HIGH CAPACITY ASSAY PLATE

Abstract: A method of making a multiwell plate is disclosed. Sections (36) of material having well imprints (24) therein are assembled by stacking, and then bonded in order to form a multiwell plate.

Description: HIGH CAPACI Y ASSAY PLATE FIELD OF THE INVENTION The invention relates to a multiple well assay plate for applications in biological or chemical assays. BACKGROUND OF THE INVENTION The relationship between structure and function of molecules is a fundamental issue in the study of biological and other chemical based systems. Structure- function relationships are important in understanding, for example, the function of enzymes, cellular communication, and cellular control and feedback mechanisms. Certain macromolecules are known to interact and bind to other molecules having a specific three dimensional spatial and electronic distribution. Any macromolecule having such specificity can be considered a receptor, whether the macromolecule is an enzyme, a protein, a glycoprotein, an antibody, an oligonucleotide sequence of DNA, RNA or the like. The various molecules to which receptors bind are known as ligands. Pharmaceutical drug discovery is one type of research that relies on the study of structure-function relationships. Much contemporary drug discovery involves discovering novel ligands with desirable patterns of specificity for biologically important receptors. Thus, the time to bring new drugs to market could be greatly reduced through the use of methods and apparatus which allow rapid generation and screening of large numbers of ligands. A common way to generate such ligands is to synthesize libraries of ligands on solid phase resins. Since the introduction of solid phase synthesis methods for peptides, oligonucleotides, and other polynucleotides, new methods employing solid phase strategies have been developed that are capable of generating thousands, and in some cases millions of individual peptide or nucleic acid polymers using automated or manual techniques. These synthesis strategies, which generate families or libraries of compounds are generally referred to as "combinatorial chemistry" or "combinatorial synthesis" strategies. The current storage format for compound libraries is a 96 well format well plate typically made from polypropylene and having rubber stopper sheets or hot seal covers. Certain processes and chemistries require that chemical reagents (which may be reactants, solvents, or reactants dissolved in solvents) be kept under inert or anhydrous conditions to prevent reactive groups from reacting with molecular oxygen, water vapor, or other agents. Examples of moisture sensitive chemistries include peptide chemistry, nucleic acid chemistry, organometallic, heterocyclic, and chemistries commonly used to construct combinatorial chemistry libraries. The solvent used for storage of synthesized chemicals is typically dimethylsulfoxide (DMSO) . Storage plates made from polymers have the disadvantage of being incapable of withstanding the extreme temperature variations that are sometimes required in combinatorial chemistry reactions and storage (between -20° and 370° C) . Creating a multiwell plate from glass is a solution to this and other problems that are inherent in using polymers, such as sample interaction with the base polymer making up the plate. Glass, however cannot be injection molded and it is extremely difficult to press glass into a 96 well plate mold. One method currently used in producing a multiwell plate from glass involves a boring process. In this process, slabs of borosilicate glass conforming to the industry standard 96 well plate footprint are machined such that 96 individual wells are bored into the slab. This approach however is extremely costly. Another method of making glass well plates involves vacuum thermoforming. By this method, small plates are produced from glass by vacuum thermoforming a thin glass sheet, as described in commonly assigned French Patent application 96-13530. This technique offers well volumes of anywhere from 200μml to O.lμml volume capacity per well. While these volumes may be convenient for high- throughput screening bioassay applications aimed at sample and reagent conservation, they are probably too small for chemical synthesis in organic solvent, the storage of drugs or drug candidates in organic solvent or long term storage where closure is required. The potential for using sealable multiwell plates made of glass extend beyond use as a storage device for combinatorial chemistry. Glass multiwell plates may also be used for such tasks as: interfacing with instruments, extraction, derivatization, synthesis, and more. SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a multiwell plate that can be manufactured from glass or an organic polymer in a cost effective way. Further, it is an object to provide a method of making a glass multiwell plate that can produce varied and unique well designs. It is another object to provide a glass multiwell plate having wells that can be sealed from the external environment, for example by means of a septa. It is yet another object of the present invention to provide a multiwell plate that can withstand great variations in temperature . The present invention relates to a method of making a glass multiwell plate for use in biological and chemical assays, experimentation and storage. The method comprises the steps of: double rolling a ribbon of heated glass such that a plurality of half-well imprints are cast into each side of the ribbon, cutting a plurality of glass sections from the ribbon, aligning and stacking the individual sections one on the other, bonding the sections of glass together, and cooling the finished ware. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a view of the double rolling assembly used in the method of the present invention. Fig. 2. is a partially exploded view of the stacking arrangement making up the glass multiwell plate of the present invention. Fig. 3 is a plan view of the glass multiwell plate of the present invention. Fig. 4 is a side view of a segment of the plate of Fig. 3. Fig. 5 is a side view of a segment of a 384 well glass assay plate that is an embodiment of the present invention. Fig. 6 is a side view of a segment of a 1536 well glass assay plate that is an embodiment of the present invention. Fig. 7 is a side view of a segment of the glass multiwell assay plate that is an embodiment of the present invention. Fig. 8 is a side view of a segment of a glass multiwell assay plate that is an embodiment of the present invention. Fig. 9 side view of a segment of a glass multiwell assay plate that is an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION Fig. 1 displays the manufacturing steps involved in constructing the multiwell plate of the present invention. The production system includes a double roller having a top casting roll 12 and bottom casting roll 14. The top and bottom rolls rotate in opposing directions and serve to draw semi-molten glass from a reservoir tank 16. Each roller is fitted with a series or plurality of dies 18, preferably shaped in the form of a bisected well from a multiwell plate, in the central casting zone portion 20 of the roller surface. As the glass ribbon 22 passes between the rollers, each side of the ribbon is molded with the shape from the dies 18. The dies 18 preferably align such that an identical imprint 24 is made at the same point on both sides of the ribbon 22. In addition, the top roller has, on the surface of the casting zone 20, an indentation 26 disposed after a predetermined number of well dies. The glass from the ribbon fills this indentation and forms a pin 28 on the surface of the molded ribbon. The lower roller has a round knob, the size of which roughly corresponds to the size of the indentation on the top roller. This knob creates an imprint in the lower surface of the ribbon which preferably aligns with the pin that is formed on the top surface of the ribbon. The casting rollers 12, 14 preferably contact one another on both sides of the casting zone. Forward of the casting rollers is arranged a support (not pictured) for the ribbon of glass 22 emerging from the casting rolls 12, 14. Horizontal rollers 30 direct the ribbon of glass 22 emerging from the casting rolls into a predetermined horizontal path prescribed for the ribbon. The horizontal rollers 30 have a smooth outer surface 32 which serves to flatten the edges 34 of the ribbon 22. Like the casting rollers, the horizontal rollers rotate in opposing directions such that the ribbon progresses forward. If glass should flow between casting rollers outside of the casting zone, edge finishing may be performed by using trimming wheels, prior to the horizontal rolling. After passing through the horizontal rollers 30, the ribbon is cut into segments 36 of predetermined size such that each segment preferably has a pin 28 and a corresponding depression on opposite surfaces at one end. Cutting may be performed by flaw propagation under cold air vents. The individual segments are then stacked, one on another as shown in Fig. 2, such that the half-well impression 24 ± from one segment matches up with a half-well impression 24 i± from a successive segment, thereby combining to form a complete well 38. The pins 28 and depressions on successive segments interlock in order to properly align and register successive segments 36. Once a proper number of segments are stacked on one another, the segments are allowed to thermally bond under light side pressure at approximately 720°C. Preferably, a final cutting step is performed after assembly to obtain smooth edging. The ends of the stack may be trimmed by diamond saw, for example. Because of its ability to withstand broad ranges of temperature, borosilicate glass is the material of choice for the plate composition. It may be desirable to fit the final bonded product into a plastic frame with a soft adhesive interface in order to minimize the potential for breakage due to robotic handling. Fig. 3 shows a plan view of a group of 9 stacked segments 45 that, taken as a whole, create a 96 well plate 40 having wells 38, each having an approximate volume of 500 μml, arranged in an 8 x 12 matrix (mutually perpendicular 8 and 12 well rows) with a spacing of approximately 9 mm between the center lines of rows both in the x and y directions. The wells are preferably 20 mm deep. The well spacing, in addition to the height, length and width dimensions of the plate itself preferably conform to industry standards for a 96 well plate. In the preferred embodiment, the standard dimensions are important because a great deal of auxiliary equipment, both robotic and manual, is designed for use with these specifically sized plates. As can be seen in Fig. 3, the outermost segments 42 on the plate 40 may be made such that only one side of the segment is molded by the dies, the non-molded side 44 forms a flat side of a finished plate 40. These end segments can either be made using the same double roller technique described previously whereby one roller is flat in the casting zone, or by using a single roller having the required casting characteristics. Fig. 4 is a side view of one segment making up the plate of Fig. 3. Twelve successive well imprints 43 are formed in the segment 45. Fig. 5 is a further embodiment of the present invention. The rollers used to create the individual segments may be modified to form a variety of different sized and shaped half-well imprints that, when combined with a mating segment, can form wells of different shapes and sizes. Fig. 5 shows a side view of a segment 47 having 24 well imprints 49, each constituting one-half of a well. When 17 of these segments are stacked, a plate having 384 wells, each well having an approximate volume of 100 μml, in a 16 x 24 row matrix, with a spacing of approximately 4.5 mm between the center lines of rows both in the x and y directions, is created. In order to create a well plate having the industry standard footprint, the segments must be approximately half the width of those used to form the 96 well plate. This is accomplished by varying the size of the cavity between the two rollers. The top and bottom segment of the stack of 17 may need to be made slightly thicker in order to create a plate having an industry standard footprint that may be manipulated by robotic means. Fig. 6 shows another embodiment of the present invention, namely a side view of a segment 51 having 48 half-well imprints 53. When 33 of these segments are stacked, a plate having 1536 wells, each well having an approximate volume of 20 μml, in a 32 x 48 row matrix, with a spacing of approximately 2.5 mm between the center lines of rows both in the x and y directions, is created. In order to create a well plate having the industry standard footprint, the segments must be approximately one-quarter the width of those used to form the 96 well plate. This is accomplished by varying the size of the cavity between the two rollers. The top and bottom, segment of the stack of 33 may need to be made slightly thicker in order to create a plate having an industry standard footprint that may be manipulated by robotic means. Alternatively, smooth surfaced segment blanks having only a pin or a pin receiving depression may be mated to the first and last stacked segment in order to properly size the plate to the desired footprint. The rolling process of the present invention allows a large freedom in well profile as demonstrated in Figs. 4- 9. While some examples of well profiles are exhibited, they are intended only as non-limiting examples of potential well designs. It can be envisioned that the rolling process for forming these glass multiwell plates may lead to any variety of well design complexities. Fig. 7 is cross section of a plate which is a further embodiment of the present invention. The walls of wells 55 of the plate narrow to a top neck 50 and opening 52. Each opening 52 is sealed closed with a mass polymer unitary septa 54 that covers wells 55 of the entire plate. Having a neck 50 on the wells adds the advantage of allowing for the attachment of dependable air tight closures, which are extremely important for long time storage, as well as for certain processes such as gas chromatography derivatization, and chemistries that require that chemical reagents be kept under inert or anhydrous atmosphere. Access to the well 55 can typically be achieved by use of a non-coring pipetting needle 56 that pierces the septa 54. The composition of the septa largely depends on the chemistry involved, but common materials include thermoplastic rubber, natural rubber, PTFE (typically used as a lining), and EPDM. Fig. 8 is yet another cross section of an embodiment of the present invention. In this embodiment, a more complex system is displayed. Each well 57 is separated into two stages, an upper stage 60 and a lower stage 62 separated by narrowing section 64 that holds a filter 66. With this type of apparatus, fluid from the upper stage 60 may be filtered into the lower stage 62. Further, the lower stage may be equipped with a small capillary 68 leading from the bottom well stage 62 to a membrane 70 coating the bottom surface of the plate. Such a plate is ideal for use in nucleic acid synthesis. The synthesis of short length nucleic acids on solid beads by addition of sequential bases, reactants, and rinsing, is facilitated by the device shown in Fig. 8. For example, the first membrane 66 holds the solid beads as each reactant is added to the top of the well 57 and then removed by vacuum through the bottom of the plate. The fluid flow is controlled by the membrane 66. The membrane stops fluid flow when the fluid level/gas interface reaches the membrane due to capillary force or "bubble point". The final cleavage and retention of the synthesized sequence is accomplished by the second membrane 70. The arrangement of the second membrane 70 and the capillary 68 serves to concentrate the nucleic acid in a relatively small area, on the membrane 70. The embodiment shown in Fig. 8 may also be used in biological synthesis. For example, bacterial viruses known as phages may be cultured in the well space above the first membrane 66. The phages are then infected with phages containing a required genetic sequence. After multiplication, the resultant phages pass through the first membrane 66, but are retained by adsorption or sieving by the second membrane 70. The capillary 68 concentrates the phages in a small area, and the phages can be eluted from the second membrane 70. Building off the general concept shown in Fig. 8, the cross sectional view of the plate in Fig. 9 contains capillaries 65 connecting wells 63 together along with filters 61, a membrane 67 along the bottom plate surface, and a metallic electrode grid 72. A plate such as this could be used in electro-activated synthesis. The use of electrochemistry for chemical synthesis of organic and inorganic compounds can be used as a tool in combinatorial chemistry. The combination of glass containers and electrodes gives a unique combination. The electrodes can become part of the synthesized compound as in the case of aluminum, or can be chemically less reactive as with gold or platinum. The use of electrochemistry can generate oxidation, reduction, or free radicals in situ at the surface of the electrode. The chemical resistance and thermal capabilities of glass as well as the lack of gas permeability provide the ability to precisely control the environment in the reaction container or well. The electrical properties of glass as well as its' ability to seal to metal electrical connections to make devices such as pH measuring electrodes, ion measuring electrodes, and microelectrodes demonstrate a feasibility for use in multiwell electrochemical devices. It should be noted, however, that although preferred, the present invention is not limited to a glass contruction. For example, segments made of a thermoplastic material may be separately molded or imprinted with well imprints and similarly assembled to obtain the same effect. Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims: WE CLAIM : 1. A method of making a multiwell plate comprising the steps of : a) providing first and second diametrically opposed rollers, each rotating in opposing directions, each roller having plurality of dies formed in its peripheral surface; b) passing a ribbon of material in a thermoplastic state between said rollers such that said dies make imprints on said material; c) cutting said ribbon in substantially equal segments; d) stacking said segments such that said imprints from successively stacked segments align; and, e) bonding said segments together. 2. The method of claim 1 wherein said dies are half cylinders imprinted such that the center line of said cylinder is perpendicular to the length of the ribbon. 3. The method of claim 1 wherein a pin and a corresponding indent are imprinted on opposing sides of said segment, said pin from one segment mating with a corresponding indent from an opposing segment during said stacking step. 4. The method of claim 1 wherein during said rolling step, said ribbon of material is contained within a casting zone as defined by said rollers. 5. A multiwell plate made substantially of glass for use in chemical or biological applications comprising: a) a plurality of wells arranged in a matrix; and, b) said wells having an opening having a diameter that is less than the diameter of the well. 6. The multiwell plate of claim 5 wherein said wells have a neck region of narrowed diameter terminating in said opening. 7. The multiwell plate of claim 5 wherein said plate has 96 wells. 8. The multiwell plate of claim 5 wherein said plate has 384 wells. 9. The multiwell plate of claim 5 wherein said plate has 1536 wells. 10. The multiwell plate of claim 5 wherein said wells further comprise a central section of narrowed diameter capable of holding a filter. 11. The multiwell plate of claim 5 wherein at least one said wells is in communication with a metallic electrode grid. 12. The multiwell plate of claim 5 wherein said glass is a borosilicate glass. 13. The multiwell plate of claim 5 wherein at least one said well is in fluid communication with a bottom surface of said plate through a capillary passage. 14. The multiwell plate of claim 5 wherein at least two wells are in fluid communication through a capillary passage . 15. The multiwell plate of claim 5 further comprising an airtight sealing member excluding at least one said well from atmosphere outside said well. 16. The multiwell plate of claim 15 wherein said sealing member is capable of penetration by a needle while still retaining said exclusion from said atmosphere. 17. A method of making a multiwell plate for use in biological or chemical analysis comprising the steps of: a) imprinting at least one surface of at least two opposing articles, b) contacting said imprinted surfaces of said articles whereby said imprints collectively form at least one well. 18. A multiwell plate for use in chemical or biological applications comprising: a plurality of substantially rectangular segments having a portion of a well formed in at least one surface thereof, said segments being stacked and bonded together to form a plate and said well portions from successive segments align to form wells. 19. The multiwell plate of claim 18 wherein said wells have a neck region of narrowed diameter terminating in said opening. 20. The multiwell plate of claim 18 wherein said plate has 96 wells. 21. The multiwell plate of claim 18 wherein said plate has 384 wells. 22. The multiwell plate of claim 18 wherein said plate has 1536 wells. 23. The multiwell plate of claim 18 wherein said wells further comprise a central section of narrowed diameter capable of holding a filter. 24. The multiwell plate of claim 18 wherein at least one said wells is in communication with a metallic electrode grid. 25. The multiwell plate of claim 18 wherein said plate is made from a glass. 26. The multiwell plate of claim 18 wherein said plate is made from a plastic. 27. The multiwell plate of claim 18 wherein at least one said well is in fluid communication with a bottom surface of said plate through a capillary passage. 28. The multiwell plate of claim 18 wherein at least two wells are in fluid communication through a capillary passage. 29. The multiwell plate of claim 18 further comprising an airtight sealing member excluding at least one said well from atmosphere outside said well. 30. The multiwell plate of claim 29 wherein said sealing member is capable of penetration by a needle while s + \i.ll retaining said exclusion from said atmosphere. 31. The multiwell plate of claim 18 wherein said multiwell plate has a smooth outer periphery.

Inventors: DANNOUX THIERRY L A, DANNOUX, THIERRY, L., A.
Applicants: CORNING INC, DANNOUX THIERRY L A, CORNING INCORPORATED, DANNOUX, THIERRY, L., A.
Application year: 1998
Main classification: C12M1
IPC codes: B01L3, B29C43, C03B13, C03B23, C12M1, G01N33