Multiwell plate device

A multiwell plate device is provided having a frame, a substantially flat substrate and a multiwell structure supported by the substrate. The multiwell structure includes multiple bottomless wells formed therein. The substrate is supported by the frame and may be processed by an automated arrayer or instrument that is used to print or spot arrays in a pattern on a reaction surface of the substrate. Thereafter, the multiwell structure may be engaged with the substrate and the multiwell structure and substrate may be engaged with the frame in an upright orientation. For scanning or other analysis, the multiwell structure and substrate may be disengaged from the frame, inverted 180°, and then reengaged with the frame in the inverted orientation.

FIELD OF THE INVENTION

This invention relates generally to a multiwell plate device and the method for scanning a reaction surface from above and/or below.

BACKGROUND OF THE INVENTION

Multiwell plate devices serve a broad spectrum of laboratory uses. Most applications involve attachment or immobilization of biological materials including, without limitation, biomolecules such as polypeptides and nucleic acids, cells, tissues or fragments biological material, to a surface within the wells (sidewall and/or bottom surface) and the performance of one or more reactions followed by some sort of quantitative and/or qualitative analytical process.

Robotic instruments have been developed for performing automated processing of multiwell plates. Such automated processes include, without limitation, deposition of biological materials (spotting, printing, etc.), addition or removal of reagents, washing, scanning and analysis. The capability of such automated instruments is typically limited to processing plates with “standard” dimensions as established by the Society of Biomolecular Sciences (SBS Standards). Thus, the “footprint” for most multiwell plates is approximately 85 mm×125 mm with wells located in a standardized format depending upon the total number of wells. The American National Standards Institute (ANSI) has published the SBS Standards for microplates as: “Footprint Dimensions” (ANSI/SBS 1-2004), “Height Dimensions” (ANSI/SBS 2-2004), “Bottom Outside Flange Dimensions” (ANSI/SBS 3-2004) and “Well Postions” (ANSI/SBS 4-2004). All of these ANSI/SBS publications are incorporated herein by reference.

Although a standard structure for multiwell plates has facilitated automatic robotic processing, at the same time the structure presents a challenge with regards to certain types of procedures, particularly as the number of wells grows beyond 96. For example, spotting or printing of a microarray on the bottom surface of a well using automatic/robotic liquid handling systems or “arrayers” requires the pin or stylus or other printing/spotting means to move significantly up and down as it arrays one well and moves to the next to print or spot another array. Such movement increases processing time and increases the risk of damage to printing pins or stylus from unwanted collision with plate features above the surface to be printed or arrayed. Therefore, a need exists for a multiwell plate device more conducive to rapid processing.

Moreover, analysis of reactions occurring in the wells of a multiwell plate presents a challenge. Often, the analysis is accomplished by detecting or measuring a change in the material attached to the bottom surface of the wells (substrate) rather than a change in a fluid reaction mixture contained within the wells, as is the case for ELISA-type assays.

Optical detection is the most commonly utilized method to detect changes in surface-localized reactions, particularly with regards to arrays representing multiple different reactions. For surface-localized reactions, the focal plane for proper measurement of the reaction is often limited to a very small range of depths, typically a range of no more than about 5 mm. Analysis, whether done via automated scanning or microscopy or other means, can be performed by directing a light or energy source from above the reaction surface of the substrate or from below (through the substrate) and focusing an optic that captures the detectable signal from above or below the reaction surface. In some cases, for example when certain types of coated substrates and/or mixtures of detection agents are used, analysis from both above and below the reaction surface is useful in order to glean optimal data. However, the design of a standard multi-well plate complicates efforts to analyze results from both above and below the reaction surface. Most automatic scanners/analyzers can scan from only above or below the reaction surface but not both. Because of the dimensions of a standard multiwell plate, the focal plane of the reaction surface (bottom surface of the wells) when the plate is upright is significantly different from the focal plane when the plate is turned over. One prior art solution has been to use two separate analysis systems wherein one is capable of scanning from above the reaction surface and the other from below. Such an approach is expensive and time-consuming. Alternatively, another solution has been use of a multiwell plate device comprising separate pieces assembled to form the plate including a substrate that is detachable from the multiwell plate structure to eliminate physical interference by the plate structure with the focal plane of the reaction surface.

U.S. patent application Ser. No. 10/739,784 to Harvey et al., incorporated herein by reference, teaches the use of 1-4 glass microscope slides placed into a frame-like holder having standard multiwell plate dimensions. The slides are spotted or printed prior to placement in the holder. Once in place in the holder, each slide is topped in a releasable manner with a separate multiwell chamber plate having bottomless wells such that the printed glass slide forms a bottom surface for the chamber plate. Finally a retention means is used to retain the slides in the holder. After processing, the chamber plates and slides are removed from the holder and separated, and each slide is analyzed. Thus, the frame-like holder is used only during the reaction phase of the process; the steps of printing arrays and analyzing results are performed on each individual slide while separated from the holder.

U.S. patent application Ser. No. 11/134,449 to Haines et al., incorporated herein by reference, teaches a device comprising a substrate with a functional coating and biomolecules attached thereto. The substrate is reversibly attached to a superstructure containing multiple openings (multiwell structure). A frame-like tray holds the substrate and serves as an alignment jig for the superstructure. After processing, the system is completely disassembled to remove the substrate for analysis. Thus, the assembled device is used during the reaction phase of the procedure and, optionally, during the step of printing arrays, but it is disassembled for analysis.

U.S. Pat. No. 7,063,979 to MacBeth et al., incorporated herein by reference, teaches a microtiter-microarray device comprising a bottomless multiwell plate structure, one or more substrates having predeposited microarrays, and one or more gaskets for sealing the substrates to the multi-well plate structure. The seal must be fluid-tight but may be either reversible or irreversible. The patent teaches use of a first aligning device to align the gasket and plate structure for attachment purposes and a second aligning device for attachment of the substrates bearing predeposited microarrays. A separate device is used to remove the substrate after processing for analysis via conventional slide scanner. Alternatively, the substrate can remain attached to the gasket and plate structure for analysis via plate scanner, for example, Tecan LS-200 scanner (Tecan, Durham, N.C.). Thus, the reaction surface in a fully assembled multiwell plate device falls within a particular focal plane when the plate is upright and a significantly different focal plane when turned over. As described in U.S. Pat. No. 7,063,979, to scan the reaction surface from the opposite side with a plate scanner, the substrate must be detached and turned over 180°.

A detachable substrate presents a challenge because it must be attached to the plate structure in such a way as to be fluid-tight during the reaction phase of processing and yet removable without a level of force that could break or otherwise damage the substrate and without leaving adhesive or other material that might interfere with analysis. A need exists for a multiwell plate device wherein the reaction surface can be scanned from above or below while maintained within the detectable focal plane of a scanning device without requiring detachment of the substrate from the multiwell plate structure.

SUMMARY OF THE INVENTION

The present invention overcomes the foregoing and other shortcomings and drawbacks of multiwell plate devices heretofore known. While the invention will be described in connection with certain embodiments, it will be understood that the invention is not limited to these embodiments. On the contrary, the invention includes all alternatives, modifications and equivalents as may be included within the spirit and scope of the present invention.

In accordance with one embodiment of the present invention, a multiwell plate device is provided having a frame, a substantially flat substrate including a reaction surface and an opposite bottom surface, and a multiwell structure supported by the substrate. The multiwell structure has multiple bottomless wells formed therein and may be engaged with the reaction surface of the substrate using an adhesive layer. In one embodiment, the frame includes a top surface and defines an opening therethrough. A substrate engaging member, such as a ledge or projection by way of example, is disposed adjacent at least a portion of the opening and has a top surface and an opposite bottom surface.

During use of the multiwell plate device, the substrate may be first placed on the substrate engaging member with the substrate contained by the opening in the frame. In one embodiment, the reaction surface of the substrate is substantially flush with a top surface of the frame so that the reaction surface of the substrate may be manually processed or processed in an automated manner by an arrayer or other instrument that is used to print or spot arrays in a pattern that matches the SBS Standard pattern of wells, or in any other desired pattern.

After the substrate has been printed or spotted or otherwise processed, the multiwell structure is attached to the reaction surface of the substrate while the substrate is retained on the frame. After the multiwell structure and substrate are attached, they are lifted from the frame and the multiwell structure is then at least partially inserted through the opening from beneath the frame with the reaction surface of the substrate engaging the bottom surface of the substrate engaging member. In this configuration, the multiwell plate device is ready for conventional use.

In accordance with one aspect of the present invention, the multiwell plate device is reconfigurable for scanning or other analysis. In particular, the multiwell structure and attached substrate may be removed from the frame through application of a manual force to the multiwell structure. Following disengagement from the frame, the multiwell structure and substrate may then be inverted 180° so that the open ends of the wells are now facing down with the multiwell structure located beneath the substrate. In this inverted orientation, the multiwell structure may be least partially inserted through the opening from above the frame so that the reaction surface of the substrate now engages the top surface of the substrate engaging member.

The frame and multiwell structure may have cooperating alignment structures to assist in aligning the multiwell structure relative to the frame while the multiwell structure is at least partially inserted through the opening from above and beneath the frame.

In one embodiment, the bottom surface of the substrate is generally flush with the top surface of the frame when the multiwell plate and substrate are inverted and engaged with the frame in the inverted orientation. The invertible configuration of the multiwell structure and substrate relative to the frame allows the reaction surface of the substrate to be scanned from both above or below while being maintained within the detectable focal plane of a scanning device without requiring detachment of the substrate from the multiwell structure.

According to another aspect of the present invention, a multiwell plate device is provided having a frame defining an opening therethrough, a substantially flat substrate including a reaction surface and an opposite bottom surface, a multiwell structure supported by the substrate, and at least one projection extending upwardly from the frame. The multiwell structure has multiple bottomless wells formed therein and may be engaged with the reaction surface of the substrate using an adhesive layer.

A substrate receiving surface is disposed adjacent at least a portion of the opening and the at least one projection is positioned outwardly of the substrate receiving surface. The multiwell structure has a pocket formed on a lower side thereof for receiving the substrate therein with the reaction surface of the substrate engaging the multiwell structure. The multiwell structure also includes at least one recess formed on an upper side thereof.

During use of the multiwell plate device according to this embodiment, the substrate is placed on the substrate receiving surface and the substrate may then be manually processed or processed in an automated manner by an arrayer or other instrument that is used to print or spot arrays in a desired pattern on the reaction surface of the substrate. Thereafter, the multiwell structure is engaged with the reaction surface of the substrate by applying downward pressure to the multiwell structure while the substrate is retained on the frame. In this upright orientation of the multiwell structure and substrate, the bottom surface of the substrate engages the substrate receiving surface with the substrate received within the pocket. In this configuration, the multiwell plate device is ready for conventional use.

In accordance with another aspect of the present invention, the multiwell plate device is reconfigurable for scanning or other analysis. In particular, the multiwell structure and attached substrate may be removed from the frame through application of a manual force to the multiwell structure and substrate. Following disengagement from the frame, the multiwell structure and substrate may then be inverted 180° so that the open ends of the wells are now facing down with the multiwell structure located beneath the substrate. In this inverted orientation, the multiwell structure may be reengaged with the frame with the least one projection received in the at least one recess formed on the upper side of the multiwell structure.

The invertible configuration of the multiwell structure and substrate relative to the frame in this embodiment allows the reaction surface of the substrate to be scanned from both above or below while being maintained within the detectable focal plane of a scanning device without requiring detachment of the substrate from the multiwell structure.

The above and other objects and advantages of the present invention shall be made apparent from the accompanying drawings and the description thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention, in one aspect, is a multiwell plate device10(seeFIG. 8) comprising at least three components: i) a substantially flat substrate12, ii) a bottomless multiwell structure14and a frame16. In another aspect, the invention is a method for scanning a reaction surface from above and/or below.

Referring toFIGS. 1 and 2, the frame16has an open area18used to contain the substrate12for processing in an automated array printer or other instrument or for manual processing. In a preferred embodiment, the frame16has a “footprint” that conforms to the standard dimensions for multiwell plates (SBS Standards), for example, 85.5×127.6 mm. The open area18in the frame16may vary depending upon the size of the substrate12but preferably should be sized so that it nearly matches the dimensions of the substrate12to prevent detrimental movement or shifting of the substrate12during processing. For example, if using a substrate12of about 75.7 mm×111.3 mm, an open area18of about 75.8 mm×111.4 mm is suitable.

The substrate12rests upon ledges20or other protrusions on the inner surfaces22of the frame16. Preferably, the depth of the ledges20from a top surface24of the frame16is approximately equal to the thickness of the substrate12such that a reaction surface26of the substrate12as it sits in the open area18is essentially flush with the top surface24of the frame16. For example, if the substrate12is 1 mm in thickness, the ledges20are preferably located about 1 mm below the top surface24of the frame16.

Once the substrate12is in place in the open area18of the frame16, an automated arrayer or instrument is used to print or spot arrays in a pattern that matches the SBS Standard pattern of wells, or any other desired pattern, without the need for wasted vertical movement typically needed when printing or spotting the bottom of a conventional multiwell plate.

Referring toFIGS. 3-6, after the substrate12is printed or spotted or otherwise processed, the bottomless multiwell structure14is attached to the reaction surface26of the substrate12while in place on the frame16. Preferably, the dimensions of the multiwell structure14are smaller than those of the substrate12so that the substrate12forms a perimeter28(as shown inFIG. 9) around the multiwell structure14. For example, if the substrate12is about 75.7 mm×11.3 mm, the multiwell structure14might be about 73 mm×108.6 mm.

In one embodiment, the multiwell structure14has two or more lateral projections30each with a downwardly extending portion serving as alignment tabs32that mate with alignment receptors34in the frame16to guide the placement of the multiwell structure14onto the substrate12so that the wells36of the multiwell structure14correspond with the printed or spotted areas of the substrate12. The size, shape and number of the lateral projections30, alignment tabs32and corresponding alignment receptors34may be varied as long as multiwell structure14can be placed onto the reaction surface26of the substrate12with sufficient accuracy in relation to the arrays or other material contained on the substrate12.

In one embodiment, the surface38(seeFIGS. 4 and 5) of the multiwell structure14that contacts the substrate12contains a pre-applied adhesive (not shown). A removable liner (not shown) may be used to protect the adhesive layer until time of use. In another embodiment, shown inFIGS. 3 and 4, a thin flat adhesive carrier layer40is used to attach the multiwell structure14to the substrate12. For example, the carrier layer40has adhesive on both sides and forms an intervening layer between the multiwell structure14and the substrate12. For convenience, the multiwell structure14can be supplied with the carrier layer40already attached to its surface38.

When the alignment tabs32are inserted into the alignment receptors34in the frame16, downward pressure may be applied to the multiwell structure14to affect a functional seal or attachment to the substrate12.

Referring toFIGS. 7 and 8, after the multiwell structure14and substrate12are attached, they are lifted from the frame16and then reinserted from underneath the frame16with the open end of the wells36facing upward through the open area18in the frame16. The lateral projections30of the multiwell structure14facilitate alignment by fitting into the alignment receptors34or other receptive features in the frame16. The multiwell structure14with attached substrate12is pushed upward through the open area18of the frame16until the reaction surface26of the substrate12abuts the underside of the ledges20provided on the inner sidewall22of the frame16.

In one embodiment, the multiwell structure14and/or the lateral projections30fit snugly to hold the multiwell structure14securely in the frame16. Optionally, features on the internal sidewalls22of the frame16(not shown) may be used to secure or enhance the fit. The multiwell plate device10assembled in this mode can be used in substantially the same way as a conventional single-piece multiwell plate device.

For scanning or other analysis, the multiwell place device10may be used as shown inFIG. 8. Alternatively, the multiwell structure14with the substrate12attached may be removed from the frame16through application of manual force to the multiwell structure14and then inverted 180° so that the open ends of the wells36are facing down and the substrate12is on top, as shown inFIG. 9.

Continuing withFIGS. 9 and 10, the multiwell structure14is inserted into the open area18in the frame16from above and lowered so that the substrate12comes to rest on the top surface of the ledges20provided on the inner sidewalls22of the frame16. The reaction surface26of the substrate12is now at the level of the top surface of the ledges20whereas in the other format or “mode” (with the multiwell structure14facing up), the reaction surface26is at the level of the bottom surface of the ledges20, thus the focal plane differs only by the thickness of the ledges20. The thickness of the ledges20is generally influenced by the material used to form the frame16since the material strength of the ledges20must be sufficient to bear the weight of the multiwell structure14and substrate12. Typically, a thickness of about 0.3-1.0 mm is adequate for most materials suitable for manufacturing the frame16.

Now referring to an alternative embodiment of the present invention, a multiwell plate device100is shown inFIGS. 12-15comprising at least three components: i) a substantially flat substrate102, ii) a bottomless multiwell structure104and a frame106. In another aspect, the invention is a method for scanning a reaction surface from above and/or below.

Referring toFIG. 12, the frame106has an open area108used to contain the substrate102for processing in an automated array printer or other instrument or for manual processing. In a preferred embodiment, the frame106has a “footprint” that conforms to the standard dimensions for multiwell plates (SBS Standards), for example, 85.5×127.6 mm. The open area108in the frame106may vary depending upon the size of the substrate102but preferably should be sized so that it nearly matches the dimensions of the substrate102. For example, as shown inFIG. 12, if using a substrate102of about 75.7 mm×111.3 mm, an open area108of about 75.5 mm×111.1 mm permits the substrate102to rest upon a receiving surface110of the frame106. In one embodiment, the receiving surface110is slightly elevated compared to the remaining outer surface112of the frame106as shown inFIG. 15.

To minimize movement of the substrate102while positioned on the frame106, one or more ridges114or other protrusions may be included on the frame106.FIG. 12shows ridges114on each of the four sides of the frame106, but variations are contemplated, including a continuous ridge surrounding the entire open area108or multiple ridges114on the same side or the ridges114may be limited to fewer than all four sides of the frame106.

In the embodiment shown inFIG. 15, the height of a ridge114is greater than the thickness of the substrate102such that the ridge114defines the receiving surface110for the substrate102adjacent the open area108. For example, if the substrate102is 1 mm in thickness, the ridges114may be about 2.8 mm in height above the outer surface112of the frame106. The receiving area110is elevated compared to the remaining outer surface112of the frame106. The ridges114also permit the multiwell structure104to fit securely to the frame106without adhesive contact between the multiwell structure104and the frame106. Further, the ridges114mate with one or more grooves116in the top surface118of the multiwell structure104when the structure104with attached substrate102is used in an inverted format.

For additional ease in assembling the device, optional structural features may be included on the substrate102and/or the frame106that permit the substrate102to fit into the frame106in only one orientation. For example, a corner of the substrate102and a corresponding corner of the frame106may be angled or notched to permit a matched fit (not shown). Alternative means of dictating orientation are contemplated.

Once the substrate102is in place in the open area108of the frame106, and optionally before the multiwell structure104is placed onto the substrate102, an automated arrayer or instrument is used to print or spot arrays in a pattern that matches the SBS Standard pattern of wells, or any other desired pattern (not shown). The positioning of the substrate102in the frame106also serves to properly locate the substrate102relative to the X, Y stops which are standard on arrayer platforms (not shown).

After the substrate102is printed or spotted or otherwise processed, the bottomless multiwell structure104is attached to a reaction surface120of the substrate102while in place on the frame106. In the embodiment shown inFIGS. 12-15, the dimensions of the multiwell structure104are greater than those of the substrate102so that the multiwell structure104fits over the ridges114on the frame106.

As shown inFIGS. 12,13and15, the multiwell structure104has the top surface118, a bottom surface124and four sidewalls126. The bottom surface124provides the surface for attachment to the reaction surface120of the substrate102. Since the sidewalls126fit flush against the frame106when assembled, a recessed area or “pocket”128is provided in the bottom surface124to accommodate the thickness of the substrate102. For example, if the substrate102is about 1 mm in thickness, the pocket128is at least 1 mm in depth to provide additional allowance for adhesive, so that the bottom surface124of the multiwell structure104makes full contact with the substrate102via an intervening adhesive layer130when assembled. The ridges114may serve as alignment guides for the multiwell structure104to guide the placement of the multiwell structure104onto the substrate102so that the wells132of the multiwell structure104correspond with the printed/spotted areas of the substrate102.

In one embodiment, the bottom surface124of the multiwell structure104that contacts the substrate102contains a pre-applied adhesive (not shown). A removable liner (not shown) may be used to protect the adhesive layer until time of use. Alternatively, a thin flat adhesive carrier layer (not shown) is used to attach the multiwell structure104to the substrate102. For example, the carrier layer has adhesive on both sides and forms an intervening layer between the multiwell structure104and the substrate102. For convenience, the multiwell structure104can be supplied with the carrier layer already attached to its bottom surface124.

When the ridges114are inserted into the pocket128in the bottom surface124of the multiwell structure104, downward pressure may be applied to the multiwell structure104to affect a functional seal or attachment to the substrate102via the adhesive130, as shown inFIG. 15.FIG. 14shows the fully assembled plate device100. The multiwell plate device100assembled in this mode can be used in substantially the same way as a conventional single-piece multiwell place device.

Alternatively, the multiwell structure104with the substrate102attached may be removed from the frame106through application of manual force to the multiwell structure104and then inverted 180° so that the open ends of the wells132are facing downward and the substrate102is on top. The multiwell structure104may then be attached to the frame106by aligning the ridges114on the frame106with the grooves116in the top surface122of the multiwell structure104so that the sidewalls126are flush against the frame106.

With regards to manufacture, the substrates12,102may be made from any substantially flat material useful for containing biological materials. In a preferred embodiment, the substrate is glass, but alternatively, silicon, quartz, plastics, metals or other materials may be used. Further, part or all of the substrates12,102may be treated and/or coated with other chemicals or compounds to enhance qualities including, without limitation, binding capacity or specificity, as is known in the art, or the substrates12,102may be uncoated/untreated. Also, the substrates12,102may be transparent, translucent or opaque or any combination of the above. While the present invention has been exemplified as having a single substrate12,102, multiple smaller substrates may be utilized (not shown). For example, multiple glass microscope slides could be substituted for a single substrate. Further, the thickness of the substrates12,102can be varied. Typically, a substrate12,102with a thickness in the range of 0.3 mm-1.0 mm is suitable for many uses but the thickness can be increased or decreased.

An optional feature of the substrate102is a bar code or other indicia134(seeFIGS. 12 and 14) to facilitate identification, inventory, tracking, processing and/or other aspects of the handling of the substrate102and/or assembled multiwell plate100. To facilitate viewing of the indicia134an aperture or window136(seeFIG. 12) may be provided in the multiwell structure104or frame106.

The multiwell structures14,104can be made from any moldable material, such as a plastic polymer, and may be rigid or flexible. Material cost may be a factor because the multiwell structures14,104are ideally disposable after use. By way of non-limiting example, polystyrene, polypropylene and the like provide suitable materials for the multiwell structures14,104. Dimensions of the multiwell structures14,104may vary depending upon the width and length of the substrates12,102or composite of multiple substrates. Further, the wells36,132of the multiwell structures14,104should be formatted to meet SBS Standards. The depth of the wells36,132may conform to SBS Standards or alternatively, shallow depths are suitable. In one embodiment, the depth of the wells36,132in the multiwell structures14,104is no more than 5 mm (more shallow than SBS Standards). Further, the shape of the wells36,132may be round as shown inFIGS. 3-10and12-14or they may be some other shape such as square-shaped as shown inFIG. 11.

The frames16,106are molded or machined from any number of materials including, without limitation, plastic polymers, acrylics and metals. The frames16,106may be disposable or reusable depending upon the durability of the material used, cost, etc. The height of the frames16,106may conform to SBS Standards or it can be varied according to the depth of the multiwell structures14,104. For example, if the multiwell structures14,104are about 4-5 mm in depth, an appropriate height for the frame16is about 13.5-14.0 mm and the appropriate height for the frame106is about 5.0-14.0 mm.

The optional adhesive carrier layer40may comprise a film or resilient gasket-like material such as silicone or closed-cell polyethylene foam and the like. Preferably the adhesive used to attach the substrates12,102to the multiwell structures14,104is irreversible but alternatively, a reversible adhesive may be more appropriate for certain uses. Likewise, a combination of irreversible adhesive on one side of the carrier layer40and reversible adhesive on the other side may be used. Adhesives of these types are known in the art.

Other embodiments of the invention may be apparent to those skilled in the art and are considered to be part of the scope and spirit of the present invention. The descriptions and examples provided herein are intended to be exemplary and not limiting with regards to the scope of the invention.