Patent Application
20060133957
This invention relates to a device and method for fragmenting material, such as large molecular weight polymers, cells, lipid particles and the like, by hydrodynamic shear.
Several methods have been described for fragmenting solute or particulate material contained in a liquid sample, for example, fragmenting DNA into smaller polynucleotide molecules for preparation of libraries and cloning for DNA sequence analysis, chromatin immunoprecipitation assay, and other biological research purposes.
Various fragmentation methods include passing the solution or suspension through a syringe or pipette, atomization, sonic treatment, and, in the case of DNA fragmentation, the use of restriction enzymes such as restriction endonucleases. While these methods have been successful in generating—DNA fragments, each method has limitations. The syringe method often fails to provide small enough fragments—for the study of DNA replication, repair, and transcription. It is also labor intensive and low throughput. Sonic treatment requires a large amount of sample material, generates a broad distribution of fragments, and is difficult to reproduce. Enzymatic methods requires a cocktail of different enzymes to generate the necessary fragments for proper sequence analysis; but do not produce random fragmentation. In addition, enzymatic methods often produce a broad distribution of fragments and a low yield of fragments of appropriate lengths for subsequent analysis.
Therefore, there exists a need in the art for an apparatus and method to efficiently produce a narrow and reproducible distribution of random fragments. There is also a need for a device and method that can efficiently extremely small sample volumes, preferably in a single pass, can be operated in a multiplexed (multi-sample) mode, and at the same time, is relatively inexpensive by virtue of utilizing existing laboratory equipment.
The invention includes, in one embodiment, a device for use with a centrifuge for fragmenting solute or particulate material contained in a liquid sample. The device includes a substrate adapted to be supported within a centrifuge tube. A microchannel formed in the substrate and extending between upper and lower channel ends defines a plurality of shear regions, each designed to subject material present in the sample liquid to a shearing force as sample liquid is forced through the shear region under the influence of a selected centrifugal force applied to the tube in which the device is supported. The device may include a plurality of microchannels in the same substrate, with each microchannel having similar or dissimilar geometries, and each microchannel having a plurality of shear regions. Material contained in a liquid sample applied to the upper end of the microchannel, with the device supported in a centrifuge tube within a centrifuge, is fragmented by shearing as the sample is forced successively through the plurality of shear regions in the microchannel, when the selected centrifugal force applied to the tube.
In various embodiments, (i) the device further includes a holder adapted to be received within a selected-size centrifuge tube, and adapted to support the substrate within the tube; (ii) the substrate includes support members constructed to support the substrate within a selected-size centrifuge tube; and (iii) the device is formed as an integral unit with a centrifuge tube.
The microchannel preferably includes at least 5 shear regions, typically 10-20 or more. The microchannel may be serpentine in shape, for example, to increase the number of shear regions that can be accommodated along the flow path.
The device may include a sample-receiving well and a fluid-flow barrier interposed between the well and the microchannel, for preventing liquid sample applied to the well from reaching the upper end of the microchannel until a selected centrifugal force is applied to the device. The barrier may include a pair of deformable members that remain interlocked at a channel-sealing condition until deformed under the selected centrifugal force. Alternatively, the barrier may include a frangible seal designed to fracture when a liquid sample is forced against the seal under the selected centrifugal force. In still another embodiment, the barrier may include an electronically controlled valve that can be activated, from a closed to an open condition, when an external electronic signal is applied to the valve.
The shear regions in the microchannel may be defined by a change in the cross-sectional area of the channel, in a direction substantially perpendicular to the direction of fluid flow in the channel. For example, a shear region may be defined by adjacent upstream and downstream channel segments having a ratio of cross-sectional areas of at least 3:1. In this embodiment, the upstream channel segment includes a central barrier which acts to prevent liquid flow through a central portion of that channel segment. This embodiment is used, for example, in fragmenting polynucleotide molecules, where the downstream segment in a microchannel has a width dimension of less than 20 microns.
The shear regions in the microchannel may be defined by changes in the direction of liquid flow in the microchannel, that is, bends or curves in the microchannel that force sample liquid to change direction as it is forced through the microchannel. In still another embodiment, the shear regions may be defined by physical barriers or baffles placed in the path of liquid flow in the microchannel.
In a related aspect, the invention includes a polymer-fragmentation kit designed for use with a centrifuge for fragmenting a sample solution of polymers, such as polynucleotides, into a plurality of polymer-fragment pools, each with a different fragment-size range. The kit includes a plurality of fragmentation devices of the type described above, where the shear regions in each device have a device-specific shear-region geometry designed to subject material present in the sample solution of polymers to a device-specific shearing force as sample solution is forced through the shear region under the influence of a selected centrifugal force applied to the tube in which the device is supported. The polymers contained in a liquid sample applied to the upper ends of different devices in the kit, with the devices supported in centrifuge tubes within a centrifuge, are fragmented by shearing as the samples are forced successively through the plurality of shear regions in each device, to produce polymer fragments having different size ranges.
In still another aspect, the invention provides a method for use with a centrifuge for fragmenting solute or particulate material contained in a liquid sample. The method includes the steps of applying the sample solution to an upstream region of a microchannel device having a microchannel defining a plurality of shear regions, and with the microchannel device supported within a centrifuge tube in the centrifuge, subjecting the tube to the selected centrifugal force. Sample material is fragmented by shearing as the sample is forced successively through the plurality of shear regions in the microchannel.
The method may be used for processing a plurality of samples at the same time, by applying one or more sample solutions to each of a plurality of such microchannel devices, each supported within a different tube in a centrifuge. For example, each of the plural microchannel devices may have a microchannel whose shear regions are defined by different, device-specific channel geometries, such that the same sample applied to different devices is subjected to different shear forces under the same centrifugal force, yielding different fragment size ranges for the different devices.
The centrifugal force applied to the microchannel is preferably between 5,000 and 20,000 G, more preferably 10,000-16,000 G, where 1 G is the gravitational acceleration at the surface of the Earth, approximately 9.81 m/sec2. The total time over which the centrifugal force is applied is preferably less than 1 minute. The sample volume added to the microchannel device may be a small as 5 and 200 μl, or less. The method may be carried out under conditions such that sample material is forced through the microchannel only when the selected centrifugal force to which the tube is subjected reaches the selected centrifugal force.
For use in fragmenting polymer molecules, the centrifugal force to which the tube is subjected may be such, in relation to the geometry of the microchannel shear regions, to fragment the polymer molecules into a desired size range under the influence of the selected force.
For use in assaying an intracellular analyte in a cell sample, movement of the cell sample through the microchannel, under the influence of a selected centrifugal force to which the tube is subjected, is effective to disrupt the cells and release intracellular contents.
For use in forming desired size lipid particles in a particle suspension, movement of the particles through the microchannel, under the influence of a selected centrifugal force to which the tube is subjected, is effective to produce the desired lipid-particle sizes. This method may be used, for example, in forming liposomes of desired size distribution or lamellar structure.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
I. Definitions
The terms below have the following meanings unless indicated otherwise;
The term “channel” and “microchannel” are used interchangeably to mean a path or conduit within which a fluid sample travels.
A “microchannel” has a cross-sectional dimension, e.g., width or height or diameter, in the direction substantially perpendicular to the direction of flow in the channel, that is less than about 500 microns, typically between 5-250 microns. These dimensions may apply along the entire length of the channel, or may be confined to shear regions of the channel; that is, the microchannel dimensions may apply only within a shear region of the channel.
The term “shear region” refers a region within a channel at which a differential flow velocity of liquid flowing through the region is effective to exert of shearing force on solute or particulate material dissolved or suspended in the liquid and on the liquid itself. Where the material is an elongate polymer, e.g., a polynucleotide, the shearing force is effective to shear the polymer into smaller fragments. Where the material is a cellular material, such as mammalian or bacterial cells, the shearing force is effecting to disrupt the cell, exposing the cells' intracellular contents. Where the material is lipid particles, such as liposomes or lipid droplets in an oil-in-water emulsion, the shear force is effective to disrupt the particles such that they reform in smaller, more uniform sizes.
A “shear region” may be formed in a microchannel by any channel geometry that causes differences in liquid flow velocities at different points within that region. In one general embodiment, a shear region is formed by a change in the cross-sectional area of the flow path, typically a change in flow-channel width of at least 2:1 or 3:1 over a short channel distance. Local flow-velocity variations that can serve as shear regions can also be produced by bends or turns in the flow path, or by placing obstacles to flow in the flow path, or a combination of two of more of these geometries.
The extent of shear occurring at a shear region will depend on the local geometry at a shear region, the speed at which liquid is flowing through the region, and the orientation of material in the liquid. Implicit in the definition of a shear region is that the liquid velocity through the region is sufficient to produce a shear force that can fragment material in the liquid flowing through the region, and this in turn, requires that the centrifugal force applied to the liquid is sufficient to produce such flow velocity. Also implicit in the definition is that the cross-sectional area of the flow channel, at least in the regions of shear, is small enough to promote fragmenting shear forces at flow velocities consistent with normal centrifugation speeds.
II. Fragmentation Device
With continued reference to
Each shear region in the microchannel has channel geometry designed to cause sharp local differences in liquid flow velocities in the liquid sample flowing through the channel. In the embodiment shown in
The ratio of W2 to W1, and therefore corresponding cross-sectional areas of segments 36, 38, is typically 2:1 or greater, e.g., 3:1 to 4:1 or larger. Typically, where the device is used for fragmenting polymers, W1 is in the range 1-50 microns, and preferably, for use in fragment polynucleotides between about 5-20 microns. Similarly, W2 dimensions are typically in the range 50-250 microns. Although smaller W1 channel dimensions are possible, a nano-scale channel cross-section may lead to channel clogging and to inability to move liquid through the channel at speeds adequate to shear the material in the liquid. Similarly, although larger W2 channel dimensions are possible, larger dimensions may inhibit the efficient processing of small volumes, e.g., in the range 1-10 μl, and may also require fluid-velocities that are impractical to achieve under normal centrifugation operation, e.g., with a standard table-top centrifuge.
Although shear regions 32, 34 described above are formed at the interface between a larger-to-smaller channel segment, it will be appreciated that the interface between a smaller-to-larger channel segments will also form a region of shear in the channel, such as region 44 at the downstream end of channel segment 38 in
Substrate 24 may be formed by any of a variety of techniques suitable for preparing microchannel and/or microfluidics devices. Preferably the substrate is formed of two layers or plates that are laminated together by conventional bonding or laminating methods such as anodic bonding in the case of a Silicon substrate. The substrate may be of any suitable material, such as polymer (plastic), silicon, fused silica, or the like, and preferably, a polymer substrate. Illustrative polymers include methyl methacrylate and copolymers thereof, dimethylsiloxanes, polystyrene, and polycarbonate. Preferred polymers are PMMA (polymethylmethacrylate) and cyclic olefin homopolymer or co-polymer thermoplastics.
The microchannel formed in the substrate may vary as to dimensions, width, depth and cross-section, as well as shape, being rounded, trapezoidal, rectangular, etc. The path of the channels may be straight, angles, serpentine, or the like, consistent with the requirement of imposing a plurality of shear regions along the microchannel path. Typical channel dimensions are as given above. The channel length will range typically from one to several centimeters in length.
In a typical substrate assembly, one of two plates is prepared to include the microchannel and a second flat-surfaced plate is laminated to the plate to enclose the channel. Various surface treatment methods are available for forming the microchannel in a plate, including injecting molding, techniques involving surface-etch techniques, surface-embossing techniques, and/or microfabrication methods. The two plate may be laminated by heat-fusion, polymer adhesives, or the like, according to known methods. Manufacturing techniques also exist that allow fabrication of the channel in a single plate so that a second plate is not needed to seal the channel along its length. Any of these techniques commonly available may be used to fabricate the channel in the substrate.
Each plate forming the substrate is preferably 0.5 to 25 mm in thickness, for a total substrate thickness of between about 1-5 mm, with the constraints that the substrate is strong enough to withstand the g forces to be applied. Once the device is formed, the microchannel may be coated with a suitable lubricant or other coating material effective to improve flow properties through the microchannel and/or to reduce sticking or aggregation at the channel walls. One exemplary coating employed in some of the examples below is a silane coating, e.g., dichlorodimethylsilane. The coating may be applied simply by running a sample of the liquid coating material through the channel under centrifugal force.
In the general embodiment of the invention illustrated in
Also shown at
As can be appreciated from
Although not shown, the device of the invention may further be formed to include a centrifuge tube. Here a preformed substrate, which may be in the shape of a plate or space-filling plug, is inserted into a centrifuge tube and the two secured together with a suitable binding agent. The upper end of the device may include an opening through which sample at the bottom of the tube can be accessed, e.g., by a micropipette. Alternatively, the channel containing substrate and centrifuge tube can be manufactured in a one step process, leaving a device where the substrate is integral to the centrifuge tube.
Various exemplary shear-region geometries that can be formed readily within a microchannel are illustrated in
Channel 82 illustrated in
The channels described above rely on a sharp change in channel cross-sectional size for producing shear. However, shear regions within a microchannel can also be formed by abrupt changes in flow direction, as illustrated in
In the channel 108 in
Finally,
As noted above, the shear force created at each shear region along the length of a microchannel will depend on channel geometry and the speed of liquid being forced through the microchannel under centrifugal force. In order to insure that liquid sample flowing through the microchannel is at a desired flow velocity, and/or at a substantially uniform velocity in each flow region, it may be desirable to restrict sample flow through the microchannel until a selected centrifugal force (centrifuge speed), e.g., 5000-15,000 G is reached. This can be done, in accordance with various embodiments of the invention shown in
Device 126 shown in
Device 134 shown in
Device 142 shown in
Although not shown here, the invention also includes a plurality of devices of the type described above, where each device may include different geometry shear-regions, such that a selected substrate can produce a desired type of fragmentation, e.g., polymer fragment size range, that is distinctive for that substrate. This allows the user to select a desired fragmentation outcome, e.g., fragment size range, or to achieve each of a plurality of different outcomes, preferably in a multiplexed operation, that is, where several samples are being processed at the same time; e.g. in different tubes in the same or multiple centrifuges.
III. Fragmentation Method
In the method of the invention, a sample material contained in a liquid solution or suspension is fragmented by applying the sample solution to an upstream region of a microchannel device defining a plurality of channel shear regions, and with the device supported in a centrifuge tube, subjecting the tube to a centrifugal force sufficient to cause liquid shear forces that fragment the material by shearing as the sample is forced successively through the shear regions of the microchannel.
The material to be fragmented may be linear or branched polymers. A polymer of particular interest is large polynucleotides, e.g., chromosomal or naked DNA obtained from biological samples. Such polynucleotide strands can have base-number sizes of a million or more, and it is often desired, for purposes of DNA cloning, sequencing, or other analysis to fragment the polynucleotide material into fragments within a desired size range, e.g., 10-20 kbases, or into different groups of desired sizes, e.g., 5-10 kbases, 8-15 kbases, 10-20 kbases, etc. The example below details DNA fragmentation results obtained in microchannels whose shear regions are formed by different, selected channel widths.
Another exemplary polymer is polyethylene glycol where it is desired to reduce the size heterogeneity of a population of polymer molecules to a desired size range, e.g., 3-5 kdalton molecular weight, for purposes of creating a more uniform size distribution of the polymers. In general, any large-molecule weight and/or polymer with size heterodispersity may be a candidate for fragmentation into smaller sizes and in a more uniform size range.
Another material suitable for fragmentation in the method are biological cells, preferably in individual cell suspension, such as a suspension of bacterial cells, or cultured mammalian or plant cells, or subcellular fractions, such a mitochochondria, nuclei, or chloroplasts. Here the purpose of the method is to disrupt cells and release subcellular and/or sub-particulate material, for purposes of analyzing or isolating one or more intracellular components. As an example, the method may be used to analyze small cellular fraction, e.g., in 1-10 μl volume. If the sample material to be analyzed is intracellular DNA, the method provides the advantage of being able to disrupt cells and nuclei, and fragment release DNA all in a single fragmentation step that may involve an extremely small or dilute cell fraction.
Another material to be fragmented, in accordance with the invention, are lipidic particles, including micelles, liposomes, triglyceride particles, lipid-in water emulsion particles, and lipoprotein particles, such as high density lipoprotein particles, obtained from a blood sample. In one embodiment, liposomes having a heterodisperse size range, e.g., 0.10 to 20 microns, are fragmented into smaller, more uniform particles sizes, e.g., liposomes having sizes in the 0.05 to 0.2 micron size range, and/or having single or few lipid lamellae.
In one general embodiment, the method is used for simultaneous processing of multiple samples, one fragmentation device for each of the up to 8 or more tube slots in a centrifuge. The method may be applied to multiple samples, each processed in an identical fragmentation device, or may be applied to a single sample, e.g., DNA sample, fragmented in different devices designed to produce different fragment size ranges at the same centrifugal force, as described above.
The centrifugal force that is applied in the method is selected to produce a desired shear force at the shear regions in the microchannel device. Typical selected centrifugal forces are between 5000 and 20,000 G, e.g., 10,000-16,000 G-. The g force applied is readily determined, for a given centrifuge, by a speed-g force conversion table provided for centrifugation instruments. In the table-top centrifuge noted above, speeds of up to 20,000 rpm are effective to produce centrifugal forces of up to about 27,000 G. As can be appreciated, the high g forces achievable in the invention are much higher than those that can be achieved by conventional pump or syringe techniques, and this contributes to both shearing efficiency and speed.
The centrifugation time may be virtually instantaneous, i.e., a few seconds, once the centrifuge as reached the desired speed, or may be up to several minutes, e.g., 30 minutes, particular where very small channel dimensions are employed. Preferred centrifugation times are less than 1 minutes, since the higher liquid velocities associated with shorter transit times contribute to higher shear forces. Multiple microchannels in a substrate can be used to reduce the centrifugation time compared to centrifugation time when using a substrate with only one microchannel. As noted above, the sample material may be released into the microchannel by suitable valving mechanisms only when the centrifuge has reached a desired rpm.
In accordance with another feature of the invention, sample, volumes may be quite small, since (i) the microchannels themselves have a low total volume, (ii) virtually all of the sample liquid is driven through the microchannel and into the bottom of the tube, leaving little or no sample residue, (iii) transit times are short so evaporation effects are minimized, there is no reprocessing of the sample, since the sample is fragmented multiple times as it is forced through the microchannel. Sample volumes may be in the nanoliter range, and typically sample volume added to the microchannel device is between 5 and 200 μl (micro-liters).
Finally, the method may be easily automated, for example, where an autosampler is used to inject samples into centrifuge tubes and to extract processed samples containing DNA fragments from centrifuge tubes. The shearing process may also be computer controlled to select run parameters such as centrifuge rotational speed and duration, and to select a shearing device of a given geometry.
From the foregoing, it can be seen how various objects and features of the invention are met. The system is capable of multiple processing of a sample in a single rapid centrifugation run, avoiding the time, expense and material loss associated with multiply processing a sample. Further, multiple sample can be processed under identical g-force conditions, allowing several samples to be processed simultaneously, or a single sample to be processed under different shear conditions. The method is amenable to small sample volumes, and provides efficient recovery of processed sample material. Finally, the method can be practiced with very little additional expense, assuming that a centrifuge and tubes are already available on site.
The following example illustrates the application of the invention to DNA shearing, for purposes of obtaining DNA fragments of a more uniform size distribution.
DNA was extracted from mouse genomic DNA, and brought to a final concentration of 10 ng/μl. Substrates having rectangular microchannel segments of different selected-size widths were prepared as described above. The substrates have 10 shear regions, and one of the following segment-widths ratios:
Substrate (A) 250 μm wide channel segment:20 μm narrow channel segment (250:20).
Substrate B: 250:75
Substrate C: 100:10
Substrate D: 100:20
Substrate E: 100-40.
Sample runs were carried out in 2 ml polypropylene microfuge tubes, employing an Eppendorf Model 5415C centrifuge. Each substrate was washed with 100 micro-liter 95% ethanol, followed by 100 μl distilled water. Where indicated, the substrate microchannels were coated by running 20 μl dichlorodimethylsilane through the wafer. After channel coating, the substrate was soaked for 1 week in 2 ml water (water was changed 3 times over the week), then washed 5 times with water, to remove all residues.
Sample of DNA, 70 μl were applied to the substrates and fragmented by running each sample at 15,000 rpm, corresponding to approximately 15,000 G, for 10 seconds, following an 8-second period needed to reach maximum speed. The samples were passed through the device under similar conditions 0 to eight times. Sample aliquots (5 μl) were applied to standard acrylamide gel slabs and fractionated by electrophoresis under standard conditions. The resulting fragment patterns were photographed under UV light in the presence of ethidium bromide.
Although the invention has been described with respect to particular geometries and applications, it will be apparent that various changes and modification may be made without departing from the invention.
This application claims priority of U.S. Ser. No. 60/440,841 filed on Jan. 17, 2003, which is incorporated in its entirety herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60440841 | Jan 2003 | US |
