The present invention relates to the field of chemical reactions requiting temperation and/or incubation of the reaction mixture and in particular chemical reactions in liquid media The present invention discloses a new device and method for efficient and homogenous mixing, temperation and/or incubation of reaction mixtures.
Many important industrial processes as well as procedures applied in laboratories of various kids are dependent on chemical reactions. Commonly the time consumed for completing a process or procedure is determined by the time it takes for some specific chemical reaction or reactions to reach equilibrium. This is often referred to as the kinetic properties of a chemical reaction or simply reaction kinetics. A host of variables influence the reaction kinetics in each case, for instance the concentrations of reactants, temperature, presence of catalytic agents etc.
Typically, increased temperature makes chemical reactions faster by speeding up key mechanisms like bringing molecules or molecule domains in contact with each other. Therefore it is common to heat the reaction vessels, for example bringing them in contact with an open flame, hot gas, hot liquid, hot sand or a solid material. This procedure is often referred to as incubation.
One typical problem involved with incubations of fluid reaction mixtures is thermal heterogeneity, because the parts of the reaction mixture being in close contact with the walls of the reaction vessel will become heated before the more central parts of the reaction mixture. In many cases there is a risk of part of the reaction mixture becoming overheated before other parts even reach the desired temperature. This leads to temperature gradients forming in the reaction mixture. Hot subsets of the reaction mixture has normally lower density than cold subsets which tend to generate temperature gradients or discrete layers of more or less isothermal bodies of liquid, so called thermoclines. Thus warm, less dense portions of the reaction mixture tend to find a position above cold, denser portions. Molecular motion and currents in the reaction mixture will eventually homogenize the reaction mixture with respect to temperature, a process referred to as temperation of the reaction mixture. The time it takes to temperate a reaction mixture may contribute substantially to the time required for the complete reaction.
However, time-consumption in itself is not the sole problem involved with temperation of chemical reaction mixtures. In certain incubation procedures such as the repetitive temperations involved in so called thermocycling processes, e.g. for performing polymerase chain reactions, also known as PCR-reactions, long temperation periods also leads to unwanted side-reactions, sometimes causing severe quality problems with respect to the accuracy and specificity of the obtained PCR-product.
In the ongoing strive to miniaturize chemical reaction volumes, as evident e.g. in the field of high throughput screening, several other problems are encountered. In a small reaction vessel, such as a well on a microtitre plate, both the mixing and temperation of sample and reagents may become severely restricted. When two or more miscible fluids are mixed, we normally assume that they first form a homogenous mixture, which then reacts. This is however rarely the case.
Conventional microtitre plates and cuvettes are often manufactured from polystyrene, a hydrophilic polymer. Without dwelling on the exact behaviour of the liquid at the vessel boundaries, it can be concluded that stagnant areas will form and insufficient mixing easily occur in a small reaction vessel, such as a well on a microtitre plate. The properties of the reactants and sample fluids also influence their interaction with each other and with the vessel boundaries. Partial segregation, the formation of layers, aggregation and so on are only a few examples of irregularities that can be encountered in a reaction vessel.
There are reasons for distinguishing between two different processes causing problems with heterogeneous temperature distribution in a reaction mixture. The process caused by the lower fluidity close to the walls of a reaction vessel is a problem increasing when reaction scale decreases. In contrast, the problem involved with central parts of the liquid body being colder than the liquid close to walls when heating a reaction vessel, increases when reaction scale increases. This is the reason why thermocycling devices for processes in which proper temperation is required (e.g. processes like PCR) have a very narrow dynamic range with respect to the reaction scale. Typically, in PCR-reactions these problems are most severe when reaction volumes are less than 5 xcexcL and larger than 50 xcexcL.
Another problem, seemingly unrelated to the mixing and temperation issues, is that of evaporation. In order to avoid evaporation, there exists a tendency to make the reaction vessels and in particular the wells on microtitre plates deeper and more narrow. Naturally, this further enhances the previously mentioned problems of insufficient mixing and temperation.
So far, temperature heterogeneity has been discussed in terms of properties in a single reaction vessel. Especially when discussing miniaturisation of assays yet another dimension of temperation heterogeneity need to be considered; that of variation between reaction vessels. In assays with comparative purposes (i.e. with or without quantitative analysis like screening for novel drug candidates, mutations in nucleic acids, single nucleotide polymorphism and so forth) it is important to consider the reproducibility, commonly referred to as well-to-well uniformity.
Since the processes leading to poor thermal uniformity are difficult to predict quantitatively, the only available solution to the problem is often to focus on means to enhance the homogenisation processes. To do this, various strategies are applied.
One is to use reaction vessels with specific flat or oblong configurations in order to minimize the distance between the central and peripheral parts of the bulk of the reaction mixture. An example of this is to perform the incubation in thin capillaries as described by Wittwer, C. T. et al. (The LightCycler(trademark): A Microvolume Multisample Fluorimeter with Rapid Temperature Control, Bio Techniques 22:176-181, January 1997). One disadvantage with this approach is that the glass capillaries, loosely attached to their plastic holding portions, require extensive manual handling. The choice of glass capillaries makes possible both rapid temperation of the reaction mixture and detection of fluorescence after amplification. However, a glass capillary tends to maximize the surface-to-fluid contact, with all the consequences this has on mixing and temperation. Further, the use of glass capillaries has the additional draw-back of obstructing the post-PCR processing of the sample. Examples of post-PCR processing include DNA sequencing procedures etc.
Other ways to solve the problem with mixing and temperature heterogeneity is to introduce perturbation by agitating or shaking the reaction vessel. Specific instruments designed for this purpose are for example various kinds of flask shakers and so called Vortex machines. A problem often encountered with this approach is that the perturbation periodicity may cause currents or standing waves and therefore incomplete homogeneity.
Another approach for homogeisation is the use of ultrasonic waves in a standard procedure called sonication. The latter procedure is, unfortunately, often difficult to combine with a number of standard incubation methods.
Yet another approach is the use of flow-through systems, where a constantly mowing liquid is subjected to heating.
Within the field of PCR, much attention has been given to heat transfer and to the question of faster ramping, i.e. how to shorten the speed for temperature adjustments in both the heating and cooling phase.
WO 98/49340 (PCT/AU98/00277) discloses a temperature cycling device and method where a reaction mixture and a sample is loaded into loading wells on a disposable rotor, which rotor is then placed into a centrifugal thermal cycling device and spun, so that the reaction mixture and sample are moved by centrifugal force to a reaction well at the periphery of the rotor. The device comprises heating means, for example infrared lights, convection heating elements or microwave sources. Interestingly, also provisions for cooling the rotor are included in the specification. According to one embodiment, the rotor speed is increased, resulting in air being drawn into the device and rapidly cooling the contents of the reaction chambers at the periphery of the rotor. In addition to ambient air, a coolant gas can be used. Refrigerated air is given as an example of coolant gases. Importantly, the disclosure of WO 98/49340 implies the use of different speeds of rotation. Further, WO 98/49340 does not address the problems of mixing and homogenous temptation For example, it does not specify the direction of heating, nor does it contemplate simultaneous heating and cooling.
DE 19501105 A1 discloses a centrifuge with a temperature control system where a circulating fluid enters the rotor from above and flows out-wards and down-wards in the direction of the radius, around the test-tubes or sample containers. The inventor of the centrifuge according to DE 19501105 criticises the hitherto known devices using a radiating source of heat and rejects them as unsatisfactory.
It was the purpose of the present invention to solve the problem with time consuming and insufficient mixing and temperation steps in procedures where incubation of small-volume chemical reaction mixtures are involved by finding means for effective mixing and homogenisation. Importantly, said means should be compatible with means for unhindered analysis of the reaction and preferably also applicable in existing and future technologies in the fields of chemical and biotechnological reactions, screening and analysis, such as high throughput screening, amplification reactions etc.
In particular, the need for a fast and accurate thermocycling device and corresponding method is nor yet satisfied by the prior art devices and methods.
This problem is solved by the invention according to the characterising portion of the attached claims, which are hereby incorporated. The invention comprises both devices and methods as disclosed in the description an defined in the attached claims. Further problems solved by the invention and advantages obtained can be derived from the description and drawings.