Patent Application
20050019224
The present invention relates generally to a virtual well plate system that orders and retains fluid drops in a defined spatial array, and more particularly, to a virtual well plate system that permits accurate and controlled joining of the plates to create the desired virtual wells.
Because of the large volumes of data, compounds and targets, screening laboratories are required to work faster than ever in order to develop new products for market. Therefore, it is necessary to use a high-throughput screening system that delivers accurate data at a fast rate.
In order to better aid in such processes, a virtual well plate system was developed, which is the subject matter of published International Application No. WO 99/39829 (PCT/US99/02300) entitled VIRTUAL WELLS FOR USE IN HIGH THROUGHPUT SCREENING ASSAYS by Tina Garyantes, the entire disclosure of which is incorporated herein by reference. Basically, microliter-like plates containing virtual wells formed by an arrangement of relatively hydrophilic domains within relatively hydrophobic fields are provided. Assay mixtures are confined to the hydrophilic domains of the virtual wells by the edges of the hydrophobic fields.
Specifically, an array of droplets is confined to the hydrophilic domains within the hydrophobic field on a glass plate. Surface tension holds the droplets on the plates. In particular, there is a base and a lid, each typically having a 1536 well array of hydrophilic spots on a hydrophobically masked glass slide. The glass slides are each framed to enable controlled docking of the lid on the base. Thus, when the base and lid are assembled together, the glass plates of each are brought into close proximity with each other, whereby the aligned droplets touch to create short liquid columns or virtual wells. The hydrophobic masked regions surrounding each virtual well ensures that the liquid stays in each well and does not migrate or travel to adjacent virtual wells. Such virtual well plate systems are well known, and for example, sold by Becton, Dickinson and Company, 1 Becton Drive, Franklin Lakes, N.J. 07417 under the trademark FALCON.
The use of virtual wells is a versatile platform for biochemical and cell-based assays, and provides distinct advantages. Specifically, the use of virtual wells permits homogeneous and high throughput screening of assays with assay mixtures having volumes on the order of about 100 nl to 10 μl, while also providing a means for easily moving fluids. This provides an extremely flexible and efficient general assay platform that can be used with, for example, a wide variety of fluorescence and luminescence-based detection modes, with minimal waste of compounds.
However, a problem with such known virtual well system is in regard to the assembly of the lid on the base to form the virtual wells. In such case, it is necessary to either manually combine the lid and base or to use expensive and complicated robotics. This is because it is necessary to maintain the glass plates of the lid and base apart a sufficient distance so that the droplets do not combine to form the columns prior to the desired time. Therefore, since the glass plates must generally be kept separate and apart from each other, this further adds to the burden of preparation, storage and assembly of the lid and base.
Further, existing virtual well plates are not directly applicable to kinetic read assays such as the Fluorometric Imaging Plate Reader system sold under the trademark AFLIPR@ by Molecular Devices Corporation, 1311 Orleans Avenue, Sunnyvale, Calif. 94089-1136 FLIPR. The AFLIPR@ and similar systems available from CyBio AG, Göschwitzer Straβe 40, D-07745 Jena, Germany, and Hamamatsu Corporation, U.S.A., 360 Foothill Road, Bridgewater, N.J. 08807-0910, include integrated pipetting to enable kinetic read assays. These are assays whose response rapidly follows the addition of a stimulant, typically an agonist, and where the time course of that response needs to be recorded from its onset through the peak response and resolution of the response.
However, the two plate virtual well plate system of prior art is a closed system in that the upper plate blocks any further addition by pipettes from above. Further addition by pipettes, such as on the AFLIPR@ system, would require the use of a single plate system, which in practice would be simply a low volume microplate.
The two plate virtual well plate could be used if the lid and base were delivered separately to the AFLIPR@ system. This would require additional plate loading robotics, and a system to keep the plates separate after the door is closed to reduce ambient light.
Accordingly, it is an object of the present invention to provide a virtual well plate system that overcomes the aforementioned problems.
It is another object of the present invention to provide a virtual well plate system that provides an accurate and controlled arrangement for preassembling a base and lid, each containing liquid-filled virtual wells without the contents of those wells joining to form columnar virtual wells, that is, the plates are assembled but not in close proximity, and a mechanism or kinematic system integral to the plate system allowing the plates carrying those virtual wells to be brought into close proximity by an external force or actuation.
It is still another object of the present invention to provide a virtual well plate system that maintains the base and lid in a spaced apart, but assembled condition, ready for formation of the virtual wells upon the application of an external force to the lid.
It is yet another object of the present invention to provide a virtual well plate system which can use a conventional pipette head or similar mechanism to move the glass plate of the lid into close proximity with the glass plate of the base to form the virtual wells, without the use of specialized robotic equipment.
It is a further object of the present invention to provide a virtual well plate system that provides near simultaneous formation of all 1536 wells.
It is a still further object of the present invention to provide a virtual well plate system that provides lateral alignment of the glass plate of the lid relative to the glass plate of the base to form the virtual wells.
It is a yet further object of the present invention to provide a virtual well plate system that can be used in an automated system.
It is another object of the present invention to provide a virtual well plate system that can simultaneously deliver 384, 1536 or higher well counts.
It is still another object of the present invention to provide a virtual well plate system in which no tip washing is required to avoid cross contamination of samples.
It is yet another object of the present invention to provide a virtual well plate system the spring-separated virtual well plate is compatible with the existing AFLIPR@ system without additional robotics, that is, without any re-engineering, although slight add-ons can be made to optimize the compatibility
It is a further object of the present invention to provide a virtual well plate system in which plate separation is inherent.
It is a further object of the present invention to provide a virtual well plate system in which even one microliter additions, which would be difficult with disposable pipette tips, are very reliable with the two plate virtual well plate system of the present invention.
In accordance with an aspect of the present invention, a virtual well plate system includes a base including a base plate having an upper surface with a hydrophobic region which defines a plurality of hydrophilic domains on the upper surface of the base plate, each hydrophilic domain adapted to hold a droplet of liquid therein; a movable lid including a lid plate having a lower surface with a hydrophobic region which defines a plurality of hydrophilic domains on the lower surface of the lid plate, each hydrophilic domain of the lid plate adapted to hold a droplet of liquid therein in a hanging manner; and a resistance arrangement mounted to at least one of the base and the lid which maintains the base and lid in an assembled condition such that the base plate and lid plate are maintained at a sufficient distance to prevent formation of virtual wells by the droplets thereon, and which permits movement of the lid toward the base upon application of an external force sufficient to overcome a resistance of the resistance arrangement in order to form the virtual wells by a combination of the droplets on the base plate and the lid plate.
In one embodiment, the resistance arrangement includes springs which support the movable lid above the base. In a particular example of this embodiment, a stationary lid is mounted to the base, and the springs are connected between the stationary lid and the movable lid for supporting the movable lid above the base. These springs can be coil, leaf or other spring elements. In such case, the stationary lid includes at least one opening through which an external pressing device can be inserted for biasing the movable lid toward the base against the force of the springs. In another example of this embodiment, the base includes upstanding side walls, and the springs include coil springs connected between upper ends of the side walls and the movable base.
In another embodiment, the resistance arrangement includes a deformable spacer between the lid and the base.
The deformable spacer can be a resilient member. As one example, the deformable spacer includes springs positioned between the base and the lid. The springs can be connected to the base or the lid, and can, for example, be cantilevered leaf springs. The cantilevered leaf spring can be positioned between the base and the lid when the lid is moved toward the base by the external force, so as to maintain the base plate and the lid plate separated by a predetermined distance sufficient to form the virtual wells. Alternatively, the base can include a recess for receiving the deformable spacer.
The deformable spacer can alternatively be a non-resilient member. As one example, the non-resilient member can be a crushable member which is crushed when the external force is applied to the lid. The crushable member can include slits for permitting easy crushing thereof.
In one embodiment, the base includes peripheral flanges having upper surfaces, and the base plate includes an upper surface which is positioned lower than the upper surfaces of the peripheral flanges such that, when the lid is moved by the application of the external force sufficient to overcome the resistance of the resistance arrangement, the peripheral flanges maintain a lower surface of the lid plate at a predetermined distance from the upper surface of the base plate for formation of the virtual wells. The peripheral flanges can include recesses in the upper surfaces thereof for holding the resistance arrangement and for permitting the resistance arrangement to collapse entirely in the recesses such that the lid rests on the upper surfaces of the peripheral flanges when the lid is moved by the application of the external force sufficient to overcome the resistance of the resistance arrangement.
Preferably, the base includes upstanding side walls and the lid is slidably positioned within the upstanding side walls. In such case, the resistance arrangement can include first detents on inner surfaces of the upstanding side walls and second detents on outer surfaces of the lid for engagement with the first detents such that the first detents support the lid plate at a sufficient distance from the base plate to prevent formation of the virtual wells by the droplets thereon, and which permit movement of the lid plate toward the base plate upon application of an external force sufficient to overcome resistance of the second detents riding over the first detents in order to form the virtual wells by a combination of the droplets on the base plate and the lid plate. In one example, there are two substantially vertically aligned first detents and one second detent which is captured between the two first detents to maintain the lid plate at a sufficient distance from the base plate to prevent formation of the virtual wells by the droplets thereon.
In another embodiment, the resistance arrangement includes at least one laterally movable spring biased element mounted to the base for applying a lateral force to the lid to maintain the lid plate at a sufficient distance from the base plate to prevent formation of the virtual wells by the droplets thereon and to also laterally align the lid plate relative to the base plate, and which permits movement of the lid plate toward the base plate with the lateral alignment upon application of an external force sufficient to overcome resistance of the laterally movable spring biased element in order to form the virtual wells by a combination of the droplets on the base plate and the lid plate.
In one embodiment, the base includes upstanding side walls, and each laterally movable spring biased element includes a cam lever pivotally mounted to at least one upstanding side wall and a spring for biasing the cam lever inwardly of the base. Each upstanding side wall to which at least one cam lever is pivotally mounted includes at least one opening therein, and each cam lever is pivotally mounted to the base and is positioned in a respective opening. Preferably, each cam lever includes a first detent on an inner facing surface thereof, and the lid includes at least one second detent on an outer facing surface thereof for engagement with each first detent.
In another embodiment, each laterally movable spring biased element includes a guide plate and springs which bias the guide plate inwardly of the base in a lateral direction so as to maintain the base and lid in an assembled condition by the force of the guide plate on the lid such that the base plate and lid plate are maintained at a sufficient distance to prevent formation of the virtual wells by the droplets thereon, and which permits movement of the lid plate toward the base plate upon application of an external force sufficient to overcome frictional resistance between the guide plate and the lid in order to form the virtual wells by a combination of the droplets on the base plate and the lid plate.
In still another embodiment, the base includes upstanding side walls, with at least one upstanding side wall including an opening therein, and each laterally movable spring biased element includes a cantilevered leaf spring hinged to the base and positioned in a respective opening. A wedge element is provided on an outer surface of the lid in association with each cantilevered leaf spring such that downward movement of the lid by the external force causes engagement between each cantilevered leaf spring and associated wedge to laterally move the lid plate into lateral alignment with the base plate.
In yet another embodiment, each laterally movable spring biased element includes an upstanding cantilevered leaf spring having an inwardly bowed configuration and extending upwardly from the base between the upstanding side wall of the base and the lid for laterally biasing the lid when the lid plate is moved toward the base plate.
Preferably, the base includes a plurality of connected upstanding side walls, and there are a plurality of the laterally movable spring biased elements mounted to two adjacent upstanding side walls for laterally aligning the lid relative to the base.
The above and other objects, features and advantages of the invention will become readily apparent from the following detailed description thereof which is to be read in connection with the accompanying drawings.
Referring to the drawings, and initially to
When base 12 and lid 14 are assembled together, as shown in
However, as discussed above, a problem with such known virtual well plate system 10 is with regard to the assembly of the base 12 and lid 14 to form virtual wells 26. In such case, it is necessary to either manually combine base 12 and lid 14, or to use expensive and complicated robotic equipment. This is because it is necessary to maintain base 12 and lid 14 apart a sufficient distance so that droplets 24 do not combine to form columns or virtual wells 26 prior to the desired time. Therefore, base 12 and lid 14 must generally be kept separate and apart from each other, which further adds to the burden of preparation, storage and assembly thereof.
Referring now to
As with virtual well plate system 10, virtual well plate system 110 includes a base 112 having a glass plate 116 provided in a frame 118 made of any suitable material, including but not limited to a metal such aluminum or steel, a plastic, a thermoplastic elastomer, etc. Although glass plate 116 and frame 118 are shown to have a generally rectangular configuration, the present invention is not limited thereby. Each side wall 120 of frame 118, as shown in cross-section, includes a long vertical wall section 122 which terminates at its lower end at a short outwardly directly horizontal wall section 124, and which in turn, terminates at its outer end at a short downwardly directed vertical foot wall section 126 that supports base 112 on a surface. Further, short horizontally oriented flanges 128 extend inwardly from the lower ends of long vertical wall sections 122 at positions higher than horizontal wall sections 124, the purpose for which will be better understood from the discussion which follows. Glass plate 116 is secured to base 112 at a position below flanges 128. In this regard, for example, glass plate 116 can be secured directly to the underside of flanges 128, as shown in
A hydrophobic field 130 is provided on glass plate 116 to define a plurality of, for example, 1536, hydrophilic domains 132. Only some of the hydrophilic domains 132 are shown in
Virtual well plate system 110 further includes a lower movable lid 114 having a glass plate 136 provided in a frame 138 made of any suitable material, including but not limited to a metal such aluminum or steel, a plastic, a thermoplastic elastomer, etc. Although glass plate 136 and frame 138 are shown to have a generally rectangular configuration, the present invention is not limited thereby. The outer dimensions of frame 138 permit lid 114 to fit within frame 118 of base 112 and to slide vertically therein. Frame 138 includes two spring retaining elements 140 on each of the two opposing short walls. Each spring retaining element 140 can be any suitable device, such as an opening in frame 138, a hook or loop on frame 138, etc. for holding one end of a coil spring. Spring retaining element 140 is shown as a hook 140a in
A hydrophobic field 142 is provided on glass plate 136 to define a plurality of, for example, 1536, hydrophilic domains 144 equal in number, dimensions and spacing to hydrophilic domains 132 on glass plate 116. Only some of hydrophilic domains 144 are shown in
Virtual well plate system 110 further includes an upper stationary lid 148 comprised of a frame 150 made of any suitable material, including but not limited to a metal such aluminum or steel, a plastic, a thermoplastic elastomer, etc., and surrounding a central opening 154. Frame 150 has the same general outer dimensions as side walls 120 of base 112, and is immovably connected to the upper end of long vertical wall sections 122 of side walls 120. Frame 150 includes two spring retaining elements 152 on each of the two opposing short walls. Each spring retaining element 152 can be any suitable device, such as an opening in frame 150, a hook or loop on frame 150, etc. for holding one end of a coil spring. Spring retaining element 152 is shown as a hook 152a in
Four coil springs 156 are connected between corresponding spring retaining elements 140 and 152, such that lower movable lid 114 is suspended above horizontally oriented flanges 128 of base 112, as shown in
The entire assembly can then move along a conveyor in an automated process or can merely be placed manually in a machine such that an upper pressing assembly 158 extends downwardly through opening 154 in upper stationary lid 148 to push down lower movable lid 114 against the force of coil springs 156, until lower movable lid 114 rests on the upper surfaces of horizontally oriented flanges 128. The thickness of flanges 128 determines the spacing between glass plates 116 and 136, for example, 0.85 mm (0.034 inch). Upper pressing assembly, in a preferred embodiment, is formed by conventional pipette tips or tip holders.
Of course, it will be appreciated that droplets 134 and 146 are in alignment with each other. This can be accomplished, for example, by configuring the dimensions of lower movable plate 114 to have little or no play when sliding within base 112. In addition, as shown in
When lower movable lid 114 is moved to the position shown in
Thus, the present invention presents a virtual well plate system 110 that provides an accurate and controlled arrangement for combining base 112 and lower movable lid 114 to form virtual wells 160, and more particularly, that maintains base 112 and lower movable lid 114 in an assembled condition, ready for formation of virtual wells 160. In this regard, this embodiment of the present invention uses a conventional pair of patterned glass plates 116 and 136, while adding a secondary or upper stationary lid 148 which permits assembly of base 112 and lower movable lid 114, but which maintains base 112 and lower movable lid 114 spaced apart by springs 156. This simplifies the mechanism required to execute a kinetic addition. Merely pressing on lower movable lid 114 with a pipette head or similar mechanism overcomes the preload force of coil springs 156, and moves glass plate 136 of lower movable lid 114 down into close proximity with glass plate 116 of base 112 to form virtual wells 160. This can easily be accomplished within the darkened enclosure of the aforementioned Fluorometric Imaging Plate Reader system sold under the trademark AFLIPR@ by Molecular Devices Corporation, or any other similar reader, without the use of a specialized robot to move lower movable lid 114. Additionally, since lower movable lid 114 is contained within the standard envelope of base 112, which is a shallow well microplate, the assembly of base 112 and lower movable lid 114 can be loaded into the Fluorometric Imaging Plate Reader system using existing stackers and mechanisms.
Since existing virtual well plates are designed with 1536 well patterns, the improved spring-spaced plate of the present invention provides 1536 capabilities to the Fluorometric Imaging Plate Reader system, and provides near simultaneous formation of all 1536 virtual wells.
It will be appreciated that various modifications within the scope of the present invention can be made to virtual well plate system 110. For example, upper stationary lid 148 can be eliminated, and the upper ends of coil springs 156 can be connected to openings 122a in long vertical wall sections 122, as shown in
It will be appreciated that the present invention is not limited to the coil spring arrangement of
For example, a virtual well plate system 210 according to a second embodiment of the present invention is shown in
However, deformable spacers 262 are provided only at a portion of the perimeter of lid 214. This is because deformable spacers 262 are flattened when a downward external pressure is applied to lid 214, and space must be provided for the lateral expansion of deformable spacers 262.
In addition, it is preferable that deformable spacers 262 be provided in recesses 264 in the upper surfaces of flanges 228, as shown in the enlarged views of
It will be appreciated that there are numerous constructions for deformable spacers 262, and some examples of deformable spacers 262 that can be used will now be provided, bearing in mind that the present invention is not limited to these specific examples.
Deformable spacers 262 can be reversible (resilient) or irreversible (non-resilient) in accordance with a third embodiment of the present invention.
It will be appreciated that cantilevered leaf spring 266 can be formed integrally with frame 238 of lid 214 instead of being formed integrally with base 212, as shown in
In addition, in the embodiment of
In order to remove lid 214 after formation of the virtual wells, a special tool (not shown) can be used. This can be, for example, a simple hook that enters an opening in frame 238, is rotated and then pulls up on lid 214. Alternatively, a vacuum gripper or the like can be used to remove lid 214.
As discussed above, deformable spacers 262 can be irreversible, that is, not resilient, so that it does not return to its initial position when the force on lid 214 is removed.
Irreversible or non-resilient, deformable spacers 262 can take other forms, such as that of a Chinese lantern projection 280, as shown in
Irreversible deformable spacers 262 can take other forms, such as that of a slitted hemispherical dome or bubble projection 284, as shown in
Referring now to
When a downward force is applied to lid 314, tabs 386 break away from frame 338 and remain in open slots 388, while lid 314 is forced down against the upper surface of flanges 328 in order to form the virtual wells, as shown in
Referring now to
In order to remove lid 414 after formation of the virtual wells, a special tool (not shown) can be used. This can be, for example, a simple hook that enters an opening in frame 438, is rotated and then pulls up on lid 414. Alternatively, a vacuum gripper or the like can be used to remove the lid.
Referring now to
In this regard, long vertical wall sections 522 of two adjacent side walls 520 of base 512 each include at least one opening 590, and a cam lever 592 is positioned in each opening 590 and pivotally mounted by a pivot pin 594 at the upper end of opening 590. A spring 596, which can be a leaf spring (as shown), coil spring, torsion spring or the like, normally biases each cam lever 592 inwardly of base 512. A detent 574a is provided on the inner surface of each cam lever 592, and a detent 576a is provided on the outer surface of frame 538 of lid 514 that faces cam lever 592, and is in vertical alignment with detent 574a. A detent 574b is provided on the inner surface of the side wall 520 which is opposite to cam lever 592, and a detent 576b is provided on the outer surface of frame 538 that faces detent 574b, and is in vertical alignment with detent 574b.
In this manner, as shown in
At the time when detents 576a and 576b pass or ride over detents 574a and 574b, the spring force of springs 596 move cam levers 592 in the counter-clockwise direction to the position shown in
It will be appreciated that cam levers 592 are preferably provided on two adjacent side walls 520, with detents 574b being provided on the opposing two side walls 520 so as to provide biasing of lid 514 in the lateral X-Y directions to obtain X-Y alignment to a zero reference position.
Although spring activated cam lever 592 is shown as being pivoted about a pivot pin 594, it will be appreciated that cam lever 592 can be pivoted at a living hinge, and thereby be integral with base 512. As a further modification, the hinge or pivot point for cam lever 592 can be at the bottom of opening 590 of base 512, as opposed to the top which is shown in
A modification of the sixth embodiment is shown in
It will be appreciated that leaf springs 692 are shown biased outwardly for the sake of better explanation, but will normally be biased inwardly. The spring force from leaf springs 692 is sufficient to hold lid 614 so that upper glass plate 636 thereof is spaced from the lower glass plate (not shown) of the base to prevent formation of the virtual wells, in the absence of an external downward actuating force on lid 614.
In addition, leaf springs 692 interact with wedges 674b to bias lid 614 in the lateral X-Y directions. Because of the increase in depth of wedges 674b, lid 614 is gradually pushed in the X and Y directions until the remaining two side walls of frame 638 abut against columnar stops 676a on the inner surfaces of side walls 620 that do not contain leaf springs 692. The downward force on lid 614 prevents lid 614 from raising up from base 612. In this position, upper glass plate 636 of lid 614 is separated from the lower glass plate (not shown) of base 612 by a predetermined distance of, for example, 0.85 mm.
A further modification of the sixth embodiment is shown in
Thus, when lid 714 is pushed down, frame 738 slides against the inner surface of guide plate 799, which by reason of coil springs 798 biases lid 714 to the right in
A further modification of the sixth embodiment is shown in
It will be appreciated that any other suitable resistance arrangement can be used which is mounted to the base and/or the lid and which maintains the base and lid in an assembled condition such that the base plate and lid plate are maintained at a sufficient distance to prevent formation of virtual wells by the droplets thereon, but which permits movement of the lid toward the base to form the virtual wells upon application of an external force sufficient to overcome the resistance of the resistance arrangement. This can even be accomplished by a simple friction fit between the lid and base.
Upper stationary lid 1048 includes an upper wall 1050 having a plurality of, for example, four, access openings 1054 which serve the same function as central opening 154 in
In addition, x-y registration spring arms 1098 are formed in a cantilevered manner at two adjacent side walls of movable lid 1014, and the free ends of which engage the inner surfaces of long vertical wall sections 1022 of side walls 1020 of base 1012. In this manner, the wells on base 1012 and lid 1014 are aligned with each other.
It will be appreciated that certain assays are near real-time kinetic, and do not require the time course to be recorded from the onset through the peak response and resolution of the response. Yet it is difficult to make additions of agonist to the plate simultaneously at 1536 and higher well counts. The spring (or otherwise separated) plate can be used with these readers, in conjunction with a simple electromechanical or pneumatic plunger device to actuate the plate just prior to the loading into the detection system. This would require the “actuate, remain actuated” embodiment.
It will be appreciated that virtual wells are not only hydrophilic wells in a hydrophobic field, but are any surface modification, etc. that orders or retains drops in a defined spatial array. Thus, the simultaneous addition of the present invention is a feature of any two plate virtual well plate system, and also applies to all plate densities. In this manner, the addition can be after assembly and upon actuation of the invention.
It will further be appreciated that in all of the above embodiments, alignment, cam operation, and detent functions can be either on the lid or the base.
Further, various features from different embodiments can be combined. For example, the X-Y alignment feature and the cam action can be combined with the detents, and are not restricted to a friction type system.
It will further be appreciated that the present invention can be made of any suitable material. For example, the entire assembly can be made entirely of plastic, with the virtual wells using textured areas of the lid. In such case, the lid would have molded-in features for the detents and springs.
Preferably, there would be one base compatible with three lids, namely, a first lid that has no resistance elements, a second lid that has a resilient element and detents and a third lid that simply has detents. This would allow the same base to be ordered for use as a standard virtual well plate, with a machine that can actively hold down the lid, and one that needs the lid to remain down after being actuated by an external device.
Having described specific preferred embodiments of the invention with reference to the accompanying drawings, it will be appreciated that the present invention is not limited to those precise embodiments and that various changes and modifications can be effected therein by one of ordinary skill in the art without departing from the scope or spirit of the invention as defined by the appended claims.
This application claims the benefit of U.S. provisional patent application No. 60/478,801, filed Jun. 16, 2003, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60478801 | Jun 2003 | US |
