Device for automatically carrying out polymerase chain reactions

The invention relates to a device for automatic performance of polymerase chain reactions in a plurality of reaction containers, each reaction container is closed with a lid and containing a predetermined volume of a liquid reaction mixture, which apparatus comprises the following components:<ul><li>a) a carrier which has an arrangement of chambers for holding the reaction container, each chamber being adapted to receive the lower part of a reaction vessel, and wherein the carrier consists of a material having a high thermal conductivity, and that it has a top surface, a bottom surface and a cylindrical outer wall has, each of the chamber of the carrier has an opening which is located in the upper surface of the support,</li><li>b) arranged in the chambers of the reaction vessel support, which are each closed by a lid,</li><li>c) a computerized control and regulating device, and</li><li>d) controlled by the control and regulating device has means for cyclic alteration of the temperature of the support.</li></ul>

More particularly, the invention relates to a device of this type, preferably as an integral part of an automatic analyzer for performing the Polymerase Chain Reaction ( "Polymerase Chain Reaction") is suitable.

A device of the type mentioned above is described in EP-A- 0,488,769 A2.

A device of the above mentioned type is also described in EP-A- 0,236,069 A2, it is pointed out in addition that means for cyclic alteration of the temperature of a carrier of the kind mentioned above may contain a Peltier element.

Devices of the aforementioned type are called "thermal cycler". This term is used in the description below.

The devices described in EP-A-0488769 A2 and EP-A- 0,236,069 A2, have the disadvantage that by the temperature changes and acting on the reaction vessel pressure, the outer walls of the reaction vessel to the walls of chamber the carrier liable. The thus procured positive connection makes it difficult to remove the reaction vessel from the carrier.

The invention is therefore based to provide a device of the type mentioned are available, with which the above-mentioned disadvantage can be solved the task.

This object according to the invention is achieved with a device of the aforementioned type which is characterized in that<ul><li>(I) the arrangement of the chamber in the carrier is annular,</li><li>(Ii) it comprises a hinged lid for holding the sealed, arranged in the reaction container carrier, and</li><li>(Iii) contains a lifting device to facilitate the removal of the reaction containers from the compartments in the carrier, which lifting device comprises an ejection lever which is connected at one end with a hinge of the hinged lid and at the other end free, and an ejection disc which with the axis of rotational symmetry of the carrier is concentrically mounted on the ejection lever and on the periphery an arrangement of recesses has, which serve for removing the reaction container from the chambers.</li></ul>

The inventive device facilitates the removal of the reaction container from the chambers of the support after performing the polymerase chain reactions.

Another advantage of the inventive device is that it resolves the above-mentioned drawback of the known devices of this type in the simplest possible manner and with little effort.

Description of an embodiment

An embodiment of the invention will be described with reference to the accompanying drawings. Show it:<dl id="dl0001"><dt>Fig. 1</dt><dd>a taken-out from an analytical device thermal cycler part 2, which contains the thermal cycler 18 and 19, wherein the thermal cycler 18 is opened and a sample taken from this tube ring 23 is shown,</dd><dt>FIG. 2</dt><dd>a section through the line II-II in Fig. 1, wherein the thermal cycler 18 is closed,</dd><dt>Fig. 3</dt><dd>a perspective view of the thermal cycler 18 according to FIG. 1, which is supplemented by a lifting device 53,</dd><dt>Fig. 4</dt><dd>a comparison with FIG. 2 enlarged section through the thermal cycler in the closed state,</dd><dt>Fig. 5</dt><dd>a thermal cycler according to FIG. 4 in the open state,</dd><dt>Fig. 6</dt><dd>a schematic representation of a "master-slave" control for controlling and monitoring the operating parameters of a thermal cycler,</dd><dt>Fig. 7</dt><dd>a temperature-time diagram of a data stored in the master processor temperature profile or the resulting temperatures the thermal block and the sample.</dd></dl>

Thermal cycler

In the following description, a device is referred to with the thermal cycler, which serves for automatic execution of temperature cycles in at least one closed with a lid sample tubes 21 containing a predetermined volume of a liquid reaction mixture.

Hereinafter, a thermal cycler is described, which is preferably suitable as a constituent of an automatic analyzer for performing the Polymerase Chain Reaction. The analyzer is designed for example for performing immunoassays.

In Fig. 1, a thermal cycler part 2 is shown removed from an analyzer 1. This thermal cycler part 2 contains, for example, two identical thermal cyclers 18, 19 and a standby position 22. The following description of the thermal cycler 18 also applies to the thermal cycler 19th

The thermal cycler 18 includes the following components:<ul><li>a) a thermal block 33 which serves as a carrier of the sample tube, and an annular array of recesses 27, with each recess serving as a chamber for receiving the lower portion of the sample tube 21,</li><li>b) shown in FIG. 6 computerized control and regulating device, and</li><li>c). through this control and regulating device controlled heating and cooling elements as a means of cyclical variation in the temperature of the thermal block 33</li></ul>

The thermal block 33 is made of a material that has a high thermal conductivity. The heater 33 is preferably a body made of aluminum or silver. The heating block 33 has a top surface, a bottom surface and a cylindrical outer wall, each of the recesses 27 of the thermoblock 33 has an opening located in the upper surface of the support.

As shown in Figures 1 and 3, for example, twelve sample tubes 21 are combined into a test tube ring 23rd The sample tubes 21 are conical, cylindrical in shape at the bottom of the top and sealed by a lid 87th As shown in Fig. 1 and 3 can be clearly seen, such a sample tube assembly 23 can be inserted into corresponding recesses 27 of the thermoblock 33 of the thermal cycler 18th

Access to the contents of a test tube

The thermal cycler 18 has a hinged lid 28, having an opening 29 a recess 27 of the thermoblock 33 which allows piercing of the closure 87 of the sample tube inserted in the recess 21 having a pipetting needle. As seen from Fig. 2, is aligned in the closed position of the hinged lid 28, each of the openings 29 with the longitudinal axis 31 of the corresponding sample tube 21st

The openings 29 of the hinged lid 28 allow access to the contents of each test tube in the closed hinged lid 28. For this purpose the pipetting needle 32 is a pipetting device inserted (not shown in Figure 3) through one of the openings 29 pierced the lid 87 of the sample tube 21 with the pipetting needle 32 and subsequently filtered off with suction a certain volume of the liquid contained in the test tube.

Heat transfer between the heating block and sample tubes

It is apparent from Fig. 2 that the recesses 27 are adapted to the conical portion of the sample tubes 21 in the thermal block 33 so that the peripheral wall of the sample tube 21 reliably to the inner wall of the recess 27 can come for the purpose of the best heat transfer to the plant. In order to increase the thermal response speed, precision and uniformity, the heater 33 is heat-insulated as possible held in a housing 34 and has little mass with good thermal conductivity.

Heating element in the hinged lid of the thermal cycler

The lid 28 preferably includes a heating element, such as an electric resistance heater 52 that serves to heat the closed, arranged in the heating block 33 sample tubes.

In a first embodiment of the thermal cycler, the electric resistance heater 52 is used in combination with a Peltier element 36 as described below in order to achieve a desired temperature profile (temperature curve over a certain time interval) in the heating block 33rd In this embodiment, the Peltier element is used as a cooling or as a heating element within a temperature profile according to the temperature to be reached.

The interaction of the electrical resistance heater 52 with the Peltier element 36 allows, to achieve the required speed of temperature change of the thermal block 33 and the required precision and homogeneity of the temperature distribution. By the action of the resistance heater 52 of the possible condensation in the cover region of the test tube 21 is also avoided.

Seaming pressing device of the hinged lid of the thermal cycler

The hinged cover 28 preferably includes a Seaming pressing means for retaining the closed, arranged in a heating block 33 sample tubes 21. For this purpose, the hinged lid 28 is a spring-held pressure plate 46 which pushes each sample tube 21 with a defined force into the recesses 27 of the thermoblock 33 , Recesses 47 for receiving the dome-shaped cover 87 of the sample tube 21 and piercing holes 48 for the pipetting needle 32 provided coaxially to the sample tube 21 in the pressure plate 46th As spring element 49 may be provided a corrugated washer. By a locking ring 51, the pressure plate 46 is secured in hinged cover 28 open against falling out.

The above-mentioned resistance heater 52 is preferably contained in the resilient platen 46th

Peltier element as a cooling or heating element

As shown in FIG. 2, a thermal cycler 18 preferably contains at least one Peltier element 36 as part of the measures provided for in the thermal cycler 18 means for cyclic change in the temperature of the thermal block 33. The Peltier element 36 with its heat transfer surface 37 over a large area with the lower surface of the thermal block 33 and thermally contacted with his other heat transfer surface 38 over a large area to a heat sink 39 for dissipating heat in contact. The cooling body 39 is preferably made of aluminum or copper. For heat dissipation, a switchable fan 45 is provided.

The schematically illustrated in Fig. 2 Peltier element 36 is preferably an array of such elements.

In the above mentioned first embodiment of the thermal cycler, the Peltier element 36 is used as a cooling or as a heating element. This mode of operation of the Peltier element 36 and its interaction with the electric resistance heater 52 makes it possible to reach the required temperature of the thermoblock within a temperature profile.

To extend the life of the Peltier element 36 that is before thermodynamically justified mechanical spike protected preferably in that the Peltier element is held 36 pressed by a central spring-biased fixing against the thermal block 33rd To this end, the Peltier element is clamped resiliently between the heat transfer surfaces of the thermal block 33 and the heat sink. 39 For example, the cooling body 39 is pressed by a compression spring 41 with its contact surface against the Peltier element 36th The spring tension can be adjusted by an adjustment screw 42, spring plate 43 and a ball joint 44, which 39 further increases the degrees of freedom of the heat sink.

Peltier element exclusively as a cooling element

In a variant of the embodiment described here, the Peltier element 36 is, that is used solely as a cold-generating element only as a cooling element. Characterized an extension of the lifetime of the Peltier element is achieved.

Additional heating to the thermal block

In a second embodiment of the thermal cycler that preferably additionally contains an electrical resistance heater 35 which is arranged around the thermal block 33 and along the periphery of its cylindrical outer wall. Using this additional Heizeleme n-th in the thermal cycler, the Peltier element 36 is used only for cooling. This has the advantage of relieving the Peltier element of thermally induced mechanical stress and thus contributes to extend help the life of the Peltier element in the thermal cycler.

Hoist

By the temperature changes and the action of the spring 49 adhere to the conical portions of the sample tubes 21 on the walls of the recesses 27 of the thermoblock 33. characterized procured frictional connection makes it difficult to remove the sample tube 21 from the thermal cycler 2. For this reason, has been in the embodiment shown in FIG. 3 to 5, a lifting device 53 proposed which greatly facilitates removal of the test-tube ring 23 from the heater 33.

As can be seen from Figures 3-5, the lifting device 53 includes a rocker 55, which serves as the ejection lever. The rocker 55 is connected at one end with a hinge of the hinged lid 28th The rocker 55 is at the other end free. The lifting device 53 further includes an ejector plate 58 which is concentric with the axis of rotational symmetry of the thermal block 33 on which the rocker is arranged 55th The ejection disc 58 has on the periphery of an array of recesses 61 which are used for removing the sample tube ring 23 from the recesses 27 of the thermoblock 33rd

As seen from Fig. 3, the rocker 55 is guided on the axis of rotation 54 of the hinged lid 28. Rotational axis side, the rocker 55 on two lugs 56 with recesses 57, in which engages the rotational axis 54th The ejection disc is screwed onto the rocker 55th The ejection disc 58 has at its peripheral edge at 59 semi-circular recesses 61, which are aligned projection exactly with the recesses 27 in the unit heater 33, and the cylindrical portions of the sample tubes 21, which are inserted in the recesses 27 (FIG. 5). The peripheral edge 59 of the ejection disc 58 thus engages under the inner flange portion 62 of the sample tube ring 23 and the flanges of the sample tube 21. The shape and function of the recess 57 in the lugs 56 of the rocker 55 in relation to the axis of rotation 54 of the hinged lid 28 as well as a the hinged lid 28 at a distance<u>e</u> attached to the control pin 63 which also engages in the recess 57, can be seen from Figures 4 and 5. FIG. With a closed hinged lid 28, the lifting device 53 is still functional. When opening the hinged lid 28 gets the pin 63 from a certain angle of opening in contact with a control surface 64 of the recess 57 and causes pivoting of the rocker 55 about the point P, which has a lifting of the sample container 21 to follow. By tilting movement of the rocker 55 about the point P and the increasing inclination of the ejection disc 58 is caused to the individual test tube 21 associated breakaway forces manifest at different times, so that the sample tube 21 is gradually released from their recesses 27th The effort and the material stress is kept at a low level and increases the ease of handling.

Control and regulation of the thermal cycler

A control and regulating device of the thermal cycler 18 via master-slave processors 72, 73 is shown in Fig. 6 schematically.

The temperature of the pressure plate 46 of the hinged lid 28, the thermal block 33 and the environment is detected by means of temperature sensors 65, 66, 67 and fed through a temperature interface 68 to the slave processor 73rd In the master processor 72 (interface to the user) among others, the temperature setpoint values, the time reference values, the number of temperature cycles and the rate of heating and cooling processes are entered.

It may already be selected and worn predetermined, stored temperature / time profiles. The entry is through the keyboard 16 or another interface, this data is supplied to the slave processor 73, which controls a power controller 71 via controller 69, which in turn controls the power supply to the heating elements 35, 52 and the Peltier element 36th The feedback (actual values) are supplied via the slave processor 73 to master processor 72 and processed there and displayed to the user. In this example the user about the current sample temperature, the temperatures reached already to the time and the still-to-reach temperatures is informed with time stamp.

The operating state of the system is continuously monitored and logged. Errors that can not be resolved by the system itself, cause an automatic shutdown or error message.

The temperature of the sample is determined from the temperature of the thermal block 33 computationally. For this, the transfer function is determined from the sample chamber to the sample in the sample tube 21st This function is essentially a low-pass with dead time.

Using appropriate control algorithms (sampled systems) is in each case calculates the manipulated variable, which is necessary to track the temperature of the sample to the predetermined setpoint temperature. These calculations are performed with a signal processor. The calculated manipulated variable is fed to the power controller 71 in the form of a pulse width. The power controller 71 is for example a power FET with a matching protective and interference suppression.

The control and regulation described above allows the use of the thermal cycler to sample in a sample tube ring used in the thermal cycler according to specific temperature profiles to heat and cool. The temperature profiles are defined by plateau temperatures of defined duration, and the gradient that defines the time at which a plateau-temperature must be reached. The condition is that all samples in the thermal cycler at the same time have the same temperatures.

In Fig. 7 Temperature gradients are, for example shown from one cycle process. Curve A shows the temperature profile on the heating block 33, the curve B shows the temperature curve of the liquid in the reaction vessel 21. The thermal cycler can temperatures between 40 and 98 degrees Celsius can be set. Typically, the lower temperatures are between 50 and 60 degrees centigrade and the upper temperatures from 90 to 96 degrees Celsius. When the average temperature is used, it is around 72 degrees Celsius. The agreement reached with the thermal cycler heating / cooling rate is 1 ° C per second. A typical cycle has a duration of 120 seconds. When the corresponding temperatures have to be kept for longer than 10 seconds, the cycle time is extended accordingly.