WO2004078352A2

WO2004078352A2 - Microtiter plate for holding small volumes of liquids

Info

Publication number: WO2004078352A2
Application number: PCT/US2004/006942
Authority: WO
Inventors: Brian Ayling
Original assignee: Amersham Biosciences (Sv) Corp
Priority date: 2003-03-05
Filing date: 2004-03-05
Publication date: 2004-09-16
Also published as: WO2004078352A3

Abstract

The current invention provides an apparatus for holding small amount of liquid at a greater depth. Preferable dimensions of the apparatus take the form of a conventional microtiter plate, with evenly spaced arrays of wells in rows and columns. The wells are designed in a slot form, wherein all the slots in a microtiter plate are parallel to either the rows or columns of the array of wells. Alternatively, the wells can take the shape of a steep cone. Microtiter plates with wells so designed enable significant reduction of dead volume in liquid transfer.

Description

MICROTITER PLATE FOR HOLDING SMALL VOLUMES OF LIQUIDS

Cross Reference to Related Applications

This application claims priority to United States provisional patent application number 60/452,194, filed March 5, 2003; the disclosures of which are incorporated herein by ^'reference in their entirety.

Field of the Invention

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

Background of the Invention

Conventional microtiter plates provide convenient vessels for performing multiple small volume, chemical/ biological reactions, as well as easy storage for large numbers of samples. Various improvements and modifications of the basic microtiter plate design have been made to better meet the demand for increased throughput . A microtiter plate commonly consists of a rectangular plate formed from injection molded polystyrene plastic. The plate contains a number of hollows arranged in a grid. These hollows are known as wells and act as reaction vessels for individual reactions, or storage containers for individual samples. The three most important standards of microtiter plates are: their outer dimensions, that allow them to fit in standard instruments; their so-called grid spacing, which is the distance between 'the center of one well to the center of the adjacent well in the same row or column in the grid; and the position of the wells in relation to each other and to the outer edges of the plate. The most widely used microtiter plate format is approximately 128 times 85 times 4 millimeters with 96 wells arranged in a grid of eight rows and 12 columns . The grid spacing between wells is about nine millimeters. However, several other divisions are found within the above-cited format. Usually encountered are also plates with 192, 384 or even more wells. Microtiter plates constituting one half of the above-cited format are also in use.

The wells of a conventional microtiter plate typically have cylindrical walls and either flat, round, or V-shaped bottoms. Microtiter plates with wells that arranged in a grid format are the most commonly used of multi-sample plate. However, other arrangements of wells, such as circular forms, are also found in multi-sample plates .

The use of even smaller volume microtiter plates is sometimes desirable. First, reagents, both biologically and chemically derived, are generally expensive and/or in very limited supply. By decreasing the assay volume, many more test components can be assayed with a given amount of biological target. Second, the use of smaller volumes is necessary for maintaining a high concentration of samples, where the concentration is critical in a reaction.

Recently, biological microarrays have been widely used to examine gene activity and to identify gene mutations . Microarrays are formed by depositing biological material such as nucleic acid fragments in a pattern on a substrate such as a glass microchip. After a hybridization reaction between the nucleic acid samples on the microarray and a fluorescence labeled nucleic acid probe, the chips may be read with high-speed fluorescent detectors and the intensity of each spot quantified. The location and intensity of each spot reveals the identity and amount of each nucleic acid sequence present in the sample. Because tens of thousands of gene fragments may be present on a single microarray, data for entire genomes may be acquired in a single experiment.

The quality of the microarray greatly influences the quality of the data obtained using microarray analysis. For example, arrays having uniform spot size provide uniform signal intensities at each pixel and result in data having greater precision. Uniform spot size is also an indication that an equal amount of array material (e.g., DNA, RNA, protein, etc.) is present across the entire spot, thus ensuring that the binding of labeled sample to array sequences will occur at the same rate across the spot. Also desirable is the ability to create a regular array pattern capable of being consistently reproduced multiple times on multiple substrates. These qualities are dependent on the means used for depositing the array material onto the substrate.

Although the typical volume transferred in creating microarray spots is in the sub-nanoliter (10^~9 L) or picoliter (10^~12 L) range, it is becoming more conventional to refer to the spot diameter which may be in the range of about 100 μm to about 220 μm. Several techniques for depositing large number of spots in this volume/diameter range have been developed, such as photolithographic techniques involving the in situ synthesis of oligonucleotides on a substrate, and ink-jet and contact printing techniques for depositing biological materials on substrates. Of these, contact printing is by far the most successful' depositing means.

Contact printing involves the use of rigid pin tools, also referred to as "pens," which are dipped into the sample solution, resulting in the transfer of small volumes of fluid onto the tip of the pins . Microarray spots are created by touching the pins or pin, samples onto the surface. Such pin tools can be solid pins or capillaries, tweezers, and split pins that hold larger f sample volumes than solid pins. US Patent numbers

5,807,522, 5,770,151 and US patent application number 10/029,737 disclose several such devices. The sample solutions used for printing are typically stored in a microtiter plate of 96 or 384 wells, and are transferred to the pens directly from the wells of such microtiter plates .

The current design of the microtiter plate requires that a large volume of sample solution be used in order for the pens to acquire a desirable amount of sample to be spotted. However, as stated above, large amounts of biological samples are difficult, if not impossible, to get. Consequently, it would be desirable to provide an improved, low-volume microtiter plate that conforms to current standards and working methods, yet offers a steeper well. Such microtiter plates will also benefit other applications in the biomedical and chemical field of which reduced reaction volume is preferred.

Summary of the Invention

It is an object of the current invention to provide an apparatus with wells that hold a small amount of liquid at a higher depth than a conventional microtiter plate well, so that sample liquid is readily reachable by a transfer means, such as a pen described in US patent application no: 10/029,737. This and other objects are accomplished in an apparatus comprising an array of sample containers (wells) connected to each other, each of the wells having a bottom, a top opening for receiving a liquid, and at least one side wall enclosing the interior of said sample container, such that the width of the interior of said well is reduced to a slot. In one embodiment of the invention, the slot of each well is about 40 degree at the base.

It is a principal object of the invention to provide a low volume microtiter plate of 96 or 384 wells with a steep well. Consequently, another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the wells are reduced to a slot of about 40 degrees at the base, and the slot of each well is arranged in parallel to the rows of the wells .

Another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the wells are reduced to a slot of about 30 degrees at the base, and the slot of each well is arranged in parallel to the rows of the wells.

Yet another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the wells are reduced to a slot of about 20 degrees at the base, and the slot of each well is arranged in parallel to the rows of the wells.

Another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the slot of each well is about 40 degrees at the base, and the slot of each well is arranged in parallel to the columns of the wells .

Still another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the wells are reduced to a slot of about 30 degrees at the base, and the slot of each well is arranged in parallel to the columns of the wells.

Another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the wells are reduced to a slot of about 20 degrees at the base, and the slot of each well is arranged in parallel to the columns of the wells.

The object of the current invention can also be met by an alternative design, in which a well is divided into two regions, joint at the interface of the two regions. The upper region is a wider slot toward the opening of the well, while the lower region has a narrower slot toward the bottom part of the well. Similar to the single slot designs described above, this design also provides an apparatus with wells that hold a small amount of liquid at a higher depth than a conventional microtiter plate well, in which sample liquid is readily reachable by a transfer means.

The objects of the invention can also be accomplished in an apparatus comprising an array of wells connected to each other. Each of the wells having a bottom, at least one side wall enclosing the interior of said well, such that the interior of said well is reduced to a steep cone of about 40 degrees at the base, and a top opening for receiving a liquid.

Another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the interior of said sample container is reduced to a steep cone of about 40-degree at the base.

Another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the interior of said well is reduced to a steep cone of about 30-degree at the base.

Yet another embodiment of the invention comprises a 96- or 384-well microtiter plate in which the interior of said well is reduced to a steep cone of about 20-degree at the base.

The microtiter plates of the structures described above offer steep wells, hence require a much lower amount of sample for each well, while satisfying conditions required for low volume liquid transfer from a sample well to a spotter pen. The instant invention also offers methods of making these microtiter plates, as well as methods of using them. The above and other objects, features and advantages of the present invention will become apparent from the following description and the appended claims, taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

The invention is described in greater detail with reference to the enclosed drawings :

FIG. 1 shows a representative well of the current invention with a liquid transfer pen inserted in it, compared to a conventional microtiter plate well outlined by the dotted lines. Note that the width of the well is reduced to a slot (solid lines) .

FIG. 2 shows a well with a 40-degree slot at the base, as presented by the solid lines. The dotted lines show the outline of a conventional microtiter plate well.

FIG. 3 shows a well with a 30-degree slot at the base, as presented by the solid lines. The dotted lines show the outline of a conventional microtiter plate well. FIG. 4 shows a well with a 20-degree slot at the base, as presented by the solid lines. The dotted lines show the outline of a conventional microtiter plate well.

FIG. 5 shows an alternative design of the current invention. The well is consisted of two regions joint at the interface: an upper region with a wider slot toward the opening of the well, and a lower region with a narrower slot toward the bottom part of the well.

Detailed Description of the Invention

The apparatus of the current invention is comprised of an array of sample containers, or wells, and is capable of holding small amount of liquids at a higher t depth than conventional microtiter plate wells. These platforms are well suited for use as a sample storage apparatus for samples being transferred to a microarray spotter pen device, but can be used for any appropriate purpose.

Generally, the multi-well platforms of the present invention comprise two regions, a well field and a border. The border can be of any dimension, shape, or thickness, but preferably forms a multi-well platform with outer dimensions that are similar to those of a standard 96- or 384-well microtiter plate, whose dimensions are approximately 85.5 mm in width by 127.75 mm in length. Multi-well platforms of the present invention having these dimensions can be compatible with robotics and instrumentation, such as multi-well platform translocators and readers as they are known in the art.

, Typically, wells will be arranged in two-dimensional linear arrays on the multi-well platform. However, the wells can be provided in any type of array, such as geometric or non-geometric arrays. The number of wells can be a multiple of 96 within these ranges, preferably the square of an integer multiplied by 96.

The wells can be placed in a configuration so that the well center-to-well center distance can be between about 0.5, 1, 2, 5, or 10 millimeters and about 1, 2, 5, 10, or 20 millimeters. Typically, the multiwell plate has wells with a well-center-to-well-center distance of less than about 10 mm, preferably less than 5 mm and some times less than about 3 mm. Smaller well-center to well- center distances are preferred for smaller volumes.

The wells can have a diameter (when the wells are circular) or maximal diagonal distance (when the wells are not circular) between about 0.2, 0.5, 1, 5, 10, or 50 millimeters and about 1, 5, 10, 20, 50, or 100 millimeters. Preferably, the well diameter is between ' about 0.5 and 100 millimeters, more preferably between about 1 and 50 millimeters, and most preferably, between about 2 and 20 millimeters.

The wells can have a depth between about 1, 5, 10, 20, or 50 millimeters and about 5, 10, 20, 50, or 100 millimeters. Preferably, the well depth is between about 1 millimeter and 100 millimeters, more preferably between about 2 millimeters and 50 millimeters, and most preferably between about 3 millimeters and 20 millimeters .

Well volumes typically can vary depending on well depth and cross sectional area. Well volumes can range between about 0.5, 1, 5, 10, 25, 50, 100 or 200 microliter and about 5, 15, 40, 80, 100, 200, 500, or 1000 microliters. Preferably, the well volume is between about 500 nanoliters and 500 microliters, more preferably between about 1 microliter and 200 microliters, and most preferably between about 0.5 microliters and 20 microliters .

Wells can be made in any cross sectional shape (in plan view) including, square, round, hexagonal, other geometric or non-geometric shapes, and combinations (intra-well and interwell) thereof. Wells can be made in any cross sectional shape (in vertical view) including shear vertical or chamfered walls, wells with flat or round bottoms, conical walls with flat or round bottoms, and curved vertical walls with flat or round bottoms, and combinations thereof.

A preferred embodiment of the current invention is shown in FIG. 1 - 4. A typical well of a microtiter plate is redesigned such that the internal shape is changed to a progressively smaller slot. These designs greatly reduce the capacity of the well. An increased sample height is achieved, with the same volume of sample, compared to a well of a conventional microtiter plate. In FIG. 1 - 4, the outer borders of the well is shown by the dotted lines, and illustrate the shape of the conventional microtiter plate well, with a cylindrical top portion, a conical bottom portion, and a small flat bottom. The solid lines represent the internal of the wells of the present invention, with two side-walls pointing to each other at an angle. The shape of the well is changed from the conventional cylindrical/conical shape to a progressively smaller slot, with the minimum distance between the two sidewalls at the bottom of the well at about 1 mm. The angle between the two side-walls is at most about 40 degrees (FIG. 2), preferably about 30 degrees (FIG. 3), and most preferably about 20 degrees (FIG. 4) . Alternatively, the two side-walls can be parallel as well. These designs allow the use of 50% or less sample solution while maintaining the same sample depth in the well, thus allowing the use of much less samples for spotting microarray slides. The slot profile of the well would also allow the spotter pen to be inserted with a degree of tolerance to changes in the exact positioning of the pen. The dead volume for effective sample filling of a spotter pen (minimum amount of sample solution that is needed for effective filling of the pen device) is reduced from 20 microliters to about 10 microliters, even 5 microliters .

An alternative embodiment of the current invention is a conical well design. Like the slot design, this design also minimizes the inside volume of the well, as compared to the conventional microtiter plate well. The angle at the base of the cone is at most about 40 degrees, and is preferably about 20 degrees. This conical well design would allow the use of less sample solution while maintaining the same sample depth, thus allows the microarray spotter pen to be inserted to a sufficient depth to fill the capillary portion of the pen, while reducing the dead volume for spotter pen sample filling.

Another embodiment of the current invention is illustrated in FIG. 5. Here a well is divided into two regions joint at the interface. The upper region is a wider slot (or cone) , while the lower region has a narrower slot (or cone) toward the bottom of the well, with an angle of at most about 40 degrees between the two sidewalls. Preferably, the narrower slot at the lower half of the well has a 20-degree angle between the two sidewalls, and the minimum distance between the two sidewalls is about 1mm. Similar to the other designs in the current invention, this design also provides an apparatus the wells of which hold a small amount of liquid at a higher depth, compared to conventional microtiter plate wells. An advantage of this design is that it provides a wider opening compared to some of the alternative designs, and easier access to the wells by a sample loading means or the spotter pen devise.

The materials for manufacturing the multi-well platform will typically be polymeric, since these materials lend themselves to mass manufacturing techniques. Preferably, polymers are selected that are known to have low fluorescence or other properties. Various methods in the art can be used to confirm that selected polymers possess the desire properties. Polymeric materials can particularly facilitate plate manufacture by molding methods known in the art and developed in the future, such as insert or injection molding.

The general apparatus and the microtiter plates according to the current invention can be used for holding small amount of liquid samples for microarray sample spotting, as well as for various chemical and/or biochemical reactions .

While the preferred embodiment of the present invention has been shown and described, it will be obvious in the art that changes and modifications may be made without departing from the teachings of the invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not as a limitation. The actual scope of the invention is intended to be defined in the following claims when viewed in their proper perspective based on the prior art.

Claims

What is claimed is :

1. An apparatus for holding a small volume of material, comprising an array of wells connected to each other, each of said wells having:

(1) a bottom;

(2) at least one side wall enclosing the interior of said well, such that the width of the interior of said well is reduced to a slot; and (3) a top opening for receiving material being held.

2. The apparatus according to claim 1 wherein:

(1) the dimensions and layout of said apparatus is in accord with a conventional microtiter plate;

(2) said slot of each well is about 40 degrees at the base; and

(3) said slot of each well is arranged in parallel to the rows of the microtiter plate.

3. The apparatus according to claim 1 wherein:

(1) the dimensions and layout of the apparatus is in accord with a conventional microtiter plate;

(2) said slot of each well is about 30 degrees at the base; and (3) said slot of each well is arranged in parallel to the rows of the microtiter plate.

4. The apparatus according to' claim 1 wherein: (1) the dimensions and layout of the apparatus is in accord with a conventional microtiter plate;

(2) said slot of each well is about 20 degrees at the base; and

5. The apparatus according to claim 1 wherein:

(1) the dimensions and layout of said apparatus is in accord with a conventional microtiter plate; (2) said slot of each well is about 40 degrees at the base; and

(3) said slot of each well is arranged in parallel to the columns of the microtiter plate.

6. The apparatus according to claim 1 wherein:

(2) said slot of each well is about 30 degrees at the base; and (3) said slot of each well is arranged in parallel to the columns of the microtiter plate.

7. The apparatus according to claim 1 wherein:

(1) the dimensions and layout of the apparatus is in accord with a conventional microtiter plate; (2) said slot of each well is about 20 degrees at the base; and (3) said slot of each well is arranged in parallel to the columns of the microtiter plate.

8. The microtiter plate according to any of claims 2-7 wherein said microtiter plate is of 96-well format.

9. The microtiter plate according to any of claims 2-7 wherein said microtiter plate is of 384-well format.

10. An apparatus for holding a small volume of material, comprising an array of wells connected to each other, each of said array of wells having:

(1) a bottom; (2) at least one side wall enclosing the interior of said well, such that the interior of said well is reduced to a cone; and (3) a top opening for receiving material being held.

11. The apparatus according to claim 10 wherein the interior of said well is reduced to a steep cone of about 40 degrees at the base.

12. The apparatus according to claim 10 wherein the interior of said well is reduced to a steep cone of about 30 degrees at the base.

13. The apparatus according to claim 10 wherein the interior of said well is reduced to a steep cone of about 20 degrees at the base.

14. The apparatus according to any of claims 10-13, wherein the dimensions of the apparatus are the same as a conventional microtiter plate.

15. The microtiter plate according to claim 14 wherein said microtiter plate is of 96-well format.

16. The microtiter plate according to claim 14 wherein said microtiter plate is of 96-well format.

17. An apparatus for holding a small volume of material, comprising an array of wells connected to each other, each of said wells having: (1) a bottom; (2) a top opening for receiving material being held; and

(3) at least one side wall enclosing and dividing the interior of said well into two regions joined at the interface, whereas the upper region toward the opening of the well is wider and the lower region toward the bottom of the well is narrower.

18. The apparatus according to claim 17 wherein:

(2) each of said well is a slot, with said upper region wider and said lower region narrower, at about 20 degrees at the base; and

19. The apparatus according to claim 17 wherein: (1) the dimensions and layout of said apparatus is in accord with a conventional microtiter plate; (2) each of said well is a slot, with said upper region wider and said lower region narrower, at about 20 degrees at the base; and (3) said slot of each well is arranged in parallel to the columns of the microtiter plate.

20. The microtiter plate according to any of claims 18- 19 wherein said microtiter plate is of 96-well format .

21. The microtiter plate according to any of claims 18- 19 wherein said microtiter plate is of 384-well format .