Device for automatically carrying out polymerase chain reactions

The invention relates to a device for automatically Conducting polymerase chain reactions in a variety of Reaction containers, each reaction container with a lid is closed and a predetermined volume of a liquid contains the reaction mixture, which apparatus comprises the following components includes:<sl><li>a) a carrier of an array of chambers for receiving Reaction vessel has, each chamber being adapted to receive the lower receive, and wherein the carrier part of a reaction container of a material having a high thermal conductivity, and a top surface, a bottom surface and a cylindrical The outer wall has, each of the chambers of the carrier has an opening, which lies in the upper surface of the support,</li><li>b) arranged in the chambers of the support reaction vessels each of are closed with a lid,</li><li>c) a computerized control and regulating device, and</li><li>d) controlled by the control and regulating device means to cyclic change of the temperature of the support.</li></sl>

More particularly, the invention relates to a device of this kind, the preferably as an integral part of an automatic Analyzing apparatus for performing the polymerase chain reaction ( "Polymerase Chain Reaction") is suitable.

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

A device of the kind mentioned above is also EP-A-described 0236069 A2, it is pointed out in addition, that means for cyclic alteration of the temperature of a wearer of may include above-mentioned type, a Peltier element.

are devices of the type mentioned in "Thermal cycler" called. This name is in the following description, used.

In EP-A-0488769 A2 and EP-A- 0,236,069 A2 -Described devices 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 container from the carrier.

Although it is known from DE-A-3024210 A1 a metal block thermostat , in which reaction vessels by means of a carrier plate simultaneously in Holes on or executable. A disadvantage is that it additional lift mechanisms for transporting the carrier plate are required, which the manufacturing cost considerably increases.

The invention is therefore based on the object a device of to provide the kind mentioned are available, with which the above mentioned drawbacks can be solved.

This object is achieved according to a device according to Claim 1 dissolved.

The inventive device facilitates the removal of Reaction vessel from the chambers of the support after performing the Polymerase chain reactions.

A further advantage of the inventive device is that it the above-mentioned drawback of the known devices of this type to the simplest possible way and fixes with little effort.

Description of an embodiment

An embodiment of the invention, with reference to the accompanying drawings. Show it:<dl tsize="6"><dt>Fig. 1</dt><dd>one taken out of an analyzer Thermal cycler part 2, which contains the thermal cycler 18 and 19, wherein the thermal cycler 18 is opened and a therefrom sample taken tube ring 23 is shown,</dd><dt>Fig.2</dt><dd>a section through the line II-II in Fig. 1, wherein the closed thermal cycler 18,</dd><dt>Fig. 3</dt><dd>according to a perspective view of the thermal cycler 18 Fig. 1, which is complemented 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 regulating and monitoring the operating parameters of a thermal cycler,</dd><dt>Fig. 7</dt><dd>a temperature-time diagram of a processor in the master stored temperature profile or that the resulting resulting temperatures the thermal block and the sample.</dd></dl>

The illustrated in Figures 1 and 2 thermal cycler part 2 as such not covered by the claim. 1

Thermal cycler

In the description below is with a thermal cycler Device referred to by the automatic execution Temperature cycles in at least one closed with a lid Sample tube 21 is that a predetermined volume of a liquid The reaction mixture contains.

Hereinafter, a thermal cycler is described which preferably as part of an automatic analysis apparatus for performing the polymerase chain reaction is useful. The analyzer is for example, designed for performing immunoassays.

In Fig. 1, a thermal cycler part 2 is made of an analyzer 1 shown removed. 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:<sl><li>a) a thermal block 33 which serves as a carrier of the sample tube, and which has an annular array of recesses 27, each Recess as a chamber for receiving the lower portion of the Sample tube 21 serves,</li><li>b) shown in FIG. 6 computerized control and Regulating device, and</li><li>c) controlled by this control and regulating device heating or Cooling elements as a means for cyclic alteration of the temperature of the Thermoblock 33rd</li></sl>

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

As shown in Figures 1 and 3, for example, twelve sample tubes 21 are a test tube ring 23 are summarized. The sample tubes 21 are conical, cylindrical in shape in the lower region in the upper region and sealed by a lid 87th As shown in FIGS. 1 and 3 good is seen, such a sample tube assembly 23 in corresponding recesses 27 of the thermoblock 33 of the thermal cycler 18 used.

Access to the contents of a test tube

The thermal cycler 18 has a hinged lid 28, the pro Recess 27 of the thermoblock 33 has an opening 29, which is a the closure of 87 set in the recess piercing Sample tube 21 allows a pipetting. As shown in FIG. 2 visible, 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 provide access to Contents of each test tube is closed hinged lid 28 is For the pipetting needle 32 (not shown in Figure 3) introduced pipetting by the openings 29, the lid 87 the test tube 21 pierced with the pipetting needle 32 and then a certain volume of contained in the test tube Fluid aspirated.

Heat transfer between the heating block and sample tubes

It is apparent from Fig. 2 that the recesses 27 in the adapted heater 33 on the conical section of the sample tube 21 are, so that the peripheral wall of the sample tube 21 to reliably the inner wall of the recess 27, the purpose best Heat transfer, can come to rest. To the thermal Reaction rate to increase precision and homogeneity, the Heater 33 possi waxed insulated mounted 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, for example a electrical resistance heater 52 for heating the closed, arranged in a heating block 33 sample tubes used.

In a first embodiment of the thermal cycler, the electrical resistance heater 52 in combination with a below described Peltier element 36 used to produce a desired Temperature profile (temperature curve over a certain time interval) in the Thermoblock achieving 33rd In this embodiment, the Peltier element depending on the temperature to be reached within a used temperature profile as a cooling or a heating element.

The interaction of the electrical resistance heater 52 with the Peltier element 36 enables the required speed of Temperature changes of the unit heater 33, and the required To achieve precision and homogeneity of the temperature distribution. By the effect of the resistance heater 52 is also a possible Condensation avoided in the cover region of the test tube 21st

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, in a heating block 33 arranged test tubes 21. For this purpose, the hinged lid 28 a resiliently held pressure plate 46 on which each sample tube 21 with a defined force into the recesses 27 of the thermoblock 33 pushes. Recesses 47 for receiving the dome Cover 87 of the sample tube 21 and puncture openings 48 for the Pipetting 32 are coaxial with the sample tube 21 in the pressure plate 46 provided. As spring element 49 may be provided a corrugated washer. By a locking ring 51, the pressure plate 46 is open, Hinged lid 28 is secured against falling.

The above-mentioned resistance heater 52 is preferably in the resilient pressure plate 46 included.

Peltier element as a cooling or heating element

As shown in Fig. 2, a thermal cycler 18 preferably comprises at least one Peltier element 36 as a part of the thermal cycler 18 means provided for cyclic change of the temperature of the Thermal block 33. The Peltier element 36 is with its one Heat transfer surface 37 with a large area of ​​the lower surface of the Thermal block 33 and with its other heat transfer surface 38 a large area is thermally coupled to a heat sink 39 for heat dissipation in liquid 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 is used as a cooling or as a heating element, the Peltier element 36th These Operation of the Peltier element 36 and its interaction with the electrical resistance heater 52 allows the required Temperature of the thermal block within a temperature profile to to accomplish.

To extend the life of the Peltier element 36 is this before thermodynamically justified mechanical stress peaks preferably protected in that the Peltier element 36 by a pressed central, spring-loaded mounting against the thermal block 33 is maintained. To this end, the Peltier element is elastically between the Heat transfer surfaces of the unit heater 33 and the heat sink 39 clamped. For this, the cooling body 39 for example by means of a pressure spring 41 pressed with its contact surface against the Peltier element 36th The Spring tension can have a set screw 42, spring plate 43 and a Ball joint 44 can be set, which of the degrees of freedom The heat sink 39 even further.

Peltier element exclusively as a cooling element

In a variant of the embodiment described here is the Peltier element 36 exclusively as a cryogenic element, ie used as a cooling element. This is an extension of the reached lifetime of the Peltier element.

Additional heating to the thermal block

In a second embodiment of the thermal cycler contains these preferably in addition an electric resistance heater 35, to the the heater 33 and along the periphery of its cylindrical, disposed outer wall. Using this additional Heating element in the thermal cycler, the Peltier element 36 only Cooling used. This has the advantage of relieving the Peltier element of thermally induced mechanical stress and contributes characterized help the lifetime of the Peltier element in thermal cycler to extend.

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 Recesses 27 of the unit heater 33. The thus procured positive 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 suggested that the removal of the sample tube 23 from the ring Heater 33 much easier.

As seen from Figures 3-5 visible, contains the lifting device 53 a Rocker 55, which serves as the ejection lever. The rocker 55 is at one end to a hinge of the hinged lid 28 is connected. The rocker 55 is on the other end free. The lifting device 53 further includes a Ejection disc 58, with the rotational symmetry axis of the thermoblock 33 is concentrically on which the rocker is arranged 55th The Ejection disc 58 has on the periphery of an array of Recesses 61 for removing the sample tube 23 from the ring Recesses 27 of the thermoblock 33 serve.

As seen from Fig. 3, the rocker 55 on the axis of rotation 54 of the is Hinged lid 28 out. Rotational axis side, the rocker 55 two tabs 56 with recesses 57, into which engages the rotational axis 54th The Ejection disc is screwed onto the rocker 55th The ejection disc 58 has, at its peripheral edge 59/2 circular recesses 61 on which projection exactly with the recesses 27 in the unit heater 33, or the cylindrical portions of the sample tube 21 in alignment, which inserted in the recesses 27 (FIG. 5). The peripheral edge 59 of the Ejection disc 58 thus engages beneath the inner flange portion 62 the sample tube ring 23 and the flanges of the test tube 21. The Shape and function of the recess 57 in the tabs 56 of the rocker 55 in connection with 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 control pin 63, which also engages in the recess 57, is from Figures 4 and 5 visible. With a closed hinged lid 28, the lifting device is 53 still function. When opening the hinged lid 28 of the pen device 63 from a certain opening angle in contact with a control surface 64 of the Recess 57 and causes swiveling of the rocker 55 around the Point P, resulting in a lifting of the sample container 21 to the sequence. By Tilting movement of the rocker 55 around the point P by increasing or Skew the ejection disc 58 causes the the offset each sample tube 21 assigned breakaway forces in time manifest, so that the sample tube 21 gradually from its Recesses are dissolved 27th The effort and the Material stress is kept at a low level and the ease of use increases.

Control and regulation of the thermal cycler

A control and regulating device of the thermal cycler 18 over 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 area is by means of temperature sensors 65, 66, 67 and detected through a temperature interface 68 to the slave processor 73 supplied. be the master processor 72 (interface to the user) among other things the temperature setpoint values, the time reference values, the number of Temperature cycles and the speed of the heating and cooling operations entered.

It can already predetermined, stored temperature / time profiles be selected and removed. The input is the keyboard 16 or other interface. This data is the slave processor 73 supplied, which a power controller 71 controls via a controller 69, which in turn the power supply of the heating elements 35, 52 and the Peltier element 36 regulates. The feedback (actual values) be the Slave processor 73 to master processor 72 is supplied to and processed or displayed to the user. In this way, the user of which is current sample temperature, which already reached temperatures Time stamp, and the still-to-reach temperatures according to the time informed.

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

The temperature of the sample is calculated from the temperature of the thermoblock 33 calculated. For this, the transfer function is the Sample space intended to sample in test tube 21st This feature is essentially a low pass with dead time.

Using appropriate control algorithms (scanned systems) is each of the calculated manipulated variable, which is necessary to maintain the temperature of the sample the predetermined setpoint temperature nachzuführen. These calculations be performed with a signal processor. The calculated manipulated variable is fed to the power controller 71 in the form of a pulse width. Of the Power controller 71 for example is a power FET with a matching Protective suppression circuit.

The control and control described above enables the use of the thermal cycler to sample in a vessel engaged in thermal cycler to heat the sample tube ring for specific temperature profiles and to cool. The temperature profiles are defined by plateau temperatures defined duration, and the gradient that defines the time, in a plateau-temperature must be reached. Condition is that all samples in the thermal cycler at the same time the same temperatures have.

In FIG. 7, for example, temperature variations of a Cycle process shown. Curve A shows the temperature profile at Heater 33, the curve B shows the temperature curve of the liquid in the reaction vessel 21. The thermal cycler Temperatures are adjustable from 40 to 98 degrees Celsius. Typically, the lower temperatures between 50 and 60 degrees Celsius and the upper Temperatures from 90 to 96 degrees Celsius. When the average Temperature is used, it is around 72 degrees Celsius. The with the Thermal cycler scored heating / cooling rate is 1 degree Celsius per second. A typical cycle has a duration of 120 seconds. If the corresponding temperatures are kept longer than 10 seconds need, the cycle time is extended accordingly.