Patent Application
20050236319
The use of multiwell plates to filter and purify various products such as proteins, DNA, RNA, plasmids and the like or for use in drug screening or drug discovery in the laboratory is widespread and growing. The advantages are many. The ability to use small volumes of samples required especially with experimental compounds or with the screening of 1000s of potential compounds reduces cost. The ability to run multiple samples at the same time reduce time and cost.
Most plate-based systems are arranged to have a filter plate positioned above a collection device such as a collection plate. A typical system is shown in
All fluid in the filter plate must pass through the filter or membrane 10 before reaching the collection plate well 14. Most filter plates 2 also contain an underdrain 18 below the filter or membrane 10. The underdrain 18 generally contains a spout 20 (as shown) to direct the fluid from the filter plate 2 to the well 14 of the collection plate 12 below it. The spout 20 also acts to hold back fluid flow through it when it is subject to simple atmospheric pressure. Flow occurs with aqueous based fluids only when a sufficient pressure differential, such as a vacuum is applied to the system. It also contains some type of sloped surface 22 to cause the fluid in the underdrain 18 to move toward the spout 20.
In practice, the system is assembled and placed on a vacuum manifold. The vacuum draws the fluid through the filter plate and underdrain and into the collection device. However, some fluid remains behind after the filtration has been completed. Typically, this fluid is found in the underdrain and as a pendant drop extending downward from the opening.
Several problems exist with leaving some sample behind.
For smaller volume application such as 384 and 1536 well systems (these systems include that number of wells on a plate that is of the same size as that used for a 96 well plate, meaning that the well size and sample size respectively 4× and 16× smaller than that of a 96 well plate system) the loss of sample can amount to 10 to 20% of the entire sample.
For all multiwell systems, the fluid in the pendant drops can often migrate to adjacent wells along adjacent surfaces or the pendant drops can be transferred to an adjacent well when the plates are taken apart to obtain the material in the collection plate. This leads to cross contamination of the sample and reduces the reliability of the system and the test that has been run. Likewise, many systems run sequential steps in the same system. The residual material can either then be present in the second step collection sample which is undesirable or it can over time migrate back or wick back through the filter or membrane and be present in the well of the filter plate from which it was removed. If for example the first step was a desalting step to remove salts or primers or other chemicals from a sample, this leads to a less pure sample and may complicate the second or later steps performed upon it. Additionally, when the filter plate is removed from the manifold, any pendant drops tend to rain down on the collection plate, equipment and adjacent laboratory surfaces thereby contaminating them.
Several approaches have been made to resolve the issue of pendant drop formation.
U.S. Pat. No. 4,902,481 uses a specially designed spout configuration having a collar which extends in a direction perpendicular to the vertical axis of the spout so that the collar and spout outer surface prevent pendant drop migration and direct any pendant drops into the collection well.
It however merely controls the pendant drop's lateral movement, not its formation or its migration into the collection plate.
In U.S. Pat. No. 4,526,690 the use of a hydrophobic porous layer at the bottom of each well prevented pendant drops from forming. However, when sufficient pressure is applied to the system the liquid overcomes the hydrophobic resistance and flows through the membrane to the collection plate. Additionally, the use of a separate grid of drop guiding projections, arranged between the two plates, is used to pull any drops that are formed along its surfaces and into the collection well.
In many applications the use of a hydrophobic membrane is not suitable. Even when they may be suitable, the vacuum required is higher than normally used as it needs to overcome the phobic resistance of the filter.
Likewise, the use of the separate grid with the hydrophobic system has not proven to be successful. Plates by different manufacturers can vary in their dimensions making such grids often plate specific. Additionally and more importantly, many plates are handled robotically and the introduction of a component that is loose and not easily gripped by robotic arms is not acceptable. Additionally, robotics are not exact and their handling often leads to overcompression of the plates which in turn leads to puncturing of the membrane by the grids which is unacceptable. To date no commercial embodiment of this design has been introduced.
US 2002/0179520A1 and 2002/0150505A1 uses the normal plate system and moves the top plate relative to the collection plate before they are completely pulled apart so as to cause any pendant drop to touch off on one or more walls of the collection wells. Preferably, this is accomplished by a movement of both plates relative to each other in a first and then in an opposite direction so there are two touch off attempts.
This idea requires specialized robotic equipment to create the relative movement between the plates. Additionally, the plate dimensions and movements need to be tightly controlled in order to ensure that the spout moves sufficiently close to the first and optimally the second wall of the well to create the touch off function while not moving the spout too close to cause an actual touching which could potentially damage the plate system.
U.S. Pat. No. 5,108,704 teaches the formation of a unique filter plate design in which the spout is located at an edge of the well beyond the point below the active filter area. The spout is designed to mate with the wall of the collection plate so that no drop is formed and all liquid flows down the wall. For plates with more than 96 wells (e.g. 384) there is not enough room on the standard plate size (as defined by the American National Standards Institute/Society for Biological Standards (ANSI/SBS) which sets industry standards for among other things, device sizes including multiwell plate dimensions standards), for the spout to be outside the active membrane area and still conform to the ANSI/SBS dimension standards. This limits that plate's applicability and acceptability.
This product has not been successfully commercialized. It requires the use of a new plate design. Moreover it requires that both the filter plate and the collection plate be made to high tolerances in order to create the exact fit required. Such a device is not acceptable in robotic applications in that the robots don't have the fine control necessary to mate and detach the plates. As such they would be continuously jammed and/or damaged making them useless.
What is desired is a device that provides the advantages of the current multiwell plate system but which reduces or eliminates the issue of pendant drops or at the very least controls them and which is robotically friendly. Moreover, it is desired to have a device that provides consistent pendant drop removal across the length and breadth of the plate. The present invention provides such a system.
The present invention relates to a multiwell plate having pendant drop control. More particularly, it relates to a multiwell plate having an opening in its bottom located so as to provide pendant drop control into the collection device downstream of the opening.
The present invention is to a filter plate and a collection system having an upper filter plate and a lower collection device. The filter plate has has two or more wells in register with the collection device. The filter plate has an underdrain having a lower opening that is in fluid communication with the collection device. It preferably contains a spout. The opening is offcenter of the centerpoint of the wells between which it resides. The opening is close to at least one wall of the well of the collection device but set off from that wall by a distance sufficient to ensure easy assembly and disassembly of the devices without contact or damage of the opening, especially when in the form of a spout within the well of the collection device. In this manner, any drop that begins to form contacts the adjacent surface of the collection device and travels down it into the collection device.
It is an object of the present invention to provide a multiple well filter plate comprising a plate having a top, a bottom and a thickness between the top and the bottom, a plurality of wells extending through the thickness, each well having an open top and at least a partially open bottom, a filter located adjacent the bottom to form a permeably selective opening to the bottom, an underdrain having a top surface, a bottom surface and a thickness in between, the top surface of the underdrain attached to the bottom of the plate, the underdrain having a series of chambers that register and mate with the bottom of the plurality of wells of the plate so as to ensure that fluid passing through the filter of a selected well enters only the respective chamber of the underdrain, each chamber having an opening through the bottom surface of the underdrain to an outside environment and each opening being offset from a centerpoint determined by the intersection of two or more diameters of the registered well of the collection device and chamber of the underdrain.
It is an object of the present invention to provide a multiple well plate filtration system comprising a filter plate having a top, a bottom and a thickness between the top and the bottom, a plurality of wells extending through the thickness, each well having an open top and at least a partially open bottom, a filter located adjacent the bottom to form a permeably selective opening to the bottom, an underdrain having a top surface, a bottom surface and a thickness in between, the top surface of the underdrain attached to the bottom of the plate, the underdrain having a series of chambers formed in its thickness that register and mate with the bottom of the plurality of wells of the plate so as to ensure that fluid passing the filter of a selected well enters only the respective chamber of the underdrain, each chamber having an opening through the bottom surface of the underdrain to an outside environment, a collection device located below the underdrain, the collection device having a top, a bottom and a thickness between the top and the bottom, a plurality of wells extending through the thickness, each well having an open top, each well of the collection plate is in align with a well of the filter plate and its associated underdrain chamber and opening and the opening of the underdrain has the ability to form a pendant drop of a radius R, the opening being located adjacent at least one wall of the collection device by a distance of between about 0.05 R and less than about 1 R.
It is another object to provide a multiple well plate filtration system comprising a filter plate having a top, a bottom and a thickness between the top and the bottom, a plurality of wells extending through the thickness, each well having an open top and at least a partially open bottom, a filter located adjacent the bottom to form a permeably selective opening to the bottom, an underdrain having a top surface, a bottom surface and a thickness in between, the top surface of the underdrain attached to the bottom of the plate, the underdrain having a series of chambers formed in its thickness that register and mate with the bottom of the plurality of wells of the plate so as to ensure that fluid passing the filter of a selected well enters only the respective chamber of the underdrain, each chamber having an opening through the bottom surface of the underdrain to an outside environment, a collection device located below the underdrain, the collection device having a top, a bottom and a thickness between the top and the bottom, a plurality of wells extending through the thickness, each well having an open top, each well of the collection plate is in align with a well of the filter plate and its associated underdrain chamber and opening and the opening is capable of forming a pendant drop of a radius of R, and wherein the opening is offset from an inner wall of the well of the collection device by a distance of from about 0.05 R to less than about 1 R.
It is an additional object to provide a device for separating a liquid sample comprising:
It is an additional object to provide a device for separating a liquid sample comprising:
It is another object of the present invention to provide a multiple well plate filtration system comprising a filter plate having a top, a bottom and a thickness between the top and the bottom, a plurality of wells extending through the thickness, each well having an open top and at least a partially open bottom, a filter sealed adjacent the bottom to form a permeably selective opening to the bottom, an underdrain having a top surface, a bottom surface and a thickness in between, the top surface of the underdrain attached to the bottom of the plate, the underdrain having a series of chambers formed in its thickness that register and mate with the bottom of the plurality of wells of the plate so as to ensure that fluid passing the filter of a selected well enters only the respective chamber of the underdrain, each chamber having an opening through the bottom surface of the underdrain to an outside environment, a collection device located below the underdrain, the collection device having a top, a bottom and a thickness between the top and the bottom, a plurality of wells extending through the thickness, each well having an open top, each well of the collection device is in align with a well of the filter plate and its associated underdrain chamber and opening, each opening being offset from a centerpoint determined by the intersection of two or more diameters of the registered well of the collection device, the vertical centerline and an inner wall of the collection plate being separated by a distance A and the opening being from about 0.05 A to less than about the distance A from the inner wall of the well of the collection plate.
The present invention relates to the control and preferably recovery of pendant drops formed on the bottom of an underdrain in a multiwell filtration plate system.
The invention can be demonstrated by the first embodiment of the present invention as shown in
As shown, the opening 38 and the spout 42 of the underdrain 34 are arranged to be offcenter of a vertical centerline 48 of the well 46 of the collection device 44. Also as shown in this embodiment they are offcenter of the same vertical centerline 48 of the well 26 of the filter plate 24 although as explained in further detail below, it need not be.
The centerline can be determined by a variety of means. One simple means is to simply take two or more diameters of the well 46, preferably three or more especially when there may be two or more different diameters in the well 46 (such as in a rectangular, oval or teardrop shaped well) and to note the point where they intersect. A vertical line can then be formed through that intersection point to yield a vertical centerline for the well. Another method is to simply determine the center or innermost radius point of the well and draw a vertical centerline through it. Other methods may also be used.
By setting the opening 38 and/or spout 42 (if used) off from the centerline of the well, they are located closer to one wall of the well than the other. In this way, a drop formed on the opening 38 or spout 42 will preferentially move toward that wall and be drawn by surface energy into the collection device 44 below.
In another embodiment of the present invention shown in
In a further embodiment shown in
Knowing this and using this, one can then position the spout 42 adjacent to but not in contact with the inner wall 50 such that a drop formed on the spout 42 will always contact the inner wall before reaching its full dimension and therefore be carried into the collection device 46. To state this in an empirical formula when the ratio of B:C of the opening 38 is ≧0.2, the drop will have a radius R for a given type of fluid, and the spout 42 and opening 38 location may be from about 0.05 R to less than about 1 R away from a surface 50 of the collection device, preferably from about 0.05 R to about 0.95 R away from the surface 50 of the collection device.
In addition to pulling pendant drops into the well of the collection plate rather than having them hang there and potentially lead to contamination or crosstalk, the present invention has other advantages. One advantage is that by offsetting the opening/spout location, one does not trap an air bubble in the well as the fluid flows into the collection plate as can occur with a centered spout/opening design, especially with the smaller well sizes such as 384 and 1536 well plates. Another advantage is that the design tends to reduce splashing and vaporization of the fluid as it flows into the well as the wall appears to act as a dampener and controls the flow of the fluid into the well in a more even and controlled manner. Other advantages of the present invention may also exist.
The underdrain can be an integral component of the filter plate, having been molded as part of the plate, overmolded on to a preformed plate or preformed separately and bonded to a preformed plate. Alternatively, it can be preformed and releasably attached to the bottom of a preexisting plate.
Suitable polymers which can be used to form the underdrain, collection plate and the filter plate include but are not limited to polycarbonates, polyesters, nylons, PTFE resins and other fluoropolymers, acrylic and methacrylic resins and copolymers, polysulphones, polyethersulphones, polyarylsulphones, polystyrenes, polyvinyl chlorides, chlorinated polyvinyl chlorides, ABS and its alloys and blends, polyolefins, preferably polyethylenes such as linear low density polyethylene, low density polyethylene, high density polyethylene, and ultrahigh molecular weight polyethylene and copolymers thereof, polypropylene and copolymers thereof and metallocene generated polyolefins.
Preferred polymers are polyolefins, in particular polyethylenes and their copolymers, polystyrenes and polycarbonates.
The underdrain, collection plate and filter plate may be made of the same polymer or different polymers as desired.
Likewise the polymers may be clear or rendered optically opaque or light impermeable. When using opaque or light impermeable polymers, it is preferred that their use be limited to the side walls so that one may use optical scanners or readers on the bottom portion to read various characteristics of the retentate. When the filter is heat bonded to the underdrain, it is preferred to use polyolefins due to their relatively low melting point and ability to form a good seal between the device and the filter.
One may use one or more filters in a given device. Typically, one filter layer is used, although some applications may require two or more filter layers (sometimes as a prefilter or to perform other desired functions). The filter(s) may be of any variety commonly used in filtering biological specimens including but not limited to microporous membranes, ultrafiltration membranes, coarse filters such as fibrous mats or papers, nanofiltration membranes, or reverse osmosis membranes. Preferably microporous membranes, ultrafiltration membranes, coarse filters or nanofiltration membranes are used. Even more preferably, microporous, coarse filters and ultrafiltration membranes are used.
Representative suitable microporous membranes include nitrocellulose, cellulose acetate, polysulphones including polyethersulphone and polyarylsulphones, polyvinylidene fluoride, polyolefins such as ultrahigh molecular weight polyethylene, low density polyethylene and polypropylene, nylon and other polyamides, PTFE, thermoplastic fluorinated polymers such as poly (TFE-co-PFAVE), polycarbonates or particle filled membranes such as EMPORE® membranes available from 3M of Minneapolis, Minn. Such membranes are well known in the art and are commercially available from a variety of sources including Millipore Corporation of Billerica, Mass. If desired these membranes may have been treated to render them hydrophilic. Such techniques are well known and include but are not limited to grafting, crosslinking or simply polymerizing hydrophilic materials or coatings to the surfaces of the membranes.
Representative ultrafiltration or nanofiltration membranes include polysulphones, including polyethersulphone and polyarylsulphones, polyvinylidene fluoride, and cellulose. These membranes typically include a support layer that is generally formed of a highly porous structure. Typical materials for these support layers include various non-woven materials such as spun bounded polyethylene or polypropylene, or glass or microporous materials formed of the same or different polymer as the membrane itself. Such membranes are well known in the art, and are commercially available from a variety of sources such as Millipore Corporation of Billerica, Mass.
Suitable coarse filters include glass mats, glass fibers, fibrous mats of cellulosic material or plastic and the like as well as filter papers such as pH papers or DEAE papers.
As described above, with the use of a plurality of wells, it is important that at least some, preferably all the wells of the first plate register with the well(s) of the collection device. Typically multiple well plates have been made in formats containing 6, 96, 384 or 1536 wells and above. The number of wells used is not critical to the invention. This invention may be used with any multiple number of wells provided that the filter is capable of being secured to the filter plate in a manner that locates it adjacent to the bottom of the well and preferably forms a liquid tight seal between the periphery of the filter and the end of the wells of the plate. The wells are typically arranged in mutually perpendicular rows. For example, a 96 well plate will have 8 rows of 12 wells. Each of the 8 rows is parallel and spaced apart from each other. Likewise, each of the 12 wells in a row is spaced apart from each other and is in parallel with the wells in the adjacent rows. A plate containing 1536 wells typically has 128 rows of 192 wells. The wells may have a shape that is round, square, rectangular, triangular other polygonal shape, oval, teardrop or any other design commonly used in such plates.
A variety of methods for forming the filter plate according to the present invention may be used. Any method which locates and preferably seals the membrane within the well of the plate or on to the bottom of the plate (in the single plate design) and on or in the well of the bottom plate (in the two plate design) such that all fluid within the well must pass through the filter before leaving the well through the bottom opening will be useful in this invention.
One method of forming such a device is to form a single plate of a suitable plastic as described above and use a mechanical seal between the well wall and the filter. In this embodiment, there is an undercut formed around the periphery of the inner wall of the well. The filter is sized so as to fit within the undercut portion of the well. The filter is placed within the well. Optionally, a sealing gasket is applied on top of the filter within the undercut. This sealing gasket applies pressure to the filter and ensures that all the fluid must pass through the filter thereby eliminating any leakage or bypass of the filter by the fluid. This gasket may be in the form of a preformed gasket such as an O-ring. Alternatively, a gasket formed of a molten or liquid material may be cast into the undercut to seal the filter in place. An example of a molten material suitable for this embodiment, are any of the well-known hot melt materials such as polyethylene or polypropylene or ethylene vinyl acetate copolymers. A liquid gasket may be formed of any curable rubber or polymer such as an epoxy, urethane or synthetic rubber.
Another method of forming such a device is to use an adhesive to bond and seal the edge of the filter within the well such as all fluid must pass through the filter before entering the opening in the bottom of the well. Adhesive may be either molten or curable as discussed above.
A further method is to use a thermal bond to secure the filter to the well. In this embodiment, a filter sealing device which has a sealing surface which is heated is brought into contact with the upper filter surface and transfer its thermal energy to the surrounding filter and well material. The energy causes either the filter material or the well materials or both to soften and or melt and fuse together forming an integral, fluid tight seal. This process may be used when either the filter material or the well material or both are formed of a thermoplastic material. It is preferred that the well as well as at least a portion of the filter material adjacent the downstream side of the filter be formed of a thermoplastic material. The sealing surface is only a portion of the filter surface and is a continuous structure so that a ring or peripheral area of the filter is sealed to the well so as to form a liquid tight seal between the filter, the well and the opening in the bottom of the well.
This application claims the benefit of U.S. Provisional Application Ser. No. 60/564,999, filed on Apr. 23, 2004.
| Number | Date | Country | |
|---|---|---|---|
| 60564999 | Apr 2004 | US |
