Cell., 5:147-160 (1994).

Selective manipulation of checkpoint control in cancer cells could afford broad utilization in cancer chemotherapeutic and radiotherapy regimens and may, in addition, offer a common hallmark of human cancer "genomic instability" to be exploited as the selective basis for the destruction of cancer cells. A number of factors place CHK1 as a pivotal target in DNA-damage checkpoint control. The elucidation of inhibitors of this and functionally related kinases such as CDS1/CHK2, a kinase recently discovered to cooperate with CHK1 in regulating S phase progression (see Zeng et a/., Nature, 395:507-510 (1998); Matsuoka, Science, 282:1893-1897 (1998)), could provide valuable new therapeutic entities for the treatment of cancer.

Another group of kinases are the tyrosine kinases. Tyrosine kinases can be of the receptor type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular). Receptor-type tyrosine kinases are comprised of a large number of transmembrane receptors with diverse biological activity. In fact, about 20 different subfamilies of receptor-type tyrosine kinases have been identified. One tyrosine kinase subfamily, designated the HER subfamily, is comprised of EGFR (HER1 ), HER2, HER3 and HER4. Ligands of this subfamily of receptors identified so far include epithelial growth factor, TGF-alpha, amphiregulin, HB-EGF, betacellulin and heregulin. Another subfamily of these receptor-type tyrosine kinases is the insulin subfamily, which includes INS-R, IGF-IR, IR, and IR-R. The PDGF subfamily includes the PDGF-alpha and beta receptors, CSFIR, c-kit and FLK-II. The FLK family is comprised of the kinase insert domain receptor (KDR), fetal liver kinase-1(FLK-1 ), fetal iiver kinase-4 (FLK-4) and the fms-ϋke tyrosine kinase-1 (flt-1 ). For detailed discussion of the receptor-type tyrosine kinases, see Plowman et ai, DN&P 7£6):334-339, 1994.

At least one of the non-receptor protein tyrosine kinases, namely, LCK, is believed to mediate the transduction in T-cells of a signal from the interaction of a cell- surface protein (Cd4) with a cross-linked anti-Cd4 antibody. A more detailed discussion of non-receptor tyrosine kinases is provided in Boien, Oncogene, 8:2025- 2031 (1993). The non-receptor type of tyrosine kinases is also comprised of numerous subfamilies, including Src, Frk, Btk, Csk, AbI ₁ Zap70, Fes/Fps, Fak, Jak,

Ack, and LIMK. Each of these subfamilies is further sub-divided into varying receptors. For example, the Src subfamily is one of the largest and includes Src, Yes, Fyn, Lyn, Lck, BIk, Hck, Fgr, and Yrk. The Src subfamily of enzymes has been linked to oncogenesis. For a more detailed discussion of the non-receptor type of tyrosine kinases, see Boten, Oncogene, 8:2025-2031 (1993).

In addition to its role in cell-cycle control, protein kinases also play a crucial role in angiogenesis, which is the mechanism by which new capillaries are formed from existing vessels. When required, the vascular system has the potential to generate new capillary networks in order to maintain the proper functioning of tissues and organs. In the adult, however, angiogenesis is fairly limited, occurring only in the process of wound healing and neovascularization of the endometrium during menstruation. On the other hand, unwanted angiogenesis is a hallmark of several diseases, such as retinopathies, psoriasis, rheumatoid arthritis, age-related macular degeneration, and cancer (solid tumors). Protein kinases which have been shown to be involved in the angiogenic process include three members of the growth factor receptor tyrosine kinase family; VEGF-R2 (vascular endothelial growth factor receptor 2, also known as KDR (kinase insert domain receptor) and as FLK 1 ); FGF-R (fibroblast growth factor receptor); and TEK (also known as Tie-2).

VEGF-R2, which is expressed only on endothelial cells, binds the potent angiogenic growth factor VEGF and mediates the subsequent signal transduction through activation of its intracellular kinase activity. Thus, it is expected that direct inhibition of the kinase activity of VEGF-R2 will result in the reduction of angiogenesis even in the presence of exogenous VEGF (see Strawn et al, Cancer Res., 56:3540- 3545 (1996)), as has been shown with mutants of VEGF-R2 which fail to mediate signal transduction. Millauer et a/, Cancer Res. , 56: 1615-1620 ( 1996). Furthermore, VEGF-R2 appears to have no function in the adult beyond that of mediating the angiogenic activity of VEGF. Therefore, a selective inhibitor of the kinase activity of VEGF-R2 would be expected to exhibit little toxicity.

Similarly, FGFR binds the angiogenic growth factors aFGF and bFGF and mediates subsequent intracellular signal transduction. Recently, ft has been suggested that growth factors such as bFGF may play a critical roie in inducing angiogenesis in solid tumors that have reached a certain size. Yoshiji et at., Cancer

Research, 57: 3924-3928 (1997). Unlike VEGF-R2, however, FGF-R is expressed in a number of different ceil types throughout the body and may or may not piay important roles in other normal physiological processes in the adult. Nonetheless, systemic administration of a small molecule inhibitor of the kinase activity of FGF-R has been reported to block bFGF-induced anglogenesis in mice without apparent toxicity. Mohammad et al., EMBO Journal, 17:5996-5904 (1998).

TEK (also known as Tie-2) is another receptor tyrosine kinase expressed only on endothelial cells which has been shown to play a role in angiogenesis. The binding of the factor angiopoietin-1 results in autophosphorylation of the kinase domain of TEK and results in a signal transduction process which appears to mediate the interaction of endothelial cells with peri-endothelial support cells, thereby facilitating the maturation of newly formed blood vessels. The factor angiopoietin-2, on the other hand, appears to antagonize the action of angiopoietin-1 on TEK and disrupts angiogenesis. Maisonpierre et al., Science, 277:55-60 (1997). The kinase, JNK, belongs to the mitogen-activated protein kinase (MAPK) superfamily. JNK plays a crucial role in inflammatory responses, stress responses, cell proliferation, apoptosis, and tumorigenesis. JNK kinase activity can be activated by various stimuli, including the proinflammatory cytokines (TNF-alpha and interleukin- 1 ), lymphocyte costimulatory receptors (CD28 and CD40), DNA-damaging chemicals, radiation, and Fas signaling. Results from the JNK knockout mice indicate that JNK is involved in apoptosis induction and T helper cell differentiation.

Pim-1 is a small serine/threonine kinase. Elevated expression levels of Pim-1 have been detected in lymphoid and myeloid malignancies, and recently Pim-1 was identified as a prognostic marker in prostate cancer. K. Peitola, "Signaling in Cancer Pim-1 Kinase and its Partners", Annaies Universitatis Turkuensis, Sarja - Ser. D Osa - Tom. 616, (August 30, 2005), http://kiriasto.utu.fi/iulkaisupalvelut/annaalit/2004/D616.h tml. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. K. Petersen Shay et al., Molecular Cancer Research 3: 170-181 (2005). Aurora kinases (Aurora-A, Aurora-B, Aurora-C) are serine/threonine protein kinases that have been implicated in human cancer, such as colon, breast and other solid tumors. Aurora-A (also sometimes referred to as AIK) is believed to be involved

in protein phosphorylation events that regulate the cell cycle. Specifically, Aurora-A may play a role in controlling the accurate segregation of chromosomes during mitosis. Misregulation of the celJ cycle can lead to cellular proliferation and other abnormalities. In human colon cancer tissue, Aurora-A, Aurora-B, Aurora-C have been found to be overexpressed (see Bischoff et a/., EMBO J., 17:3052-3065 (1998); Schumacher et a!., J. Cell Biol. 143:1635-1646 (1998); Kimura et al., J. Biol. Chem., 272:13766-13771 (1997)). c-Met is a proto-oncogene that encodes for a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF). The c-Met protein is expressed mostly in epithelial cells, and due to its function it is also known as hepatocyte growth factor receptor, or HGFR. When HGF/SF activates c-Met, the latter in turn may activate a number of kinase pathways, including the pathway from Ras to Raf to Mek to the mitogen-activated protein kinase ERK 1 to the transcription factor ETS 1. Met signaling has been implicated in the etiology and malignant progression of human cancers (see Birchmeier et a/., Nature Reviews Molecular Cell Biology, 4;915-925 (2003); Zhang etal., Journal of Cellular Biochemistry, 88:408-417 (2003); and Paumelle et a/., Oncogene, 21:2309-2319 (2002)).

Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP K2 or MK2) mediates multiple p38 MAPK-dependent cellular responses. MK2 is an important intracellular regulator of the production of cytokines, such as tumor necrosis factor alpha (TNFa), interleukin 6 (IL-6) and interferon gamma (IFNg), that are involved in many acute and chronic inflammatory diseases, e.g. rheumatoid arthritis and inflammatory bowel disease. MK2 resides in the nucleus of non-stimulated cells and upon stimulation, it translocates to the cytoplasm and phosphorylates and activates tuberin and HSP27. MK2 is also implicated in heart failure, brain ischemic injury, the regulation of stress resistance and the production of TNF-α (see Deak et a/., EMBO. 17:4426-4441 (1998); Shi et al., Biol. Chem. 383:1519-1536 (2002); Staklatvala., Curr. Opin. Pharmacol. 4:372-377 (2004); and Shiroto et a/., J. MoI. Cell Cardiol. 38:93-97 (2005)). There Is a need for effective inhibitors of protein kinases in order to treat or prevent disease states associated with abnormal cell proliferation. Moreover, it is desirable for kinase inhibitors to possess both high affinities for the target kinase as

single-chain antibody molecule, multispecitic antibody, and/or other antigen binding sequences of an antibody. In another embodiment, the glass fin is coated with lectins, which bind to carbohydrates found in bacteria coats and can be used to capture bacteria in a sample. In another embodiment, the sample tiller 20 is made of a porous glass monolith, a porous glass-ceramic, or porous monolithic polymers. Porous glass monolith may be produced using the sol-gel methods described in U.S. Patent Nos. 4,810.674 and 4,765,818, which are hereby incorporated by reference. Porous glass-ceramic may be produced by controlled crystallization of a porous glass monolith. Porous monolithic polymers are a new category of materials developed during the last decade. In contrast to polymers composed of very small beads, a monolith is a single, continuous piece of a polymer prepared using a simple molding process. In one embodiment, the housing 10 serves as the mold for die porous monolithic polymers. Briefly, a section of the passage way 12 of the housing 10 is filled with a liquid mixture of monomers and porogens. Next, a mask that is opaque to ultraviolet light is placed over the filed section. The mask has a small slit that exposes a small portion of the filled section. Finally, the monomers/porogens mixture in the filled section is irradiated with ultraviolet light through the tiny opening on the mask. The UV irradiation triggers a polymerization process mat produces a solid but porous monolithic material in the filled section of the passage way 12.

In yet another embodiment, the sample filter 20 is made of a hydrophilic matrix with impregnated chemicals that lyses cell membranes, denaturing proteins, and traps nucleic acids. In one embodiment, the hydrophilic matrix is FTA paper® (Whatman, Florham Park, NJ). Biological samples are applied to the FTA * paper and cells contained in the sample are lysed on the paper. The paper is washed to remove any non-DNA material (the DNA remains entangled within the paper). The DNA is then eluted for subsequent analysis.

The sample filter 20 is shaped to fit tightly into the passage way 12 to prevent the sample from channeling or bypassing the sample filter 20 during operation. In one embodiment, the filter 20 is fitted into the passage way 12 through mechanical means such as crimping, press fitting, and heat shrinking the housing 10 or a portion thereof. In another embodiment, the filter 20 is attached to the interior of passage way 12 through an adhesive. In yet another embodiment, the side of the frit is tapered to the contour of the passage way 12 In the embodiments shown in Figures IA- ID, the housing 10 has the

shape of a frustocønical pipette tip with the first opening 14 dimensioned to fit on the end of a liquid delivery system, such as a manual pipettor or an electronic pipetting device. Samples are taken up though the second opening 16, passed through the sample filter 20 and then retained in the section of the housing IO that is above the sample filter 20. In one embodiment, the liquid delivery system is an electronic pipetting device, such as an electronic pipettor or a robotic pipetting station.

In one embodiment, the sample filter 20 includes at least two sections, a first section 22 that binds specifically to nucleic acids and a second section 24 that specifically binds to another analyte of interest, such as proteins (Figure 1 B). In another embodiment, the housing 10 contains a pre-tilter 30 placed between the second opening 16 and the sample filter 20 (Figure 1C). The pre-filter 30 has a pore size that is larger than the pore size of the sample filter 20 and does not bind specifically to nucleic acids. In yet another embodiment, the housing contains an aerosol filter 40 in the proximity of the first opening 14 to prevent contamination from the pumping device (Figure 1 D). In another embodiment, the housing 10 further contains a plurality of mechanical lysing beads, such as glass beads, in the space between the sample filter 20 and the aerosol filter 40. The mechanical Iy sing beads are used to disrupt the cells and release the nucleic acid by vortexing the entire sample preparation device 100. In this embodiment, the second opening 16 may be covered with a cap during vortexing to prevent the liquid from escaping from the second opening 16.

Another aspect of the present invention relates to an integrated sample preparation cartridge. Referring now to Figures 2A-2C, an embodiment 200 of the integrated sample preparation cartridge includes a base 50 and the sample preparation device 100. The base 50 contains a first sample port 51 and a second sample port 52 on the top surface 61, a third sample port 53 and a fourth sample port 54 at the bottom surface 62. a first channel 55 connecting the first sample port 51 to the third sample port 53, a second channel 56 connecting the second sample port 52 to the fourth sample port 54, and a third channel 57 connecting the first channel 55 and the second channel 56.

The first sample port 51 is configured to receive the sample preparation device 100, so that ihe sample preparation device 100, whether in a column configuration or pipette tip configuration, can be easily inserted into the first sample port 51 and form a liquid-tight seal with the base 50.

Once attached to the first sample port 51, the sample preparation device 100 maintains a vertical position A sample may be loaded onto the sample preparation

device 100 from either the first opening 14 (i.e., going down the sample passage way 12) or the second opening 16 (i.e., going the sample passage way 12). Alternatively, the sample preparation device 100 may be attached to the first sample port 51 with a preloaded sample. The second sample port 52 can also be used to introduce a liquid into the integrated sample preparation cartridge 200 or to take out a liquid from the integrated sample preparation cartridge 200. The second sample port 52 is configured to receive the tip 26 of a liquid delivering device, such as a pipettor or a robotic pipetting station. In one embodiment, the second sample port 52 is a self-sealing inlet containing a seal 58 that can be punctured by a pipette tip and seals after the removal of the tip. Such a self- sealing entry port for a pipette allows easy introduction of the sample without the risk of opening caps, which are often a cause of contamination. In one embodiment, the seal 58 is a Multisip™ split septum plug from Abgene (Epsom, UK). In another embodiment, the seal 58 is a port valve, such as the Duckbill valves and dome valves from Mini valve International (Yellow Springs, OH). In another embodiment, the first sample port 51 also contains a self-sealing device, such as a dome valve or a septum, that is receptive to the sample preparation device 100.

In another embodiment, either the first sample port 51 or the second sample port 52 or both ports can be sealed with a screw cap or a press fit cap to allow the introduction and removal of samples. The ports can also be sealed with a tape seal to prevent leaking during the automation process.

The first channel 55, the second channel 56 and the third channel 57 connect the first sample port 51 io the second sample port 52 so that ihe nucleic acid purifiaclion process can be completed within the integrated sample preparation cartridge 200. The third sample port 53 and the fourth sample port 54 may be connected to waste bottles to collect the flow-through from the sample preparation device 100.

The integrated sample preparation cartridge 200 can be configured to be compatible with fluidic control systems, such as the Flow Pro Fluidic Handling System (Global FIA, Fox Island, WA). In one embodiment, the first sample outlet 53 and the second sample outlet 54 are fitted with Luer-activated valves 59. The Luer-activated valves 59 are normally closed valves that may be opened only upon insertion of a luer- type fitting. The Luer-activated valves 59 allow easy insertion into the fluidic control system and prevent leaking of sample from the sample preparation cartridge 200 after the sample preparation cartridge 200 is removed from the fluidic control system. In one

embodiment, the integrated sample preparation cartridge 200 is designed to be plugged into a flυidic control system without the need for tightening screws or adjusting bolts.

A person of ordinary skill in the art understands that the general layout of the integrated sample preparation cartridge 200 allows for other sample introduction and elution withdrawal strategies. In one embodiment, the sample preparation cartridge 200 is connected to a flυidic control system. The sample preparation device 100 is inserted into the first sample port 51. A sample is introduced into the integrated sample preparation cartridge 200 through the second sample port 52, which is sealed off after the introduction of the sample. A chaotrophe, such as guanidine, is introduced into the integrated sample preparation cartridge 200 through the fourth sample port 54 by the flυidic control system and mixed with the sample within the integrated sample preparation cartridge 200. The sample/chaotrophe mixture is then pushed into the sample preparation device 100 from the second opening 16 of the sample preparation device 100, passing the filler 20 and entering the section of the housing 10 that h above the sample filter 20. The sample/ehaotrophe mixture is then withdrawn from the integrated sample preparation cartridge 200 through the fourth sample port 54 and discarded as waste. A washing buffer is introduced into the integrated sample preparation cartridge 200 through the third sample pott 53 by the fluidic control system. Similar to the movement of the sample/ehaotrophe mixture, the washing buffer is forced into and then withdrawn from the sample preparation device 100, passing the filter 20 twice during the process. The washing step may be repeated several times. Finally, an eluting buffer is introduced into the sample preparation device 100 through the second opening 16, eluting the bound nucleic acids into the section of the housing 10 that is above the sample filter 20, from where the elυant is removed for further analysis. In another embodiment, the sample is introduced through the first opening 14 of the sample preparation device 100 which is attached to the first sample port 51, and the eluant is removed from the second sample port 52. In another embodiment, the sample is introduced through the first opening 14 of sample preparation device 100, which is attached to the first sample port 51, and the eluant is removed from the first opening 14 of sample preparation device 100. In another embodiment, the sample is introduced onto the sample preparation device 100, which is attached to the first sample port 51, through the second sample port 52, and the eluani is removed from ihe second sample port 52. In another embodiment, the sample is pre-loaded into the sample preparation device 100 before the sample preparation device 100 is inserted into the first sample port 51 of the

integrated sample preparation cartridge 200. After the washing and eluting steps, the elυant is removed from the second sample port 52. In yet another embodiment, the sample is pre-loaded into the sample preparation device 100 before the sample preparation device 100 is inserted into the first sample port 51 of the integrated sample preparation cartridge 200. After the washing step, the anaiyte bound on the sample filler 20 are elυted into the sample preparation device 100, which is then removed from the first sample port 51 with the purified analyle in the elution buffer within the space between the sample filter 20 and the aerosol filter 40.

The integrated sample preparation cartridge 200 is easy to use. First, this device does not require centrifugation and thus eliminates the complexity associated with transferring samples from tubes to spin columns as well as simplifies the instrumentation required. Additionally a self-sealing entry port for a pipettor allows easy introduction of the sample without the risk of opening caps, which are often a cause of contamination. Additionally, the Luer-activated valves make cartridge insertion and removal simple and easy without the risk of losing sample due to leakage after the process is complete.

In addition, the vertical orientation of the sample preparation device 100 forces bubbles to rise to the top of the device from the sample filter 20, which improves tluidic control and enhances anaiyte binding and elution. Additionally, the small pores of the sample filter 20 reduces large air boluses into small bubbles which migrate to the top of the liquid column inside the sample passage way 12, creating a vibrant mixing effect of the chaotrophe with the sample. Ii should be noted that the pipeite tip configuration of the sample preparation device 100 allows bidirectional flow of the sample/washing/elution liquids through the sample filter 20, while most sample preparation approaches rely on flow in only one direction through the filter. The bidirectional flow feature not only allows the sample liquid to be taken into the sample preparation device 100 and eluted out of the sample preparation device 100 from the same opening (e.g., the second opening 16), but also permits a user to pipette a sample up and down for a number of cycles, thus providing the capability to process sample volumes larger than that of the sample preparation device 100. In one embodiment, the channels 55, 56 and 57 are designed to have the shortest possible length to reduce unwanted biomolecular (nucleic acid) adsorption to the interior surfaces of the integrated sample preparation cartridge 200. The channels may also be surface coated to reduce unwanted biomolecule adsorption. In one embodiment, the

channels 55, 56 and 57 have diameters in the range of 0.1-5 mm to reduce the surface-to- volume ratios and therefore reduce unwanted nucleic acid adsorption.

After the removal of the eluani the integrated sample preparation cartridge 200 is removed from the Flow Control Station and discarded. The base 50 of lhe integrated sample preparation cartridge 200 can be made of any material that is resistant to the chemicals commonly used in the sample solubilization/washing/eluting process. Iλ one embodiment, the base 50 is made of a transparent material. Examples of the base 50 materials include, but are not limited to, polycarbonate and polypropylene. The fluidic control system can be any fluidic control system that is capable of providing the desired flow rate in the integrated sample preparation cartridge 200. In one embodiment, lhe fluidic control system is the Flow Pro Fluidic Handling System by Global FIA (Fox Island, WA). EXAMPLES Example J: Purification of nucleic acids from blood sample containing Bacillus anthracis using 2.0 ml Rainiu filtered pipette tip and 3 mm glass frit

In this experiment, nucleic acids were purified from a blood sample containing Bacillus amhracis using a 2.0 ml Rainin filtered pipette tip and a 3 mm glass frit (ROBU Glasfilter-Geraete GmbH, Germany) with the following protocol: 1. Label one 15 ml conical tube as: Flow Through, and four 1.5 ml centrifuge tubes as: ϊithanol 1, Ethanol 2, Hthanol 3 _V and Hluant.

2. Mix 360 μl of blood with 40 μl of Bacillus amhracis ( 10* colony forming unit (cfu) / ml in waier) in the Flow Through tube (final concentration 10 ⁴ cfu/røl).

3. Add 1120 μl of Qiagen AL Lysis Buffer to the mixture. 4. Add 80 μl of Proteinase K (20mg/ml).

5. Add 400 μl of lysozyme, vortex and spin down.

6. Incubate the sample mixture at 55 ⁰ C for 1 hour.

7. Add 2000 μl of 96-100% ethanol to the Flow Through lube. Vortex and pulse spin down the mixture. 8. Aliquot 100 μl of elution buffer (10 m.M Tris. pi I 8.0) into the Eluant tube.

Place the tube on the heat block set at 70°C. (Elution buffer must be heated at 70°C for at least 5 minutes. Keep the buffer on the heal block until step 13.) 9. Aliquot 1 ml of 70% Ethanol into each of the three Ethanol tubes.

10. Pipette sample mixture into the Flow Through tube using frit tip (medium porosity) with a Rainin Electronic Pipeltor. Pipette for 5 cycles (cycle - aspirate + dispense).

11. Wash the bound nucleic acids by pipetting the 70% EtOH in Ethanol 1 tube for 10 cycles using a Rabύn electronic Pipettor. Repeat the wash with the

70% EtOH in Ethanol 2 tube and Ethanol 3 tube (three washes total).

12. Purge the EtOH from the frit tip by pipetting air for 20 cycles. Wipe the outside of the tip and tap the tip gently if a noticeable amount of ethanol is left. 13. Elute the nucleic acids on the frit by pipetting the 70 ⁰ C elution buffer of step 8 for 10 cycles. The buffer may start to bubble but continue pipetting and spin down the microcentrifuge tube once complete. Make sure all the buffer has been purged from the tip. 14. Collect the etuant and discard the frit tip 15. Quantitating the eluted nucleic acids with real time PCR.

As shown in Figure 3, nucleic acids from Bacillus imihracis are detected in the eluant.

Example 2: Purification of nucleic acids from Streptococcus pyogenes using 2.0 ml Rainin filtered pipette tip and 2 mm glass frit. In this experiment, nucleic acids were purified from Streptococcus pyogenes suspensions of various concentrations using a 2.0 ml Rainin filtered pipette tip and a 2 mm glass frit (ROBU Glasfilter-Geraete GmbH, Germany) with the following protocol:

1. Label three 1.5 ml centrifuge tubes as: Sample ^• * ^• Guanidine, Ethanof, and Eluant,

2. Lyse by vortexing with glass beads. 3. Mix 500 μl of Streptococcm pyogenes sample with 500 μl of 6M guanidine, pH

6.5, by vortexing. An aliquot of unprocessed (i.e.. the pre-guanidine) Streptococcus pyogenes were also saved as a control for real-time PCR analysis in step 10.

4. Aliquot 100 μl elution buffer (1 inM NaOH) into the eluant tube. Pipette the sample/guanidine mixture with a frit tip (medium porosity) and a Rainin

Electronic Pipettor. Pipette for 5 cycles (cycle - aspirate -*- dispense).

5. Pipette 1 ml 70% EtOB to wash bound nucleic acids using the Rainin electronic Pipettor for 5 cycles.

6. Pass air through the frit tip to purge EtOH using the electronic pipettor. Repeat pipetting for 20 cycles Io remove traces of EtOH. Tap the frit tip gently if a noticeable amount of ethanol is left. Remove the EtOH on the outside of the tip with Kimwipe®. 7. Elute the nucleic acids from the frit with the 70 ⁰ C elution buffer from step 4 by pipetting for IO cycles. Make sure all the elυtion buffer has been purged from the tip.

8. Collect the eluant and discard the frit tip

9. Quantitating the eluted nucleic acids with real time PCR. As shown in Figure 4, Streptococcus pyogenes nucleic acids are detected in samples prepared by the frit tip.

Example 3: Purification of nucleic acids from nasopharyngeal sample containing

Bacillus anthraeis using 2.0 nil Rainin filtered pipette tip and 2 mm glass frit

In this experiment, nucleic acids were purified from nasopharyngeal sample containing Bacillus anthraeis using a 2.0 ml Rainin filtered pipette tip and a 2 mm frit

(ROBU Glasfilter-Geraete GmbH, Germany) with the following protocol:

1. Label three 1.5 ml centrifuge tubes as: Sample -f Guanidine, Ethanol, and Eluant.

2. Prepare nasopharyngeal sample by mixing 450 μl of nasopharyngeal with 50 μl of Bacillus anthraeis ( 10 ⁵ colony forming unit (cfu) / ml in water) in the Sample + Guanidine tube (final concentration 10 ¹ cfu/ml). Save an aliquot of the nasopharyngeal sample as control in the real-time PCR analysis of Step 1 1.

3. Add 500 μl of 6M guanidine, pH 6.5, into the Sample + Guanidine tube and vortex.

4. Aliquot 100 μl of elution buffer (10 mM Tris, pH 8.0) into the Eluant tube. Place the tube on the heat block set at 7OX {elution buffer must heat at 7O ⁰ C for at least

5 minutes). Keep the tube on the heat block until step 9.

5. Pipette the sample/guanidine mixture using a frit tip (medium porosity) with a Rainin Electronic Pipettor. Pipette for 5 cycles (cycle - aspirate ^• * ^• dispense).

6. Pipette 1 ml 70% EtOH to wash bound nucleic acids using the Rainin electronic Pipetior.

7. Pass air through the frit tip to purge EtOH using the electronic pipettor. Repeat pipetting for 20 cycles to remove traces of HtOH. Tap the frit tip gently if a noticeable amount of ethanol is left. Remove the EtOH on the outside of the tip with Kimwipe®

8. Elute the nucleic acids from the frit with the 70°C elυtion buffer from step 4 by pipetting for IO cycles. Make sure all the elution buffer has been purged from the tip.

9. Collect the eluant and discard the frit tip 10. Quantitating the eluted nucleic acids with real time PCR.

As shown in Figure 5, nucleic acids from Bacillus anthracis are detected in the eluant.

Example 4: Purification of nucleic acids from Blood sample containing Venezuela Equine Encephalitis virus using 1.2 ml Gilson filtered pipette tip and S mm glass frit In this experiment, nucleic acids were purified from blood sample containing

Venezuela Equine Encephalitis virus using a 2.0 ml Rainin filtered pipette tip and a 5 mm glass frit (ROBU GlasilUer-Geraete GmbH, Germany) with the following protocol: 1. Label six 1.5 ml centrifuge tubes as: Flow Through, Ethanol L Ethanol 2,

Ethanol 3. Eluant 1 and Eluant 2. 2. Mix 90 μl of blood with 10 μl of Venezuela Equine Encephalitis virus ( 10" plaque forming unit (pfu) / ml in water) in the Flow Through tube (final concentration 10 ⁴ pfu/ml).

3. Add 280 μl of Qiagen AL Lysis Buffer to the mixture.

4. Add 40 μl of Proteinase K (20mg/ml). 5. Add 100 μl of lysozyme, vortex and spin down.

6. Incubate the sample mixture at 55°C for I hour.

7. Add 500 μl of 96-100% ethanol to the Flow Through Tube. Vortex and pulse spin down the mixture.

8. Aliquot 100 μl of elution buffer ( 10 mM Tris, pH 8.0} into the Eluant tubes. Place the tubes on the heat block set at 70X. (Elution buffer must be heated at 70°C for at least 5 minutes. Keep the tubes on the heat block until step 13.)

9. Aliquot 1 ml of 70% Ethanol into each of the three Ethanol tubes.

10. Pipette sample mixture into the Flow Through tube using frit tip (medium porosity) with a Gilson Electronic Pipettor. Pipette for 5 cycles (cycle - aspirate t dispense). Wash the bound nucleic acids by pipetting the 70% EtOH in Ethanol

1 tube for 10 cycles using the electronic Pipettor. Repeat the wash with the 70% EtOM in Ethanol 2 lube and Ethanol 3 tube (three washes total) Purge the EiOH from the frit tip by pipetting air for 20 cycles. Wipe the outside of the tip and tap the tip gently if a noticeable amount of ethanol is left.

11. Elute the nucleic acids on the frit by pipetting the 70 ⁰ C elution buffer of step 8 for IO cycles. Remove the elution buffer from the heat block once the cycles are completed. Make sure all the buffer has been purged from the tip.

12. Collect the elυant in Etuant I tube. 13. Repeat lhe step 13 with the same frit tip, collect the eluatU in Biuant 2 tube, and discard the frit tip 14, Quantitating the eluted nucleic acids with real time PCR.

As shown in Figure 6, nucleic acids from Venezuela Equine Encephalitis vims are detected in both the first and second eluant. The first eluant, however, contains nucleic acids of Venezuela Equine Encephalitis at a much higher concentration.

Example 5: Automatic sample preparation using flυidic control system and the integrated sample preparation system

A prototype of the integrated sample preparation device shown in Figure 2A was connected to a Flow Pro Fluidic Handling System (Global FIA, Fox Island, VVA). Nucleic acids were purified from Yersinia pestis suspension with the following protocol:

1. Label two 1.5 ml centrifuge tubes as: Sample and Eluant.

2. Aliquot 150 μi of 1 mM NaOH into a tube designated "Elution Buffer" which is located on the Global FIA system.

3. Aliquot 500 μl 70% EtOH into a Wash tube which is located on the Global FIA system.

4. Mix the 500 μl of Yersinia pestis suspension (10 ⁴ CfWmI in water) with 500 μl of 6M guanidine.. pH 6.5, in the "sample" tube (step 1) and vortex. Save some un-mixed sample for analysis later.

5. Pipette the sample/guanidine mixture into frit tip (medium porosity) with a Rainin Electronic Pipettor.

6. Place the frit tip (with sample inside) onto the Sample Prep Cartridge located on the Global FIA device.

7. Perform the "Frit Tip Sample Toggle ^** sequence (Figure 7) using the FIoLV Software. 8. Perform the "Frit Tip EtOH Wash" sequence using the FIoLV Software.

9, Perform the "Frit Tip EtOH Dry" sequence using the FIoLV Software.

10. Perform the "FrU Tip Elutiorf sequence using the FIoLV Software.

11. Once the sequence is completed, remove the frit tip from the Global FtA system, attach the frit tip to a Rainin Electronic Pipetlor and dispense the eluant into the 1.5 ml centrifuge tube labeled 'εluanf . 1 1. Discard the frit tip 12. Quantitating the eluted nucleic acids with real time PCR.

As shown in Figure 8, nucleic acids from Yersinia jxtstis are detected in the eluant.

The terms and descriptions used herein are set forth by way of illustration only and are not meant as limitations. Those skilled in the art will recognize that many variations are possible within the spirit and scope of the invention as defined in the following claims, and their equivalents, in which all terms are to be understood in their broadest possible sense unless otherwise indicated.