US20040200909A1 - Apparatus and method for cell disruption - Google Patents

Abstract

An apparatus for disrupting cells or viruses comprises a container having a chamber for holding the cells or viruses. The container includes at least one flexible wall defining the chamber. The apparatus also includes a transducer for impacting an external surface of the flexible wall to generate pressure waves in the chamber. The apparatus also includes a pressure source for increasing the pressure in the chamber. The pressurization of the chamber ensures effective coupling between the transducer and the flexible wall. The apparatus may also include beads in the chamber for rupturing the cells or viruses.

Description

RELATED APPLICATION INFORMATION
• This application claims priority from provisional application Ser. No. 60/136,703 filed May 28, 1999. This application is also a continuation in part of U.S. application Ser. No. 09/469,724 filed Dec. 21, 1999. These applications are incorporated by reference herein for all purposes.[0001]
FIELD OF THE INVENTION
• The present invention relates to an apparatus and method for rapidly disrupting cells or viruses.[0002]
BACKGROUND OF THE INVENTION
• The extraction of nucleic acid from cells or viruses is a necessary task for many applications in the fields of molecular biology and biomedical diagnostics. Once released from the cells, the nucleic acid may be used for genetic analysis, e.g., sequencing, pathogen identification and quantification, nucleic acid mutation analysis, genome analysis, gene expression studies, pharmacological monitoring, storing of DNA libraries for drug discovery, etc. The genetic analysis typically involves nucleic acid amplification and detection using known techniques. For example, known polynucleotide amplification reactions include polymerase chain reaction (PCR), ligase chain reaction (LCR), QB replicase amplification (QBR), self-sustained sequence replication (3SR), strand-displacement amplification (SDA), “branched chain” DNA amplification, ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), and cycling probe reaction (CPR).[0003]
• The extraction of nucleic acids from cells or viruses is generally performed by physical or chemical methods. Chemical methods typically employ lysing agents (e.g., detergents, enzymes, or strong organics) to disrupt the cells and release the nucleic acid, followed by treatment of the extract with chaotropic salts to denature any contaminating or potentially interfering proteins. Such chemical methods are described in U.S. Pat. No. 5,652,141 to Henco et al. and U.S. Pat. No. 5,856,174 to Lipshutz et al. One disadvantage to the use of harsh chemicals for disrupting cells is that the chemicals are inhibitory to subsequent amplification of the nucleic acid. In using chemical disruption methods, therefore, it is typically necessary to purify the nucleic acid released from the cells before proceeding with further analysis. Such purification steps are time consuming, expensive, and reduce the amount of nucleic acid recovered for analysis.[0004]
• Physical methods for disrupting cells often do not require harsh chemicals that are inhibitory to nucleic acid amplification (e.g., PCR). These physical methods, however, also have their disadvantages. For example, one physical method for disrupting cells involves placing the cells in a solution and heating the solution to a boil to break open the cell walls. Unfortunately, the heat will often denature proteins and cause the proteins to stick to the released nucleic acid. The proteins then interfere with subsequent attempts to amplify the nucleic acid. Another physical method is freeze thawing in which the cells are repeatedly frozen and thawed until the cells walls are broken. Unfortunately, freeze thawing often fails to break open many structures, most notably certain spores and viruses that have extremely tough outer layers.[0005]
• Another physical method for disrupting cells is the use of a pressure instrument. With this method, a solution of mycobacterial microorganisms is passed through a very small diameter hole under high pressure. During passage through the hole, the mycobacteria are broken open by the mechanical forces and their internal contents are spilled into solution. Such a system, however, is large, expensive and requires a cooling system to prevent excessive heat from building up and damaging the contents of the lysed cells. Moreover, the instrument needs to be cleaned and decontaminated between runs and a large containment system is required when infectious material is handled. A further disadvantage to this system is that the solution must contain only particles having substantially the same size, so that it may not be used to process many untreated clinical or biological specimens.[0006]
• It is also known that cells can be lysed by subjecting the cells to ultrasonic agitation. Typically, the cells are disrupted by placing an ultrasonic probe directly into a volume of liquid containing the cells. Since the probe is in direct contact with a sample liquid, cross contamination and cavitation-induced foaming present serious complications.[0007]
• Another method for cell disruption is disclosed by Murphy et al. in U.S. Pat. No. 5,374,522. According to the method, solutions or suspensions of cells are placed in a container with small beads. The container is then placed in an ultrasound bath until the cells disrupt, releasing their cellular components. This method has several disadvantages. First, the distribution of ultrasonic energy in the bath is not uniform, so that a technician must locate a high energy area within the bath and place the container into that area. The non-uniform distribution of ultrasonic energy also produces inconsistent results. Second, the ultrasound bath does not focus energy into the container so that the disruption of the cells often takes several minutes to complete, a relatively long period of time when compared to the method of the present invention. Third, it is not practical to carry an ultrasound bath into the field for use in biowarfare detection, forensic analysis, or on-site testing of environmental samples.[0008]
SUMMARY
• The present invention overcomes the disadvantages of the prior art by providing an improved apparatus and method for disrupting cells or viruses. In contrast to the prior art methods described above, the present invention provides for the rapid and effective disruption of cells or viruses, including tough spores, without requiring the use of harsh chemicals. The disruption of the cells or viruses can often be completed in 5 to 10 seconds. In addition, the apparatus and method of the present invention provide for highly consistent and repeatable lysis of cells or viruses, so that consistent results are achieved from one use of the apparatus to the next.[0009]
• According to a first embodiment, the apparatus comprises a container having a chamber for holding the cells or viruses. The container includes at least one flexible wall defining the chamber. The apparatus also includes a transducer, such as an ultrasonic horn, for impacting an external surface of the flexible wall to generate dynamic pressure pulses or pressure waves in the chamber. The apparatus also includes a pressure source for increasing the pressure in the chamber. The pressurization of the chamber ensures effective coupling between the transducer and the flexible wall. The apparatus may also include beads in the chamber for rupturing the cells or viruses.[0010]
• In operation, the cells or viruses to be disrupted are placed in the chamber of the container. A liquid is also placed in the chamber. In one embodiment, the cells or viruses are placed in the chamber by capturing the cells or viruses on at least one filter positioned in the chamber. In this embodiment, the liquid placed in the chamber is usually a lysis buffer added to the chamber after the cells or viruses have been captured. In an alternative embodiment, the liquid placed in the chamber contains the cells or viruses to be disrupted (e.g., the liquid is a sample containing the cells or viruses) so that the liquid and cells are placed in the chamber simultaneously. In either embodiment, the transducer is placed against the external surface of the flexible wall, and the static pressure in the chamber is increased. Disruption of the cells is accomplished by impacting the flexible wall with the transducer to generate dynamic pressure pulses or pressure waves in the chamber. Beads may also be agitated in the chamber to rupture the cells or viruses.[0011]
• A greater understanding of the invention may be gained by considering the following detailed description and the accompanying drawings.[0012]
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1 is an isometric view of a cartridge for analyzing a fluid sample according to a first embodiment of the invention.[0013]
• FIG. 2 is a lower isometric view of the cartridge of FIG. 1.[0014]
• FIG. 3 is an exploded view of the cartridge of FIG. 1.[0015]
• FIG. 4 is another exploded view of the cartridge of FIG. 1.[0016]
• FIG. 5 is a partially cut away view of an ultrasonic horn coupled to a wall of a lysing chamber formed in the cartridge of FIG. 1.[0017]
• FIG. 6 is an exploded view of a filter stack positioned in the lysing chamber of the cartridge of FIG. 1.[0018]
• FIG. 7 is a top plan view of the cartridge of FIG. 1.[0019]
• FIG. 8 is a bottom plan view of the cartridge of FIG. 1.[0020]
• FIG. 9 is a schematic block diagram of the cartridge of FIG. 1.[0021]
• FIG. 10 is an isometric view of an instrument into which the cartridge of FIG. 1 is placed for processing.[0022]
• FIG. 11 is an isometric view of the cartridge of FIG. 1 in the instrument of FIG. 10.[0023]
• FIG. 12 is a partially cut-away view of the cartridge of FIG. 1 in the instrument of FIG. 10.[0024]
• FIG. 13 is a schematic, plan view of optical sensors positioned to detect liquid levels in the cartridge of FIG. 1.[0025]
• FIG. 14 is a partially cut away, schematic, side view of a slotted optical sensor positioned to detect the liquid level in a sensor chamber of the cartridge of FIG. 1.[0026]
• FIG. 15A is a cross-sectional view of a portion of the body of the cartridge of FIG. 1 illustrating two different types of valves in the cartridge.[0027]
• FIG. 15B is a cross-sectional view of the valves of FIG. 15A in a closed position.[0028]
• FIG. 16A is another cross-sectional view of one of the valves of FIG. 15A in an open position.[0029]
• FIG. 16B is a cross-sectional view of the valve of FIG. 16A in a closed position.[0030]
• FIGS. 17-19 illustrate a valve actuation system for opening and closing the valves of FIG. 15A.[0031]
• FIG. 20 is a cross sectional view of alternative valve actuators for opening and closing the valves in the cartridge of FIG. 1. FIG. 20 also shows a pressure delivery nozzle sealed to a pressure port formed in the cartridge of FIG. 1.[0032]
• FIG. 21 is a partially exploded, isometric view of a reaction vessel of the cartridge of FIG. 1.[0033]
• FIG. 22 is a front view of the vessel of FIG. 21.[0034]
• FIG. 23 is a side view of the vessel of FIG. 21 inserted between two heater plates.[0035]
• FIG. 24 is a front view of one of the heater plates of FIG. 23.[0036]
• FIG. 25 is a front view of an alternative reaction vessel according to the present invention.[0037]
• FIG. 26 is a front view of another reaction vessel according to the present invention.[0038]
• FIG. 27 is another front view of the vessel of FIG. 21.[0039]
• FIG. 28 is a front view of the vessel of FIG. 21 inserted into a heat-exchanging module of the instrument of FIG. 10.[0040]
• FIG. 29 is an exploded view of a support structure for holding the plates of FIG. 23.[0041]
• FIGS. 30-31 are assembled views of the support structure of FIG. 29.[0042]
• FIG. 32 is an isometric view showing the exterior of one the optics assemblies in the heat-exchanging module of FIG. 28.[0043]
• FIG. 33 is an isometric view of the plates of FIG. 23 in contact with the optics assembly of FIG. 32.[0044]
• FIG. 34 is a partially cut away, isometric view of the reaction vessel of FIG. 21 inserted between the plates of FIG. 23. Only the lower portion of the vessel is included in the figure.[0045]
• FIG. 35 is a schematic block diagram of the electronics of the heat-exchanging module of FIG. 28.[0046]
• FIG. 36 is an isometric view of an apparatus for disrupting cells or viruses according to another embodiment of the invention.[0047]
• FIG. 37 is a cross sectional view of the apparatus of FIG. 36.[0048]
• FIG. 38 is an exploded view of a container used in the apparatus of FIG. 36.[0049]
• FIG. 39 is a cross sectional view of the container of FIG. 38.[0050]
• FIG. 40 is a schematic block diagram of a fluidic system incorporating the apparatus of FIG. 36.[0051]
• FIG. 41 is a cross sectional view of another container for use in the apparatus of FIG. 36. An ultrasonic horn is in contact with a wall of the container that curves outwardly towards the horn.[0052]
• FIG. 42 is a cross-sectional view of the wall of FIG. 41.[0053]
• FIGS. 43A-43B are isometric views of opposite sides of another wall suitable for use in a container for holding cells or viruses to be disrupted.[0054]
• FIG. 44 is a partially cut-away, isometric view of a container incorporating the wall of FIGS. 43A-43B.[0055]
• FIG. 45 is a bottom plan view of the container of FIG. 44.[0056]
• FIG. 46 is a partially exploded, isometric view of a container for holding cells or viruses to be disrupted according to another embodiment of the invention.[0057]
• FIG. 47 is a front view of the container of FIG. 46.[0058]
• FIG. 48 is another schematic, front view of the container of FIG. 46.[0059]
• FIG. 49 is a side view of the container of FIG. 46.[0060]
• FIG. 50 is a view of a pipette inserted into the container of FIG. 46. The container is holding beads for rupturing cells or viruses.[0061]
• FIG. 51 is an isometric view of the container of FIG. 46 inserted into an apparatus for disrupting cells or viruses.[0062]
• FIG. 52 is a different isometric view of the container of FIG. 46 inserted into the apparatus of FIG. 51.[0063]
• FIG. 53 is a partially cut-away, isometric view of the apparatus of FIG. 51.[0064]
• FIG. 54 is an isometric view of a holder for holding the container of FIG. 46.[0065]
• FIG. 55 is another isometric view of the apparatus of FIG. 51 in which several parts of the apparatus have been removed to show an ultrasonic horn contacting the container of FIG. 46.[0066]
• FIG. 56 is a schematic side view of the container of FIG. 46 inserted into the apparatus of FIG. 51 for disruption of the cells or viruses contained in the container.[0067]
DETAILED DESCRIPTION
• The present invention provides an apparatus and method for analyzing a fluid sample. In a first embodiment, the invention provides a cartridge for separating a desired analyte from a fluid sample and for holding the analyte for a chemical reaction. The-fluid sample may be a solution or suspension. In a particular use, the sample may be a bodily fluid (e.g., blood, urine, saliva, sputum, seminal fluid, spinal fluid, mucus, or other bodily fluids). Alternatively, the sample may be a solid made soluble or suspended in a liquid or the sample may be an environmental sample such as ground or waste water, soil extracts, pesticide residues, or airborne spores placed in a fluid. Further, the sample may be mixed with one or more chemicals, reagents, diluents, or buffers. The sample may be pretreated, for example, mixed with chemicals, centrifuged, pelleted, etc., or the sample may be in a raw form.[0068]
• The desired analyte is typically intracellular material (e.g., nucleic acid, proteins, carbohydrates, lipids, bacteria, or intracellular parasites). In a preferred use, the analyte is nucleic acid which the cartridge separates from the fluid sample and holds for amplification (e.g., using PCR) and optical detection. As used herein, the term “nucleic acid” refers to any synthetic or naturally occurring nucleic acid, such as DNA or RNA, in any possible configuration, i.e., in the form of double-stranded nucleic acid, single-stranded nucleic acid, or any combination thereof.[0069]
• FIG. 1 shows an isometric view of a[0070]cartridge20 according to the preferred embodiment. Thecartridge20 is designed to separate nucleic acid from a fluid sample and to hold the nucleic acid for amplification and detection. Thecartridge20 has a body comprising atop piece22, amiddle piece24, and abottom piece26. An inlet port for introducing a fluid sample into the cartridge is formed in thetop piece22 and sealed by acap30. Sixpressure ports32 are also formed in thetop piece22. Thepressure ports32 are for receiving nozzles from pressure sources, e.g., pumps or vacuums. The cartridge also includesalignment legs28 extending from thebottom piece26 for positioning thecartridge20 in an instrument (described below with reference to FIG. 10). Indentations ordepressions38A,38B, and38C are formed in the top andmiddle pieces22,24. The indentations are for receiving optical sensors that detect fluid flow in thecartridge20. Thecartridge20 further includesvents34,36. Each pressure port and vent preferably includes a hydrophobic membrane that allows the passage of gas but not liquid into or out of the vents and pressure ports. Modified acrylic copolymer membranes are commercially available from, e.g., Gelman Sciences (Ann Arbor, Mich.) and particle-track etched polycarbonate membranes are available from Poretics, Inc. (Livermore, Calif.).
• FIG. 2 is an isometric view showing the underside of the[0071]cartridge20. Nineholes60 are formed in thebottom piece26 for receiving valve actuators that open and close valves in thecartridge20. Ahole62 is also formed in thebottom piece26 for receiving a transducer (described in detail below with reference to FIG. 5). Thecartridge20 also includes areaction vessel40 extending outwardly from the body of the cartridge. Thevessel40 has areaction chamber42 for holding a reaction mixture (e.g., nucleic acid mixed with amplification reagents and fluorescent probes) for chemical reaction and optical detection. One of the flow paths in the cartridge carries the reaction mixture to thechamber42 for chemical reaction and optical detection. Thevessel40 extends outwardly from the body of thecartridge20 so that thevessel40 may be inserted between a pair of opposing thermal plates (for heating and cooling the chamber42) without the need for decoupling thevessel40 from the rest of thecartridge20. This greatly reduces the risk of contamination and/or spilling. Thevessel40 may be integrally formed with the body of the cartridge (e.g., integrally molded with middle piece24). It is presently preferred, however, to produce thevessel40 as a separate element that is coupled to the body during manufacture of the cartridge.
• FIGS. 3-4 show exploded views of the cartridge. As shown in FIG. 3, the[0072]middle piece24 has multiple chambers formed therein. In particular, themiddle piece24 includes asample chamber65 for holding a fluid sample introduced through theinlet port64, awash chamber66 for holding a wash solution, areagent chamber67 for holding a lysing reagent, awaste chamber68 for receiving used sample and wash solution, aneutralizer chamber70 for holding a neutralizer, and amaster mix chamber71 for holding a master mix (e.g., amplification reagents and fluorescent probes) and for mixing the reagents and probes with analyte separated from the fluid sample. Thesample chamber65 optionally includes aside compartment155 having slightly lower walls than thesample chamber65. Theside compartment155 is for visually indicating to a user when sufficient sample has been added to thesample chamber65, i.e., when the liquid level in thechamber65 is high enough to spill over into thecompartment155.
• The[0073]top piece22 includes thevents34,36 and the sixpressure ports32, as previously described. An elastomeric membrane orgasket61 is positioned and squeezed between thepieces22,24 to seal the various channels and chambers formed in the pieces. Themiddle piece24 preferably includes multiple sealing lips to ensure that thegasket61 forms an adequate seal. In particular, themiddle piece24 preferably includes sealinglips73 surrounding each of thechambers65,66,67,68.70, and71. Themiddle piece24 also includessupport walls75 around the perimeter, andintermediate sealing lips76. The sealinglips73,76 andsupport walls75 locally compress thegasket61 and achieve a seal.
• As shown in FIG. 4, the[0074]middle piece24 has formed in its underside various channels, one of which leads to a lysingchamber86. Thechamber86 is aligned with thehole62 in thebottom piece26 so that a transducer (e.g., an ultrasonic horn) may be inserted through thehole62 to generate dynamic pressure pulses or pressure waves in the lysingchamber86. Themiddle piece24 also has ninevalve seats84 formed in its bottom surface. The valve seats84 are aligned with the nineholes60 in thebottom piece26 so that valve actuators may be inserted through theholes60 into the valve seats84.
• An elastomeric membrane or[0075]gasket61 is positioned and squeezed between thepieces24,26 to seal the various channels, valve seats, and chamber formed in themiddle piece24. Themiddle piece24 preferably includes multiple sealing lips to ensure that thegasket63 forms an adequate seal. In particular, themiddle piece24 preferably includes sealinglips73 surrounding the lysingchamber86, valve seats84, and various channels. Themiddle piece24 also includessupport walls75 around its perimeter, andintermediate sealing lips76. The sealinglips73,76 andsupport walls75 locally compress thegasket63 and achieve a seal. In addition to sealing various channels and chambers, thegasket63 also functions as a valve stem by compressing, when actuated through one of theholes60, into acorresponding valve seat84, thus shutting one of the flow channels in themiddle piece24. This valve action is discussed in greater detail below with reference to FIGS. 15-16.
• The[0076]gasket63 also forms the bottom wall of the lysingchamber86 against which a transducer is placed to effect disruption of cells or viruses in thechamber86. Each of thegaskets61,63 is preferably composed of an elastomer. Suitable gasket materials are silicone rubber, neoprene, EPDM, or any other compliant material. Each of thegaskets61,63 preferably has a thickness in the range of 0.005 to 0.125 inches (0.125 to 3.175 mm), and more preferably in the range of 0.01 to 0.06 inches (0.25 to 1.5 mm), with a presently preferred thickness of 0.031 inches (0.79 mm). The thickness is selected to ensure that the gasket is sufficiently compliant to seal the channels and chambers, to compress into the valve seats84 when forced, and to expand under pressure to contact the transducer.
• As shown in FIG. 3, the[0077]middle piece24 includes aslot79 through which thereaction vessel40 is inserted during assembly of the cartridge. Thevessel40 has twofluid ports41,43 for adding and removing fluid from the vessel. When thetop piece22 is sealed to themiddle piece24 via thegasket61, theports41,43 are placed into fluidic communication withchannels80,81, respectively, that are formed in the top piece22 (see FIG. 4). Thegasket61 seals the respective fluidic interfaces between theports41,43 and thechannels80,81. The top, middle, andbottom pieces22,24,26 are preferably injection molded parts made of a polymeric material such as polypropylene, polycarbonate, or acrylic. Although molding is preferred for mass production, it also possible to machine the top, middle, andbottom pieces22,24,26. Thepieces22,24,26 may be held together by screws or fasteners. Alternatively, ultrasonic bonding, solvent bonding, or snap fit designs could be used to assemble the cartridge.
• FIG. 4 also shows a[0078]filter ring88. Thefilter ring88 compresses and holds a stack of filters in the lysingchamber86. FIG. 6 shows an exploded view of afilter stack87. The purpose of thefilter stack87 is to capture cells or viruses from a fluid sample as the sample flows through the lysingchamber86. The captured cells or viruses are then disrupted (lysed) in thechamber86. The cells may be animal or plant cells, spores, bacteria, or microorganisms. The viruses may be any type of infective agents having a protein coat surrounding an RNA or DNA core.
• The[0079]filter stack87 comprises agasket93, afirst filter94, agasket95, asecond filter97 having a smaller pore size than thefirst filter94, agasket98, athird filter100 having a smaller pore size than thesecond filter97, agasket101, awoven mesh102, and agasket103. The filter stack also preferably includes a first set ofbeads96 disposed between the first andsecond filters94 and97 and a second set ofbeads99 disposed between the second andthird filters97 and100. Thefilter ring88 compresses thefilter stack87 into the lysingchamber86 so that thegasket93 is pressed against thefilter94, thefilter94 is pressed against thegasket95, thegasket95 is pressed against thefilter97, thefilter97 is pressed against thegasket98, thegasket98 is pressed against thefilter100, thefilter100 is pressed against thegasket101, thegasket101 is pressed against themesh102, themesh102 is pressed against thegasket103, and thegasket103 is pressed against the outer perimeter of the bottom wall of the lysingchamber86. Thegasket95 is thicker than the average diameter of thebeads96 so that the beads are free to move in the space between thefilters94 and97. Similarly, thegasket98 is thicker than the average diameter of thebeads99 so that thebeads99 are free to move in the space between thefilters97 and100. A fluid sample flowing through thechannel106 into the lysingchamber86 first flows throughfilter94, then throughfilter97, next throughfilter100, and lastly through themesh102. After flowing through thefilter stack87, the sample flows along flow ribs.91 formed in the top of the lysingchamber86 and through an outlet channel (not shown in FIG. 6).
• Referring to FIG. 5, the cells or viruses captured in the filter stack (not shown in FIG. 5 for illustrative clarity) are lysed by coupling a transducer[0080]92 (e.g., an ultrasonic horn) directly to the wall of the lysingchamber86. In this embodiment, the wall of the lysingchamber86 is formed by theflexible gasket63. Thetransducer92 should directly contact an external surface of the wall. The term “external surface” is intended to mean a surface of the wall that is external to the lysingchamber86. Thetransducer92 is a vibrating or oscillating device that is activated to generate dynamic pressure pulses or pressure waves in thechamber86. The pressure waves agitate thebeads96,99 (FIG. 6), and the movement of the beads ruptures the captured cells or viruses. In general, the transducer for contacting the wall of the lysingchamber86 may be an ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducer. The transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device. It is presently preferred that the actuator be an ultrasonic transducer, such as an ultrasonic horn. Suitable horns are commercially available from Sonics & Materials, Inc. having an office at 53 Church Hill, Newton., Conn. 06470-1614 USA. Alternatively, the ultrasonic transducer may comprise a piezoelectric disk or any other type of ultrasonic transducer that may be coupled to the container. It is presently preferred to use an ultrasonic horn because the horn structure is highly resonant and provides for repeatable and sharp frequency of excitation and large motion of the horn tip.
• As previously described in FIG. 6, the filter stack includes a gasket at both of its ends. As shown in FIG. 5, the[0081]middle cartridge piece24 has a sealinglip90 against which the gasket at one end of the filter stack is compressed. The gasket at the other end of the filter stack is compressed by thefilter ring88 to form a seal. The gasket material may expand into the relief area outside of the sealinglip90. The width of the sealinglip90 is small (typically 0.5 mm) so that an excessive amount of force is not required to achieve a sufficient seal.
• The[0082]filter ring88 is held between the filter stack and thecartridge gasket63. Thecartridge gasket63 is held between themiddle piece24 and thebottom piece26 by a sealinglip406. Force is therefore transferred from thebottom piece26 through thegasket63 to thefilter ring88 and finally to the filter stack. Thefilter ring88 contains acontact lip404 that contacts thegasket63. Thecontact lip404 is not a primary sealing lip (though it will seal) but a force transfer mechanism. The width of thecontact lip404 is larger than the width of the sealinglip90 to ensure that deformation and sealing action occurs in the filter stack and not taken up in squeezing thecartridge gasket63. Thecartridge middle piece24 also has a sealinglip406 that surrounds thefilter ring88. This is an active sealing area that should not be compromised by the presence of thefilter ring88. For this reason, there is agap407 between the sealinglip406 and thecontact lip404 on thefilter ring88. Thegap407 is provided to allow thegasket63 to extrude into thegap407 as it is compressed by the sealinglip406 and thecontact lip404. If thecontact lip404 comes to a different elevation than the sealinglip406, the seal will not be compromised because of thegap407 and the distance between thelips404 and406.
• Referring again to FIG. 6, the[0083]filter stack87 is effective for capturing cells or viruses as a fluid sample flows through thestack87 without clogging of any of thefilters94,97,100 in the stack. The first filter94 (having the largest pore size) filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc. The second filter97 (having the medium pore size) captures cells or viruses in the fluid sample. The third filter100 (having the smallest pore size) captures smaller cells or viruses in the sample. Thefilter stack87 thus enables the simultaneous capture of differently sized sample components without clogging of the filters. The average pore size of thefirst filter94 is selected to be small enough to filter coarse material from the fluid sample (e.g., salt crystals, cellular debris, hair, tissue) yet large enough to allow the passage of the target cells or viruses containing the desired analyte (e.g., nucleic acid or proteins). In general, the pore size of thefirst filter94 should be in the range of about 2 to 25 μm, with a presently preferred pore size of about 5 μm.
• The average pore sizes of the second and third filters are selected in dependence upon the average size of the target cells or viruses that contain the desired analyte(s). For example, in one embodiment, the[0084]filter stack87 is used to capture gonorrhea (GC) and chlamydia (Ct) organisms to determine the presence of the diseases in the fluid sample. The GC and Ct organisms have different average diameters, about 1 to 2 μm for GC organisms and about 0.3 μm for Ct organisms. In this embodiment, thesecond filter97 has an average pore size of about 1.2 μm while thethird filter100 has an average pore size of about 0.22 μm so that most of the GC organisms are captured by thesecond filter97 while most of the Ct organisms are captured by thethird filter100. The filter stack thus enables the simultaneous capture of differently sized target organisms and does so without clogging of the filters. The pore sizes of thefilters97,100 may be selected to capture desired cells or viruses of any size, and the scope of the invention is not limited to the specific example given.
• The[0085]filter stack87 is also useful for disrupting the captured cells or viruses to release the intracellular material (e.g., nucleic acid) therefrom. The first and second sets ofbeads96,99 serve two useful purposes in this regard. First, the beads are agitated by dynamic pressure pulses or pressure waves generated by the transducer. The movement of the beads ruptures the captured cells or viruses. Second, the beads may shear the nucleic acid released from the lysed cells or viruses so that the strands of nucleic acid are sufficiently short to flow through the filters and out of the lysingchamber86. Suitable beads for rupturing cells or viruses include borosilicate glass, lime glass, silica, and polystyrene beads.
• The beads may be porous or non-porous and preferably have an average diameter in the range of 1 to 200 μm. The average diameter of the[0086]beads96,99 is selected in dependence upon the intended target cells or viruses to be ruptured by the beads. The average diameter of thebeads96 in the first set may be equal to the average diameter of thebeads99 in the second set. Alternatively, when the first set ofbeads96 is used to rupture a type of target cell or virus that differs from the type of cell or virus to be ruptured by the second set ofbeads99, it is advantageous to select the average diameter of the beads such that the average diameter of thebeads96 in the first set differs from the average diameter of thebeads99 in the second set. For example, when the filter stack is used to capture GC and Ct cells as described above, thebeads96 are 20 μm diameter borosilicate glass beads for rupturing the GC organisms and thebeads99 are 106 μm diameter soda lime glass beads for rupturing the Ct organisms. Each of thesilicone gaskets95,98 should be sufficiently thick to allow room for thebeads96,99 to move and rupture the cells or viruses.
• The[0087]mesh102 also serves two useful purposes. First the mesh provides support to thefilter stack87. Second, the mesh breaks up air bubbles so that the bubbles can be channeled through theflow ribs91 and out of the lysingchamber86. To effectively break up or reduce the size of the air bubbles, themesh102 preferably has a small pore size. Preferably, it is a woven polypropylene mesh having an average pore size of about 25 μm. To ensure that the air bubbles can escape from the lysingchamber86, it is desirable to use the cartridge in an orientation in which liquid flows up (relative to gravity) through thefilter stack87 and the lysingchamber86. The upward flow through thechamber86 aids the flow of air bubbles out of thechamber86. Thus, the inlet port for entry of fluids into thechamber86 should generally be at the lowest point in the chamber, while the exit port should be at the highest.
• Many different embodiments of the filter stack are possible. For example, in one alternative embodiment, the filter stack has only two filters and one set of beads disposed between the filters. The first filter has the largest pore size (e.g., 5 μm) and filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc. The second filter has a pore size smaller than the first filter and slightly smaller than the target cells or viruses to be captured. Such a filter stack is described below with reference to FIG. 38. In another embodiment of the cartridge, the filter having the largest pore size (for filtering the coarse material) is positioned in a filter chamber (not shown) that is positioned upstream of the lysing[0088]chamber86. A channel connects to the filter chamber to the lysingchamber86. In this embodiment, a fluid sample flows first through the coarse filter in the filter chamber and then through a second filter in the lysing chamber to trap the target cells or viruses in the lysing chamber.
• Further, the beads in the filter stack may have a binding affinity for target cells or viruses in the fluid sample to facilitate capture of the target cells or viruses. For example, antibodies or certain receptors may be coated onto the surface of the beads to bind target cells in the sample. Moreover, the lysing[0089]chamber86 may contain two different types of beads for interacting with target cells or viruses. For example, the lysing chamber may contain a first set of beads coated with antibodies or receptors for binding target cells or viruses and a second set of beads (intermixed with the first set) for rupturing the captured cells or viruses. The beads in the lysingchamber86 may also have a binding affinity for the intracellular material (e.g., nucleic acid) released from the ruptured cells or viruses. Such beads are useful for isolating target nucleic acid for subsequent elution and analysis. For example, the lysing chamber may contain silica beads to isolate DNA or cellulose beads with oligo dT to isolate messenger RNA for RT-PCR. The lysingchamber86 may also contain beads for removing unwanted material (e.g., proteins, peptides) or chemicals (e.g., salts, metal ions, or-detergents) from the sample that might inhibit PCR. For example, thechamber86 may contain ion exchange beads for removing proteins. Alternatively beads having metal ion chelators such as iminodiacetic acid will remove metal ions from biological samples.
• FIGS. 21-22 illustrate the[0090]reaction vessel40 in greater detail. FIG. 21 shows a partially exploded view of thevessel40, and FIG. 22 shows a front view of thevessel40. Thevessel40 includes the reaction chamber42 (diamond-shaped in this embodiment) for holding a reaction mixture. Thevessel40 is designed for optimal heat transfer to and from the reaction mixture and for efficient optical viewing of the mixture. The thin shape of the vessel contributes to optimal thermal kinetics by providing large surfaces for thermal conduction and for contacting thermal plates. In addition, the walls of the vessel provide optical windows into thechamber42 so that the entire reaction mixture can be optically interrogated. In more detail to FIGS. 21-22, thereaction vessel40 includes arigid frame46 that defines theside walls57A,57B,59A,59B of thereaction chamber42. Theframe46 also defines aninlet port41 and achannel50 connecting theport41 to thechamber42. Theframe46 also defines anoutlet port43 and achannel52 connecting theport43 to thechamber42. Theinlet port41 andchannel50 are used to add fluid to thechamber42, and thechannel52 andoutlet port43 are used for exit of fluid from thechamber42. Alignment prongs44A,44B are used to position thevessel40 correctly during assembly of the cartridge.
• As shown in FIG. 21, the[0091]vessel40 also includes thin, flexible sheets attached to opposite sides of therigid frame46 to form opposingmajor walls48 of the chamber. (Themajor walls48 are shown in FIG. 1 exploded from therigid frame46 for illustrative clarity). Thereaction chamber42 is thus defined by therigid side walls57A,57B,59A,59B of theframe46 and by the opposing major walls.48. The opposingmajor walls48 are sealed to opposite sides of theframe46 such that theside walls57A,57B,59A,59B connect themajor walls48 to each other. Thewalls48 facilitate optimal thermal conductance to the reaction mixture contained in thechamber42. Each of thewalls48 is sufficiently flexible to contact and conform to a respective thermal surface, thus providing for optimal thermal contact and heat transfer between the thermal surface and the reaction mixture contained in thechamber42. Furthermore, theflexible walls48 continue to conform to the thermal surfaces if the shape of the surfaces changes due to thermal expansion or contraction during the course of the heat-exchanging operation.
• As shown in FIG. 23, the thermal surfaces for contacting the[0092]flexible walls48 are preferably formed by a pair of opposingplates190A,190B positioned to receive thechamber42 between them. When thechamber42 of thevessel40 is inserted between theplates190A,190B, the inner surfaces of the plates contact thewalls48 and the flexible walls conform to the surfaces of the plates. The plates are preferably spaced a distance from each other equal to the thickness T of thechamber42 as defined by the thickness of theframe46. In this position, minimal or no gaps are found between the plate surfaces and thewalls48. The plates may be heated and cooled by various thermal elements to induce temperature changes within thechamber42, as is described in greater detail below.
• The[0093]walls48 are preferably flexible films of polymeric material such as polypropylene, polyethylene, polyester, or other polymers. The films may either be layered, e.g., laminates, or the films may be homogeneous. Layered films are preferred because they generally have better strength and structural integrity than homogeneous films. In particular, layered polypropylene films are presently preferred because polypropylene is not inhibitory to PCR. Alternatively, thewalls48 may comprise any other material that may be formed into a thin, flexible sheet and that permits rapid heat transfer. For good thermal conductance, the thickness of eachwall48 is preferably between about 0.003 to 0.5 mm, more preferably between 0.01 to 0.15 mm, and most preferably between 0.025 to 0.08 mm.
• Referring again to FIG. 22, the[0094]vessel40 also preferably includes optical windows for in situ optical interrogation of the reaction mixture in thechamber42. In the preferred embodiment, the optical windows are theside walls57A,57B of therigid frame46. Theside walls57A,57B are optically transmissive to permit excitation of the reaction mixture in thechamber42 through theside wall57A and detection of light emitted from thechamber42 through theside wall57B. Arrows A represent illumination beams entering thechamber42 through theside wall57A and arrows B represent emitted light (e.g., fluorescent emission from labeled analytes in the reaction mixture) exiting thechamber42 through theside wall57B.
• The[0095]side walls57A,57B are preferably angularly offset from each other. It is usually preferred that thewalls57A,57B are offset from each other by an angle of about 90° . A 90° angle between excitation and detection paths assures that a minimum amount of excitation radiation entering through thewall57A will exit throughwall57B. In addition, the 90° angle permits a maximum amount of emitted light (e.g. fluorescence) to be collected throughwall57B. Thewalls57A,57B are preferably joined to each other to form a “V” shaped intersection at the bottom of thechamber42. Alternatively, theangled walls57A,57B need not be directly joined to each other, but may be separated by an intermediary portion, such as another wall or various mechanical or fluidic features which do not interfere with the thermal and optical performance of the vessel. For example, thewalls57A,57B may meet at a port which leads to another processing area in communication with thechamber42, such as an integrated capillary electrophoresis area. In the presently preferred embodiment, a locatingtab58 extends from theframe46 below the intersection ofwalls57A,57B. Thetab58 is used to properly position thevessel40 in a heat-exchanging module described below with reference to FIG. 28.
• Optimum optical sensitivity may be attained by maximizing the optical path length of the light beams exciting the labeled analyte in the reaction mixture and the emitted light that is detected, as represented by the equation:[0096]
• I_o/I_i=C*L*A,
• where I[0097]_ois the illumination output of the emitted light in volts, photons or the like, C is the concentration of analyte to be detected, I_iis the input illumination, L is the path length, and A is the intrinsic absorptivity of the dye used to label the analyte.
• The thin,[0098]flat reaction vessel40 of the present invention optimizes detection sensitivity by providing maximum optical path length per unit analyte volume. Referring to FIGS. 23 and 27, thevessel40 is preferably constructed such that each of thesides walls57A,57B,59A,59B of thechamber42 has a length L in the range of 1 to 15 mm, the chamber has a width W in the range of 1.4 to 20 mm, the chamber has a thickness T in the range of 0.5 to 5 mm, and the ratio of the width W of the chamber to the thickness T of the chamber is at least 2:1. These parameters are presently preferred to provide a vessel having a relatively large average optical path length through the chamber, i.e. 1 to 15 mm on average, while still keeping the chamber sufficiently thin to allow for extremely rapid heating and cooling of the reaction mixture contained therein. The average optical path length of thechamber42 is the distance from the center of theside wall57A to the center of thechamber42 plus the distance from the center of thechamber42 to the center of theside wall57B.
• More preferably, the[0099]vessel40 is constructed such that each of thesides walls57A,57B,59A,59B of thechamber42 has a length L in the range of 5 to 12 mm, the chamber has a width W in the range of 7 to 17 mm, the chamber has a thickness T in the range of 0.5 to 2 mm, and the ratio of the width W of the chamber to the thickness T of the chamber is at least 4:1. These ranges are more preferable because they provide a vessel having both a larger average optical path length (i.e., 5 to 12 mm) and a volume capacity in the range of 12 to 100 μl while still maintaining a chamber sufficiently thin to permit extremely rapid heating and cooling of a reaction mixture. The relatively large volume capacity provides for increased sensitivity in the detection of low concentration analytes, such as nucleic acids.
• In the preferred embodiment, the[0100]reaction vessel40 has a diamond-shapedchamber42 defined by theside walls57A,57B,59A,59B, each of the side walls has a length of about 10 mm, the chamber has a width of about 14 mm, the chamber has a thickness T of 1 mm as defined by the thickness of theframe46, and the chamber has a volume capacity of about 100 μl. This reaction vessel provides a relatively large average optical path length of 10 mm through thechamber42. Additionally, the thin chamber allows for extremely rapid heating and/or cooling of the reaction mixture contained therein. The diamond-shape of thechamber42 helps prevent air bubbles from forming in the chamber as it is filled with the reaction mixture and also aids in optical interrogation of the mixture.
• Referring again to FIG. 22, the[0101]frame46 is preferably made of an optically transmissive material, e.g., a polycarbonate or clarified polypropylene, so that theside walls57A,57B are optically transmissive. As used herein, the term optically transmissive means that one or more wavelengths of light may be transmitted through the walls. In the preferred embodiment, the opticallytransmissive walls57A,57B are substantially transparent. In addition, one or more optical elements may be present on the opticallytransmissive side walls57A,57B. The optical elements may be designed, for example, to maximize the total volume of solution which is illuminated by a light source, to focus excitation light on a specific region of thechamber42, or to collect as much fluorescence signal from as large a fraction of the chamber volume as possible.
• In alternative embodiments, the optical elements may comprise gratings for selecting specific wavelengths, filters for allowing only certain wavelengths to pass, or colored lenses to provide filtering functions. The wall surfaces may be coated or comprise materials such as liquid crystal for augmenting the absorption of certain wavelengths. In the presently preferred embodiment, the optically[0102]transmissive walls57A,57B are substantially clear, flat windows having a thickness of about 1 mm.
• The[0103]side walls59A,59B preferably includes reflective faces56 which internally reflect light trying to exit thechamber42 through theside walls59A,59B. The reflective faces56 are arranged such that adjacent faces are angularly offset from each other by about 90°. In addition, theframe46 defines open spaces between theside walls59A,59B and thesupport ribs53. The open spaces are occupied by ambient air that has a different refractive index than the material composing the frame (e.g., plastic). Due to the difference in the refractive indexes, the reflective faces56 are effective for internally reflecting light trying to exit the chamber through thewalls59A,59B and provide for increased detection of optical signal through thewalls57A,57B. Preferably, the opticallytransmissive side walls57A,57B define the bottom portion of the diamond-shapedchamber42, and the retro-reflective side walls59A,59B define the top portion of the chamber.
• A preferred method for fabricating the[0104]reaction vessel40 will now be described with reference to FIGS. 21-22. Thereaction vessel40 may be fabricated by first molding therigid frame46 using known injection molding techniques. Theframe46 is preferably molded as a single piece of polymeric material, e.g., clarified polypropylene. After theframe46 is produced, thin, flexible sheets are cut to size and sealed to opposite sides of theframe46 to form themajor walls48 of thechamber42. Themajor walls48 are preferably cast or extruded films of polymeric material, e.g., polypropylene films, that are cut to size and attached to theframe46 using the following procedure. A first piece of film is placed over one side of theframe46. Theframe46 preferably includes atack bar47 for aligning the top edge of the film. The film is placed over the bottom portion of theframe46 such that the top edge of the film is aligned with thetack bar47 and such that the film completely covers the bottom portion of theframe46 below thetack bar47. The film should be larger than the bottom portion of theframe46 so that it may be easily held and stretched flat across the frame. The film is then cut to size to match the outline of the frame by clamping to the frame the portion of the film that covers the frame and cutting away the portions of the film that extend past the perimeter of the frame using, e.g., a laser or die. The film is then tack welded to the frame, preferably using a laser.
• The film is then sealed to the[0105]frame46, preferably by heat sealing. Heat sealing is presently preferred because it produces a strong seal without introducing potential contaminants to the vessel as the use of adhesive or solvent bonding techniques might do. Heat sealing is also simple and inexpensive. The heat sealing may be performed using, e.g., a heated platen. An identical procedure may be used to cut and seal a second sheet to the opposite side of theframe46 to complete thechamber42. Many variations to this fabrication procedure are possible. For example, in an alternative embodiment, the film is stretched across the bottom portion of theframe46 and then sealed to the frame prior to cutting the film to size. After sealing the film to the frame, the portions of the film that extend past the perimeter of the frame are cut away using, e.g., a laser or die.
• Although it is presently preferred to mold the[0106]frame46 as a single piece, it is also possible to fabricate the frame from multiple pieces. For example, theside walls57A,57B forming the angled optical windows may be molded from polycarbonate, which has good optical transparency, while the rest of the frame is molded from polypropylene, which is inexpensive and compatible with PCR. The separate pieces can be attached together in a secondary step. For example, theside walls57A,57B may be press-fitted and/or bonded to the remaining portion of theframe46. Theflexible walls48 may then be attached to opposite sides of theframe46 as previously described.
• Referring again to FIG. 3, it is presently preferred to use a[0107]gasket61 to seal theports41,43 of thevessel40 to correspondingchannels80,81 (FIG. 4) in the cartridge body. Alternatively, fluidic seals may be established using a luer fitting, compression fitting, or swaged fitting. In another embodiment, the cartridge body and frame of thevessel40 are molded as a single part, and the flexible major walls of the vessel are heat-sealed to opposite sides of the frame.
• Referring again to FIG. 22, the[0108]chamber42 is filled by forcing liquid (e.g., a reaction mixture) to flow through theport41 and thechannel50 into thechamber42. The liquid may be forced to flow into thechamber42 using differential pressure (i.e., either pushing the liquid through theinlet port41 or aspirating the liquid by applying a vacuum to the outlet port43). As the liquid fills thechamber42, it displaces air in the chamber. The displaced air exits thechamber42 through thechannel52 and theport43. For optimal detection of analyte in thechamber42, the chamber should not contain air bubbles. To help prevent the trapping of air bubbles in thechamber42, the connection between thechamber42 and theoutlet channel52 should be at the highest point (with respect to gravity) in thechamber42. This allows air bubbles in thechamber42 to escape without being trapped. Thus, thevessel40 is designed to be used in the vertical orientation shown in FIG. 22.
• FIG. 25 shows another[0109]vessel206 designed to be used in a horizontal orientation. Thevessel206 has aninlet port41 and aninlet channel50 connecting theinlet port41 to the bottom of thechamber42. The vessel also has anoutlet port43 and anoutlet channel50 connecting theoutlet port43 to the top of thechamber42. Thus, any air bubbles in thechamber42 may escape through theoutlet channel52 without becoming trapped. FIG. 26 shows anothervessel207 having twoinlet ports41,45 and oneoutlet port43.Inlet channels50,54 connect therespective inlet ports41,45 to thechamber42, andoutlet channel52 connects thechamber42 tooutlet port43. Many other different embodiments of the vessel are also possible. In each embodiment, it is desirable to evacuate thechamber42 from the highest point (with respect to gravity) in the chamber and to introduce liquid into the chamber from a lower point.
• FIGS. 15A-15B illustrate two types of valves used in the cartridge. As shown in FIG. 15A, there are two types of fundamental concepts to the valve action, and hence two types of valves. The first valve uses a cone-shaped or[0110]conical valve seat160 formed in themiddle cartridge piece24. Thevalve seat160 is a depression, recess, or cavity molded or machined in themiddle piece24. Thevalve seat160 is in fluid communication with achamber167 through a port orchannel157 that intersects the center of theconical valve seat160. As shown in FIG. 15B, avalve actuator164 having a spherical surface is forced against theelastic membrane63 and into thevalve seat160, establishing a circular ring of contact between themembrane63 and thevalve seat160. The kinematic principle is that of a ball seated into a cone. The circular seal formed by themembrane63 andvalve seat160 prevents flow between the channel157 (and hence the chamber167) and aside channel158 extending from a side of thevalve seat160. Theside channel158 is defined by themembrane63 and themiddle cartridge piece24.
• As shown in FIG. 15A, the other type of valve controls the cross flow between the[0111]channel158 and anotherside channel159 formed between themembrane63 and themiddle cartridge piece24. In this case, a circular ring of contact would be ineffective. Instead, the second valve comprises a recess depression orcavity161 formed in themiddle cartridge piece24. Thecavity161 separates thechannels158,159 from each other. An end of thechannel158 is positioned on one side of thecavity161, and an end of thechannel159 is positioned on the opposite side of thecavity161. Thecavity161 is defined by a firstcurved surface162A positioned adjacent the end of thechannel158, a secondcurved surface162B positioned adjacent the end of thechannel159, and athird surface163 between the first and secondcurved surfaces162A,162B. As shown in FIG. 15B, the curved surfaces provide two valve seats that are the primary contact area for themembrane63 to seal off the flow between thechannels158 and159. The kinematic principle is that of a ball (or spherical end on a valve actuator) held by three contact points, the upward force on the actuator and the twovalve seats162A,162B.
• As shown in FIG. 16A, the first and second[0112]curved surfaces162A,162B are preferably concentric spherical surfaces. Thevalve actuator164 has also has a spherical surface for pressing themembrane63 tightly against thesurfaces162A,162B. In addition, each of thesurfaces162A,162B preferably has a spherical radius of curvature R1 equal to the combined radius of curvature R2 of thevalve actuator164 plus the thickness T of themembrane63. For example, if the radius of curvature R2 of the surface of thevalve actuator164 is 0.094 inches and themembrane63 has a thickness T of 0.031 inches, then the radius of curvature R1 of each of thesurfaces162A,162B is 0.125 inches. In general, the size and radius of curvature of the valve seats is dependent upon the size of the channels in the cartridge. The valves are preferably made just large enough to effectively seal the channels but no larger so that dead volume in the cartridge is minimized.
• As shown in FIG. 16B, the[0113]third surface163 is recessed from the first andsecond surfaces162A,162B to provide agap166 between themembrane63 and thethird surface163 when themembrane63 is pressed against the first andsecond surfaces162A,162B. Stated another way, thesurfaces162A,162B are raised or elevated from thesurface163. Thegap166 ensures that themembrane63 contacts primarily the valve seats.162A,162B rather than the entire surface of thecavity161 so that maximum pressure is applied to thevalve seats162A and162B by themembrane63. This provides a very strong seal with minimal actuator force required.
• Referring again to FIG. 15B, in both types of valves the respective kinematic principle defines the location of the mating parts. In both the ball-in-cone concept and the ball-against-two-spherical-surfaces concept, the ball or spherical shaped valve actuator is permitted to seek its own location as it is forced against the valve seat(s). There is a deliberate clearance (e.g., 0.01 to 0.03 inches) between the valve actuator and the hole in the[0114]bottom cartridge piece26 in which theactuator164 travels so that only the valve seat action defines the location of the mating pieces.
• The valve actuators can be controlled by a variety of mechanisms. FIGS. 17-19 illustrate one such mechanism. As shown in FIG. 17, a[0115]valve actuator172 has a spherical surface for pressing thegasket63 into a valve seat. Theactuator172 also has aflange177 on its bottom portion. The cartridge includes an elastic body, such as aspring174, that pushes against a ledge in thelower cartridge piece26 to bias the valve actuator against thegasket63. Thespring174 is sufficiently strong to close the valve unless a deliberate force is applied to pull down theactuator172. The valves in the cartridge may be kept closed in this manner for shipping and storage before the cartridge is used. Thus, the cartridge may be preloaded during manufacture with the necessary reagents and wash solutions to analyze a fluid sample without the fluids leaking out of the cartridge during shipping and storage.
• The actuator pull-down mechanism is usually located in an instrument into which the cartridge is placed for sample. analysis (one such instrument is described in detail below with reference to FIG. 10). The mechanism comprises a sliding[0116]guide175 that rotates a hinged pull-down member180 having ajaw181 for receiving theflange177 of theactuator172. As shown in FIG. 18, the slidingguide175 rotates the hinged pull-down member180 until theflange177 is positioned within thejaw181. As shown in FIG. 19, asolenoid146 pulls down themember180 and thus thevalve actuator172 so that thegasket63 is released from the valve seat, thus opening the valve and permitting fluid flow between thechannels170 and171.
• FIG. 20 illustrates the manner in which fluid flow into and out of the sample chamber, wash chamber, neutralizer chamber, and reagent chambers is controlled in the cartridge. Each of these chambers, as illustrated by a[0117]chamber414 in FIG. 20, is covered by ahydrophobic membrane410 that allows the passage of gas but not liquid therethrough. Thehydrophobic membrane410 is positioned between thechamber414 and apressure port32. Thepressure port32 is formed in theupper cartridge piece22 and positioned over thechamber414. Themembrane410 holds liquids in thechamber414 during shipping and storage of the cartridge, even if the cartridge is turned upside down. Thepressure port32 is sized to receive apressure nozzle182 that is connected to a pressure source (e.g., a vacuum or pneumatic pump) usually located in the external instrument. Thenozzle182 includes an o-ring184 and aflange415. Aspring185 pushes against theflange415 to force thenozzle182 into thepressure port32 so that the o-ring184 establishes a seal around theport32. In operation, positive air pressure or a vacuum is applied to thechamber414 through thepressure port32 to force liquids out of or into, respectively, thechamber414.
• A conical valve seat[0118]160 (previously described with reference to FIGS. 15A-15B) is formed in themiddle cartridge piece24 below thechamber414 to control the flow of liquid between thechamber414 and a connectingchannel411. The valve is opened and closed by avalve actuator188 having aflange187 and aspring188 pressing against the flange to hold the valve closed until a downward force is applied to theactuator186. The downward force is preferably supplied by a solenoid that pulls down theactuator186 to open the valve. Thevalve actuator186 and solenoid are preferably located in the instrument.
• FIGS. 7-8 show top and bottom plan views, respectively, of the cartridge. FIG. 9 is a schematic block diagram of the cartridge. As shown in any of FIGS. 7-9, the cartridge includes a[0119]sample chamber65 having a port for adding a fluid sample to the cartridge and a sample flow path extending from thesample chamber65. The sample flow path extends from thesample chamber65 through avalve107 and into achannel106. Thechannel106 includes asensor region136 in which thechannel106 has a flat bottom enabling easy optical detection of the presence of liquid in the channel. The sample flow path continues from thechannel106 into the lysingchamber86 and through thefilter stack87. The sample flow path also includes achannel109 for exit of fluid from the lysingchamber86, achannel110 having a flat-bottomeddetection region137, avalve111, and achannel112 leading to the ventedwaste chamber68 through avalve114.
• The cartridge also includes the[0120]wash chamber66 for holding wash solution and thereagent chamber67 for holding lysing reagent. Thewash chamber66 is connected to the lysingchamber86 through avalve115,channel117, andchannel106. Thereagent chamber67 is connected to the lysing chamber.86 through avalve119,channel117, andchannel106. Sample components (e.g., cells or viruses in the sample) are captured in thefilter stack87 and lysed in thechamber86 to release target analyte (e.g., nucleic acid) from the sample components. The cartridge also includes an analyte flow path extending from the lysingchamber86 for carrying the analyte separated from the fluid sample to thereaction vessel40 for chemical reaction and optical detection. The analyte flow path extends from thechamber86 through thechannel109,channel110, andvalve111. After passing through thevalve111, the analyte flow path diverges from the sample flow path. While the sample flow path extends thoughchannel112 into thewaste chamber68, the analyte flow path diverges into theU-shaped channel122. The analyte flow path then extends into and out of theneutralizer chamber70 through avalve124. The analyte flow path also passes into and out of themaster mix chamber71 through avalve126. From themaster mix chamber71, the analyte flow path extends along thechannel122, through avalve127, throughchannel80, and into thereaction vessel40 through theport41.
• The[0121]reaction vessel40 includes theport41 for adding a reaction mixture to the vessel, and theport43 for exit of fluids (e.g., air or excess reaction mixture) from the vessel. The cartridge also includeschannel81 in fluid communication with theport43. Thechannel81 includes a flat-bottomeddetection region130 for detecting the presence of liquid in the channel. Thechannel81 connects to a channel131 (channel131 extends straight down perpendicular to the page in the top plan view of FIG. 7).Channel131 connects to achannel132 which in turn connects to achannel134 through a valve133 (channel134 extends straight up perpendicular to the page in the top plan view of FIG. 7). Thechannel134 leads to thevent36 which has a hydrophobic membrane to permit the escape of gas but not liquid from the cartridge. The channels, vent and valve positioned downstream from thereaction vessel40 are used to pressurize thechamber42 of the vessel, as is described in the operation section below.
• The cartridge also includes a[0122]first pressure port105 positioned above thesample chamber65, asecond pressure port116 positioned above thewash chamber66, athird pressure port118 positioned above thereagent chamber67, afourth pressure port123 positioned above theneutralizer chamber70, afifth pressure port125 positioned above themaster mix chamber71, and asixth pressure port128 positioned at the end of theU-shaped channel122. The cartridge further includessensor chambers120 and121 in fluid communication with thewaste chamber68. Thesensor chambers120 and121 indicate when predetermined volumes of liquid have been received in thewaste chamber68, as is described in detail below.
• Referring to FIG. 10, the cartridge is preferably used in combination with an[0123]instrument140 designed to accept one or more of the cartridges. For clarity of illustration, theinstrument140 shown in FIG. 10 accepts just one cartridge. It is to be understood, however, that the instrument may be designed to process multiple cartridges simultaneously. Theinstrument140 includes acartridge nest141 into which the cartridge is placed for processing. Theinstrument140 also includes the transducer92 (e.g., an ultrasonic horn) for generating dynamic pressure pulses or pressure waves in the lysing chamber of the cartridge, ninevalve actuators142 for actuating the nine valves in the cartridge, nine correspondingsolenoids146 for pulling down the valve actuators, and sixpressure nozzles145 for interfacing with six corresponding pressure ports formed in the cartridge. In addition, the instrument includes or is connected to one or more regulated pressure sources for supplying pressure to the cartridge through thepressure nozzles145. Suitable pressure sources include syringe pumps, compressed air sources, pneumatic pumps, or connections to external sources of pressure. The instrument further includes three slottedoptical sensors143 and three reflectiveoptical sensors144.
• FIG. 13 illustrates the slotted[0124]optical sensors143 positioned to detect liquid in thesensor chambers120,121 and in thereagent chamber67. Eachsensor143 includes a built in LED and photodiode positioned on opposite sides of the sensor. The LED emits a beam that is detected by the photodiode if the beam is not substantially refracted. Such slotted optical sensors are commercially available from a number of suppliers. The cartridge is shaped so that the slotted optical sensors fit around thechambers67,120, and121. The operation of each sensor is as follows. If liquid is not present in the chamber the sensor surrounds, the beam from the LED is substantially refracted by air in the chamber and the curved inner walls of the chamber and only a weak signal, if any, is detected by the photodiode since air has an index of refraction that does not closely match that of the plastic cartridge. If there is liquid present in the chamber, however, the beam from the LED does not refract or is only slightly refracted and produces a much stronger signal detected by the photodiode since the liquid has an index of refraction closely matching that of the plastic cartridge. Theoptical sensors143 are therefore useful for determining the presence or absence of liquid in thechambers67,120, and121.
• FIG. 14 shows a cut-away, schematic side view of the[0125]sensor chamber120 in fluid communication with thewaste chamber68 and surrounded by the slottedoptical sensor143. Thesensor chamber120 andsensor143 are used to indicate when a predetermined volume of liquid is present in thewaste chamber68. Thesensor chamber120 is partially separated from thewaste chamber68 by awall151 having aspillover rim152. The height of the wall is selected so that when the predetermined volume of liquid is received in thewaste chamber68, the liquid spills over thespillover rim152 and into thesensor chamber120. The liquid in thesensor chamber120 is then detected by thesensor143.
• Referring again to FIG. 13, the cartridge may also include a[0126]second sensor chamber121 in fluid communication with thewaste chamber68. Thesecond sensor chamber121 is also separated from thewaste chamber68 by awall153 having a spillover rim. Thewall153 is taller than thewall152 so that liquid does not spill over thewall153 until a second predetermined volume of fluid in addition to the first predetermined volume of fluid has been received in thewaste chamber68. Thesensor chambers120,121 and theoptical sensors143 are useful for controlling the operation of the cartridge. The height of thewall152 is preferably selected such that when a fixed volume of fluid sample from thesample chamber65 has flowed through the sample flow path to thewaste chamber68, the sample liquid spills over into thesensor chamber120 and is detected. The detection inchamber120 triggers the release of wash solution from thewash chamber66 which flows through the sample flow path to thewaste chamber68. When an incremental volume of the wash solution is received in thechamber68, liquid spills over thewall153 into thesensor chamber121 and is detected. The detection of liquid in thechamber121 then triggers the release of lysing reagent from thechamber67. Thesensor143 surrounding thechamber67 may then be used to indicate when thechamber67 is empty, triggering the start of ultrasonic lysis. In an alternative embodiment, the cartridge may have two waste chambers, one for sample and one for wash, with each waste chamber having a respective sensor chamber connected thereto.
• In-line reflective[0127]optical sensors144 are used to determine the presence or absence of liquid in the flat-bottomeddetection regions130,136,137, ofchannels81,106, and110, respectively (FIG. 7). Eachsensor144 has a built in emitter and detector that is positioned over a flat-bottomed detection region. The emitter emits a beam that is reflected from the cartridge and detected by the detector. The sensor detects a change in signal when as an air/liquid interface passes through the detection region. Optionally, dual emitter reflective optical sensors may be used for a more reliable detection operation. Both types of reflective optical sensors are well known in the art and commercially available.
• Referring again to FIG. 10, the[0128]instrument140 also includes a heat-exchangingmodule147 having aslot148 for receiving the reaction vessel of the cartridge. Themodule147 is described in detail below with reference to FIG. 28. Theinstrument140 further includes alatch mechanism149 for latching alid150 over a cartridge. Thecartridge nest141 includes alignment holes401 for receiving the legs of the cartridge. The alignment holes401 ensure proper positioning of the cartridge in thenest141 so that thepressure nozzles145,transducer92, andvalve actuators142 fit into the corresponding ports in the cartridge and so that the reaction vessel fits into theslot148. Thetransducer92 should be positioned in theinstrument140 such that when the cartridge is placed in thenest141, the transducer contacts the bottom wall of the lysingchamber86, as shown in the cut-away view of FIG. 5. In addition, the instrument may include a spring or similar mechanism to bias thetransducer92 against the wall of the lysingchamber86.
• The[0129]instrument140 also includes various conventional equipment not shown in FIG. 10 including a main logic board having a microcontroller for controlling the operation of thesolenoids146,transducer92, heat-exchangingmodule147, andoptical sensors143,144. The instrument also includes or is connected to a power supply for powering the instrument and a pneumatic pump for supplying air pressure through thenozzles145. Theinstrument140 is preferably computer-controlled using, e.g., the microcontroller which is programmed to perform the functions described in the operation section below. Alternatively, the instrument may controlled by a separate computer, or controlled by a combination of a separate computer and an on-board microcontroller.
• FIG. 11 shows an isometric view of the[0130]cartridge20 placed in theinstrument140 for processing. FIG. 11 shows a partial cut-away view of theinstrument140 with thelid150 closed. Referring again to FIG. 11, a memory or microprocessor chip may optionally be incorporated as part of thecartridge20. This chip preferably contains information such as the type of cartridge, program information such as specific protocols for the processing of the cartridge, tolerances for accept and reject, serial numbers and lot codes for quality tracking, and provision for storing the results of the processing. Integrated electronic memory on thecartridge20 allows for rapid, easy, and error-free set-up of theinstrument140 for different fluidic processing protocols. When thecartridge20 is inserted into theinstrument140, the instrument may electronically address the memory on the cartridge, and thus automatically receive the appropriate set of instructions for controlling the time-sequence of fluidic operations to be carried out with the inserted cartridge. Theinstrument140 may simply sequentially retrieve and execute each step in the cartridge's memory, or download its contents so that the user may edit the sequence using, e.g., the controller computer.
• If suitable memory is included on the cartridge, such as writable memory (e.g., erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), etc., intermediate and final results, based on the sample introduced into the cartridge, could be written by the instrument into the cartridge's memory for co-located storage with the physical sample after processing. This is particularly advantageous in applications where archiving of samples and results is necessary, such as forensics. In addition, other information can be stored in the memory on the cartridge, in unalterable (or alterable) forms. For example, cartridge serial number, lot manufacture information, and related information could be pre-programmed and unalterable. User data, technician identification number, date of test, location of test and instrument serial number could be unalterably written into the cartridge. This allows for easy identification of the “chain of custody” in the handling of a specimen. Engineers skilled in the art of data storage will recognize that other memory means than electronic can be used, such as optically-addressed printed regions (e.g., ink-jet or thermal), magnetic strips, etc.[0131]
• FIG. 28 shows the heat-exchanging[0132]module147 of the instrument into which thereaction vessel40 is inserted for thermal processing and optical detection of target analyte(s) in the reaction mixture. Themodule147 preferably includes ahousing208 for holding the various components of the module. Themodule147 also includes thethermal plates190 described above. Thehousing208 includes a slot (not shown in FIG. 28) above theplates190 so that the reaction chamber of thevessel40 may be inserted through the slot and between the plates. The heat-exchangingmodule147 also preferably includes a cooling system, such as afan212. Thefan212 is positioned to blow cooling air past the surfaces of theplates190 to cool the plates and hence cool the reaction mixture in thevessel40. Thehousing208 preferably defines channels for directing the cooling air past theplates190 and out of themodule147.
• The heat-exchanging[0133]module147 further includes anoptical excitation assembly216 and anoptical detection assembly218 for optically interrogating the reaction mixture contained in thevessel40. Theexcitation assembly216 includes afirst circuit board220 for holding its electronic components, and thedetection assembly216 includes asecond circuit board222 for holding its electronic components. Theexcitation assembly216 includes one or more light sources (e.g., an LED. laser, or light bulb) for exciting fluorescently-labeled analytes in thevessel40. Theexcitation assembly216 also includes one or more lenses for collimating the light from the light sources, as well as filters for selecting the excitation wavelength ranges of interest. Thedetection assembly218 includes one or more detectors (e.g., a photodiode, photomultiplier tube, or CCD) for detecting the light emitted from thevessel40. Thedetection assembly218 also includes one or more lenses for focusing and collimating the emitted light, as well as filters for selecting the emission wavelength ranges of interest. Suitable optical excitation and detection assemblies for use in the heat-exchangingmodule147 are described in International Publication Number WO 99/60380 (International Application Number PCT/US99/11182) published Nov. 25, 1999, the disclosure of which is incorporated by reference herein.
• The[0134]optics assemblies216,218 are positioned in thehousing208 such that when the chamber of thevessel40 is inserted between theplates190, theexcitation assembly216 is in optical communication with thechamber42 through the opticallytransmissive side wall57A (see FIG. 22) and thedetection assembly218 is in optical communication with the chamber through the opticallytransmissive side wall57B (FIG. 22). In the preferred embodiment, theoptics assemblies216,218 are placed into optical communication with the optically transmissive side walls by simply locating theoptics assemblies216,218 next to the bottom edges of theplates190 so that when the chamber of the vessel is placed between the plates, theoptics assemblies216,218 directly contact, or are in close proximity to, the side walls.
• FIG. 34 shows a partially cut-away, isometric view of the chamber of the vessel inserted between the[0135]plates190A,190B (the top portion of the vessel is cut away). The vessel preferably has an angled bottom portion (e.g., triangular) formed by the opticallytransmissive side walls57A,57B. Each of theplates190A,190B has a correspondingly shaped bottom portion. The bottom portion of thefirst plate190A has a first bottom edge.250A and a second bottom edge2190B. Similarly, the bottom portion of thesecond plate190B has a firstbottom edge252A and a secondbottom edge252B. The first and second bottom edges of each plate are preferably angularly offset from each other by the same angle that theside walls57A,57B are offset from each other (e.g., 90°). Additionally, theplates190A,190B are preferably positioned to receive the chamber of the vessel between them such that thefirst side wall57A is positioned substantially adjacent and parallel to each of the firstbottom edges250A,252A and such that thesecond side wall57B is positioned substantially adjacent and parallel to each of thesecond bottom edges2190B,252B. This arrangement provides for easy optical access to the opticallytransmissive side walls57A,57B and hence to the chamber of the vessel. A gel or fluid may optionally be used to establish or improve optical communication between each optics assembly and theside walls57A,57B. The gel or fluid should have a refractive index close to the refractive indexes of the elements that it is coupling.
• Referring again to FIG. 28, the[0136]optics assemblies216,218 are preferably arranged to provide a 90° angle between excitation and detection paths. The 90° angle between excitation and detection paths assures that a minimum amount of excitation radiation entering through the first side wall of the chamber exits through the second side wall. Also, the 90° angle permits a maximum amount of emitted radiation to be collected through the second side wall. In the preferred embodiment, thevessel40 includes a locating tab58 (see FIG. 22) that fits into a slot formed between theoptics assemblies216,218 to ensure proper positioning of thevessel40 for optical detection. For improved detection, themodule147 also preferably includes a light-tight lid (not shown) that is placed over the top of thevessel40 and made light-tight to thehousing208 after the vessel is inserted between theplates190.
• Although it is presently preferred to locate the[0137]optics assemblies216,218 next to the bottom edges of theplates190, many other arrangements are possible. For example, optical communication may be established between theoptics assemblies216,218 and the walls of thevessel40 via optical fibers, light pipes, wave guides, or similar devices. One advantage of these devices is that they eliminate the need to locate theoptics assemblies216,218 physically adjacent to theplates190. This leaves more room around the plates in which to circulate cooling air or refrigerant, so that cooling may be improved.
• The heat-exchanging[0138]module147 also includes aPC board226 for holding the electronic components of the module and anedge connector224 for connecting themodule147 to the instrument140 (FIG. 10). The heating elements and temperature sensors on theplates190, as well as theoptical boards220,222, are connected to thePC board226 by flex cables (not shown in FIG. 28 for clarity of illustration). Themodule147 may also include agrounding trace228 for shielding the optical detection circuit. Themodule147 may optionally include an indicator, such as anLED214, for indicating to a user the current status of the module such as “heating,” “cooling,” “finished,” or “fault”.
• The[0139]housing208 may be molded from a rigid, high-performance plastic, or other conventional material. The primary functions of thehousing208 are to provide a frame for holding theplates190,optics assemblies216,218,fan212, andPC board226. Thehousing208 also preferably provides flow channels and ports for directing cooling air from thefan212 across the surfaces of theplates190 and out of the housing. In the preferred embodiment, thehousing208 comprises complementary pieces (only one piece shown in the schematic side view of FIG. 28) that fit together to enclose the components of themodule147 between them.
• Referring again to FIG. 23, the[0140]plates190A,190B may be made of various thermally conductive materials including ceramics or metals. Suitable ceramic materials include aluminum nitride, aluminum oxide, beryllium oxide, and silicon nitride. Other materials from which the plates may be made include, e.g., gallium arsenide, silicon, silicon nitride, silicon dioxide., quartz, glass, diamond, polyacrylics, polyamides, polycarbonates, polyesters, polyimides, vinyl polymers, and halogenated vinyl polymers, such as polytetrafluoroethylenes. Other possible plate materials include chrome/aluminum, superalloys, zircaloy, aluminum, steel, gold, silver, copper, tungsten, molybdenum, tantalum, brass, sapphire, or any of the other numerous ceramic, metal, or polymeric materials available in the art.
• Ceramic plates are presently preferred because their inside surfaces may be conveniently machined to very high smoothness for high wear resistance, high chemical resistance, and good thermal contact to the flexible walls of the reaction vessel. Ceramic plates can also be made very thin, preferably between about 0.6 and 1.3 mm, for low thermal mass to provide for extremely rapid temperature changes. A plate made from ceramic is also both a good thermal conductor and an electrical insulator, so that the temperature of the plate may be well controlled using a resistive heating element coupled to the plate.[0141]
• Various thermal elements may be employed to heat and/or cool the[0142]plates190A,190B and thus control the temperature of the reaction mixture in thechamber42. In general, suitable heating elements for heating the plate include conductive heaters, convection heaters, or radiation heaters. Examples of conductive heaters include resistive or inductive heating elements coupled to the plates, e.g., resistors or thermoelectric devices. Suitable convection heaters include forced air heaters or fluid heat-exchangers for flowing fluids past the plates. Suitable radiation heaters include infrared or microwave heaters. Similarly, various cooling elements may be used to cool the plates. For example, various convection cooling elements may be employed such as a fan, peltier device, refrigeration device, or jet nozzle for flowing cooling fluids past the surfaces of the plates. Alternatively, various conductive cooling elements may be used, such as a heat sink, e.g. a cooled metal block, in direct contact with the plates.
• Referring to FIG. 24, each[0143]plate190 preferably has aresistive heating element206 disposed on its outer surface. Theresistive heating element206 is preferably a thick or thin film and may be directly screen printed onto eachplate190, particularly plates comprising a ceramic material, such as aluminum nitride or aluminum oxide. Screen-printing provides high reliability and low cross-section for efficient transfer of heat into the reaction chamber. Thick or thin film resistors of varying geometric patterns may be deposited on the outer surfaces of the plates to provide more uniform heating, for example by having denser resistors at the extremities and thinner resistors in the middle. Although it is presently preferred to deposit a heating element on the outer surface of each plate, a heating element may alternatively be baked inside of each plate, particularly if the plates are ceramic. Theheating element206 may comprise metals, tungsten, polysilicon, or other materials that heat when a voltage difference is applied across the material. Theheating element206 has two ends which are connected torespective contacts204 which are in turn connected to a voltage source (not shown in FIG. 24) to cause a current to flow through the heating element. Eachplate190 also preferably includes atemperature sensor192, such as a thermocouple, thermistor, or RTD, which is connected by twotraces202 to respective ones of thecontacts204. Thetemperature sensor192 is be used to monitor the temperature of theplate190 in a controlled feedback loop.
• The plates have a low thermal mass to enable rapid heating and cooling of the plates. In particular, it is presently preferred that each of the plates has a thermal mass less than about 5 J/° C., more preferably less than 3 J/° C., and most preferably less than 1 J/° C. As used herein, the term thermal mass of a plate is defined as the specific heat of the plate multiplied by the mass of the plate. In addition, each plate should be large enough to cover a respective major wall of the reaction chamber. In the presently preferred embodiment, for example, each of the plates has a width X in the range of 2 to 22 mm, a length Y in the range of 2 to 22 mm, and a thickness in the range of 0.5 to 5 mm. The width X and length Y of each plate is selected to be slightly larger than the width and length of the reaction chamber. Moreover, each plate preferably has an angled bottom portion matching the geometry of the bottom portion of the reaction chamber, as previously described with reference to FIG. 34. Also in the preferred embodiment, each of the plates is made of aluminum nitride having a specific heat of about 0.75 J/g° C. The mass of each plate is preferably in the range of 0.005 to 5.0 g so that each plate has a thermal mass in the range of 0.00375 to 3.75 J/° C.[0144]
• The opposing[0145]plates190 are positioned to receive the chamber of thevessel40 between them such that the flexible major walls of the chamber contact and conform to the inner surfaces of the plates. It is presently preferred that theplates190 be held in an opposing relationship to each other using, e.g., brackets, supports, or retainers. Alternatively, theplates190 may be spring-biased towards each other as described in International Publication Number WO 98/38487, the disclosure of which is incorporated by reference herein. In another embodiment of the invention, one of the plates is held in a fixed position, and the second plate is spring-biased towards the first plate. If one or more springs are used to bias the plates towards each other, the springs should be sufficiently stiff to ensure that the plates are pressed against the flexible walls of the vessel with sufficient force to cause the walls to conform to the inner surfaces of the plates.
• FIGS. 29-30 illustrate a[0146]preferred support structure209 for holding theplates190A,190B in an opposing relationship to each other. FIG. 29 shows an exploded view of the structure, and FIG. 30 shows an assembled view of the structure. For clarity of illustration, thesupport structure209 andplates190A,190B are shown upside down relative to their normal orientation in the heat-exchanging module of FIG. 28. Referring to FIG. 29, thesupport structure209 includes a mountingplate210 having theslot148 formed therein. Theslot148 is sufficiently large to enable the chamber of the vessel to be inserted through it. Spacing posts230A,230B extend from the mountingplate210 on opposite sides of theslot148. Spacing post230A hasindentations232 formed on opposite sides thereof (only one side visible in the isometric view of FIG. 29), and spacingpost230B hasindentations234 formed on opposite sides thereof (only one side visible in the isometric view of FIG. 29). Theindentations232,234 in the spacing posts are for receiving the edges of theplates190A,190B. To assemble the structure, theplates190A,190B are placed against opposite sides of the spacing posts230A,230B such that the edges of the plates are positioned in theindentations232,234. The edges of the plates are then held in the indentations using a suitable retention means. In the preferred embodiment, the plates are retained byretention clips236A,236B. Alternatively, theplates190A,190B may be retained by adhesive bonds, screws, bolts, clamps, or any other suitable means.
• The mounting[0147]plate210 andspacing posts230A,230B are preferably integrally formed as a single molded piece of plastic. The plastic should be a high temperature plastic, such as polyetherimide, which will not deform of melt when theplates190A,190B are heated. The retention clips230A,230B are preferably stainless steel. The mountingplate210 may optionally includeindentations240A,240B for receivingflex cables238A,238B, respectively, that connect the heating elements and temperature sensors disposed on theplates190A,190B to thePC board226 of the heat-exchanging module147 (FIG. 28). The portion of theflex cables238A adjacent theplate190A is held in theindentation240A by a piece oftape242A, and the portion of theflex cables238B adjacent theplate190B is held in theindentation240B by a piece oftape242B.
• FIG. 31 is an isometric view of the assembled[0148]support structure209. The mountingplate210 preferably includestabs246 extending from opposite sides thereof for securing thestructure209 to the housing of the heat-exchanging module. Referring again to FIG. 28, thehousing208 preferably includes slots for receiving the tabs to hold the mountingplate210 securely in place. Alternatively, the mountingplate210 may be attached to thehousing208 using, e.g., adhesive bonding, screws, bolts, clamps, or any other conventional means of attachment.
• Referring again to FIG. 29, the[0149]support structure209 preferably holds theplates190A,190B so that their inner surfaces are angled very slightly towards each other. In the preferred embodiment, each of the spacing posts230A,230B has awall244 that is slightly tapered so that when theplates190A,190B are pressed against opposite sides of the wall, the inner surfaces of the plates are angled slightly towards each other. As best shown in FIG. 23, the inner surfaces of theplates190A,190B angle towards each other to form a slightly V-shaped slot into which thechamber42 is inserted. The amount by which the inner surfaces are angled towards each other is very slight, preferably about 1° from parallel. The surfaces are angled towards each other so that, prior to the insertion of thechamber42 between theplates190A,190B, the bottoms of the plates are slightly closer to each other than the tops. This slight angling of the inner surfaces enables thechamber42 of the vessel to be inserted between the plates and withdrawn from the plates more easily. Alternatively, the inner surfaces of theplates190A,190B could be held parallel to each other, but insertion and removal of thevessel40 would be more difficult.
• In addition, the inner surfaces of the[0150]plates190A,190B are preferably spaced from each other a distance equal to the thickness of theframe46. In embodiments in which the inner surfaces are angled towards each other, the centers of the inner surfaces are preferably spaced a distance equal to the thickness of theframe46 and the bottoms of the plates are initially spaced a distance that is slightly less than the thickness of theframe46. When thechamber42 is inserted between theplates190A,190B, therigid frame46 forces the bottom portions of the plates apart so that thechamber42 is firmly sandwiched between the plates. The distance that theplates190A,190B are wedged apart by theframe46 is usually very small, e.g., about 0.035 mm if the thickness of the frame is 1 mm and the inner surfaces are angled towards each other by 1°.
• Referring again to FIG. 30, the retention clips[0151]236A,236B should be sufficiently flexible to accommodate this slight outward movement of theplates190A,190B, yet sufficiently stiff to hold the plates within the recesses in the spacing posts230A,230B during insertion and removal of the vessel. The wedging of the vessel between theplates190A,190B provides an initial preload against the chamber and ensures that the flexible major walls of the chamber, when pressurized, establish good thermal contact with the inner surfaces of the plates.
• Referring again to FIG. 28, to limit the amount that the[0152]plates190 can spread apart due to the pressurization of thevessel40, stops may be molded into the housings ofoptics assemblies216,218. As shown in FIG. 32, thehousing249 of theoptics assembly218 includes claw-like stops247A,247B that extend outwardly from the housing. As shown in FIG. 33, thehousing249 is positioned such that the bottom edges of theplates190A,190B are inserted between thestops247A,247B. Thestops247A,247B thus prevent theplates190A,190B from spreading farther than a predetermined maximum distance from each other. Although not shown in FIG. 33 for illustrative clarity, the optics assembly216 (see FIG. 28) has a housing with corresponding stops for preventing the other halves of the plates from spreading farther than the predetermined maximum distance from each other. Referring again to FIG. 23, the maximum distance that stops permit the inner surfaces of theplates190A,190B to be spaced from each other should closely match the thickness of theframe46. Preferably, the maximum spacing of the inner surfaces of theplates190A,190B is slightly larger than the thickness of theframe46 to accommodate tolerance variations in thevessel40 andplates190A,190B. For example, the maximum spacing is preferably about 0.1 to 0.3 mm greater than the thickness of theframe46.
• FIG. 35 is a schematic, block diagram of the electronic components of the heat-exchanging[0153]module147. The module includes aconnector224 or flex cable for connection to the main logic board of the instrument. The module also includesheater plates190A,190B each having a resistive heating element as described above. Theplates190A,190B are wired in parallel to receivepower input253 from the instrument. Theplates190A,190B also includetemperature sensors192A,192B that output analog temperature signals to an analog-to-digital converter264. Theconverter264 converts the analog signals to digital signals and routes them to the microcontroller in the instrument through theconnector224.
• The heat-exchanging module also includes a cooling system, such as a[0154]fan212, for cooling theplates190A,190B and the reaction mixture contained in the vessel inserted between the plates. Thefan212 is activated by switching apower switch272, which is in turn controlled by acontrol logic block270 that receives control signals from the microcontroller. The module further includes four light sources, such asLEDs200, for excitation of labeled analytes in the reaction mixture and fourdetectors198, preferably photodiodes, for detecting fluorescent emissions from the reaction mixture. The module also includes an adjustablecurrent source255 for supplying a variable amount of current (e.g., in the range of 0 to 30 mA) to each LED to vary the brightness of the LED. A digital-to-analog converter260 is connected between the adjustablecurrent source255 and the microcontroller to permit the microcontroller to adjust the current source digitally.
• The adjustable[0155]current source255 is preferably used to ensure that each LED has about the same brightness when activated. Due to manufacturing variances, many LEDs have different brightnesses when provided with the same amount of current. Therefore, it is presently preferred to test the brightness of each LED during manufacture of the heat-exchanging module and to store calibration data in amemory268 of the module. The calibration data indicates the correct amount of current to provide to each LED. The microcontroller reads the calibration data from thememory268 and controls thecurrent source255 accordingly.
• The module additionally includes a signal conditioning/gain select/offset adjust[0156]block262 comprised of amplifiers, switches, electronic filters, and a digital-to-analog converter. Theblock262 adjusts the signals from thedetectors198 to increase gain, offset, and reduce noise. The microcontroller controls block262 through adigital output register266. Theoutput register266 receives data from the microcontroller and outputs control voltages to theblock262. Theblock262 outputs the adjusted detector signals to the microcontroller through the analog-to-digital converter264 and theconnector224. The module also includes thememory268, preferably a serial EEPROM, for storing data specific to the module, such as calibration data for theLEDs200,thermal plates190A,190B, andtemperature sensors192A,192B.
• The operation of the cartridge and instrument will now be described. As shown in FIG. 3, a fluid sample to be analyzed is added to the[0157]sample chamber65 through thesample port64 and thecap30 screwed into theport64 to seal the port shut. Referring to FIG. 10, thecartridge20 is then placed into thecartridge nest141 of theinstrument140 for processing. All valves in the cartridge are initially closed when the cartridge is placed into theinstrument140. When the cartridge is placed in the instrument, thetransducer92 contacts an external surface of theflexible gasket63 forming the bottom wall of the lysingchamber86 , as shown in FIG. 5.
• Referring again to FIG. 10, the[0158]instrument140 is preferably computer-controlled to perform the functions described in the following section, e.g., opening and closing valves in the cartridge usingvalve actuators142, providing pressure to the cartridge throughnozzles145, activating thetransducer92, sensing liquid presence or liquid levels usingoptical sensors143 and144, and controlling the heat-exchanging andoptical detection module147. A programmer having ordinary skill in the art will be able to program a microcontroller and/or computer to perform these functions based upon the following description.
• Referring to FIG. 9, liquids are preferably forced to flow through the cartridge using differential pressure. Although positive pressure is described herein, negative pressure (vacuum) may also be used to control fluid flow in the cartridge. The maximum amount of positive pressure that can be applied is usually limited by the hydrophobic membranes which may reach liquid break-through pressure above[0159]30 pounds per square inch (psi). The lower limit of pressure is limited by the need to move sample and other fluids through the cartridge sufficiently quickly to meet assay goals. Below 1 psi, for example, sample may not flow efficiently through thefilter stack87. Pressure in the range of 6 to 20 psi is generally adequate. The sample flow rate through the cartridge is preferably in the range of 10 to 30 ml/minute. The wash flow rate may be slower, e.g. 6 to 18 ml/minute so that the wash effectively washes the lysingchamber86.
• A specific protocol will now be described with reference to FIG. 9 to illustrate the operation of the cartridge. It is to be understood that this is merely an example of one possible protocol and is not intended to limit the scope of the invention. To begin, the cartridge is preferably primed with wash solution from the[0160]wash chamber66 before the fluid sample is forced to flow from thesample chamber65. To prime the cartridge,valves111 and115 are opened and a pressure of 10 psi is applied to thechamber66 through thepressure port116 for about two seconds. A small portion of the wash solution flows through thechannels117 and106, through the lysingchamber86, through thechannels109 and110, into theU-shaped channel122, and all the way to the hydrophobic membrane below thepressure port128.
• Following priming,[0161]valve115 andpressure port116 are closed andvalves107 and114 are opened. At the same time, a pressure of 20 psi is applied to thesample chamber65 through thepressure port105 for about 15 seconds to force the sample to flow through thechannel106, through thefilter stack87 in thechamber87, through thechannels110,111,112 and into the ventedwaste chamber68. As the sample passes thedetection region136 in thechannel106, the reflective optical sensor144 (FIG. 13) may be used to determine when thesample chamber65 has been emptied. As the sample liquid flows through thefilter stack87, target cells or viruses in the sample are captured. When a predetermined volume of sample reaches thewaste chamber68, some of the liquid spills over into thesensor chamber120, triggering the next step in the protocol. Alternatively, instead of using feedback from optical sensors to trigger events, the steps in a predetermined protocol may simply be timed, e.g., applying predetermined pressures for predetermined durations of time to move known volumes of fluid at known flow rates.
• The flow-through design of the lysing[0162]chamber86 permits target cells or viruses from a relatively large sample volume to be concentrated into a much smaller volume for amplification and detection. This is important for the detection of low concentration analyte in the sample, such as nucleic acid. In particular, the ratio of the volume of the sample forced to flow through the lysingchamber86 to the volume capacity of thechamber86 is preferably at least 2:1, and more preferably at least 5:1. The volume of sample forced to flow through thechamber86 is preferably at least 100 μl, and more preferably at least 1 ml. In the presently preferred embodiment, a sample volume of 5 ml is forced to flow through the lysingchamber86, and thechamber86 has a volume capacity of about 0.5 ml, so that the ratio is 10:1. In addition, the lysingchamber86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) as the sample is forced to flow through the chamber. Sonicating thechamber86 helps to prevent clogging of thefilter stack87, providing for more uniform flow through thechamber86. In particular, the sound waves help keep particulate matter or the beads in the filter stack from clogging one or more filters.
• In the next step,[0163]valves111,114,115 are opened and a pressure of 20 psi is applied to thewash chamber66 for about seven seconds to force the wash solution to flow through thechannels117 and106 into the lysingchamber86. The washing solution washes away PCR inhibitors and contaminants from the lysingchamber86 and carries then through thechannels109,110, and112 into thewaste chamber68. A variety of suitable wash solutions of varying pH, solvent composition, and ionic strength may be used for this purpose and are well known in the art. For example, a suitable washing reagent is a solution of 80 mM potassium acetate, 8.3 mM Tris-HCl, pH 7.5, 40 uM EDTA, and 55% ethanol. The lysingchamber86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) while the wash solution is forced to flow through the chamber. Sonicating thechamber86 helps to prevent clogging of thefilter stack87, providing for more uniform flow through thechamber86 as previously described. In addition, the sound waves may help loosen the material to be washed away. When the incremental volume of wash solution reaches thewaste chamber68, some of the liquid spills over into thesensor chamber121, triggering the next step in the protocol.
• In the next step,[0164]valve115 is closed andvalve119 is opened while a pressure of 15 psi is applied to thereagent chamber67 through thepressure port118 for about three seconds. The pressure forces lysing reagent to flow from thechamber67 through thechannels117,106 into the lysingchamber86, and into thechannel110. Thechamber86 is thus filled with liquid. Suitable lysing reagents include, e.g., solutions containing a chaotropic salt, such as guanidine HCl, guanidine thiocyanate, guanidine isothiocyanate, sodium iodide, urea, sodium perchlorate, and potassium bromide. In the presently preferred embodiment, a lysing reagent that is not inhibitory to PCR is used. The lysing reagent comprises 10 mM tris, 5% tween-20, 1 mM tris (2-carboxyethyl phosphine hydrochloride), 0.1 mM Ethylene Glycol-bis (b-amino-ethyl ether)—N,N,N′,N′—tetracetic acid. After the lysingchamber86 is filled with lysing reagent, thevalves111,114 are closed.Valve119 remains open and a pressure of 20 psi is applied topressure port118. The static pressure in thelysis chamber86 is therefore increased to 20 psi in preparation for the lysis of the cells or viruses trapped in thefilter stack87.
• Referring again to FIG. 5, the pressurization of the lysing[0165]chamber86 is important because it ensures effective coupling between thetransducer92 and theflexible wall63 of the lysingchamber86. To disrupt the cells or viruses in thechamber86, thetransducer92 is activated (i.e., set into vibratory motion). Theflexible wall63 of the lysingchamber86 transfers the vibratory motion of thetransducer92 to the liquid in thechamber86 by allowing slight deflections without creating high stresses in the wall. Thewall63 may be formed by the elastomeric membrane as previously described. Alternatively, the wall may be a film or sheet of polymeric material (e.g., a polypropylene film) preferably having a thickness in the range of 0.025 to 0.1 mm. Thetransducer92 is preferably an ultrasonic horn for sonicating thechamber86. Thechamber86 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 60 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 47 kHz. The amplitude of the horn tip is preferably in the range of 20 to 25 μm (measured peak to peak).
• As the tip of the[0166]transducer92 vibrates, it repeatedly impacts theflexible wall63. On its forward stroke (in the upward direction in FIG. 6), the tip of thetransducer92 pushes thewall63 and creates a pressure pulse or pressure wave in thechamber86. On its retreating stroke (downward in FIG. 5), the tip of thetransducer92 usually separates from theflexible wall63 because theflexible wall63 cannot move at the same frequency as the transducer. On its next forward stroke, the tip of thetransducer92 once again impacts thewall63 in a head-on collision as the tip and wall speed towards each other. Because thetransducer92 and thewall63 separate as thetransducer92 vibrates, the effective forward stroke of the transducer is less than its peak-to-peak amplitude. The effective forward stroke determines the level of sonication in thechamber86. It is therefore important to increase the static pressure in the lysingchamber86 so that when the tip of thetransducer92 retreats, theflexible wall63 is forced outwardly to meet the tip on its return stroke. The static pressure in thechamber86 should be sufficient to ensure that the effective forward stroke of thetransducer92 generates pressure pulses or pressure waves in thechamber86. It is presently preferred to increase the static pressure in thechamber86 to at least 5 psi above the ambient pressure external to the cartridge, and more preferably to a pressure in the range of 15 to 25 psi above the ambient pressure.
• On each forward stroke, the[0167]transducer92 imparts a velocity to the liquid in thechamber86, thus creating a pressure pulse that quickly sweeps across thechamber86. The beads in the filter stack87 (FIG. 6) are agitated by the pressure pulses in thechamber86. The pressure pulses propel the beads into violent motion in thechamber86, and the beads mechanically rupture the cells or viruses to release the material (e.g., nucleic acid) therefrom. It should be noted that some types of cells, such as blood cells, are relatively weak and may be disrupted using only pressure pulses without the use of beads. Other types of cells (particularly spores) have highly resistant cell walls and beads are generally required for effective lysis.
• Referring again to FIG. 9, following disruption of the cells or viruses,[0168]valves111,124 are opened and a pressure of 12 psi is delivered for about 4 seconds to thereagent chamber67 through thepressure port118. The pressure forces the lysis reagent to elute the nucleic acid from thefilter stack87 and to flow with the nucleic acid into theneutralization chamber70. The lysingchamber86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) while the eluting the nucleic acid. Sonicating thechamber86 may help prevent clogging of thefilter stack87, as previously described. The chamber420 is partially filled (e.g., half-filled) with neutralizer, such as detergent, for neutralizing the lysing reagent. If a lysing reagent non-inhibitory to PCR is used, the neutralizer is optional.
• In the next step, the[0169]valve124 is closed to hold the lysing reagent, analyte, and neutralizer in thechamber70. Thevalve114 is opened and a pressure of 15 psi is applied for about three seconds through thepressure port128 to force any liquid in theU-shaped channel122 to flow into thewaste chamber68. Next,valves124 and126 are opened and a pressure of 15 psi is applied for about five seconds through thepressure port123 on top of theneutralizer chamber70. The pressure forces the neutralized lysing reagent and nucleic acid in thechamber70 to flow into thechannel122 and into themaster mix chamber71. Thevalve126 to themaster mix chamber71 is then closed. The master mix chamber contains PCR reagents and fluorescent probes that mix with the neutralized lysing reagent and nucleic acid to form a reaction mixture.
• In the next step, the[0170]channel122 is cleared by openingvalve114 to wastechamber68 and applying a pressure of 15 psi for about one second to pressureport128. In the next step, the reaction mixture formed in themaster mix chamber71 is moved into thereaction vessel40 as follows.Valves126,127, and133 are opened and a pressure of 15 psi is applied for about six seconds to thepressure port125 on top of themaster mix chamber71 to force the reaction mixture to flow through thechannel122,valve127, andchannel80 into thereaction vessel40 through theport41. The reaction mixture fills thechamber42 of the vessel, displacing air in the chamber which exits through theoutlet channel52. The air escaping through theoutlet channel52 travels inchannel81past sensor region130 and intochannel131. Fromchannel131, the air flows intochannel132, throughvalve133,channel134, and exits the cartridge through thevent36. When a volume of reaction mixture sufficient to fill thechamber42 has flowed into the vessel, excess reaction mixture exits the vessel through theoutlet channel52. The excess reaction mixture flows intochannel81 and is optically detected in thesensor region130. When the reaction mixture is detected,valve133 is closed while pressure from thepressure port125 is applied to pressurize thereaction chamber42.
• Referring again to FIG. 23, the pressurization of the[0171]chamber42 expands the flexiblemajor walls48 of the vessel. In particular the pressure forces themajor walls48 to contact and conform to the inner surfaces of theplates190A,190B. This ensures optimal thermal conductance between theplates190A,190B and the reaction mixture in thechamber42. It is presently preferred to pressurize thechamber42 to a pressure in the range of 2 to 30 psi above ambient pressure. This range is presently preferred because 2 psi is generally enough pressure to ensure conformity between thewalls48 and the surfaces of theplates190A,190B, while pressures above 30 psi may cause bursting of thewalls48, deformation of theframe46 orplates190A,190B, or bursting of the hydrophobic membranes in the cartridge. More preferably, thechamber42 is pressurized to a pressure in the range of 8 to 15 psi above ambient pressure. This range is more preferred because it is safely within the practical limits described above. When thechamber42 is pressurized, the reaction mixture in thevessel40 is thermally processed and optically interrogated to determine the presence or absence of a target analyte in the mixture.
• Referring again to FIG. 35, the reaction mixture is thermally processed between the[0172]plates190A,190B using standard proportional-integral-derivative (PID) control using target temperatures and feedback signals from thetemperature sensors192A,192B. Proportioning may be accomplished either by varying the ratio of “on” time to “off” time, or, preferably with proportional analog outputs which decrease the average power being supplied either to the heating elements on theplates190A,190B or to thefan212 as the actual temperature of theplates190A,190B approaches the desired set point temperature. PID control combines the proportional mode with an automatic reset function (integrating the deviation signal with respect to time) and rate action (summing the integral and deviation signal to shift the proportional band). Standard PID control is well known in the art and need not be described further herein. Alternatively, the reaction mixture may be thermally processed using a modified version of PID control described in International Publication Number WO 99/48608 (Application Number PCT/US99/06628) the disclosure of which is incorporated by reference herein.
• As the reaction mixture is thermally cycled between the[0173]heater plates190A,190B to amplify one or more target nucleic acid sequences in the mixture, the mixture is optically interrogated, preferably at the lowest temperature point in each cycle. Optical interrogation is accomplished by sequentially activating each of theLEDs200 to excite different fluorescently-labeled analytes in the mixture and by detecting light emitted (fluorescent output) from thechamber42 using detectors the198. Referring again to FIG. 22, excitation beams are preferably transmitted to thechamber42 through the opticallytransmissive side wall57A, while fluorescent emission is detected through theside wall57B.
• One advantage of the cartridge of the present invention is that it allows the intracellular material from a relatively large volume of fluid sample, e.g. several milliliters or more, to be separated from the sample and concentrated into a much smaller volume of reaction fluid, e.g., 100 μL or less. The cartridge permits extraordinary concentration factors by efficiently extracting material from milliliter quantities of fluid sample. In particular, the[0174]sample chamber65 preferably has a volume capacity in the range of 100 μl to 12 ml. More preferably, thesample chamber65 has a volume capacity of at least 1 ml. The lower limit of 1 ml is preferred because at least 1 ml of sample should be analyzed to detect low concentration analytes such as nucleic acid. The upper limit of 12 ml is preferred because a sample volume greater than 12 ml would require a much larger cartridge and likely clog the filter stack. In the presently preferred embodiment, the sample chamber has a volume capacity of 5.5 ml for holding 5 ml of sample.
• The[0175]wash chamber66 has a volume capacity proportional to the volume of the lysingchamber86. In particular, thewash chamber66 preferably holds a volume of wash that is at least one to two times the volume of the lysingchamber86 to ensure that there is enough wash solution to wash out PCR inhibitors and debris from thechamber86. In the presently preferred embodiment, the volume of the lysingchamber86 is about 0.5 ml and the volume of thewash chamber66 is 2.5 ml for holding 2 ml of wash solution. The lysing chamber volume of 0.5 ml is a compromise between a size large enough to avoid clogging of thefilter stack87 and a size small enough to concentrate analyte into a small volume for improved amplification and detection.
• The[0176]reagent chamber67 preferably holds a volume of lysing reagent that is at least one to two times the volume of the lysingchamber86 so that there is sufficient lysing reagent to pressurize the chamber and to elute nucleic acid from the chamber. In the presently preferred embodiment, thechamber67 has a volume capacity of 1.5 ml for holding about 1 to 1.5 ml of lysing reagent. Thewaste chamber68 has a volume capacity sufficient to hold the sample, wash solution, and unused lysing reagent. Thewaste chamber68 is sized at 9.5 ml volume capacity in the preferred embodiment.
• The size of the[0177]neutralization chamber70 is dependent upon the volume of the lysingchamber86 since the neutralizer in thechamber70 neutralizes the volume of lysing reagent that fills the lysingchamber86. It is currently preferred that the lysing chamber have a volume if 0.5 ml, so thechamber70 has a volume capacity of 10.0 ml for holding about 0.5 ml of neutralizer that is mixed with 0.5 ml of the lysing reagent and eluted analyte. The volume capacity of themaster mix chamber71 should be sufficient to produce a reaction mixture to fill thevessel40 and thechannels122,127 leading to the vessel. In the presently preferred embodiment, the master mix chamber has a volume capacity of 200 μl for holding an initial load of 100 μl of master mix to which is added 100 μl of neutralized lysing reagent and eluted analyte to form the reaction mixture.
• The flow channels in the cartridge are generally D-shaped in cross section (with the[0178]gasket63 forming the flat side of the channel) and preferably have a width or diameter in the range of {fraction (1/64)} to ⅛ of an inch (0.4 to 3.2 mm), and more preferably a width of {fraction (1/32)} to {fraction (1/16)} of an inch (0.8 to 1.6 mm). These ranges are presently preferred to avoid having channels to narrow (which creates flow restriction) and to avoid having channels too wide (which yields unused volumes of liquid sitting in the flow path).
• Many modifications to the structure and operation of the cartridge and instrument are possible in alternative embodiments. For example, although amplification by PCR is presently preferred, the cartridge and instrument may be used to amplify nucleic acid sequences using any amplification method, including both thermal cycling amplification methods and isothermal amplification methods. Suitable thermal cycling methods include, but are not limited to, the Polymerase Chain Reaction (PCR; U.S Pat. Nos. 4,683,202, 4,683,195 and 4,965,188); Reverse Transcriptase PCR (RT-PCR); DNA Ligase Chain Reaction (LCR; International Patent Application No. WO 89/09835); and transcription-based amplification (D. Y. Kwoh et al. 1989, Proc. Natl.[0179]Acad. Sci. USA 86, 1173-1177). Suitable isothermal amplification methods useful in the practice of the present invention include, but are not limited to, Rolling Circle Amplification; Strand Displacement Amplification (SDA; Walker et al. 1992, Proc. Nati. Acad. Sci. USA 89, 392-396); Q-.beta. replicase (Lizardi et al. 1988, Bio/Technology 6, 1197-1202); Nucleic Acid-Based Sequence Amplification (NASBA; R. Sooknanan and L. Malek 1995, Bio/Technology 13, 563-65); and Self-Sustained Sequence Replication (3SR; Guatelli et al. 1990, Proc. Nati.Acad. Sci. USA 87, 1874-1878).
• Moreover, the cartridge and instrument may be used to conduct chemical reactions other than nucleic acid amplification. Further, although fluorescence excitation and emission detection is preferred, optical detection methods such as those used in direct absorption and/or transmission with on-axis geometries may also be used to detect analyte in the cartridge. Another possible detection method is time decay fluorescence. Additionally, the cartridge is not limited to detection based upon fluorescent labels. For example, detection may be based upon phosphorescent labels, chemiluminescent labels, or electrochemiluminescent labels.[0180]
• A fluid sample may be introduced into the cartridge by a variety of means, manual or automated. For manual addition, a measured volume of material may be placed into a receiving area of the cartridge through an input port and a cap is then placed over the port. Alternatively, a greater amount of sample material than required for the analysis can be added to the cartridge and mechanisms within the cartridge can effect the precise measuring and aliquoting of the sample needed for the specified protocol. It may be desirable to place certain samples, such as tissue biopsy material, soil, feces, exudates, and other complex material into another device or accessory and then place the secondary device or accessory into the cartridge. For example, a piece of tissue may be placed into the lumen of a secondary device that serves as the cap to the input port of the cartridge. When the cap is pressed into the port, the tissue is forced through a mesh that slices or otherwise divides the tissue.[0181]
• For automated sample introduction, additional design features of the cartridge are employed and, in many cases, impart specimen accession functionality directly into the cartridge. With certain samples, such as those presenting a risk of hazard to the operator or the environment, such as human retrovirus pathogens, the transfer of the sample to the cartridge may pose a risk. Thus, in one embodiment, a syringe may be integrated into a device to provide a means for moving external fluidic samples directly into the cartridge. Alternatively, a venous puncture needle and an evacuated blood tube can be attached to the cartridge forming an assembly that can be used to acquire a sample of blood. After collection, the tube and needle are removed and discarded, and the cartridge is then placed in an instrument to effect processing. The advantage of such an approach is that the operator or the environment is not exposed to pathogens.[0182]
• The input port can be designed with a consideration of appropriate human factors as a function of the nature of the intended specimen. For example, respiratory specimens may be acquired from the lower respiratory tract as expectorants from coughing, or as swab or brush samples from the back of the throat or the nares. In the former case, the input port can be designed to allow the patient to cough directly into the cartridge or to otherwise facilitate spitting of the expectorated sample into the cartridge. For brush or swab specimens, the specimen is placed into the input port where features of the port and closure facilitate the breaking off and retaining of the end of the swab or brush in the cartridge receiving area.[0183]
• In another embodiment, the cartridge includes input and output tubes that may be positioned in a sample pool of very large volume, such as a flowing stream of water, so that the sample material flows through the cartridge. Alternatively, a hydrophilic wicking material can serve as an interactive region so that the entire cartridge can be immersed directly into the specimen, and a sufficient amount of specimen is absorbed into the wicking material. The cartridge is then removed, and can be transported to the laboratory or analyzed directly using a portable instrument. In another embodiment, tubing can be utilized so that one end of the tube is in direct communication with the cartridge to provide a fluidic interface with at least one interactive region and the other end is accessible to the external environment to serve as a receiver for sample. The tube can then be placed into a specimen and serve as a sipper. The cartridge itself may also serve as the actual specimen collection device, thereby reducing handling and inconvenience. In the case of specimens involved in legal disputes or criminal investigations, the direct accessing of the test material into the fluidic cartridge is advantageous because the chain of custody is conveniently and reliably preserved.[0184]
• Referring again to FIG. 9, reagents may be exogenously introduced into the cartridge before use, e.g., through sealable openings in the[0185]reagent chamber67,neutralizer chamber70, andmaster mix chamber71. Alternatively, the reagents may be placed in the cartridge during manufacture, e.g., as aqueous solutions or dried reagents requiring reconstitution. The particular format is selected based on a variety of parameters, including whether the interaction is solution-phase or solid-phase, the inherent thermal stability of the reagent, speed of reconstitution, and reaction kinetics. Reagents containing compounds that are thermally unstable when in solution can be stabilized by drying using techniques such as lyophilization. Additives, such as simple alcohol sugars, methylcelluloses, and bulking proteins may be added to the reagent before drying to increase stability or reconstitutability.
• Referring again to FIG. 21, the[0186]reaction vessel40 does not require two flexible sheets forming opposingmajor walls48 of thereaction chamber42. For example, in one alternative embodiment, thevessel40 has only one flexible sheet forming a major wall of the chamber. Therigid frame46 defines the other major wall of the chamber, as well as the side walls of the chamber. In this embodiment, the major wall formed by theframe46 should have a minimum thickness of about 0.05 inches (1.25 mm) which is typically the practical minimum thickness for injection molding, while the flexible sheet may be as thin as 0.0005 inches (0.0125 mm). The advantage to this embodiment is that the manufacturing of thereaction vessel40 is simplified, and hence less expensive, since only one flexible sheet need be attached to theframe46. The disadvantage is that the heating and cooling rates of the reaction mixture are likely to be slower since the major wall formed by theframe46 will probably not permit as high a rate of heat transfer as the thin, flexible sheet.
• Referring to FIG. 28, the heat-exchanging[0187]module147 only requires one thermal surface for contacting a flexible wall of thereaction vessel40 and one thermal element for heating and/or cooling the thermal surface. The advantage to using one thermal surface and one thermal element is that the apparatus may be manufactured less expensively. The disadvantage is that the heating and cooling rates are likely to be about twice as slow. Further, although it is presently preferred that the thermal surfaces be formed by the thermallyconductive plates190, each thermal surface may be provided by any rigid structure having a contact area for contacting a wall of thevessel40. The thermal surface preferably comprises a material having a high thermal conductivity, such as ceramic or metal. Moreover, the thermal surface may comprise the surface of the thermal element itself. For example, the thermal surface may be the surface of a thermoelectric device that contacts the wall to heat and/or cool the chamber.
• It is presently preferred to build the transducer into the[0188]instrument140. In another embodiment, however, the transducer may be built into the cartridge. For example, a piezoelectric disk may be built into the cartridge for sonicating the lysing chamber. Alternatively, a speaker or electromagnetic coil device may be built into the cartridge. In these embodiments, the cartridge includes suitable electrical connectors for connecting the transducer to a power supply. In embodiments in which the transducer is built into the cartridge, the transducer should be prevented from contacting the fluid sample directly, e.g., the transducer should be laminated or separated from the sample by a chamber wall. Further, lysis of the cells or viruses may be performed using a heater in place of or in combination with a transducer. The heater may be a resistive heating element that is part of cartridge, or the heater could be built into the instrument that receives the cartridge. In this embodiment, the cells or viruses are disrupted by heating the lysis chamber to a high temperature (e.g., 95° C.) to disrupt the cell walls.
• FIGS. 36-46 show another[0189]apparatus350 for disrupting cells or viruses according to the present invention. FIG. 36 shows an isometric view of theapparatus350, and FIG. 37 shows a cross sectional view of theapparatus350. As shown in FIGS. 36-37, theapparatus350 includes a cartridge orcontainer358 having achamber367 for holding the cells or viruses. The container includes aflexible wall440 defining thechamber367. In this embodiment, theflexible wall440 is the bottom wall of thechamber367. Theflexible wall440 is preferably a sheet or film of polymeric material (e.g., a polypropylene film) and thewall440 preferably has a thickness in the range of 0.025 to 0.1 mm. Theapparatus350 also includes atransducer314, such as an ultrasonic horn, for contacting an external surface of the flexible wall440 (i.e., a surface of thewall440 that is external to the chamber367). Thetransducer314 should be capable of vibratory motion sufficient to create pressure pulses in thechamber367. Suitable transducers include ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducers. The transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device.
• The[0190]apparatus350 further includes asupport structure352 for holding thecontainer358 and thetransducer314 against each other such that thetransducer314 contacts thewall440 of thechamber367 and for applying a substantially constant force to thecontainer358 or to thetransducer314 to press together thetransducer314 and thewall440 of the chamber. Thesupport structure352 includes abase structure354 having astand356. Thetransducer314 is slidably mounted to thebase structure354 by aguide364. Theguide364 is either integrally formed with thebase structure354 or fixedly attached to the base structure. Thesupport structure352 also includes aholder360 attached to thebase structure354 for holding thecontainer358. Theholder360 has a U-shaped bottom portion providing access to theflexible wall440 of thechamber367. Theguide364 and theholder360 are arranged to hold thetransducer314 and thecontainer358, respectively, such that the external surface of thewall440 contacts thetransducer314. Thesupport structure352 also includes atop retainer362 for thecontainer358. Theretainer362 is U-shaped to allow access to anexit port444 formed in thecontainer358.
• The[0191]support structure352 further includes an elastic body, such as aspring366, for applying a force to thetransducer314 to press thetransducer314 against thewall440. When thetransducer314 is in contact with thewall440, the force provided by the spring.366 is constant, providing for consistent coupling between thetransducer314 and thewall440. Thespring366 is positioned between a spring guide.372 and the base of acoupler368 that supports the bottom of thetransducer314. As shown in FIG. 36, thecoupler370 preferably has awindow370 through which the power cord (not shown) of thetransducer314 may be placed. Bolts or screws376 hold thespring guide372 inadjustment grooves374 formed in thebase structure354. The magnitude of the force provided by thespring366 may be adjusted by changing the preload on the spring. To adjust the preload on thespring366, thebolts376 holding thespring guide372 are loosened, theguide372 is moved to a new position, and thebolts376 are retightened to hold theguide372 in the new position. Once the preload on thespring366 is adjusted to provide a suitable coupling force between thetransducer314 and thewall440, it is desirable to keep the preload constant from one use of theapparatus350 to the next so that valid comparisons can be made between different samples disrupted by the apparatus.
• The magnitude of the force provided by the[0192]spring366 to press together thetransducer314 and thewall440 is important for achieving a consistent transfer of energy between thetransducer314 and thechamber367. If the force is too light, thetransducer314 will only be held lightly against thewall440, leading to poor translation of vibratory movement from thetransducer314 to thewall440. If the force is too strong, thecontainer358 orwall440 may be damaged during sonication. An intermediate force results in the most consistent and repeatable transfer of vibratory motion from thetransducer314 to thewall440. It is presently preferred that thespring366 provide a force in the range of 2 to 5 lbs.
• FIG. 38 shows an exploded view of the[0193]container358, and FIG. 39 shows an assembled view of thecontainer358. As shown in FIGS. 38-39, thecontainer358 has a body comprising a top piece448, amiddle piece450 , and abottom piece452. Themiddle piece450 defines aninlet port442 to thechamber367, and thetop piece448 defines anoutlet port444 to the chamber. Theports442,444 are positioned to permit the continuous flow of a fluid sample through thechamber367. Theflexible wall440 is held between the middle andbottom pieces450,452 usinggaskets453,454. Alternatively, theflexible wall440 may simply be heat sealed to themiddle piece450 so that thebottom piece452 andgaskets453,454 may be eliminated.
• The[0194]container358 also includes afilter stack446 in thechamber367 for capturing sample components (e.g., target cells or viruses) as the sample flows through thechamber367. The filter stack comprises (from bottom to top in FIGS. 38-39) agasket456, afirst filter458, agasket460, asecond filter464 having a smaller average pore size than thefirst filter458, and agasket466. The filter stack is held between the top andmiddle pieces448,450 of thecontainer358. The filter stack also includesbeads462 disposed between the first andsecond filters458 and464. Thegasket460 spaces thefirst filter458 from thesecond filter464. Thegasket460 should be thick enough to permit the beads to move freely in the space between thefilters458,464. A fluid sample flowing through thechamber367 first flows through thefilter458 and then through thefilter466. After flowing through the filter stack, the sample flows along flow ribs468 (FIG. 38) formed in the portion of thetop piece448 that defines the top of the chamber and through the outlet port444 (FIG. 39).
• The filter stack is effective for capturing cells or viruses as a fluid sample flows through the[0195]chamber367 without clogging of the. The first filter458 (having the largest pore size) filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc. The second filter464 (having a smaller pore size) captures target cells or viruses in the fluid sample. The average pore size of thefirst filter458 is selected to be small enough to filter coarse material from the fluid sample (e.g., salt crystals, cellular debris, hair, tissue) yet large enough to allow the passage of the target cells or viruses. In general, the average pore size of thefirst filter458 should be in the range of about 2 to 25 μm, with a presently preferred pore size of about 5 μm. The average pore size of thesecond filter464 is selected to be slightly smaller than the average size of the target cells or viruses to be captured (typically in the range of 0.2 to 5 μm).
• The[0196]beads462 are useful for disrupting the captured cells or viruses to release the intracellular material (e g., nucleic acid) therefrom. Movement of thebeads462 ruptures the cells or viruses captured on thefilter464. Suitable beads for rupturing cells or viruses include borosilicate glass, lime glass, silica, and polystyrene beads. The beads may be porous or non-porous and preferably have an average diameter in the range of 1 to 200 μm. In the presently preferred embodiment, the beads.462 are polystyrene beads having an average diameter of about 100 μm.
• The[0197]beads462 may have a binding affinity for target cells or viruses in the fluid sample to facilitate capture of the target cells or viruses. For example, antibodies or certain receptors may be coated onto the surface of thebeads462 to bind target cells in the sample. Moreover, thechamber367 may contain two different types of beads for interacting with target cells or viruses. For example, the chamber may contain a first set of beads coated with antibodies or receptors for binding target cells or viruses and a second set of beads (intermixed with the first set) for rupturing the captured cells or viruses. The beads in the chamber may also have a binding affinity for the intracellular material (e.g., nucleic acid) released from the ruptured cells or viruses. Such beads may be useful for isolating target nucleic acid for subsequent elution and analysis. For example, thechamber367 may contain silica beads to isolate DNA or cellulose beads with oligo dT to isolate messenger RNA for RT-PCR. Thechamber367 may also contain beads for removing unwanted material (e.g., proteins, peptides) or chemicals (e.g., salts, metal ions, or detergents) from the sample that might inhibit PCR.
• To ensure that the air bubbles can escape from the[0198]chamber367, it is desirable to use thecontainer358 in an orientation in which liquid flows up (relative to gravity) through thefilters458,464 and thechamber367. The upward flow through thechamber367 aids the flow of air bubbles out of the chamber. Thus, theinlet port442 for entry of fluids into thechamber367 should generally be at a lower elevation than theoutlet port444. The volume capacity of thechamber367 is usually in the range of 50 to 500 μl. The volume capacity of thechamber367 is selected to provide for concentration of analyte separated from a fluid sample without the chamber being so small that thefilters458,464 become clogged.
• The[0199]pieces448,450,452 forming the body of thecontainer358 are preferably molded polymeric parts (e.g., polypropylene, polycarbonate, acrylic, etc.). Although molding is preferred for mass production, it also possible to machine the top, middle, andbottom pieces448,450,452. Thepieces448,450,452 may be held together by screws or fasteners. Alternatively, ultrasonic bonding, solvent bonding, or snap fit designs could be used to assemble thecontainer358. Another method for fabricating thecontainer358 is to mold the body as a single piece and heat seal theflexible wall440 and thefilters458,464 to the body.
• FIG. 40 shows a fluidic system for use with the apparatus. The system includes a[0200]bottle470 for holding lysis buffer, abottle472 containing wash solution, and asample container474 for holding a fluid sample. Thebottles470,472 andsample container474 are connected via tubing to the valve ports of asyringe pump476. The inlet port of thecontainer358 is also connected to thesyringe pump476. The outlet port of thecontainer358 is connected to the common port of adistribution valve478. The system also includes acollection tube480 for receiving intracellular material removed from the sample, awaste container482 for receiving waste, and a pressure source, such as apump484. Thecollection tube480,waste container482, and pump484 are connected to respective peripheral ports of thedistribution valve478. Apressure regulator486 regulates the pressure supplied by thepump484.
• A specific protocol will now be described with reference to FIGS. 39-40 to illustrate the operation of the[0201]container358. It is to be understood that this is merely an example of one possible protocol and is not intended to limit the scope of the invention. Thesyringe pump476 pumps a fluid sample from thesample container474 through thecontainer358 and into thewaste container482. As the fluid sample is forced to flow through the filters in thechamber367, coarse material is filtered by thefilter458 and target cells or viruses in the sample are captured by thefilter464. Thechamber367 may be sonicated as the sample is forced to flow through the chamber to help prevent clogging of the filters. Next, thesyringe pump476 pumps wash solution from thebottle472 through thecontainer358 and into thewaste container482. The washing solution washes away PCR inhibitors and contaminants from thechamber367.
• In the next step, the[0202]syringe pump476 pumps lysis buffer from thebottle470 into thecontainer358 so that thechamber367 is filled with liquid. The lysis buffer should be a medium through which dynamic pressure pulses or pressure waves can be transmitted. For example, the lysis buffer may comprise deionized water for holding the cells or viruses in suspension or solution. Alternatively, the lysis buffer may include one or more lysing agents to aid in the disruption of the cells or viruses. One of the advantages of the present invention, however, is that harsh lysing agents are not required for successful disruption of the cells or viruses. Next, the distribution valve of thesyringe pump476 is closed upstream of thecontainer358, and thedistribution valve478 is opened. Thepump484 then pressurized thechamber367 through theoutlet port444, preferably to about 20 psi above the ambient pressure. Thedistribution valve478 downstream of thecontainer358 is then closed. The static pressure in thechamber367 is therefore increased to about 20 psi in preparation for the disruption of the cells or viruses trapped on thefilter464.
• Referring again to FIG. 37, the pressurization of the[0203]chamber367 is important because it ensures effective coupling between thetransducer314 and theflexible wall440. To disrupt the cells or viruses in thechamber367, thetransducer314 is activated (i.e., set into vibratory motion). Theflexible wall440 transfers the vibrational motion of thetransducer314 to the liquid in thechamber367 by allowing slight deflections without creating high stresses in the wall. Thetransducer314 is preferably an ultrasonic horn for sonicating thechamber367. Thechamber367 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 60 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 40 kHz. The amplitude of the horn tip is preferably in the range of 20 to 25 μm (measured peak to peak).
• As the tip of the[0204]transducer314 vibrates, it repeatedly impacts theflexible wall440. On its forward stroke (in the upward direction in FIG. 37), the tip of thetransducer314 pushes thewall440 and creates a pressure pulse or pressure wave in thechamber367. On its retreating stroke (downward in FIG. 37), the tip of thetransducer314 usually separates from theflexible wall440 because theflexible wall440 cannot move at the same frequency as the transducer. On its next forward stroke, the tip of thetransducer314 once again impacts thewall440 in a head-on collision as the tip and wall speed towards each other. Because thetransducer314 and thewall440 separate as thetransducer314 vibrates, the effective forward stroke of the transducer is less than its peak-to-peak amplitude. The effective forward stroke determines the level of sonication in thechamber367. It is therefore important to increase the static pressure in thechamber367 so that when the tip of thetransducer314 retreats, theflexible wall440 is forced outwardly to meet the tip on its return stroke. The static pressure in thechamber367 should be sufficient to ensure that the effective forward stroke of thetransducer314 generates the necessary pressure pulses or pressure waves in the chamber to effect cell disruption. It is presently preferred to increase the static pressure in thechamber367 to at least 5 psi above the ambient pressure, and more preferably to a pressure in the range of 15 to 25 psi above the ambient pressure.
• On each forward stroke, the[0205]transducer314 imparts a velocity to the liquid in thechamber367, thus creating a pressure pulse or pressure wave that quickly sweeps across the chamber. Thebeads462 in the filter stack446 (FIG. 38) are agitated by the pressure pulses in thechamber367. The pressure pulses propel the beads into violent motion, and the beads mechanically rupture the cells or viruses to release the analyte (e.g., nucleic acid) therefrom. Referring again to FIG. 40, following disruption of the cells or viruses, thesyringe pump476 pumps the released intracellular material from thecontainer358 into thecollection tube480.
• FIG. 41 shows another embodiment of the invention in which the[0206]container358 has asolid wall488 for contacting thetransducer314. Thesolid wall488 differs from theflexible wall440 previously described with reference to FIG. 37. Whereas the flexible wall is typically a thin film that bends under its own weight and does not hold its shape unless held on its edges, thesolid wall488 holds it shape when unsupported. The advantage of using a solid wall to contact thetransducer314 is that there is no need to pressurize thechamber367 to ensure effective coupling between thewall488 and thetransducer314. The elastic restoring force of thesolid wall488 provides the necessary coupling between the wall and thetransducer314. However, the proper design of thesolid wall488 is necessary so that the wall is not damaged (e.g., melted) by the vibratory movements of thetransducer314.
• In particular, the[0207]solid wall488 should have a natural frequency that is higher than the vibrating frequency at which thetransducer314 is operated. Preferably, the ratio of the natural frequency of thewall488 to the vibrating frequency is at least 2:1, and more preferably the ratio is at least 4:1. In addition, thewall488 should not be so rigid that it cannot transfer the vibratory motion of the transducer to the liquid in thechamber367. It is preferred that thewall488 be capable of deflecting a distance in the range of 5 to 40 μm, and more preferably about 20 μm peak to peak when thetransducer314 applies a force in the range of 1 to 10 lbs. to the external surface of thewall488. It is more preferable that thewall488 be capable of deflecting a distance in the range of 5 to 40 μm, and more preferably about 20 μm peak to peak when thetransducer314 applies a force in the range of 2 to 5 lbs. To achieve these criteria, thewall488 is dome-shaped and convex with respect to the transducer314(i.e., thewall488 curves outwardly towards the transducer). The advantage to the dome-shaped design of thewall488 is that the dome shape increases the natural frequency of the wall (compared to a flat wall) without causing the wall to be so stiff that it cannot transfer the vibratory movements of thetransducer314 to thechamber367.
• FIG. 42 shows a cross sectional view of the[0208]wall488. The dome-shapedportion495 of the wall preferably has a radius of curvature R in the range of 6.3 to 12.7 mm when the diameter D of the dome-shaped portion is about 11.1 mm. More preferably, the dome-shapedportion495 of the wall preferably has a radius of curvature R of about 9.5 mm when the diameter D of the dome-shaped portion is about 11.1 mm. Thewall488 also includes a flatouter rim497 for clamping thewall488 in thecontainer358. Alternatively, thewall488 may be integrally molded with either ofpieces450,452 (FIG. 41). The thickness T of the wall is preferably in the range of 0.25 to 1 mm. If it is less than 0.25 mm thick, thewall488 may be too weak. If the wall has a thickness greater than 1 mm, the wall may be too stiff to deflect properly in response to the vibratory movements of the transducer. In the presently preferred embodiment, thewall488 has a thickness T of about 0.5 mm. Thewall488 is preferably a molded plastic part. Suitable materials for thewall488 include Delrin® (acetal resins or polymethylene oxide), polypropylene, or polycarbonate.
• The interaction of the[0209]transducer314 with thesolid wall488 will now be described with reference to FIG. 41. Prior to activating the transducer, target cells or viruses are captured on thefilter490 by forcing a fluid sample to flow though the chamber367 (e.g., using the fluidic system previously described with reference to FIG. 40). In addition, thechamber367 is filled with a liquid (e.g., lysis buffer) as previously described. Unlike the previously described embodiments, however, thechamber367 does not require pressurization. Instead, it is preferred that ambient pressure is maintained in the chamber. Thetransducer314 is placed in contact with the external surface of thewall488, preferably using a support structure as previously described with reference to FIG. 37. In particular, a spring preferably pushes the transducer against thewall488 with a force in the range of 1 to 10 lbs., and more preferably in the range of 2 to 5 lbs.
• To disrupt the cells or viruses in the[0210]chamber367, thetransducer314 is activated (i.e., induced into vibratory motion). As the tip of thetransducer314 vibrates, it deflects thewall488. On its forward stroke (in the upward direction in FIG. 41), the tip of thetransducer314 pushes thewall488 and creates a pressure pulse or pressure wave in thechamber367. On its retreating stroke (downward in FIG. 41), thewall488 remains in contact with the tip of thetransducer314 because thewall488 has a natural frequency higher than the vibrating frequency of the transducer. In embodiments in which the transducer is an ultrasonic horn for sonicating thechamber367, thechamber367 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 40 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 40 kHz. The amplitude of the horn tip is preferably in the range of 20 to 25 μm (measured peak to peak), and the natural frequency of thewall488 should be greater than 40 kHz, preferably at least 80 kHz, and more preferably at least 160 kHz.
• One advantage to using the[0211]solid interface wall488 is that strong pressure drops can be achieved in thechamber367 as long as the static pressure in the chamber is low. For example, at atmospheric pressure, cavitation (the making and breaking of microscopic bubbles) can occur in thechamber367. As these bubbles or cavities grow to resonant size, they collapse violently, producing very high local pressure changes. The pressure changes provide a mechanical shock to the cells or viruses, resulting in their disruption. The disruption of the cells or viruses may also be caused by sharp pressure rises resulting from the vibratory movement of thetransducer314. In addition, the disruption of the cells or viruses may be caused by the violent motion of thebeads462 in thechamber367. The beads are agitated by the dynamic pressure pulses in the chamber and rupture the cells or viruses. In experimental testing, the applicants have found that it is usually necessary to use beads to disrupt certain types of cells (particularly spores) having highly resistant cell walls. Other types of cells, such as blood cells, are easier to disrupt and may often be disrupted without the use of thebeads462.
• Although the use of an ultrasonic transducer has been described as a preferred embodiment, it is to be understood that different types of transducers may be employed in the practice of the present invention. The transducer should be capable of creating pressure pulses or pressure waves in the[0212]chamber367. In addition, the transducer should be capable of providing high velocity impacts to the liquid in the chamber. Suitable transducers include ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducer. The transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device. The vibrating frequency of the transducer may be ultrasonic (i.e., above 20 kHz) or below ultrasonic (e.g., in the range of 60 to 20,000 Hz). The advantage to using higher frequencies is that cell disruption is very rapid and can often be completed in 10 to 20 seconds. The disadvantage is that ultrasonic transducers are often more expensive than a simple mechanical vibrator, e.g., a speaker or electromagnetic coil device. In one alternative embodiment, for example, thesolid wall488 is used in combination with a speaker or electromagnetic coil device that vibrates at an operating frequency in the range of 5 to 10 kHz.
• FIGS. 43A-43B illustrate another[0213]solid wall500 for contacting a transducer according to the present invention. As shown in FIG. 43A, one side of thewall500 has acentral portion502 and a plurality of stiffeningribs504 extending radially from thecentral portion502. The wall also hasrecesses506 formed between theribs504. As shown in FIG. 43B, the other side of thewall500 has aflat surface508. FIG. 44 shows a partially-cut away isometric view of thecontainer358 with thewall500. Thewall500 is preferably positioned so that the side of the wall having the flat surface is internal to thechamber367 and such that the side of the wall having theribs504 is external to the chamber. Theribs504 are advantageous because they increase the natural frequency of the wall without causing the wall to be so stiff that it cannot transfer the vibratory movements of the transducer to thechamber367.
• FIG. 45 shows a bottom plan view of the[0214]container358 having thewall500. Thecentral portion502 provides the external surface of thewall500 for contacting a transducer. The interaction of thewall500 with the transducer is analogous to the interaction of thewall488 with the transducer previously described with reference to FIG. 41. In particular, thewall500 remains in contact with the tip of the transducer because thewall500 has a natural frequency higher than the vibrating frequency of the transducer. Consequently, pressurization is not required, and cavitation may be achieved. Thesolid walls488,500 described with reference to FIGS. 41-45 may be used in thecontainer358 or thewalls488,500 may be used in a fully integrated cartridge, such as the cartridge shown in FIG. 1.
• FIG. 46 shows a partially exploded view of a[0215]container274 for holding cells or viruses to be disrupted according to another embodiment of the invention. FIG. 47 shows a front view of thecontainer274. As shown in FIGS. 46-47, thecontainer274 has achamber277 for holding a liquid containing cells or viruses to be disrupted. Thecontainer274 has arigid frame278 that defines theside walls282A,282B,282C,282D of thechamber277. Therigid frame278 also defines aport276 and achannel288 that connects theport276 to thechamber277. The container also includes thin, flexible sheets attached to opposite sides of therigid frame278 to form two spaced-apart, opposingmajor walls280A,280B of the chamber. The flexiblemajor walls280A,280B are shown in FIG. 46 exploded from therigid frame278 for illustrative clarity. When thecontainer274 is assembled, themajor walls280A,280B are sealed to opposite sides of theframe278, as is described in detail below. Thechamber277 is thus defined by the spaced apart, opposingmajor walls280A,202B and by the rigid sidewalls side walls282A,282B,282C,282D that connect the major walls to each other.
• The[0216]container274 also includes aplunger284 that is inserted into thechannel288 after adding the cells or viruses to thechamber277. Theplunger284 compresses gas in thecontainer274 thereby increasing pressure in thechamber277. The gas compressed by theplunger284 is typically air filling thechannel288. The pressurization of thechamber277 forces theflexible wall280A to conform to the surface of the transducer (not shown in FIGS. 46-47), as is discussed in greater detail below. Theplunger284 also closes theport276 and seals thechamber277 from the environment external to the container.
• In general, the plunger may comprise any device capable of establishing a seal with the walls of the[0217]channel288 and of compressing gas in the container. Such devices include, but are not limited to, pistons, plugs, or stoppers. Theplunger284 of the preferred embodiment includes astem290 and apiston292 on the stem. When theplunger284 is inserted into thechannel288, thepiston292 establishes a seal with the inner walls of the channel and compresses air in the channel. Thepiston292 is preferably a cup integrally formed (e.g., molded) with thestem290. Alternatively, thepiston292 may be a separate elastomeric piece attached to the stem.
• The[0218]plunger284 also preferably includes analignment ring294 encircling the stem for maintaining theplunger284 in coaxial alignment with thechannel288 as the plunger is inserted into the channel. Thealignment ring294 is preferably integrally formed (e.g., molded) with thestem290. Thestem290 may optionally includessupport ribs293 for stiffening and strengthening the stem. Theplunger284 also includes aplunger cap296 attached to thestem290. As shown in FIG. 47, thecap296 includes asnap ring297 and the container includes anannular recess279 encircling theport276 for receiving thesnap ring297. Thecap296 may optionally include alever portion298 which is lifted to remove theplunger284 from thechannel288. Thecontainer274 may also include finger grips287 for manual handling of the container.
• FIG. 51 shows an isometric view of an[0219]apparatus304 for disrupting cells or viruses. Theapparatus304 includes atransducer314, preferably an ultrasonic horn, for generating pressure pulses the chamber of thecontainer274. Theapparatus304 also includes asupport structure306 for holding thetransducer314 and thecontainer274 against each other. Thesupport structure306 includes abase308 and afirst holder310 attached to the base for holding the outer housing of thetransducer314. Theholder310 includes a bore for receiving thetransducer314 and screws orbolts312 that are tightened to clamp the outer housing of the horn firmly in the holder. The base308 may optionally include bolt holes320 for bolting thesupport structure306 to a surface, e.g., a counter or bench top.
• As shown in FIG.52, the[0220]support structure306 also includes aholder316 for holding thecontainer274. Theholder316 is slidably mounted to thebase308 by means of aguide318. Theguide318 may be fixedly attached to the base308 or integrally formed with the base. Theguide318 has twoguide pins322, and theholder316 has twoguide slots324 for receiving the guide pins322. Theholder316 may thus slide on the guide pins322. As shown in the partially cut-away view of FIG. 53, theholder316 is designed to hold thecontainer274 such that the external surface of theflexible wall280A is exposed and accessible to thetip326 of thetransducer314. Theguide318 is appropriately aligned with thetransducer314 to slide theholder316 into a position in which the external surface of theflexible wall280A contacts thetip326.
• FIG. 54 shows an isometric view of the[0221]holder316. Theholder316 has abody317 in which are formed theguide slots324 for receiving the guide pins. The body also has arecess334 for receiving thecontainer274. The shape of therecess334 matches the shape of the lower portion of theframe278 so that the frame fits securely in therecess334. Theholder316 also includes a retainingmember328 attached to thebody317 by screws orbolts330. The retainingmember328 andbody317 define aslot332 through which theframe278 is inserted when the frame is placed in therecess334. The retainingmember328 holds theframe278 in the recess. Thebody317 also has anopening336 adjacent therecess334. The shape of theopening336 corresponds to the shape of thechamber277.
• As shown in the cross sectional view of FIG. 56, when the[0222]container274 is inserted into theholder316, theopening336 is positioned next to theflexible wall280B. Theopening336 is thus positioned to permit theflexible wall280B to expand outwardly into the opening. Theholder316 holds only the frame of thecontainer274 so that theflexible walls280A,280B are unrestrained by the holder. Theflexible wall280A is therefore free to move inwardly and outwardly with thehorn tip326 as vibrational motion is transmitted from thetip326 to thewall280A. Theflexible wall280B is also free to move inwardly or outwardly as pressure pulses sweep through thechamber277. This permits the liquid within thechamber277 to move more freely as it receives the dynamic pressure pulses and thus enhances the cell disruption in thechamber277. Venting of theopening336 is provided by first andsecond bores338,344 formed in the body of theholder316. One end of thenarrower bore338 is connected to theopening336 and the other end is connected to thelarger bore344. Thebore344 extends through the body of theholder316 to permit the escape of gas (e.g., air) from theopening336. The venting prevents pressure from building in theopening336 when theflexible wall280B expands into the opening. Such pressure would restrict the motion of thewall280B.
• Referring again to FIG. 54, the[0223]container274 has a bulb-shapedtab275 extending from the bottom of theframe278. Theholder316 hasholes340 formed in thebody317 adjacent therecess334. When theframe278 is inserted into therecess334, thetab275 is positioned between theholes340. Theholes340 are for receiving retaining pins. As shown in FIG. 55, the retaining pins342 extend from the guide318 (from which the guide pins have been removed for clarity in FIG. 55) and are positioned on opposite sides of the bulb-shapedtab275 when thecontainer274 is moved into contact with thehorn tip326. The spacing of thepins342 is less than the width of the bulb so that thepins342 hold down thetab275, and thus thecontainer274, as ultrasonic energy is transmitted into the container from thetransducer314. This ensures that thecontainer274 does not rise out of position due to the motion of thehorn tip326. Alternatively, a collar or other suitable retention mechanism may be used to hold thecontainer274 in position.
• Referring to FIG. 56, the[0224]support structure306 also includes an elastic body, such as aspring366, for applying a force to theholder316 to press thewall280A of thechamber277 against thehorn tip326. When thewall280A is in contact with thehorn tip326, the force provided by the spring is constant, providing for consistent coupling and transfer of power between thetransducer314 and thecontainer274. Thespring366 is positioned in thebore344. Theholder316 has an inner surface surrounding the junction of thelarger bore344 and thenarrower bore338. One end of thespring366 contacts the inner surface, and the other end of the spring contacts arod348 that extends from theguide318. Thespring366 is thus compressed between the surface of theholder316 and therod348 so that it pushes theholder316, and thus theflexible wall280A of thecontainer274, against thetip326.
• The magnitude of the force provided by the[0225]spring366 may be adjusted by changing the preload on the spring. Thesupport structure306 includes arod348 that contacts one end of the spring. Theguide318 includes a first bore for receiving therod348 and a second bore for receiving aset screw349 that holds therod348 in a fixed position. To adjust the preload on thespring366, thescrew349 is loosened, therod348 is moved to a new position, and thescrew349 is retightened to hold therod348 in the new position. Therod348 and setscrew349 thus provide a simple mechanism for adjusting the preload on thespring366. Once the preload on thespring366 is adjusted to provide a suitable coupling force between thewall280A and thehorn tip326, it is desirable to keep the preload constant from one use of the apparatus to the next so that valid comparisons can be made between different samples disrupted by the apparatus.
• The[0226]flexible wall280A facilitates the transfer of vibrating motion from thetransducer314 to thechamber277. Thewall280A is sufficiently flexible to conform to the surface of thetip326 of the transducer, ensuring good coupling between thetip326 and thewall280A. The surface of thetip326 that contacts thewall280A is preferably planar (e.g., flat) to ensure power coupling over the entire area of the surface. Alternatively, thetip326 may have a slightly curved (e.g., spherical) surface for contacting thewall280A Theopposite wall280B is preferably sufficiently flexible to move inwardly and outwardly as dynamic pressure pulses are generated in thechamber277. This permits the liquid within thechamber277 greater freedom of movement as it receives the pressure pulses and thus enhances the action in thechamber277.
• Referring again to FIG. 46, the[0227]walls280A,280B are preferably flexible sheets or films of polymeric material such as polypropylene, polyethylene, polyester, or other polymers. The films may either be layered, e.g., laminates, or the films may be homogeneous. Layered films are preferred because they generally have better strength and structural integrity than homogeneous films. Alternatively, thewalls280A,280B may comprise any other material that may be formed into a thin, flexible sheet. For good flexibility and energy transfer, the thickness of each wall is preferably in the range of 0.01 to 0.2 mm, and more preferably in the range of 0.025 to 0.1 mm. As previously described, theplunger284 is inserted into thechannel288 after, adding the cells or viruses to thechamber277. Theplunger284 compresses air in thechannel288, thereby increasing pressure in thechamber277. The pressurization of thechamber277 ensures effective coupling between thewall280A and the tip of thetransducer314.
• It is presently preferred to pressurize the[0228]chamber277 to a pressure in the range of 2 to 50 psi above ambient pressure. This range is presently preferred because 2 psi is generally enough pressure to ensure effective coupling between theflexible wall280A and thetransducer314, while pressures above 50 psi may cause bursting of thewalls280A,280B or deformation of the frame of thecontainer274. More preferably, thechamber277 is pressurized to a pressure in the range of 8 to 15 psi above ambient pressure. This range is more preferred because it is safely within the practical limits described above.
• A preferred method for disrupting cells or viruses using the[0229]apparatus304 will now be described with reference to FIGS. 46-56. Referring to FIG. 50,beads301 are placed in thechamber277 of the container to enhance the disruption of the cells or viruses. In general, thebeads301 may be composed of glass, plastic, polystyrene, latex, crystals, metals, metal oxides, or non-glass silicates. Thebeads301 may be porous or non-porous and preferably have a diameter in the range of 1 to 200 μm. More preferably, thebeads301 are either polystyrene beads, borosilicate glass beads, or soda lime glass beads having an average diameter of about 100 μm. Thebeads301 may be placed in thechamber277 using a funnel. The funnel should be sufficiently long to extend from theport276 through thechannel288 and into thechamber277. After inserting the funnel into thecontainer274, thebeads301 are placed in the funnel and thecontainer274 is tapped lightly (e.g., against a bench top) until thebeads301 settle into the bottom of thechamber277. It is preferred that the funnel extend through thechannel288 and into thechamber277 as thebeads301 are added to the chamber to prevent the beads from contaminating the channel. The presence of beads in thechannel288 would interfere with the subsequent stroke of the plunger into the channel. The quantity ofbeads301 added to thechamber277 is preferably sufficient to fill about 10% to 40% of the volume capacity of the chamber. For example, in the presently preferred embodiment, thechamber277 has a volume capacity of about 100 μl, and 30 to 40 mg of beads are placed into the chamber.
• After the[0230]beads301 are placed in thechamber277, the chamber is filled with a liquid containing the cells or viruses to be disrupted. Thechamber277 may be filled using a pipette having a pipette tip300 (e.g., a standard 200 μl loading tip). Alternatively, thechamber277 may be filled using a syringe or any other suitable injection system. The liquid should be a medium through which pressure waves or pressure pulses can be transmitted. For example, the liquid may comprise deionized water or ultrasonic gel for holding the cells or viruses in suspension or solution. Alternatively, the liquid may comprise a biological sample containing the cells or viruses. Suitable samples include bodily fluids (e.g., blood, urine, saliva, sputum, seminal fluid, spinal fluid, mucus, etc) or environmental samples such as ground or waste water. The sample may be in raw form or mixed with diluents or buffers. The liquid or gel may also include one or more lysing agents to aid in the disruption of the cells or viruses. One of the advantages of the present invention, however, is that harsh lysing agents are not required for successful disruption of the cells or viruses.
• After the[0231]container274 is filled with the liquid, theplunger284 is inserted into thechannel288 to seal and pressurize thecontainer274. As theplunger284 is inserted, thepiston292 compresses gas in thechannel288 to increase pressure in thechamber277, preferably to about 8 to 15 psi above ambient pressure, as previously described.
• Referring to FIG. 56, the[0232]holder316 is then pushed or pulled away from thetip326 of the transducer314 (in the direction of the rod348) so that thecontainer274 can be inserted into the holder. Thecontainer274 is then placed in theholder316. During the insertion of thecontainer274, theholder316 should be held a sufficient distance from the retainingpins342 to provide clearance between thepins342 and thetab275. After thecontainer274 is inserted into theholder316, the holder is gently released and thespring366 pushes theholder316 along theguide318 until thewall280A contacts and conforms to the surface of thehorn tip326. When thewall280A is coupled to thehorn tip326, thespring366 applies to theholder316, and thus to thecontainer274, a substantially constant force to press thewall280A against thehorn tip326. The force provided by thespring366 ensures effective coupling between thewall280A andhorn tip326 as energy is transmitted to thechamber277. As shown in FIG. 55, when thecontainer274 is moved into contact with thetip326, thetab275 slides between the retaining pins342. Thepins342 prevent the container from sliding upward in response to the vibratory motion of thetip326.
• Referring again to FIG. 56, the cells or viruses in the[0233]chamber277 are then disrupted by the pressure pulses and resulting bead movement in thechamber277 generated by the vibration of thetip326 against theflexible wall280A. The magnitude of the force provided by thespring366 to press together thewall280A and thetip326 is important for achieving a consistent transfer of energy between thetransducer314 and thechamber277. If the force is too light, thewall280A will only be held lightly against thetip326, leading to poor transmission of the vibratory movement of thetransducer314. If the force is too strong, thecontainer274 orwall280A may be damaged during sonication. An intermediate force results in the most consistent and repeatable transfer of energy from thetransducer314 to thechamber277. It is presently preferred that thespring366 provide a force in the range of 0.25 to 4 lbs., with a force of about 1 lb. being the most preferred.
• When the[0234]transducer314 is activated, thetip326 vibrates to transmit ultrasonic energy into thechamber277. There is a relationship between the coupling force between thewall280A and thetip326 and the desired amplitude of the vibratory movements of thetip326. A balance can be sought between the coupling force and the amplitude. Generally, a light coupling force requires a greater amplitude to effect disruption of the cells or viruses, while a stronger coupling force requires less amplitude to effect disruption. For the range of coupling forces presently preferred (0.25 to 4 lbs.), the peak-to-peak amplitude of the vibratory movements should be in the range of 4 to 40 μm, with a preferred peak-to-peak amplitude of amount 15 μm.
• Ultrasonic waves are preferably transmitted to the[0235]chamber277 at a frequency in the range of 20 to 50 kHz, with a frequency of about 40 kHz being preferred. The duration of time for which thechamber277 is sonicated is preferably in the range of 5 to 30 seconds. This range is preferred because it usually takes at least 5 seconds to disrupt the cells or viruses in the chamber, while sonicating the chamber for longer than 30 seconds will most likely denature or shear the nucleic acid released from the disrupted cells or viruses. Extensive shearing of the nucleic acid could interfere with subsequent amplification or detection. More preferably, the chamber is sonicated for about 10-20 seconds to fall safely within the practical limits stated above. The optimal time that a particular type of cell sample should be subjected to ultrasonic energy may be determined empirically.
• Following disruption of the cells or viruses, the[0236]container274 is removed from theholder316 by pulling theholder316 away from thetip326 and withdrawing the container from the holder. The liquid or gel containing the disrupted cells and released nucleic acid is then removed from thecontainer274. This may be accomplished by centrifuging thecontainer274 and removing the supernatant using, e.g., a pipette or syringe. Alternatively, the liquid may be removed from thecontainer274 by setting the container on edge and at an incline until the beads precipitate. The beads usually settle in about 15 to 20 seconds. When the beads have settled, the plunger is withdrawn from thecontainer274 and the liquid is removed using a syringe or pipette. The released nucleic acid contained in the liquid may then be amplified and detected using techniques well known in the art.
• One advantage of the apparatus and method of the present invention is that it provides for the rapid and effective disruption of cells or viruses, including tough spores, without requiring the use of harsh chemicals. In addition, the apparatus and method provide for highly consistent and repeatable lysis of cells or viruses, so that consistent results are achieved from one use of the apparatus to the next. The amount of energy that is absorbed by the liquid and beads held in the[0237]chamber277 depends on the amplitude of the oscillations of thetip326, the mass of the contents of thechamber277, the pressure in thechamber277, and the coupling force between thetip326 and thewall280A. All four of these parameters should be held substantially constant from one use of the apparatus to the next in order to achieve the same amount of disruption repeatably.
• Many different modifications to the apparatus shown in FIG. 56 are possible. For example, the[0238]holder316 may be slidably mounted to thebase308 by a variety of means, including rails, wheels, sliding in a groove, sliding in a cylinder, etc. Alternatively, theholder316 may be fixedly attached to thebase308 and thetransducer314 slidably mounted to the base. In this embodiment, an elastic body is positioned to apply a force to the transducer314 (either directly or to a holder holding the horn) to press together thehorn tip326 and thewall280A. In addition, in each of these embodiments, the elastic body may be positioned to either push or pull thetransducer314 or thecontainer274 towards each other. For example, thespring366 may be positioned to push or pull theholder316 towards thehorn tip326 or to push or pull thetransducer314 towards theholder316. Further, multiple elastic bodies may be employed to apply forces to both thecontainer274 and thetransducer314 to push or pull them towards each other. All of these embodiments are intended to fall within the scope of the present invention.
• Although a[0239]coil spring366 is shown in FIGS. 37 and 56, it is to be understood that any type of elastic body may be used. Suitable elastic bodies include, but are not limited to, coil springs, wave springs, torsion springs, spiral springs, leaf spring, elliptic springs, half-elliptic springs, rubber springs, and atmospheric springs. The elastic body may also be compressed air or rubber. Preferably, the elastic body is a coil spring. Coil springs are preferred because they are simple and inexpensive to place in the apparatus and because the have a low spring rate. A compressed air system is also effective, but considerably more expensive. In embodiments in which the elastic body is a spring, the spring should have a low spring rate, preferably less than 4 lb./in. A low spring rate minimizes the effect that any variations in the thickness of the chamber277 (due to small variations in manufacturing, filling, or pressurizing the container) will have on the magnitude of the force provided by the spring to press together thewall280A and thehorn tip326.
• Another advantage of the[0240]container274 is that thechamber277 holds the cells or viruses in a thin volume of liquid that can be uniformly sonicated easily. Referring to FIGS. 48-49, it is presently preferred to construct thecontainer274 such that each of thesides walls282A,282B,282C,282D of the chamber has a length L in the range of 5 to 20 mm, the chamber has a width W in the range of 7 to 30 mm, and the chamber has a thickness T in the range of 0.5 to 5 mm. In addition, thechamber277 preferably has a width W greater than its thickness T. In particular, the ratio of the width W of the chamber to the thickness T of the chamber is preferably at least 2:1. More preferably, the ratio of the width W of the chamber to the thickness T of the chamber is at least 4:1. These ratios are preferred to enable the entire volume of thechamber277 to be rapidly and uniformly sonicated. In general, the volume capacity of thechamber277 is preferably in the range of 0.02 to 1 ml.
• Referring again to FIG. 56, the[0241]transducer314 is preferably an ultrasonic horn. The thickness of the chamber277 (and thus the spacing between thewalls280A and280B) is preferably less than half of the diameter of thetip326 of the horn. This relationship between the thickness of thechamber277 and the diameter of thetip326 ensures that the ultrasonic energy received from thetransducer314 is substantially uniform throughout the volume of thechamber277. As a specific example, in the presently preferred embodiment, thetip326 has a diameter of 6.35 mm and thechamber277 has a thickness of about 1.0 mm. In addition, themajor wall280A should be slightly larger than the surface of thehorn tip326 that presses against thewall280A. This allows theflexible wall280A to flex in response to the vibratory motion of thehorn tip326.
• A preferred method for fabricating the[0242]container274 will now be described with reference to FIGS. 46-47. Thecontainer274 may be fabricated by first molding therigid frame278 using known injection molding techniques. Theframe278 is preferably molded as a single piece of polymeric material, e.g., polypropylene or polycarbonate. After theframe278 is produced, thin, flexible sheets are cut to size and sealed to opposite sides of theframe278 to form themajor walls282A,282B of thechamber277.
• The[0243]major walls282A,282B are preferably cast or extruded films of polymeric material, e.g., polypropylene films, that are cut to size and attached to theframe278 using the following procedure. A first piece of film is placed over one side of the bottom portion of theframe278. Theframe278 preferably includes atack bar299 for aligning the top edge of the film. The film is placed over the bottom portion of theframe278 such that the top edge of the film is aligned with thetack bar299 and such that the film completely covers the bottom portion of theframe278 below thetack bar299. The film should be larger than the bottom portion of theframe278 so that it may be easily held and stretched flat across the frame. The film is then cut to size to match the outline of the frame by clamping to the frame the portion of the film that covers the frame and cutting away the portions of the film that extend past the perimeter of the frame using, e.g., a laser or die. The film is then tack welded to the frame, preferably using a laser.
• The film is then sealed to the[0244]frame278, preferably by heat sealing. Heat sealing is presently preferred because it produces a strong seal without introducing potential contaminants to the container as the use of adhesive or solvent bonding techniques might do. Heat sealing is also simple and inexpensive. At a minimum, the film should be completely sealed to the surfaces of theside walls282A,282B,282C,282D. More preferably, the film is additionally sealed to the surfaces of thesupport ribs295 andtack bar299. The heat sealing may be performed using, e.g., a heated platen. An identical procedure may be used to cut and seal a second sheet to the opposite side of theframe278 to complete thechamber277.
• The[0245]plunger284 is also preferably molded from polymeric material (e.g., polypropylene or polycarbonate) using known injection molding techniques. As shown in FIG. 46, theframe278,plunger284, andleash286 connecting the plunger to the frame may all be formed in the same mold to form a one-piece part. This embodiment of the container is especially suitable for manual use in which a human operator fills the container and inserts theplunger284 into thechannel288. Theleash286 ensures that theplunger284 is not lost or dropped on the floor.
• The[0246]plunger284 is presently preferred as a simple, effective, and inexpensive mechanism for increasing pressure in thechamber277 and for sealing thechamber277 from the external environment. It is to be understood, however, that the scope of the invention is not limited to this embodiment. There are many other suitable techniques for sealing and pressurizing the container. In addition, any suitable pressure source may be used to pressurize the chamber. Suitable pressure sources include syringe pumps, compressed air sources, pneumatic pumps, or connections to external sources of pressure.
SUMMARY, RAMIFICATIONS, AND SCOPE
• Although the above description contains many specificities, these should not be construed as limitations on the scope of the invention, but merely as examples of some of the presently preferred embodiments. Many modifications or substitutions may be made to the apparatus and methods described without departing from the scope of the invention. For example, the container for holding the cells or viruses need not be one of the specialized containers described in the various embodiments above. Any type of container having a chamber for holding the cells or viruses may be used to practice the invention. Suitable containers include, but are not limited to, reaction vessels, cuvettes, cassettes, and cartridges. The container may have multiple chambers and/or channels for performing multiple sample preparation functions, or the container may have only a single chamber for holding cells or viruses for disruption.[0247]
• Further, the support structure for pressing the transducer and the wall of the container against each other may have many alternative forms. For example, in one alternative embodiment, the support structure includes a vise or clamp for pressing the transducer and container against each other. In another embodiment, the apparatus includes a pressure system for applying air pressure to press together the transducer and the container. Alternatively, magnetic or gravitational force may be used to press together the transducer and the container. In each embodiment of the invention, force may be applied to the transducer, to the container, or to both the transducer and the container.[0248]
• Therefore, the scope of the invention should be determined by the following claims and their legal equivalents.[0249]

Claims (4)

1-82. (canceled)

83. A micro-sonicator for spore or cell lysis comprising:

a container having a cavity therein adapted to contain spore or cell samples,

a quantity of ultrasonic transmission media in said cavity,

at least a membrane positioned to cover said cavity,

piezoelectric material positioned on said membrane, and

means for causing flexing of said piezoelectric material and vibration of said membrane thereby causing ultrasonic excitation of the transmission media adapted to cause lysis of spore or cell samples in said cavity.

84. In a micro-fluidic system, the improvement comprising:

a micro-sonicator for spore and cell lysis using ultrasonic excitation of the spore or cell, said micro-sonicator including a container having a cavity therein and containing ultrasonic transmission media, a membrane positioned over said cavity, and means for causing vibration of said membrane producing ultrasonic excitation of spore or cell located in said cavity.

85. A device for spore or cell lysis comprising:

a container having a chamber for holding spore or cell samples,

a quantity of ultrasonic transmission media in said chamber,

a membrane forming at least one wall of the chamber, and

an ultrasonic transducer in contact with said membrane for transmitting ultrasonic waves to the chamber.

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