WO1999049974A1

WO1999049974A1 - Plate apparatus for holding small volumes of liquids

Info

Publication number: WO1999049974A1
Application number: PCT/US1998/006368
Authority: WO
Inventors: Kevin R. Oldenburg; Gregory F. Hollis; Thomas D. Y. Chung
Original assignee: The Du Pont Merck Pharmaceutical Company
Priority date: 1998-04-01
Filing date: 1998-04-01
Publication date: 1999-10-07
Also published as: AU6791198A

Abstract

An apparatus for holding liquid, such as a microtiter plate, in which a plurality of wells are formed. Each of the wells have sidewalls that intersect in an edge that defines the boundary between adjacent wells. The edge boundary prevents liquid from collecting between the wells, thereby causing all of the liquid applied to the plate to enter one of the wells. The microtiter plate can be formed from a material having a reflectance and/or fluorescence that is optimized for the particular application in which the microtiter plate will be used. Microtiter plates for use in fluorescence measurement applications are made from material having low fluorescence, such as a liquid crystal polymer, while microtiter plates for use in luminescence measurements are made from material having a high reflectance. Microtiter plates for spectrophotometric applications are made with wells having clear bottoms and opaque walls. Such clear bottomed wells can be made by incorporating a photobleachable dye into the plate material so as to render it essentially opaque and then irradiating the bottoms of the wells so as to render them transparent. Such wells can also be made by forming the plate from an essentially transparent material and then irradiating the sidewalls with a beam of light so as to render them essentially opaque.

Description

APPARATUS FOR HOLDING SMALL VOLUMES OF LIQUIDS

Field of the Invention

The cuπent invention is directed to an apparatus for holding small quantities of liquids, such as a microtiter plate.

Back.ground of the Invention

Traditionally, screening of agents for biological activity is accomplished by placing small amounts of the compound to be tested, either in liquid or solid form, in a number of wells formed in a microtiter plate. As used herein, the term "liquid" refers to pure liquids, as well as liquids containing paniculate matter and solvents containing solute. The compound is then exposed to the target of interest, usually_^ a purified protein such as an enzyme or receptor but also possibly a whole cell or non-biologically derived catalyst. The interaction of the test compound with the target is generally measured radiochemically, spectrophotometrically, or fluorometrically. For example, fluorescent probes have been developed which are substrates for enzymes or calcium indicators, pH indicators, amine-reactive or carboxylic acid-reactive, as discussed in the Handbook of Fluorescent Probes and Research Chemicals, 5th ed., R. Haugland and Karen Larison, editor, published by Molecular Probes, Inc., 1994.

Radiochemical measurement is usually considered the most sensitive of the detection methods, followed closely by fluorescence. However, the problems 2 associated with using radioactive material, such as exposure limits, record keeping, and waste management, make this detection method significantly less attractive than detection by florescence. Consequently, the fluorescence measurement technique has gained wide spread acceptance. In the fluorescence measurement technique, light of a given wave length is directed onto a sample within the well of a microtiter plate. A portion of this light is absorbed by the sample and reemitted at a different, typically longer, wave length, which is then measured. Instrumentation for fluorescence detection is based on conventional 96-well plates. Such instrumentation is available from Dynatech Laboratories, 14340 Sullyfield Circle, Chentilly, Virgina 22021, and

Packard Instrument Co., 800 Research Park, Meriden CT 06450.

The wells of conventional 96-well plates typically have volumes of approximately 400 microliters each. The wells typically have cylindrical walls and either flat, round, or V-shaped bottoms. The plates .are conventionally made from a white or black plastic, such as polystrene, polypropelene, or ABS, that has relatively low intrinsic fluorescent properties. While this low level background fluorescence from the plate material is undesirable, it usually presents no major problems in fluorescence detection studies since the fluorescence from the sample in the well is generally orders of magnitude greater than the background fluorescence from the plate. This difference in fluorescence between the plate material and the sample is due both to the large volume of the sample in the well, usually 50-200 microliters, as well as the low surface area to volume ratio of the well in the plate.

The larger the quantity of wells that can be processed in a given batch, the higher the efficiency of the screening process. Consequently, it is desirable to concentrate a large number of wells in each microtiter plate by using microwells, rather than conventional wells. Such concentration of wells also permits very dense storage of collectives of discrete compounds for later testing as films in addressable grid positions, thus reducing the number of plates that must be tested in a complete collection of compounds. The use of micro volumes in biological screening is also desirable for reasons other than increased throughput. First, reagents, both biologically =>-.d chemically derived, are generally expensive and in very limited supply. By decreasing the assay volume, many more test components can be assayed with a given amount of biological target. Second, combinatorial chemistry libraries are made by the sequential addition of small organic building blocks onto an organic scaffold. The scaffold is covalently linked to a solid support structure, such as a Tentagel resin, via an acid, base, or photo-cleavable linker. Such solid supports structures are commonly referred to as "beads" and encompass structures having a variety shapes and sizes. In general, each bead, which is approximately 130 microns in diameter, contains 100 to 200 picomoles of compound. The small amounts of compound found on a single bead requires that the assay of the compound on that bead be perfo.rmed in small volume. For example, if all 100 picomoles of compound were cleaved from a single bead into the standard 200 microliter assay volume deposited in the 400 microliter well of a 96 well plate, the concentration would be 500 nanomolar, assuming a molecular weight of 500 daltons. This concentration is significantly below the ideal concentration of 10 micromolar that is generally used for screening compounds for biological activity. Also, it is generally desirable to be able to screen the compound at least twice so that the results CM be confirmed if the compound tests active in the first assay. In order to reach die 10 micromolar concentration or to screen the compound at least twice, and have enough left over for determination by mass spectroscopy, the compound should be cleaved into less th.an 5 microliter.

Unfortunately, assay miniatu.rization creates a number of problems. Reducing the size of the wells increases the difficulty associated with accurately dispensing liquids into diem because it becomes increasingly difficult to locate the dispensing device precisely over the center of each well. Inaccurate locating of the dispensing device will result in liquid being dispemed onto the boundary between wells, rather than into the wells themselves. Unfoitunately, the wells of conventional microtiter plates are separated by flat, horizontal surfaces upon which liquid can collect if it is not accurately dispensed into the wells. The collection of liquid between wells can create a variety of problems, including partial filling of wells, loss of reagents, and inaccurate mixing and concentration of components. Although collection of liquid between wells can be minimized by me use of dispensing devices capable of highly accurate positioning (e.g. , Packard Nanodrop™), such devices are 4 very slow, rendering impractical kinetic assays that require near simultaneous dispensing of agents into each well. Consequently, the difficulty of accurately dispensing liquids into very small wells has limited me ability to incorporate large numbers of wells into a single microtiter plate. Another problem associated with the use of small volume microwells is that pipetting into each well must be done sufficiently quickly so that evaporation does not significantly change d e volume in the well. Conventional liquid handling devices, such as the Tomtech or Sagian, are capable of pipetting volumes as small as 1 microliter and placing me liquid at defined positions. However, it would take several tens of minutes to fill a microwell plate containing 2400 wells to 9600 wells using these devices, by which time me first filled wells would have experienced significant evaporation. Other liquid h-andling devices based on inkjet printer head technology, available from BioDot and Packard, are capable of pipetting nanoliter volumes but likewise require significant time to pipet directly into a small microwell. Another problem associated with the use of small microwells arises in connection with fluorescence detection. The use of very small assay volumes results in significantly reduced reemitted light signals, m.aking die technique extremely sensitive to signal detection errors. For ex.ample, a microwell having a volume of 0.5 microliters will produce a signal that is only 0.1 to 0.2% of the signal resulting from the use of the well of a conventional 96-well plate. Accurate measurement of fluorescence is also complicated by me intrinsic fluorescence, in at least one region of the spectrum that is useful for detection of biological reactions, of the plastics from whϊcTT microtiter plates have conventionally been made, as previously discussed. The effect of .such background fluorescence is exacerbated in small volume microwells because the well surface area to volume ratio is significantly greater than in conventional 96-well plates. ConsequenUy, wlύle a given level of background fluorescence might be tolerated in a 96-well plate design, it could potentially be larger dian the total signal if the well size was reduced to that of a small microwell. In addition to me problem of background fluorescence associated with the materials from which conventional microtiter plates are made, the geometry of such plates also creates problems in signal detection. In fluorescence measurement techniques, the detection of the reemitted light from the sample within microtiter 5 plate wells is generally done with a charged coupled device (CCD) camera. This technique requires that the surface of the plate be as flat as possible so that the entire surface is in the same focal plane of the camera lens. If a plate were not flat, the wells across the plate would not be in the same focal plane and, consequently, light detection from the wells would not be uniform. This would, in tuπi, result in errors in determining me relative activity of the assay components in each well. Although conventional microtiter plates feature wells having a variety of bottoms (e.g., flat, V-shaped, round), me well walls of conventionally 96-well microtiter plates are cylindrical. When imaged witii a CCD camera, such a cylindrical well act as a lens, which tends to focus the reemitted light from the s.ample into the center of the well resulting in a gradient in die signal across the well. The signal gradient across the well results in signal deterioration and, hence, causes error in determining the relative activity in wells across the plate.

Although, as discussed above, fluorescence measurements benefit from plate materials having minimum intrinsic fluorescence, different screening technique benefit from the optimization of other properties of the microtiter plate material. Such optimization is important when using small microwells. In spectrophotometric techniques, light of a given wave length is directed onto the s.ample -and die .amount of light diat passes through the s.ample is detected. Consequently, in d is application, it is desirable for the microtiter plate wells to be as tr∑uispjirent as possible so as to minimize the interference with the transmitted light. Luminescence measurements are also used to perform biological assays. In this technique, die light generated by die s£imple^_is^' etected. Since the amount of light generated is relatively small, it is desirable that the microtiter plate material provide as high a reflectance as possible so as to maximize the signal.

Consequently, it would be desirable to provide an apparatus for holding liquids d at allowed increased diroughput screening by incorporating a large number of small wells but in which liquid was prevented from collecting at ie bowuiaries between adjacent wells. It would also be desirable to provide an apparatus for holding liquids having optimal properties for me particular screening application. Summary of the Invention

It is an object of the cuiτent invention to provide an apparatus for holding liquid in wells at incorporated a large number of small wells but in which liquid is prevented from collecting at die bound-aries between adjacent wells. This and odier objects is accomplished in an apparatus comprising a plate in which a plurality of adjacent microwells are formed, each of the microwells having (i) a bottom, (ii) at least one side wall, and (iii) an opening for receiving a liquid. The walls of each two adjacent microwells intersect so as to form an upward facing edge, the edge defining d e boundary between the openings of me adjacent microwells. In a preferred embodiment of ie invention, the widdi of each edge is no greater than approximately 250 microns, the radius of curvature is no greater than approximately 150 microns, and the maximum widtii of any hoiizontal surfaces formed on the edges is less dun approximately 80 microns, and the plate is formed from a liquid crystal polymer. The invention also encompasses a mediod of screening .an agent for biological activity comprising die steps of (i) suspending a plurality of beads in a solvent so as to form a bead containing suspension, (ii) pouring the suspension onto a plate having a plurality of microwells, each of which has a bottom, at least one side wall, widi side walls of adjacent microwells intersecting so as to form an upward facing edge, and an opening for receiving the suspension, with openings of adjacent microwells separated by a boundary defined by die edge, whereby a portion of the solvent enters each of d e microwells, (iii) allowing the suspended beads to settle into the microwells so that at least one of the beads is suspended in me portion of the solvent in each of die microwells, (iv) removing the solvent, and (v) applying die agent onto die plate.

It is anodier object of the invention to prove a microtiter plate having improved imagining capability. Consequendy, .another embodiment of the invention comprises a microtiter plate in which each of die side walls of die wells has a first portion that forms .an opening for receiving liquid .and diat is inclined at an angle to die vertical direction, and a second portion diat extends essentially in the vertical direction. 7

It is also an object of the invention to provide a method for m.aking microtiter plates, especially microtiter plates for use in spectrophotometric assays. Consequently, one embodiment of me invention encompasses a me od of making microtiter plates comprising the steps of (i) incorporating a photobleachable dye into a material so as to render ie material essentially opaque, (ii) forming the essentially opaque material into a microtiter plate having a plurality of wells formed therein, each of d e wells having a bottom formed from a portion of the essentially opaque material, and (iii) irradiating d e bottoms of the wells so as to render the material forming die bottoms transparent. In another embodiment, d e mediod of making microtiter plates comprises die steps of (i) forming a plate having a plurality of wells from an essentially transparent material, each of the wells having a bottom and a sidewall, and (ii) iιτadiating the sidewalls widi a beam of light so as to render die sidewalls essentially opaque.

Brief Dtescription of the Drawings

Figure 1 is plan view of a microtiter plate according to die current invention.

Figure 2 is a detailed view of die portion of Figure 1 enclosed by die circle indicated by II.

Figure 3 is a cross section taken through line III-III shown in Figure 2.

Figure 4 is a detailed plan view of one of die microwells shown in Figured.^'

Figure 5 is a detailed view of die portion of Figure 3 enclosed by die circle indicated by V, showing an enlargement of the boundary between microwells.

Figure 6 is a view showing a further enlargement of the microwell boundary shown in Figure 5.

Figures 7(a) and (b) are cross sectional views showing two alternate embodiments of die microwells according to die current invention. Figure 8 is a plan view of an alternate arrangement of microwells according to die current invention. In order to avoid confusion due to unnecessary 8 complexity of the drawing, the bottom is shown in only one of the microwells shown in Figure 8.

Figure 9 is a cross section taken along line IX-IX shown in Figure 8.

Figure 10 is a cross section taken along line X-X shown in Figure 8. Figure 11 is a plan view of another alternate arrangement of microwells according to the current invention.

Figure 12 is a cross section taken along line XII-XII shown in Figure 11.

Description of the Preferred Embodiment

A microtiter plate 1 according to die current invention is shown in Figure 1. As is conventional, the microtiter plate 1 is preferably rectangular, being , approximately 125 mm long, 85 mm wide, and 4 mm thick. Use of these dimensions allows the plate to be handled and indexed by currently available devices for automated microtiter plate handling. However, unlike conventional microtiter plates, the microtiter plate 1 according to die current invention may contain a very large number of very small microwells 2. Preferably, each of the microwells 2 has a depdi of approximately 1 mm and a volume of approximately 0.5 microliters or less. In one embodiment of die invention, 9600 microwells, each having a volume of approximately 0.4 microliters, are arranged in 120 rows and 80 columns. Each of die microwells 2 has an inlet 6 that forms an approximately 1 mm square. However, a lesser number of larger microwells, for example 2400 microwells, each having a depdi of approximately 3 mm and a volume of approximately 5 microliters, arranged in 60 rows and 40 columns, may also be preferred. In this embodiment, each microwell has an inlet mat forms an approximately 2 mm square. Aldiough a rectangular microtiter plate 1 is shown, it should be understood diat die microtiter plate according to die current invention could be fashioned in other shapes as well, for example a circular plate having a 125 mm diameter containing 14,500 wells arranged in a honeycomb pattern could be constructed. Such a circular arrangement will maximize the use of a circular imagining field.

As shown in Figure 1 , d e microtiter plate 1 has a border 4 diat surrounds a working portion 3 of the plate. As shown in Figures 2-4, the working 9 poπion 3 of the plate 1 consists of microwells 2 having square inlets 6 formed on die upper surface 6 of die plate. In the preferred embodiment of the invention, die body of each microwell 2 is formed by four walls, each of which extends downward from the inlet 6. The four walls consist of a first pair of opposing walls 10 and 12 and a second pair of opposing walls 11 and 13. According to one important aspect of the invention, the walls 10-13 are inclined at an angle A to die vertical direction - diat is, an angle with respect to a line perpendicular to d e plane of the plate ~ as shown best in Figure 5. Thus, the microwells 2 have ie sliape of an inverted four sided pyramid. Preferably, the walls 10-13 are steep, so d at me angle A is no greater than approximately 45°. Most preferably, die angle A is approximately 30° or less. As a result of the inclined walls 10-13, essentially all of die light incident upon the wells that is not absorbed by die plate material is either reflected away from the lens of the CCD camera or o er detection device, or reflected to die opposite side of die well, thereby preventing the aforementioned lens effect. As shown in Figure 3, die bottoms 14 of die microwells 2 are preferably flat. However, other shape bottoms, such as arcuate or conical bottoms, could also be utilized.

According to an important aspect of die invention, die walls 11 and 13 of adjacent microwells 2 intersect along edges 16, as shown best in Figures 3 .and 4. Similarly, the walls 10 and 12 of adjacent microwells 2 intersect along edges 18.

The edges 16 and 18 form the inlets 6 of the microwells 2. Thus, die boundary between die inlets 6 of adjacent microwells 2 is formed entirely by edges 16 and 18 so as to avoid die formation of flat, horizontal surfaces that would allow liquid to collect between microwells. Although in the embodiment shown in Figures 1-4, the microwells have the sliape of four side pyramids, odier sliapes could also be utilized provided diat die boundary between the inlet of adjacent microwells is formed by an edge without any intervening flat, horizontal surfaces diat would allow liquid to collect. As shown in Figure 7(a), microwells could be formed with walls having a first portion 40, adjacent die inlet edge, diat is inclined or conical, and a second portion 42, adjacent die well bottom, d at is vertical or cylindrical. By increasing die depdi of die vertical portion of the walls, die depdi of the microwell 40 10 and, therefore, its volume, can be increased wi iout increasing the size of its inlet. Thus, this geometry allows increasing the volume of die microwells without reducing dieir density. Aldiough the walls in Figures 3 and 7 are shown as being straight, arcuate walls, having eiuier convex or concave curvature, could also be utilized, provided diat diey formed sufficiently sharp edges, as discussed below. As shown in

Figure 7(b), microwells 43 with arcuate bottoms 44 could also be formed.

Figures 8-10 show an alternate embodiment in which me microtiter plate 1 ' has hexagonal microwells 32 arranged in a honeycomb configuration. In mis embodiment, die microwells 32 have six inclined walls 20-25 that intersect with the walls of die adjacent microwells along edges 25-30.

Figures 11 and 12 show yet anodier embodiment in which conical microwells 40 are utilized. In iis embodiment, me inlets of the microwells .are circles and die bottoms come to a point. However, flat or arcuate bottoms, as previously discussed, could also be utilized. The included angle B of die cone is preferably no greater ttian approximately 90°, and, more preferably, no greater than approximately 60°. In order to ensure that liquid cannot collect between adjacent microwells 40, diree faceted projections 43 are formed between each diree adjacent microwells, with each facet facing one of die microwells. The projections 43 form points mat are connected by edges 42, diereby ensuring ϋiat liquid cannot collect between microwells 40.

Although it is preferable to form the edges between adjacent microwells. such as edges 16 and 18 in die embodiment shown in Figures 1-4, in as sharp a manner as possible, it must be realized diat it is impossible to form a perfect edge in any material. This is especially so with respect to the plastics from which the microtiter plates 1 according to the current invention are preferably formed, as discussed further below. With reference to the embodiment shown in Figures 1-4, for example, when enlarged widi sufficient magnification, the edges 16 and 18 will typically appear rounded, as shown in Figures 5 and 6, raύier than perfectly sharp. Nevertheless, collection of liquid on die edges 16 and 18 can be prevented by ensuring that die edges have eidier a widdi or a radius of curvature, or both, that are sufficiently small. Preferably, die edges 16 and 18 have a width W (diat is, the widdi of die rounded portion connecting the walls 11 and 13 and 10 and 12) no 11 greater than approximately 250 microns and a radius of curvature R no greater than approximately 150 microns.

Moreover, aldiough it is preferable to avoid die formation of any flat, horizontal surfaces, it must be realized diat upon sufficient magnification, minimal flat, horizontal surfaces may be visible on the edges 16 and 18, as shown in Figure

6, widiout impairing die functioning of die microtiter plate according to ie principles of die current invention. Consequently, the widdi F of any flat, horizontal surfaces on the edges 16 and 18 should preferably be less than approximately 80 microns.

Microwells constructed according to die current invention ensure that all of the liquid deposited on d e worl ing portion 3 will find its way into one of die microwells since there are no flat surfaces between microwells that would allow liquid to collect and the edges 16 and 18 and die walls 10-13 tend to divert any liquid dispensed between microwells toward die well cavity. Thus, extremely accurate positioning of a dispensing orifice to dead center of the microwells is unnecessary, thereby permitting the use of a large number of very small microwells 2. When using microwells according to die cuirent invention, die wells can be filled widi liquid much die same way that paint is applied to a wall widi a sprayer. Liquid hitting the surface of the plate must go into a well and will not accumulate on die interwell surface. The use of the microtiter plate according to die current invention will now be discussed in connection widi a combinatorial chemistry application. First, a library of approximately 20,000 beads, each of which may be only approximately 130 microns m size, is suspended in absolute ethanol and sonicated briefly to make a suspension. This suspension is then poured onto die surface of a plate having 9600 microwells formed according to die current invention. Note that accurate, or even discrete, dispensing of die suspension into die wells is unnecessary and, in fact, die suspension can be poured over die plate in a continuous fashion.

After the application of d e suspension to die plate, die beads, which are randomly distributed over the worlcing surface of die plate, are dien allowed to settle. When a bead comes in contact with die surface of die plate, die geometry of the well inlets ensures diat each of die beads rolls into one of die wells. Consequently, if 20,000 beads are applied to the surface of die plate, each well will 12 contain on average two beads as defined by a Poisson distribution. The edianol is then removed via evaporation and a small amount of compound is cleaved from the beads. The target, usually an enzyme or protein receptor, to be tested is dien applied to die surface of the plate using die inkjet printer head technology described above. Microtiter plates according to die current invention are preferably formed by injection molding a plastic. Suitable injection molders include Atlantis Industries, Inc., Federal and Park, Milton, Delaware. In diis regard, any one of a large number of plastics, such a polypropolyene, nylon, or polystyrene could be utilized. While such plastics are suitable for radiochemical or spectrophotometric assays, their intrinsic fluorescence causes problems in fluorescence detection, as previously discussed. According to one embodiment of the current invention, this problem is solved by forming die microtiter plate 1 from a liquid crystal polymer, which has essentially no fluorescence with respect to wave lenguis in the 300 nm to 650 nm range, which is the range of interest for most biological screening applications. As used herein, die term "essentially no fluorescence" refers to materials whose fluorescence cannot be detected using a CCD camera. Preferably pigments are added to die liquid crystal polymer so as to obtain an opaque black material, thereby minimizing reflectance. The use of an intrinsically non-fluorescent and non-reflective liquid crystal polymer minimizes interference from the microtiter plate material in fluorescent measurement applications. In addition, liquid crystal polymers mold uniformly and are very chemically resistant. Suitable liquid crystal polymers include, but are not limited to, glass reinforced and mineral filled polymers, such as ose available from Hoechst Celanese, including, but not limited to grades A115, A130, A150, A230, A410, A420, A422, A430, A435, A440, A515, A530, A540, A625, A700, B230, C115, C130, C150, C550, C810, E130i, K130, K140,

L130, V140, and 8130. Most preferably, die liquid crystal polymer is grade A530D.

According to anodier embodiment of die current invention, die microtiter plate is formed from a highly reflective material, such as white polystyrene, polycarbonate, or acrylic, so as to enhance die performance of the microtiter plate when used for measurement of luminescence. In diis embodiment, sufficient reflectance is obtained by adding pigments, such as zinc or tungsten oxide, to die material forming the microtiter plate so as to yield, for example, a white 13 opaque material. Alternatively, the walls and bottoms of die microwells could be coated widi a reflective film by vapor deposition of metal particles, direct spraying, or lamination under vacuum of mirrored Mylar™ film.

Spectrophotometric assays require that die bottom of die well be optically clear. Consequently, in still ano ier embodiment, suitable for use in such assays, the bottoms of d e microwells are transparent, while die walls are non- reflective, as previously discussed. This embodiment may be formed by incorporating a photobleachable dye into a liquid crystal polymer, thereby resulting in material having an opacity greater than approximately 2 absorbance units (1 % transmittance), and preferably greater than approximately 3 absorbance units (0.1 % transmittance). Suitable photobleachable dyes include methyl violgens, paraquat, and nitrophenol based dyes, which are added in sufficient quantity to achieve die specified opacity singly or in combination. After injection molding, die microwell bottoms are irradiated with a fine, intense laser light of proper wave length, preferably of several millivolts in a 0.5 mm widdi, diereby bleaching die materi∑d iat forms the bottoms of the wells so as to result in transparent bottoms of preferably less than approximately 0.01 absorbance units (99.95 % transmittance) and, more preferably, less man approximately 0.001 absorbance units (99.99 % transmittance). Such bottoms facilitate the transmission of light through the microwell, while die dye in die material forming the walls of the wells prevents die piping of light widϋn die plate material between adjacent wells, thereby avoiding undesirable "cross-talk." Consequently, the accuracy of spectrophotometric measurements is enhanced.

Alternatively, this embodiment could be formed by injection molding die microtiter plate from a clear material, such as polystrene or a liquid crystal polymer, and uien bonding a photochromic film, such as DuPont Dylux™, onto the side walls of die microwells using heat lamination (shrink wrap), chemisorption, or an adhesive. Yet anodier alternative is to mold die microtiter plate from a clear material, such as polycarbonate, polystrene, or acrylic, diat lias been doped widi photochromic dyes or films but then render die walls of die wells opaque by application of high intensity light to only the walls, for example by irradiating die wall material with near ultraviolet light from 300-400 nm for several minutes to hours, as necessary to achieve die desired opacity. This could be accomplished by 14 the use of positionally accurate laser beams or by first masking the bottoms of die wells and using standard photoresists and optical masking techniques well known in die semiconductor industry.

The present invention may be embodied in ouier specific forms without departing from the spirit or essential attributes iereof and, accordingly, reference should be made to die appended claims, rather dian to die foregoing specification, as indicating the scope of die invention.

Claims

15What is Cl-aimed:

1. An apparatus for holding a liquid during assay, comprising a plate in which a plurality of adjacent wells are formed, each of said wells having (i) a bottom, (ii) at least one side wall, said side walls of adjacent wells intersecting so as to form an upward facing edge, and (iii) an opening for receiving a liquid, said openings of adjacent wells being separated by a boundary defined by said edge.

2. The apparatus according to claim 1, wherein the widdi of each of said edges is no greater ian approximately 250 microns.

3. The apparatus according to claim 2, wherein each of said edges forms an arcuate surface having a radius of curvature no greater dian approximately 150 microns.

4. The apparatus according to claim 1, wherein the maximum widdi of any horizontal surface formed on said edges is less dian approximately 80 microns.

5. The apparatus according to claim 1, wherein a first portion of each of said side walls is disposed adjacent said upward facing edge, and wherein said first side wall portions are inclined at an angle widi respect to the vertical direction.

6. The apparatus according to claim 5, wherein a second portion of each of said side walls extends in essentially die vertical direction.

7. The apparatus according to claim 5, wherein each of said first portions of said well side walls forms a conical surface.

8. The apparatus according to claim 7, wherein each of said side walls further comprises a second portion, said second side wall portions being cylindrical.

9. The apparatus according to claim 7, wherein said conical surface forms an included angle of no greater dian approximately 90┬░. 16

10. The apparatus according to claim 9, wherein said included angle is no greater man approximately 60┬░.

11. The apparatus according to claim 5, wherein said first side wall portions of each of said wells forms an angle of no greater than approximately 45┬░ with respect to the vertical direction.

12. The apparatus according to claim 11, wherein said angle said first side wall portions form with respect to die vertical direction is no greater dian 30┬░.

13. The apparatus according to claim 1, wherein each of said wells is a microwell.

14. The apparatus according to claim 13, wherein die volume of each of said microwells is no greater dian approximately 5 microliters.

15. The apparatus according to claim 14, wherein die volume of each of said microwells is no greater than approximately 0.5 microliters.

16. The apparatus according to claim 1, wherein at least approximately 2400 microwells are formed in said plate.

17. The apparatus according to claim 16, wherein at least approximately 9600 microwells are formed in said plate.

18. The apparatus according to claim 1, wherein said plate is formed from a liquid crystal polymer.

19. The apparatus according to claim 1, wherein each of said well bottoms is flat. 17

20. The apparatus according to claim 1, wherein each of said well bottoms is arcuate.

21. The apparatus according to claim 1, wherein said plate is formed from a transparent material, and further comprising a photochromic film bonded to said side walls.

22. An apparatus for holding a liquid during assay, comprising a plate in which a plurality of wells are formed, each of said wells comprising: a) a bottom; and b) side walls, each of said side walls having (i) a first portion, said first side wall portions forming an opening for receiving a liquid, . said first side wall portions being inclined at an angle to die vertical direction, and (ii) a second portion, said second side wall portions extending in essentially die vertical direction.

23. The apparatus according to claim 23, wherein said angle is no greater dian approximately 45┬░.

24. The apparatus according to claim 23, wherein said first portions of said side walls are conical and said second portions of said side walls are cylindrical.

25. An apparatus for holding a liquid during assay, comprising a plate in which a plurality of wells are formed, said plate formed from a material having essentially no fluorescence in the range of wave lenguis in die 300 nm to 650 nm range.

26. The apparatus according to claim 25, wherein said material is a liquid crystal polymer. 18

27. A method of screening an agent for biological activity, comprising the steps of: a) suspending a plurality of beads in a solvent so as to form a bead containing suspension; b) pouring said suspension onto a plate having a plurality of microwells formed dierein, each of said microwells having (i) a bottom, (ii) at least one side wall, said side walls of adjacent microwells intersecting so as to form an upward facing edge, and (iii) an opening for receiving said suspension, said openings of adjacent microwells being separated by a boundary defined by said edge, whereby a portion of said solvent enters each of said microwells; c) allowing said suspended beads to settle into said microwells so that each of said beads is suspended in said portion of said solvent in one of said microwells; d) removing said solvent; and e) applying said agent onto said plate.

28. The method according to claim 27, wherein said beads are from a combinatorial chemistry library.

29. A method of m-aking a microtiter plate, comprising die steps of:

(a) incorporating a photobleachable dye into a material so as to render said material essentially opaque;

(b) fo╧Çning said essentially opaque material into a microtiter plate having a plurality of wells formed dierein, each of said wells having a bottom formed from a portion of said essentially opaque material; and

(c) irradiating said bottoms of said wells so as to render said material forming said bottoms essentially transparent. 19

30. The method according to claim 29, wherein said material has an opacity of greater than approximately 2 absorbance units after said incorporation of said photobleachable dye but prior to said irradiation.

31. The method according to claim 29, wherein said portion of said material forming said well bottoms has an opacity of less th.an 0.01 absorbance units after said irradiation.

32. The method according to claim 29, wherein said material is a plastic, and wherein die step of forming said microtiter plate comprises molding.

33. A method of making a microtiter plate, comprising the steps of:

(a) forming a plate having a plurality of wells from an essentially transparent material, each of said wells having a bottom and a sidewall, each of said sidewalls being essentially transparent as initially formed; and

(b) rendering said initially transp.arent sidewalls essentially opaque.

34. The method according to claim 33, further comprising die step of doping said transparent material with a photochromic dye prior to forming said plate, and wherein die step of rendering said sidewalls essentially opaque comprises irradiafing said sidewalls widi a beam of light.

35. The method according to claim 33, wherein die step of rendering said sidewalls essentially opaque comprises bonding a photochromic film onto said sidewalls.

36. The method according to claim 33, wherein said sidewalls have an opacity of greater than 2 absorbance units after being rendered essentially opaque. 20

37. A method of malcing a microtiter plate, comprising the steps of:

(a) forming a plate having a plurality of wells from an essentially transparent material, each of said wells having a bottom and a sidewall; and

(b) coating said side walls widi a reflective film.