Abstract:
A filter plate for use in biological or chemical applications, and its method of manufacture, are disclosed. The filter plate comprises a plurality of structurally interconnected wells which comprise a matrix of wells having a uniform diameter, each well having a side wall which defines a vertically extending, generally cylindrical cavity; a bottom wall which closes the cavity, the bottom wall having a drainage opening formed therein; a filter sheet extending across and resting on top of the bottom wall; the filter sheet being irremovably fixed in position as a result of engagement with the side wall; a conical nozzle having an external surface and an internal passage communicating with the drainage opening in the bottom wall; and a membrane supporting surface across the internal passage extending from the walls of the internal passage to a plane normal to the bottom wall.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    The invention relates to a disposable multiwell filter apparatus for use in biological and biochemical assays that can be used and is compatible with existing equipment.  
           [0002]    In pharmaceutical and biological research laboratories, plates with a multitude of wells have replaced traditional test tubes for assay and analysis. For many years, multi-well laboratory plates have been manufactured in configurations ranging from 1 well to 384 wells, and beyond. The wells of multi-well plates are typically used as reaction vessels in which various assays are performed. The types of analytical and diagnostic assays are numerous. The typical areas of use include cell culture, drug discovery research, immunology, and molecular biology, among others. Current industry standard multi-well plates are laid out with 96 wells in an 8×12 matrix (mutually perpendicular 8 and 12 well rows). In addition, the height, length and width of the 96-well plates are standardized. This standardization has resulted in the development of a large array of auxiliary equipment specifically developed for 96-well formats.  
           [0003]    Many assays or tests require a mixture of particulate or cellular matter in a fluid medium. The mixture is then subjected to combination with reagents, separation steps and washing steps. The end product of such analysis is often a residue of solid matter which may be extracted for further analysis.  
           [0004]    Separation of solids from fluid medium is often accomplished by filtration. The separation is accomplished in or on the filter material by passing the liquid through it. The liquid can be propelled through the membrane either by a pressure differential or by centrifugal force. Filter plates that conform to a 96 well standardized format are known as disclosed in U.S. Pat. Nos. 4,427,415 and 5,047,215. One significant problem that has been encountered with filter plates adapted for use with a 96 well plate is that cross contamination may occur between wells. When a unitary filter sheet is sandwiched between two pieces of plastic molded in a 96 well format, liquid from one well, upon wetting the filter material, may wick through the paper to neighboring wells thereby contaminating the sample contained within that well. One solution to this problem is offered in U.S. Pat. Nos. 4,948,442 and 5,047,215. In these patents, a 96 well filter plate is disclosed comprising a filter sheet placed between two plastic plates. One of the plates has a series of ridges that cut the filter sheet when the plates are ultrasonically welded together. By cutting the filter sheet around each well, the possibility of wicking between neighboring wells is effectively eliminated. A problem with this design is that it limits the product offering to membranes that can be cut by the process and to plate materials that can be ultrasonically welded. In fact, the potential for wicking and cross contamination still exists when the filter material is not completely severed in the welding process.  
           [0005]    U.S. Pat. No. 4,427,415 discloses a filter plate of one piece construction having wells with drain holes in the bottom and capable of receiving filter discs into the wells. Wicking is obviously not a problem in this plate because individual filter discs are used as opposed to a unitary sheet of filter paper. The filter discs used in this plate are put into each well individually and are not secured to the bottom of the well. A danger exists with a filter disc that has not been secured down in that some liquid from the well could pass under the filter and thereby escape filtration, resulting in contamination of the filtrate.  
           [0006]    Our invention solves several problems of prior art filter plate designs by providing a multiwell filter plate in which 1) filters are securely fastened to the plate without the use of glue or other potentially contaminant chemical adhesives 2) an expansive variety of filter materials may be used, 3) a large number of thermoplastic components may be employed in its construction, and 4) no cross contamination through liquid wicking occurs between neighboring wells. The preferred embodiment of the present invention also offers a conical nozzle designed to cause exiting fluid to create droplets rather than lateral flow along the bottom of the plate. Further, a ring or skirt will preferably circumscribe the underside of each filter well. The skirt fits into a corresponding well of a receiver plate and is designed to prevent cross contamination that may otherwise occur by splashing of filtrate.  
         SUMMARY OF THE INVENTION  
         [0007]    It is therefore an object of the present invention to provide a disposable filtration device for chemical and biological tests in which a large number of samples may be tested simultaneously. Further objects of the present invention are: to provide a filter plate that will be compatible with existing 96 well cluster plate formats as standardized by the industry; to provide a filter plate that can be handled by automated robotic assay equipment; to provide a filter plate having individual wells having a support grid on the bottom to help support the filter element, prevent tearing, and allow for an even distribution of filtered material on the filter; to provide a filter plate in which liquid from one well can not mix with liquid from a neighboring well (the filter plate of the present design prevents lateral flow or cross-talk of liquid through the membrane to other wells); to prevent cross contamination of filtrate after passing through the filter and passing to a receiver plate; to provide a filter plate of two part construction in which each individual well filter is securely pinned between opposing plates that are insert molded against each other; and to provide a unique method for the manufacture of filter plates.  
           [0008]    Briefly, the present invention relates to an improved filter plate and its method of manufacture. The filter plate is a two part construction. It comprises a well plate preferably with 96 wells, each well being open on both ends, molded against a harvester plate insert preferably having 96 counter-bores, each containing a filter disc, whereby each counter-bore aligns with a corresponding and respective well from the well plate, and whereby the diameter of the counter-bore is greater than the diameter of the well, such that the well bonds with the outer rim of the counterbore thereby creating a lap joint. The lap joint also serves the purpose of fixing the filter disc securely to the insert without the need for glue or chemical adhesives. During the injection molding process, extremely high pressures in the mold ensure that the edges of the filter disc are pressed against the insert.  
           [0009]    The assembled filter plate product has a plurality of interconnected wells of uniform diameter, each well being defined by a circular side wall, each of the side walls being interconnected to the side wall of at least two adjacent wells, each of the wells being open at one end. Further, the plate has a bottom wall at the bottom of each of the wells, which is connected to the side wall, each of the bottom walls having an opening therein. A conical drainage nozzle having an external surface and an internal passage communicating with the opening in the bottom wall, extends downwardly from the bottom wall from a point radially inward from the side wall. Finally, a filter disc is positioned on top of the bottom walls of the wells, the peripheries of each filter being sandwiched between a bottom portion of the side wall of each well and a top portion of the bottom wall of each well. The bottom walls have an opening therein, the opening preferably taking the form of a funnel shaped nozzle. A support grid preferably extends across the opening in order to provide support for the filter disc.  
           [0010]    The method of manufacturing the plate comprises several steps, namely: forming an insert having a plurality of counter-bores; punching filter discs into the bottom surface of the counter-bore; and insert molding a well plate against the insert and filters such that wells from the well plate align with corresponding counter-bores from the insert thereby forming a lap joint that effectively secures the filter disc in place. The method can be extended for use in the manufacture of multiwell plates which do not have a filter, but require a well bottom of a different material than the side walls. 
       
    
    
     DESCRIPTION OF THE FIGURES  
       [0011]    [0011]FIG. 1 is a plan view of the insert of the present invention.  
         [0012]    [0012]FIG. 2 is a side view of the insert of the present invention.  
         [0013]    [0013]FIG. 3 is a fragmentary cross sectional view of the insert of FIG. 1, taken along the section line  3 - 3  in FIG. 1.  
         [0014]    [0014]FIG. 4 is a three dimensional view of the insert of the present invention.  
         [0015]    [0015]FIG. 5 is an enlarged view of the corner of the insert of FIG. 4.  
         [0016]    FIGS.  6 A- 6 C are cross sectional views of a three step process for punching filter discs from a unitary sheet of filter paper, and inserting the discs into the insert.  
         [0017]    FIGS.  7 A- 7 D are cross sectional three dimensional views of the molding process of the current invention whereby a well plate is molded against an insert.  
         [0018]    FIGS.  8 A- 8 D are cross sectional two dimensional views of the molding process shown in FIG. 7.  
         [0019]    [0019]FIG. 9 is a multiwell filter plate of the present invention having a corner section extracted.  
         [0020]    [0020]FIG. 10 is an enlargement of the corner of the multiwell filter plate of FIG. 9 showing a cross section of two adjacent wells.  
         [0021]    [0021]FIG. 11 is a multiwell plate of the present invention having a corner section extracted.  
         [0022]    [0022]FIG. 12 is an enlargement of the corner of the multiwell plate of FIG. 9 showing a cross section of two adjacent wells.  
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0023]    Shown in FIG. 1 is an insert  10  of the present invention. The term insert is defined as a harvester plate capable of holding filter elements. The insert  10  is molded of a preferably hydrophobic thermoplastic material and preferably has  96  separate and distinct counter-bores  12  within it. Ideally, the spacings from the center point of each counter-bore  12  will conform to spacings between the centers of wells of the industry standardized 96 well cluster plate. Each counter-bore  12  has an annular lip or rim  14  around its outer periphery. The individual counter-bores  12  are joined together by adjoining the peripheries of adjacent counter-bores. Within the periphery of the rim  14 , each counter-bore has a substantially flat bottom wall  16  capable of seating a filter disc and a depressed center area that forms the conical drain funnel  25 . Further, each counter-bore preferably has a support grid  18  partially covering the drain hole, provided to prevent filter material that is seated on the flat bottom wall  16  of a counterbore from tearing during filtration while maximizing the open filter area for fluid flow.  
         [0024]    [0024]FIG. 2 shows a side view of the insert of the present invention. Each counter-bore has a funnel shaped drain hole therethrough. Preferably, below the flat surface area of the counterbore, is an annular skirt  20 . The annular skirt serves two functions. First, the annular skirt  20  serves as a guidance system when aligning the filter plate with a 96 well receiving plate. The skirt  20  fits into a corresponding well in the 96 well plate into which filtrate is to be transferred. Any lateral movement of the filter plate, once engaged with the receiving plate, is repressed by the plurality of skirts sitting in the respective wells of the receiver plate. Second, the skirt  20  serves to minimize any contamination between wells of a receiver plate by guarding against aerosols or splashing of liquid filtrate as it transfers into the receiver plate.  
         [0025]    [0025]FIG. 3 shows a cross sectional view of one counter-bore  12  from an insert of the present invention. The counter-bore has a substantially flat bottom wall- 16  to support a filter disc, an annular rim  14  around the periphery, a grid support  18 , an annular skirt  20  and a conical nozzle serving as a drain hole  22  and extending downwardly from the bottom wall  16 , preferably terminating at a point above the termination point of the skirt. The nozzle has an external surface  24 , and an internal passage  25  that communicates with the bottom wall  16  of the counterbore  12 . The internal passage  25  is preferably funnel shaped. The opening or drain hole  22  in the nozzle, where the internal passage  25  and external surface  24  of the nozzle meet, will preferably be quite small relative to the diameter of the bottom surface of the counter-bore. The small diameter and material surface energy are intended to keep the contents of a filter well from flowing until a significant driving force is applied. The conical external surface  24  of the nozzle is designed so that its surface intersects the internal passage  25  to form a sharp edge. The purpose of the sharp edge is to cause the draining fluid to form a droplet, rather than to allow flow laterally to any adjacent well thereby causing fluid cross-contamination of the filtrate along the under surface of the insert portion of the filter plate. Additionally, the edge will cause smaller droplets to form at the opening than would otherwise form without an edge. Ideally, a chamfered edge will be provided on the bottom of the skirt (not shown). The purpose of this chamfer is to guide the filter plate into the correct location over the receiver plate. This design is intended to make the plate easy to handle by a robotic placement system.  
         [0026]    [0026]FIGS. 4 and 5 show the insert  10  from above and in a three-dimensional view. The insert  10  contains a matrix of counter-bores  12  based upon the standard 96-well standard plate. Each counterbore  12  has an annular rim  14  around its periphery. A grid system  18  provides support over each drain hole. The grid system is comprised of a series of molded supports  15  that extend across the opening in the bottom wall  16  of the counterbore  12 . The supports  15  extend across the internal passage  25  of the nozzle, are attached to the walls of the internal passage and project upward to a plane normal the top surface of the bottom wall of the counterbore. The grid system creates a substantially flat surface entirely across the bottom wall of the counterbore. The bottom wall is therefore able to provide support for a filter disc, and prevent any tearing of the disc, while still allowing filtrate to be drawn into the funnel shaped passage. The grid system further allows liquid to be drawn through the filter disc from a greater surface area than the prior art devices. This creates a more uniform distribution of filtered material on the disc and allows for a smoother flow of liquid through the plate.  
         [0027]    FIGS.  6 A- 6 C show the process of punching and inserting a filter disc into a counterbore of the insert. A molded insert  10  is placed within a punch machine preferably having 96 punches 26 sized to cut membranes that will fit into the corresponding  96  counter-bores  12  of the insert. A filter sheet  28  of the desired material is placed between the insert  10  and the punch mechanism  26 . A series of aligned bores  30  from the die side of the punch will be placed between the filter sheet  28  and each counter-bore  12  of the insert. The insertion of the filter discs preferably takes place in a two step process, first a punch, then an insertion.  
         [0028]    For clarity, FIG. 6A shows only a single counter-bore  12 . A bore  30  preferably made of hardened steel is located between the counter-bore  12  and a filter sheet  28 . Positioned above the filter sheet  28  is a cylindrical plunger  32 . The plunger  32  has a bottom wall and is surrounded by a cylindrical punch  26 . The plunger  32  is slideably mounted within the punch  26 . The punch  26  terminates at its base in a radial cutting edge  34 . The punch and plunger together make up a punch unit and are surrounded by a sleeve  36 . The outer diameter of the punch  26  is approximately the same as the inner diameter of the bore  30  such that the punch fits snugly into the bore. The diameter of the bore  30  is approximately identical to the diameter of the counter-bore  12 . FIG. 6B shows the plunger  32  having been thrust downward into the bore  30 . The cutting edge  34  of the punch has severed the filter sheet  28  such that a filter disc  38  has been cut and pushed into the bore  30 . In FIG. 6C, the punch  26  has stopped extending into the bore  30 , while the plunger  32  has continued pushing the filter disc  38  down into the counter-bore  12  and against its bottom wall  16 . The plunger  32  and the punch  26  are then retracted, leaving an insert  10  having a filter disk  38  positioned along the bottom wall  16  of the counterbore  12 . Of course, it will be appreciated that as indicated, the described sequence will be performed simultaneously on a multiplicity of wells, e.g. 96 wells. The counterbore  12  as shown in FIGS.  6 A- 6 C is only one from a matrix of counterbores making up an insert  10 . Further, bore  30  is only one bore from a die having a matrix of bores that positionally align with the insert. Likewise, the punch unit comprising a plunger  32  surrounded by a cylindrical punch  26 , is one of a matrix of punch units that positionally align with individual bores of the bore plate and individual counterbores of the insert. Preferably, sleeve  36 , which is one sleeve from a precision carrier or guide plate, will encapsulate each punch unit as a protective measure.  
         [0029]    FIGS.  7 A- 7 D and FIGS.  8 A- 8 D show the insert molding technique that may be employed to obtain the filter plate of the present invention. FIGS.  7 A- 7 D show the molding technique of one filter well, a portion of a plate of preferably  96  interconnected filter wells, in three-dimensional view. FIGS.  8 A- 8 D show the same steps in cross sectional views. The mold which will accept this insert will have a cavity geometry that will form a standard 96 well plate against the insert, with the insert forming the bottom of the plate. The mold of FIG. 7A has two parts, an upper mold  40  and a lower mold  42 . The lower mold  42  is designed to form a nest  44  for the pre-molded insert  10 , as well as create external molded surfaces of the finished part. The upper mold  40  has a set of  96  core pins  46  that serve both to form the inside surfaces of the wells and to protect and hold each filter disc  38  in place while the material flows into the mold. The diameter of the core pins  46  are preferably smaller than the diameter of the filter discs  38  so that, when the mold closes, the outer edges of the filter discs will be exposed to the mold cavity and thus wiLl also be exposed to material flowing into the mold. FIGS. 7B and 8B show the mold closed with the upper part  40  and lower part  42  of the mold pressed together. The core pin  46  is pressing the filter disc  38  in place. Material flows into the mold through a gate and flows across the cavity, thereby forming the well plate  48 . The gate is located in such a position as to optimize mold flow. The formed well plate is a plate preferably having 96 wells that extend through the plate, each well having open ends on each of its top and bottom surfaces. FIGS. 7C and 8C show the mold after the thermoplastic material has filled the mold and formed the well plate  48 . FIGS. 7D and 8D show the finished ware after it has been removed from the mold. The flange  56  would, of course, connect to corresponding flanges on adjacent wells. The well plate  48  contacts the filter disc  38  around the entire periphery of each well wall  50 . The outer rim  14  of each counter-bore  12  and the lower wall  50  of each well actually bond together during the molding process and form lap joints  52  along their entire periphery. Anywhere the new material contacts the insert directly, the materials will be bonded. The well plate  48  and insert  10  are effectively bonded at each well along the lap joints  52 . The well plate  48  is molded against the outer periphery of the filter disc  38  so as to position it securely against the bottom wall  16  of the insert  10 . In some cases, depending on the membrane material, the filter disc  38  will bond to the material forming the well wall  50  thereby further securing the membrane in place.  
         [0030]    The insert molding technique as described lends a further advantage over press fitting techniques or techniques that require ultrasonically welding two plates together. Thermoplastic materials have a tendency to change shape slightly upon cooling. Alignment between two separately molded parts can be compromised by this cooling process resulting, at times, in an improper fit between parts. However, in the present invention, since the well plate is molded against the insert, a reproducible dependable fit is guaranteed. Thereby, the fit between plates as described is inherently superior to a fit obtained by matching together two separately molded pieces.  
         [0031]    Referring to FIG. 9 and  10 , the resultant filter plate  60  has a plurality of wells  62  arranged in an 8×12 matrix. Each individual well is separated from the other, each containing a separate filter disc  38 . No wicking or cross contamination between wells  62  in the filter plate  60  is possible because filter discs  38  are cut from the filter sheet before molding, not as part of the molding process. Each individual well is sealed from neighboring wells and no liquid transfer is possible through the overlapping and material bonded joint  52  formed between the well plate  48  and the insert  10 .  
         [0032]    It should be noted that the process for manufacturing filter plates can also be employed in the manufacture of 1×N well filter strips or individual filters. Further, filter plates can have wells of any number, for example 384 wells arranged in a 16×24 matrix.  
         [0033]    It should also be noted that the process for manufacturing filter plates is not limited to wells that have a circular cross section. The counterbores of the insert and wells of the well plate may be oval, square, rectangular, etc. The discs that are punched from the sheet of material will, of course correspond to the shape of the well and therefore likewise may be oval, square, rectangular, etc. as punched from an accordingly shaped punch unit.  
         [0034]    The process for manufacturing filter plates can also be employed for producing other plates that require a well bottom of a different material than the side walls. For example, for the production of a multiwell plate having wells having opaque side walls and transparent bottoms, a transparent sheet or film such as a fluoropolymer film, may be substituted for the filter membrane material herein before described. In this embodiment and referring to FIGS. 11 and 12, the insert  60  consists of a molded support having a matrix of rings  62  corresponding to the desired multiwell plate  61 . The rings  62 , instead of having funnel shaped nozzles extending downwardly from the insert as described in the filter plate manufacturing process, are open throughout the center  64 . Each ring  62  preferably has a flat support portion  66  in a plane parallel to the plane of the insert  60 , and a substantially perpendicular annular rim  68  circumscribing the outer periphery of the flat support portion  66 . The film is then punched by the method previously discussed, and individual discs of the film material are placed against the flat support portion of the ring of the insert. The punch mechanism is preferably sized such that a punched disk of transparent film will be supported by the flat portion and will fit against the annular rim. A well plate is then molded against the insert as previously described. The material of each annular rim bonds with the material of the well plate and each disc of transparent film is pinned between the flat support portion of each ring and the wall of each well. The resultant plate has wells  74  with bottoms  70  consisting of the transparent film material and sidewalls  72  of a different material, for example, opaque polystyrene. Punching individual discs from the transparent sheet also serves the purpose of preventing optical crosstalk between wells that might otherwise occur through a unitary sheet. The rings  62  of the insert may also be opaque and extend below the surface of the well bottom  70 , thereby further preventing optical crosstalk between the wells  74 .  
         [0035]    Although preferred embodiments of the invention have been disclosed, other embodiments may be perceived without departing from the scope of the invention, as defined by the appended claims.