Patent Application
20100028988
1. Field of the Invention
This invention relates to laboratory equipment used for performing sequential chemical reactions of which the polymerase chain reaction (PCR) is an example. In particular, this invention relates to the reaction vessels used in conjunction with thermal cyclers.
2. Description of the Prior Art
PCR is one of many examples of chemical processes that require precise temperature control of reaction mixtures with rapid and precise temperature changes between different stages of the process. PCR itself is a process for amplifying DNA, i.e., producing multiple copies of a DNA sequence from a single strand bearing the sequence. PCR is typically performed in instruments that provide reagent transfer, temperature control, and optical detection in a multitude of reaction vessels such as wells, tubes, or capillaries. The process includes a sequence of steps that are temperature-sensitive, different steps being performed at different temperatures in a sequence that is repeated a multitude of times to obtain a quantity large enough for analysis and study from a starting sample of extremely small volume.
While PCR can be performed in any reaction vessel, multi-well plates are the vessels of choice. In many applications, PCR is performed in “real-time” and the reaction mixtures are repeatedly analyzed throughout the process, using the detection of light from fluorescently-tagged species in the reaction medium as a means of analysis. In other applications, DNA is withdrawn from the medium for separate amplification and analysis. Multiple-sample PCR processes in which the process is performed concurrently in a number of samples offer significant advantages, including high efficiency in treating a large number of samples simultaneously and the ability to compare and combine results from different samples for a variety of analytical, diagnostic, and research purposes. Concurrent processing is achieved by using a multi-well plate with one sample per well. The entire plate including all samples therein is simultaneously equilibrated to a common thermal environment in each step of the process. Multi-well plates are particularly useful in automated PCR procedures. Plates with 96 wells in an 8×12 array are typical, but plates with up to 1536 wells are also used.
To perform temperature cycling, the plate is placed in contact with a metal block, known in the industry as a “thermal block,” which is heated and cooled either by Peltier heating/cooling modules or by a closed-loop liquid heating/cooling system that circulates heat transfer fluid through channels machined into the block. The heating and cooling of the thermal block are typically under the control of a computer with input from the operator. The thermal block has a contour complementary to that of the plate wells to achieve full surface contact and hence intimate thermal contact and maximal heat transfer, between the block and each well.
The plate is typically of plastic formed by injection molding. Unfortunately, plastic is not a medium of high thermal conductivity and this causes the plate to present thermal resistance to heat transfer between the thermal block and the samples in each well. The plastic itself is thus a rate-limiting factor in the speed with which the temperature can be raised and lowered in the PCR process. The resistance of the plate to heat transfer can be lowered by reducing the plate thickness, but the typical injection molding process is limited in terms of how thin a plate can be formed thereby. Recognition of this limitation is found in Turner, United States Patent Application Publication No. US 2007/0059219 A1, publication date Mar. 15, 2007. The solution offered by Turner is the use of a two-stage molding process, the first stage involving injection of the resin into a mold cavity and the second involving moving the parts of the mold after it is closed to compress the resin and displace it within the closed cavity.
Plates that are formed with ultra-thin walls tend to have low rigidity, which leads to dimensional instability. In commercially viable PCR procedures, the plates and the samples that are retained in the plate wells must be manipulated by automation, and dimensional stability is required for reliable movement and positioning of the plates as well as the accurate movement of samples and reagents into and out of the individual wells. One solution is offered by Hans-Knöll Institut, European Patent Application Publication No. EP 1 000 551 A1, publication date May 17, 2005, and its counterpart, United States Patent Application Publication No. US 2004/0214315 A1, publication date Oct. 8, 2004. The plate in the Hans-Knöll Institut document is constructed with a rigid frame that surrounds the central area occupied by the wells and is joined to the central area by heat bonding. This design is of limited effect since the rigid frame occupies only the periphery of the plate, leaving the relatively large center section vulnerable to buckling. A further difficulty is that heat bonding is of limited reliability as a means of keeping the sections of the plate properly joined.
The present invention resides in a method of manufacturing multi-well plates that are sufficiently rigid to achieve dimensional stability and yet have wells with ultra-thin walls for maximal heat transfer. The invention also resides in the plates formed by the aforementioned method. In certain embodiments of the invention, the plate is a unitary structure formed from a single shot of resin by a succession of molding steps. In certain other embodiments, the plate is a composite plate that includes a multi-well plate with wells of ultra-thin walls and a supporting plate to further rigidify the remainder of the multi-well plate other than the wells. The method for forming the multi-well plate in all such embodiments includes injection molding followed by forming by way of a pressure differential, such as by vacuum forming, both steps performed on a single continuous mass of resin. The pressure differential forming can be concentrated on the sections of the plate that form the walls of the individual wells, allowing the remainder of the plate to retain a relatively rigid character produced by the injection molding. The rigidity can thereby extend to the planar horizontal areas between adjacent wells and to a peripheral rim or shoulder surrounding the well array for those plates that contain such a rim or shoulder. The pressure differential forming results in the stretching of sections of the injection-molded plate to form the thin walls of the well, and can be concentrated on portions of the walls of each well rather than on the entire well. In either case, the plate is provided with wells that have ultra-thin walls or wall segments. For example, wells can be formed in which the ultra-thin-walled portions of the wells are the lateral walls only and not the floors of the wells, or the lower parts of the lateral walls, leaving a thicker-walled neck or upper portion connecting the thin-walled lower portions to the deck.
The invention also resides in a novel multi-well plate that has a deck and an array of wells, each well having a neck portion adjacent to the deck, and a lower portion adjacent to the deck terminating in a well floor, in which the lower portion has an ultra-thin wall relative to the deck, neck, and floor. A deck of this configuration is formable from a single continuous mass of resin by injection molding followed by vacuum forming, as described above, but the plate prior to vacuum forming and the mold in which vacuum-forming is performed can be configured such that the elongation occurs only in the portion of each well between the neck and the floor. Finally, multi-well plates of different wall thicknesses can be formed by forming the injection molded plate with different thicknesses at different well locations, or by using an appropriately shaped vacuum-forming mold that will produce greater elongation in some wells than in others, or by both in combination.
The term “multi-well plates” is used herein to include any structure that has two or more wells joined to each other through connecting segments. These structures thus include strip tubes, i.e., strips with a single row of wells (or “tubes”), as well as plates with two-dimensional arrays of wells. Examples of strip tubes are those with eight tubes and those with twelve tubes; examples of plates with two-dimensional arrays are those with 48 wells in a 6×8 array, those with 96 wells in a 9×12 array, those with 384 wells in a 16×24 array, and those with still greater numbers of wells.
These and other features of the invention and of individual embodiments of the invention are described below.
a is an enlarged cross section of a portion of the plate blank of
a is an enlarged cross section of a portion of an alternative plate blank, showing a single rudimentary well, in a vacuum forming mold prior to any forming in the mold.
A variety of resins can be used for manufacturing multi-well plates in accordance with the present invention. Examples are polyolefins (including metallocene-catalyzed polyolefins), polyvinyl chloride, polycarbonate, polyesters and other thermoplastic materials. Resins that are the most compatible with the PCR reaction are the most preferred, and notable among these are polypropylene, including unoriented polypropylene, and polypropylene-polyethylene copolymer. The preliminary plate that is formed by injection molding but prior to pressure differential forming is referred to herein as the “molded part” and also as a “blank.” The injection molding step is performed in a conventional two-part mold by injecting the resin in molten form into the closed mold and allowing the resin to cool and solidify within the mold to form the molded part. The temperature of the molten resin will be that which will allow the resin to flow inside the mold, and the optimal such temperature will vary with the choice of resin and will be known to those skilled in the art or readily determined by known methods. In most cases, this temperature will be within the range of about 70° C. to about 310° C. The thickness of the molded part is determined by the mold itself, and the minimal thickness will depend on the flowability of the resin within the mold cavity. In many cases, the molded part will be of uniform thickness; in other cases, the mold can be designed to produce a part with different thicknesses in different sections. For molded parts of uniform thickness, the optimal thickness will be the minimum thickness that will provide the desired degree of rigidity. For molded parts in which certain regions are thicker than others, the thicker regions may be those that retain their thickness through the vacuum forming step and serve to provide the finished product with the desired rigidity. In preferred embodiments, each segment or wall of the molded part will have a thickness within the range of about 0.025 mm to about 3 mm, and preferably about 0.1 mm to about 2 mm.
The molded part serves as the blank for the pressure differential forming and can be a flat plate or a plate with rudimentary (partially formed) wells at the locations of the wells ultimately sought in the finished product. In the case of a flat plate, the pressure differential forming step will then draw out sections of the plate from the wells, while in the case of a plate with rudimentary wells, the pressure differential forming will extend the depths of the rudimentary wells. In both cases, the pressure differential results in stretching of sections of the blank to produce full-sized wells whose walls are thinner than the unstretched sections of the blank. These unstretched sections can either be the flat portions of the plate connecting the wells, which portions are collectively referred to herein as the “deck,” or portions of the walls of the wells themselves. The shapes of the rudimentary wells and of the mold in which pressure differential forming is performed will determine which regions of the blank will undergo stretching and thus be thinned.
The pressure differential forming step is preferably enhanced by heating the molded part to a temperature at or above the heat deflection temperature of the resin for the load that will be applied (i.e., for the pressure differential), but below the melting temperature of the resin. Heat deflection temperatures for given loads are known in the art for particular resins, and can also be determined by the standard technique designated as ASTM D 648 which uses loads of either 66 psi (0.46 MPa) or 264 psi (1.8 MPa). For molded polypropylene, for example, the average heat deflection temperature is approximately 100° C. for a load of 66 psi, and approximately 70° C. for a load of 264 psi. The optimal temperature for the pressure differential forming step will depend on the configuration of the forming equipment, taking into account the heat loss when the heated part is transferred from the heating area to the forming area of the apparatus. Pressure differential forming can be performed by vacuum forming, by blowing into a mold cavity, or by the application of a mechanical pressure, or by a two or more of these methods in succession. Mechanical pressure can be applied by pressing a male forming tool against the heated part, and is preferably performed in combination with vacuum forming as a preliminary step. Regardless of the pressure differential method used, the degree of stretching of the injection molded plastic material during pressure differential forming can vary, but in most cases the desired results will be achieved by lengthening the material in the sections being stretched by a factor within the range of about 2 to about 5, to achieve a wall thickness of from about 0.025 mm to about 0.17 mm (0.00098 inch to 0.0067 inch).
One method in accordance with the invention for forming the plate of
b shows the blank 21 in position in a vacuum forming mold 27 prior to the application of the vacuum. The mold 27 has a depression 28 or mold cavity aligned with each well 22 of the blank 21. The depression 28 and the well 22 are complementary in shape except that the depression 28 is deeper with a lower portion 29 extending beneath the floor 24 of the well of the molded part. Vacuum is applied in this lower portion 29 while the vacuum mold and the blank are heated, thereby drawing the softened resin of the blank into the lower portion, and stretching the portions of the resin that are not in contact with the mold wall. The result is shown in
a,
3
b, and 3c illustrate an alternative method, still within the scope of the invention, for forming the plate of
An alternative to the multi-well plate shown in
In the claims appended hereto, the term “a” or “an” is intended to mean “one or more.” The term “comprise” and variations thereof such as “comprises” and “comprising,” when preceding the recitation of a step or an element, are intended to mean that the addition of further steps or elements is optional and not excluded. All patents, patent applications, and other published reference materials cited in this specification are hereby incorporated herein by reference in their entirety. Any discrepancy between any reference material cited herein or any prior art in general and an explicit teaching of this specification is intended to be resolved in favor of the teaching in this specification. This includes any discrepancy between an art-understood definition of a word or phrase and a definition explicitly provided in this specification of the same word or phrase.
This application claims the benefit of U.S. Provisional Patent Application No. 61/085,674, filed Aug. 1, 2008, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61085674 | Aug 2008 | US |
