GB2176601A - Test plate assembly defining discrete regions on a microporous membrane with low boundary distortion - Google Patents

Abstract

A biochemical test plate assembly for use in multiple simultaneous contact tests arranged in a fixed array of discrete regions on a single microporous membrane sheet provides less distortion of the boundaries of these regions than do pre-existing assemblies. The assembly contains two apertured plates 11, 13; an apertured gasket sheet 17 and a microporous membrane 19, both placed between the apertured plates; and a third plate 15 for placement beneath the apertured plates, having a recess which serves as a reservoir for liquids passing through the membrane. The improvement resides in the enclosure of the lower apertured plate by the upper apertured plate and a recess in the base plate, thereby sealing the lower apertured plate and the microporous membrane off entirely from the atmosphere. This eliminates evaporation of moisture from the edges of the membrane, and any lateral diffusion of biochemical species which might occur as a result due to capillary attraction. The floor of the base plate may slope towards a vacuum port 46. <IMAGE>

Description

SPECIFICATION Test plate assembly defining discrete regions on a microporous membrane with a low boundary distortion BACKGROUND OF THE INVENTION This invention relates to an apparatus for biochemical testing and screening procedures involving the use of a microporous membrane.

A biochemical test plate assembly capable of handling multiple simultaneous tests involving a single microporous membrane is disclosed in Fernwood et al., U.S. Patent No.

4,493,815, Bio-Rad Laboratories, Inc., January 15, 1985. The assembly provides a standard 8-by-12 rectangular array of cylindrical wells, with the bottom of each well sealed by a common microporous membrane. The membrane in turn rests above a recess forming an enclosed chamber from which a vacuum may be drawn or which may be completely sealed against air loss thereby providing a static air cushion beneath the membrane. The device may thus be used for either (a) drawing a fluid containing biochemical species through the microporous membrane, or (b) supporting a static fluid above the membrane for an indefinite length of time. The exposed membrane regions collectively provide. an array of discrete test regions with highly defined boundaries.

Accurate automated detections can then be performed on the membrane after it is removed from the assembly.

The assembly generally consists of two apertured plates (an upper and a lower) and a base plate containing a recess to form the vacuum chamber beneath the wells. The microporous membrane and an apertured gasket are placed between the two apertured plates.

The membrane is thus the only obstacle between the upper plate apertures and the vacuum chamber, thereby permitting both flowthrough and static contact procedures, depending on the air pressure in the chamber.

The wells and flow passages are sealed from the surrounding room atmosphere by the apertured gasket between the two apertured plates, and a further gasket between the lower apertured plate and base plate.

It is critical that these seals be perfectly airtight so that prolonged tests can be done without loss of chamber pressure. This requires highly polished finishes at the surfaces where the seals are made, which adds considerably to the cost of manufacturing.

Furthermore, since the membrane must be fully moistened before assembly of the parts, the exposure of its outer edges to the atmosphere during the test procedures raises another disadvantage --- evaporation from these edges. This induces outward migration of the biochemical species which have contacted the membrane through the outermost wells. The result is distortion of the outermost test regions on the membrane. This is a serious failing, since the lack of uniform contact areas obscures the test results in a number of ways.

SUMMARY OF THE INVENTION An improvement over the device described above is offered by the present invention, in which the lower of the two apertured plates is fully enclosed by the remaining two plates.

This reduces the number of seals which have atmospheric contact to a single seal between the two enclosing plates. The microporous membrane is thus sealed off from the atmosphere entirely, and evaporation from the membrane itself is eliminated as well as any lateral diffusion driven by the resulting capillary attraction. With these features, the assembly of the present invention overcomes both of the problems mentioned above, while still retaining the same versatility of use and function. The result is a test plate assembly which provides even greater accuracy and reproducibility.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an expanded view of one embodiment of a test plate assembly according to the present invention; FIG. 2A and FIG. 2B are a plan view and end view with cutaway, respectively, of the lower apertured plate shown as one of the components in FIG. 1; FIG. 3A and FIG. 3B are a plan view and a side sectional view, respectively, of the bottom plate shown in FIG. 1; and FIG. 4 is a side view in partial cutaway of the assembled parts of the embodiment shown in FIG. 1.

DETAILED DESCRIPTION OF THE DRAWINGS AND PREFERRED EMBODIMENTS As in Fernwood et al., referenced above, the test plate assembly of the present invention is intended to accommodate a multitude of simultaneous biochemical tests, each in one of a series of discrete wells or reservoirs arranged in a horizontal array. Although the number, size and spacing of the wells may vary, the most common and versatile arrangement is one comprising 96 circular wells in an 8-by-12 rectangular array, with a center-tocenter spacing of approximately 9mm, an arrangement used by a large variety of associated laboratory equipment. Other examples include oval or slotshaped wells with associated apertures of appropriate shape. For convenience, the drawings and the remainder of the description herein refer to a standard 96-well array.

FIG. 1 illustrates one embodiment of the test plate assembly of the invention. The assembly is designated by the numeral 10, its primary parts consisting of an upper plate 11 having a plurality of apertures 12 arranged in the aforementioned array; a middle plate 13, also with a plurality of apertures 14 aligned with those of the upper plate 11; a lower plate 15 containing a recess 16 sufficiently large to receive the middle plate 13; a gasket sheet 17, having apertures in the identical array and alignment of the upper and middle plates 11, 13; and a microporous membrane 19 of sufficient length and width to cover all of the apertures in the array.

The assembly is held together with four captive manually operated screws 20, with coil springs 21 for holding the screws in a raised position until they are pushed down and screwed into the lower plate 15. This keeps the screw tips from interfering with the alignment of the porous membrane and gasket during assembly and disassembly. For convenience, the screws are captive in the upper plate 11, the threaded ends (not shown) mating with threaded holes 22 in the bottom plate 15 after passing through holes 23 in the gasket sheet 17. Proper placement of the middle plate 13 inside the lower plate 15 is ensured by chamfering of the middle plate 13 at one corner 24 to mate with an angled corner segment 25 of the inner wall of the recess 16.Proper orientation of the upper and lower plates is achieved by a pair of guideposts 26 along one side of the upper surface of the lower plate 15 to fit into corresponding holes (not shown) in the underside of the upper plate 11.

The middle plate 13 is shown in detail in FIGS. 2A and 2B. Surrounding each aperture 14 is a boss 30 extending upward from the plate. The upper surface of each boss is flat and coplanar with each of the remaining bosses. The result is an even and concentrated pressure on the gasket sheet 17 resting on top of the middle plate (see FIG. 1) when the assembly is secured together, the pressure being concentrated around the rims of the apertures.

The underside of the plate has an array of protruding ribs 31, which add structural strength to the plate and also keep the lower opening 32 of each aperture clear, avoiding stoppage of liquid. As mentioned above, one corner 24 of the plate is chamfered for purposes of proper orientation.

A detailed look at the lower plate 15 is offered in FIGS. 3A and 3B. It will be noted that the recess 16 is similar in lateral dimensions yet slightly larger than the middle plate 13, to snugly accommodate the latter. The angled wall 25 at one corner mates with the chamfered corner 24 of the middle plate to ensure that the middle plate is inserted with the bosses facing upward.

The recess is surrounded by a ledge 40.

When the parts of the assembly are secured together under the tension of the tightening screws, the ledge 40 is forced against the outer edge of the lower surface of the upper plate 11 through an intervening gasket, thus sealing the interior of the recess 16 from the laboratory environment. In the embodiment shown, the seal is achieved by a perimeter gasket (not shown) which rests in a machined groove 42 (shown in FIG. 3B) which completely encircles the recess 16. The gasket may assume any of a variety of conventional forms such as, for example, a large O-ring of circular cross-section (to mate with the curved groove 42 shown) or rectangular cross-section. Alternatively, the gasket may be combined with the gasket sheet 17 (see FIG. 1) as an extension thereof in the form of a protruding ridge. The latter is illustrated in FIG. 4, discussed in detail below.

Returning to FIG. 3B, the recess has a slanting floor 43 and a series of support posts 44 extending upward from the floor, on which the downwardly protruding ribs 31 of the middle plate 13 rest. This holds the middle plate above the sloping floor 43, defining an open space 45 below the middle plate to allow drainage of any liquids passing through the apertures in the middle plate. The sloping floor 43 promotes liquid drainage toward a port 46 at one side. The port may be connected to a vacuum line (not shown) for use of the assembly in performing a filter assay.

Alternatively, the port may be closed by a valve (also not shown) to close off the inner space 45 of the base plate when a static blot assay is to be performed.

The assembled structure is shown in FIG. 4.

It will be noted that the peripheral seal is formed by a protruding ridge 47 on the gasket sheet 17, extending downward into the groove 42 which has been machined into the lower plate 15. The microporous membrane 19 lies entirely within the area defined by this protruding ridge 47. Accordingly, when the entire apparatus is assembled, and the apertures 12 in the upper plate are occupied by liquid samples, the microporous membrane has no contact with the external atmosphere.

Evaporation from the side edges and the resulting distortion of the test areas in the membrane is thereby avoided.

It is preferred that the range of compression of the peripheral seal exceed the range of compression of the gasket sheet by the bosses 30 around each set of aligned apertures. The peripheral seal will then be formed first as the securing screws are tightened, and less pressure is exerted on the individual aperture seals at the top of each boss. This minimizes distortion of the contact areas on the microporous membrane. This difference in the range of compression may be achieved in any of a variety of ways, but is most conveniently achieved by selecting an appropriate height for the ridge 47 and thickness for the remainder of the gasket sheet 17.

Further features of the drawing show preferred embodiments of the structure. The apertures 12 in the upper plate, for instance, are generally cylindrical, the diameter of each undergoing a reduction from the upper surface of the plate to the lower surface. This is useful in concentrating the biochemical species as it passes through the well and is deposited on the microporous membrane, improving the ease of detection and subsequent processing steps. To maximize the well capacity, the tapering portion is located toward the bottom of the aperture, providing a well capacity ranging from about 100 to about 1,000 microliters in volume.

As another feature, the apertures 18 in the sheet gasket 17 are of slightly smaller diameter than both those of the upper plate (at the narrower end) and the lower plate. In this way, the defined test area on the microporous membrane 19 is slightly smaller than the diameter of the apertures in the plates, and slight misalignments of either the plates or the gasket sheet will not affect the size of the test area, since it will still be in full contact with the liquid either held in or passing through the wells.

The plates may be constructed of any rigid inert material, preferably transparent so that the test fluids may be observed. Conventional materials will suffice, notably acrylic, polycarbonate, polypropylene or polysulfone. A convenient means of forming the plates is by injection molding. Since this avoids the need for machining of the plates individually, open spaces or gaps are easily incorporated into the structures to reduce the weight and the amount of plastic required. The embodiments shown in the drawings are simplified, however, for a better understanding of the functional aspects of the construction.

As in the structure disclosed in Fernwood et al., referenced above, the test plate assembly of the present invention may be used for two basic modes of operation --- forcibly drawing a fluid through the membrane, and retaining a fluid above the membrane for a prolonged period of time. The former may be achieved by drawing a vacuum through the vacuum port 46, while the latter is achieved by sealing the port 46 from the atmosphere and retaining a slight positive pressure in the recess of the base plate below the middle plate. These functions may be performed either individually or sequentially in a wide variety of biochemical laboratory procedures, with improved results in terms of accuracy, reproducibility, and lack of distortion.

The foregoing description is offered primarily for purposes of illustration. Although a variety of embodiments has been disclosed, it is not intended that the present invention be limited to the particular structures or methods of operation set forth above. It will be readily apparent to those skilled in the art that numerous modifications and variations not mentioned here can still be made without parting from the spirit and scope of the invention as claimed hereinbelow.

Claims (12)

1. A biochemical test plate assembly for use in both filter assays and static blot assays, said assembly comprising: an upper plate having a plurality of apertures; a middle plate having a plurality of apertures aligned with the apertures of said upper plate; a lower plate having a recess which, when said lower plate is covered by said upper plate, defines an enclosed chamber of sufficient size to contain said middle plate; means for forming an air-tight peripheral seal between said upper plate and said lower plate around said enclosed chamber; a microporous film of sufficient size to span the apertures of said upper plate when placed between said upper plate and said middle plate, yet lie within the area defined by said peripheral seal; and a gasket sheet having a plurality of apertures aligned with the apertures of said upper plate and adapted to form a lateral seal around the adjoining edges of each aligned pair of apertures when compressed between said upper plate and said middle plate.

2. A biochemical test plate assembly according to claim 1 further comprising means for applying a vacuum to said enclosed chamber.

3. A biochemical test plate assembly according to claim 1 in which said recess includes a floor and means for supporting said middle plate above said floor, thereby defining a space between said middle plate and said floor to receive liquid passing through the apertures in said middle plate.

4. A biochemical test plate assembly according to claim 3 further comprising a vacuum port communicating said space with the exterior of said lower plate, and wherein said floor slopes toward said vacuum port.

5. A biochemical test plate assembly according to claim 3 in which said supporting means is comprised of a plurality of posts extending up from said floor.

6. A biochemical test plate assembly according to claim 1 further comprising means for securing said upper plate, said microporous film, said gasket sheet, said middle plate and said lower plate together to compress said gasket sheet and said peripheral seal forming means sufficiently to form said lateral seals and said peripheral seals, respectively.

7. A biochemical test plate assembly according to claim 1 in which said peripheral seal forming means is comprised of a compressible gasket.

8. A biochemical test plate assembly according to claim 1 in which said peripheral seal is a compressible gasket, and the range of compression of said peripheral seal exceeds that of each said lateral sheet around the ad joining edges of said apertures.

9. A biochemical test plate assembly according to claim 1 in which all said apertures are of circular cross section, and each aperture in said gasket sheet is of smaller diameter than the apertures in both said upper plate and said middle plate with which said gasket sheet aperture is aligned.

10. A biochemical test plate assembly according to claim 1 in which each of the apertures in said middle plate terminates at its upper end in a flat boss extending upward from said middle plate, the uppermost suraces of said bosses being coplanar.

11. A biochemical test plate assembly for use in both filter assays and static blot assays, said assembly comprising: an upper plate having a plurality of cylindrical apertures of uniform size and spacing; a middle plate having a plurality of cylindrical apertures of uniform size and spacing and aligned with the apertures of said upper plate; a lower plate having a recess which, when said lower plate is covered by said upper plate, defines an enclosed chamber of sufficient size to contain said middle plate, said recess including a floor and means for supporting said middle plate above said floor to define a space below said middle plate for receiving liquid, said floor being sloped toward one inner wall of said recess; a port in said inner well of said recess for communicating said space with the exterior of said lower plate; ; a microporous film of sufficient size to span the apertures of said upper plate when placed between said upper plate and said middle plate yet lie within said recess; a gasket sheet having a plurality of apertures aligned with the apertures of said upper plate and adapted to form a lateral seal around the adjoining edges of each aligned pair of apertures when compressed between said upper plate and said middle plate; and a perimeter gasket adapted to form a seal between said upper plate and said lower plate encircling said recess, the range of compression of said perimeter seal exceeding that of said gasket sheet.

12. A biochemical test plate as claimed in Claim 1 substantially as herein described with reference to the accompanying drawings.