HK1114809A

HK1114809A - Temperature sensor element for monitoring heating and cooling

Info

Publication number: HK1114809A
Application number: HK08109976.1A
Authority: HK
Inventors: Sarofim Emad; Savatic Goran
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2006-07-14
Filing date: 2008-09-08
Publication date: 2008-11-14

Description

Temperature sensor element for monitoring heating and cooling

Technical Field

The subject of the present invention is a system comprising a cartridge and a device for heating and cooling a mixture in a controlled manner, a device for heating a cartridge comprising a chamber, a method for achieving a thermal profile in a device and a method for amplifying nucleic acids.

Background

The present invention is particularly useful in the field of health care where reliable sample analysis of components contained therein is required. Chemical reactions require heating, as is known, for example, from molecular diagnostics, where it is known that nucleic acids will be denatured, i.e., changed from double-stranded hybrids to single-stranded, by heating above the melting temperature of the hybrids. An important aspect here is the control and monitoring of the heating and cooling of the sample, since the precision in these steps is a prerequisite for the method to be accurate.

One method that utilizes the reaction cycle, including the denaturation step, is the Polymerase Chain Reaction (PCR). This technology revolutionized the field of nucleic acid processing, particularly nucleic acid analysis, by providing a means to increase the amount of nucleic acid of a particular sequence from a negligible amount to a detectable amount. PCR is described in EP0201184 and EP 0200362. An instrument that utilizes heating and cooling of an extended metal block to thermally cycle a sample in a tube in a controlled manner is disclosed in EP 0236069.

It is known in the art to monitor the heating and cooling of the cartridge and reaction chamber with thermal sensors.

US patent application US2003/0008286 discloses a device consisting of a plastic chip containing an array of reaction chambers. After all chambers are filled with reagents, the chip is pressed up against the substrate with a set of temperature balances between the chip and the substrate. Separately controlled heaters and sensors located between the block and the substrate allow each chamber to follow its own thermal behavior while being well insulated from the other chambers and substrates. Thus, the heater and sensor may be located at the bottom of the block and not facing the reaction chamber, or at the top of the block, with a small block of high thermal conductivity mounted on top of the first block. A disadvantage of such an arrangement of heater and sensor is that the temperature of the liquid in the chamber can only be determined indirectly by measuring the temperature of the thermally conductive mass. Furthermore, the temperature is not measured over the entire cross-section of the chamber.

WO98/38487 discloses an assembly having a chemical reaction chamber for receiving a sample and allowing the sample to undergo a chemical reaction, and a thermal sleeve having a heating element for making effective thermal contact with the reaction chamber. The temperature of the chamber may be monitored by one or more temperature sensors located on the thermal sleeve and on the trailing edge. However, a disadvantage of such an arrangement of heaters and sensors is that the temperature is determined locally in a small portion of the reaction chamber, rather than over the entire cross-section of the chamber, and therefore the result of the measurement is not representative of the temperature prevailing in the reaction chamber.

Thus, in the field of monitoring temperatures in reactions and/or thermal cycles, which typically involve heating and/or cooling of a liquid, it is the use of thermal sensors (which measure temperature outside of a sample-containing chamber) and algorithms (which are used to interpolate and correlate the measured temperature of the thermal sensor and the temperature in the sample) to determine the temperature of the liquid indirectly. It is therefore an object of the present invention to provide a system and device comprising a thermal sensor element having improved properties for determining the temperature in a liquid sample.

Disclosure of Invention

A first subject of the invention is a system for heating and cooling a mixture in a controlled manner, comprising a cartridge and a device,

the cartridge comprises at least:

a chamber for containing the mixture; and

a contact surface for contacting the device, having a chamber contact surface and a cartridge

A body-contacting surface;

the device comprises at least one stack of layers on top of each other from top to bottom in the following order:

a first substantially flat temperature sensor element;

a heat conductive substrate; and

a heater layer;

characterized in that the sensor element is positioned on a surface of the heat conducting substrate of the device and is directed towards the contact surface of the cartridge when the device and the cartridge are in physical contact, thereby enabling the first sensor element of the device to physically interact with the cartridge body contact surface or the chamber contact surface of the cartridge.

A second subject of the invention is a device for heating in a controlled manner a cartridge comprising a chamber, said device comprising at least, from top to bottom, one on top of the other in the following order:

at least one substantially flat temperature sensor element;

a heat conductive substrate; and

a heater layer;

the method is characterized in that: the sensor element is positioned on a surface of the thermally conductive substrate, the sensor element forming a surface area allowing the sensor element to physically interact with a contact surface of a cartridge when the sensor element is brought into physical contact with the cartridge.

A third subject of the invention is a method for implementing and controlling a thermal profile in a system comprising:

-heating a cartridge containing a mixture in a chamber by means of a system according to any one of claims 1 to 13; and

-controlling the heating process and the temperature of the mixture with the first and/or second sensor element of the apparatus.

A fourth subject of the invention is a method for amplifying nucleic acids using a system according to any one of claims 1 to 13, comprising:

a) providing a sample comprising nucleic acids in a chamber of the cartridge;

b) subjecting the sample in the chamber of the cartridge to thermal cycling.

A fifth subject of the invention is an instrument for performing biological assays comprising heating a sample in a controlled manner, comprising at least a system according to any one of claims 1 to 13, wherein said device is located within the instrument to allow a determined and predetermined physical interaction with said cartridge when said cartridge is inserted into the instrument and brought into contact with said device.

Drawings

Fig. 1 shows a cross-sectional view of the basic components of an exemplary system C of the present invention, which includes a cartridge a and a device B.

Fig. 2 shows a cross-sectional view through the basic components of an exemplary system C, wherein a cartridge a contains a plurality of cartridge chambers 2 for containing a mixture, and a device B comprises a first sensor element 12 and a second sensor element 13 for each chamber of the cartridge.

Fig. 3 shows a cross-section through two different exemplary embodiments of the device according to the invention.

FIG. 4 illustrates a top view of various exemplary embodiments of an apparatus.

FIG. 5 illustrates a closed loop PID (proportional-integral-derivative) control algorithm adjustment (with z-transform equation) required to compare the measured temperature to the specified temperature.

FIG. 6 shows the temperature profiling setup used to generate the PCR curve to perform PCR by the commercially available LightCycler ParvoB19 toolkit (Roche Diagnostics GmbH, Germany).

Figure 7 shows the results of two experiments using the Light Cycler ParvoB19 kit.

An example of all required temperatures for the regulation algorithm is shown in fig. 8.

Reference numbers:

box 1 cover layer 3 of box 2 for containing mixture and box 4

Contact surface 5 for contacting a device a chamber contact surface 6

The cartridge body contact surface 7 means 11 the first sensor element 12

The second sensor element 13 is used as a cover layer 14 for the sensor element

Heat conducting substrate 15 heater layer 16 conductors 17

Sensor element 18 combined sensor/heater element 19

Detailed Description

Methods for amplifying nucleic acids are known. They will generate a large amount of nucleic acid based on the originally present target nucleic acid as a template because the activity of the enzyme is able to replicate the base sequence in the target nucleic acid. The replicon itself serves as a target for a replication sequence (preferably a base sequence that has been replicated for the first time). Thus, a huge amount of nucleic acids having the same sequence are produced.

A particularly well-known method for amplifying nucleic acids is the Polymerase Chain Reaction (PCR) method, which is disclosed in EP 0200362. In this method, a reaction mixture is subjected to repeated cycles of a thermal profile at a temperature suitable for annealing primers to the target nucleic acid, extending the annealed primers using said target nucleic acid as a template, and separating the extended products from their templates.

In a first step, a liquid comprising nucleic acids is provided. The liquid may be any liquid containing the nucleic acid to be amplified. Furthermore, the liquid contains reagents required for amplifying the nucleic acid. For each amplification method, these reagents are well known and preferably include reagents for extending the primer, preferably a template dependent DNA-or RNA-polymerase and a building block (e.g., a nucleotide) to which the primer will be attached for extension. Furthermore, the mixture will contain reagents for establishing the extension reaction conditions, such as buffers for the enzymes used and co-factors, such as salts.

In a further step, the temperature is adjusted to denature double-stranded nucleic acids, a primer is annealed to the single strand, and the annealed primer is extended. The extension reaction will be carried out at a temperature at which the polymerase is active. Preferably, a thermostable and thermoactive polymerase is used. The double strands formed are separated by denaturation as described above.

In diagnostic applications of PCR methods, in particular in rapid PCR methods, there are very high requirements on the precision and accuracy of these methods and the instruments used for these methods. Therefore, for the instrument, the accuracy of the sample temperature in the sample chamber during repeated cycling of the thermal profile should be closely monitored using thermal sensors, and the rapid, sufficient and accurate heating and/or cooling of the sample, particularly in rapid PCR methods.

The present invention provides a system, an apparatus, an instrument and methods having improved characteristics for monitoring the temperature in a liquid mixture. To this end, the mixture (e.g. comprising nucleic acids) is contained in a chamber of a cartridge which has been or is to be brought into contact with a device and which is an object for performing cooling and heating sequences, which comprises at least one thermal sensor for monitoring these cooling and heating sequences.

Such a system for heating and cooling a mixture in a controlled manner according to the invention comprises at least a cartridge and a device. The cartridge and the device are formed to move relative to each other to allow a defined and predetermined physical interaction of the cartridge and the device.

Here, the cartridge comprises a chamber for containing the mixture and a contact surface for contacting the device. A portion of the contact surface serves as a chamber contact surface. The chamber contact surface of the device is located at a position where the cartridge chamber makes the predetermined physical interaction with the device. The other part of the contact surface serves as a cartridge contact surface. The cartridge body contacting surface of the device conveys the physical interaction of the device with the body of the cartridge and/or the shelves and is located outside the chamber contacting surface.

The device (as an object for performing a cooling and heating sequence) comprises at least a first substantially flat temperature sensor element, a heat conducting substrate and a heater layer. The heater layer of the present invention comprises a substantially flat resistive heater. Such heaters are well known in the art. The heater layer is preferably made of a material with a high electrical resistance, for example selected from the group of: ruthenium oxide, silver, copper, gold, platinum, palladium, or other compatible metals, electrical conductors, or alloys thereof. The most preferred material is ruthenium oxide. Preferably, the thickness of this layer is between 10 μm and 30 μm, more preferably between 15 μm and 20 μm. The heating layer is preferably prepared by coating or screen printing a paste of the material in a specific shape and heating the composition to a temperature sufficient to sinter the specific material. Preferably, the material thus adheres to the layer on which it is sintered.

Preferably, the heater element is protected against mechanical and chemical damage by a cover layer. The cover layer is preferably made of glass or glass ceramic and is preferably between 1 μm and 25 μm thick. It is preferably manufactured by thick film deposition as is well known in the art. In addition, the layer preferably has a relatively low electrical conductivity and a relatively high thermal conductivity.

In this context, the substantially flat temperature sensor element is designed to measure the temperature at the location where it is arranged. These elements are well known to those skilled in the art and are preferably resistive elements composed of a material having a relatively high resistance, such as ruthenium oxide, platinum, gold, silver, nickel or palladium.

Particularly advantageous sensors have a thickness of between 0.01 μm and 10 μm, preferably between 0.8 μm and 1.2 μm. One example Sensor element available on the market is 1 μm thick and is available from manufacturers that manufacture thin film temperature sensors, such as Heraeus Sensor Technology (Kleinostheim, germany) or JUMO GmbH & co. These elements have connectors for permanently or reversibly connecting them to the electrical wires leading to the control unit. The sensor element can be manufactured according to known methods, for example thin-layer technology. It can be manufactured separately and then fixed to the other component by known means, for example gluing. Preferably, the sensor element is manufactured by sputtering a layer of material onto the satellite layer. This method for applying thin layers is also known. Preferred materials for the sensor element are nickel and platinum. Preferably, it is made from platinum or a mixture of platinum with other precious metals. In a preferred embodiment, the sensor element has a bifilar structure. The temperature sensor mainly comprises a long resistance wire. In this context, a bifilar structure means that the wire is bent such that two adjacent and substantially parallel portions of the wire conduct electricity in opposite directions. Thus, the current in both directions will have the same intensity. The superposition of the two opposing magnetic fields around two adjacent portions of the line is zero. So no magnetic field is emitted or absorbed.

Preferably, the temperature sensor element is protected against mechanical and chemical damage by a cover layer. In addition, the cover layer preferably has low electrical conductivity and high thermal conductivity. The cover layer is preferably made of glass, preferably with a thickness between 1 μm and 25 μm. It is preferably manufactured by thick film technology. Thus, the interaction of the sensor element of the device with the contact surface of the cartridge may be performed directly between the material forming the sensor element and the material of the cartridge or indirectly when the sensor element and/or the cartridge is covered with a cover layer.

The temperature sensor element is preferably designed to be sufficiently correlated with the temperature of the sample. This can be achieved by designing the shape of the element such that it closely resembles the shape of the sample-containing chamber of the cartridge. Preferably, in a particular embodiment comprising a protective cover layer, the contact surface of the sensor element is in close contact with the contact surface of the device. Due to the defined arrangement of the cartridge and the device, the temperature in the sample can be very determined and accurately deduced from the temperature measured in the sensor element. The temperature measurements are used to control the heating and cooling process in an instrument comprising the cartridge and the device.

The apparatus of the present invention further comprises a substantially flat, rigid and thermally conductive substrate. The substrate is preferably made of a material having a thermal conductivity of 2 x 10³And 5X 10⁶W/m²And K is formed by materials. And, saidThe substrate is flat because its thickness is preferably between 0.1 and 10mm, more preferably between 0.25 and 2 mm. The substrate has rigid characteristics, i.e., is stable to significant mechanical distortion. Furthermore, the heat conductive substrate is preferably made of a material having an electrical conductivity of less than 0.1 Ω^-1m^-1Is manufactured from the electrically isolating material of (1). Furthermore, it is preferred that the substrate has characteristics with a low thermal time constant (density x heat capacity/thermal conductivity), preferably less than 10⁵s/m². Suitable materials are selected from the group consisting of: aluminum, copper, aluminum oxide, aluminum nitride, silicon carbide, sapphire, copper, silver, gold, molybdenum, and brass. More preferred are materials having a lower conductivity, e.g. electrically isolating materials, e.g. having a conductivity of less than 10^-9Ω^-1m^-1The material of (1). Particularly advantageous materials are therefore ceramic materials, such as aluminum oxide, aluminum nitride, silicon carbide and sapphire. The substrate may also be manufactured according to known methods. Preferably, the substrate is made by sintering a ceramic. The substrate may be prepared in a form similar to the shape of the substrate, preferably in a reusable form, or may be broken into pieces of appropriate size after the sintering process.

The heat conducting substrate of the device of the invention has the advantage of increased flexibility in order to tailor the thermal behaviour of the device. For example, the thermally conductive substrate may be selected to be thermally or thermally insulating and to affect electrical conductivity and/or mechanical stability. Mechanical stability will be important in view of the forces to be applied in order to enable good thermal contact between the sensor element and the cartridge to be measured. In particular, the heat conducting substrate may also be made of an electrically isolating material. In some embodiments, the sensor elements are located on a surface of the thermally conductive substrate facing the contact surface of the cartridge. In other embodiments, the sensor element of the device of the system described above may also be used as a heater element. Here, the sensor element acts as a combined sensor/heater element and is able to detect the temperature in the chamber and to apply heat to the chamber with a short delay when the temperature in the chamber is below a specified temperature. Preferably, such a combined sensor/heater element is made of platinum or nickel. However, these combined sensor/heater elements typically have a lower heat capacity than the dedicated heater layers described above, because the combined sensor/heater layer is thinner than the thick film heater, i.e. results in a proportionately reduced cross-section and therefore a limited current density. At excessive currents, the combined sensor/heater element wire may break or fall off the substrate. These embodiments are suitable for applications where the temperature remains substantially stable throughout the application (e.g., isothermal applications). Furthermore, these embodiments have the advantage that the lateral heat density distribution can be measured and the same active correction is performed more or less simultaneously on the same area.

In a first embodiment of the invention, a system for heating and cooling a mixture in a controlled manner includes the cartridge and apparatus described above. Here, the sensor element is located on a surface of the device facing the cartridge contact surface, such that the cartridge body contact surface is able to interact with the sensor element.

In another embodiment of the system according to the invention the sensor element is located on a surface of the device facing the cartridge contact surface, such that the chamber contact surface is capable of interacting with the sensor element. In a particular embodiment, the shape of the sensor element is very similar to the shape of the cartridge chamber containing the sample. This embodiment enables the measurement of thermal images of the cartridge chamber by the sensor element of the device and enables the averaging of the temperature at the interface between the cartridge chamber and the chamber contact surface of the device. Furthermore, this embodiment enables monitoring whether the contact between the cartridge chamber and the chamber contact surface of the device extends over the entire interface or whether a part of the cartridge chamber is not in physical contact with the chamber contact surface of the device.

In a particular embodiment of the system according to the invention, the sensor element is located on a surface of the device such that the entire surface of the sensor element is in contact with the cartridge in the region of the chamber contact surface of said cartridge, and the surface of the sensor element represents at least 10%, preferably at least 25%, more preferably at least 40% of the chamber contact surface. In yet another embodiment, the sensor element surface in contact with the chamber contact surface may not be entirely filled with the sensor structure, but may also be formed as a ring or other shape suitable for imaging and averaging the temperature within the liquid in terms of a laterally extending thermal intensity distribution, geometrical characteristics and mechanical rigidity or deformability of the chamber seal. The surface of the sensor element is here considered to be the upper part of the sensor element, which upper part faces the cartridge and essentially forms a physical interaction with the cartridge when the device and the cartridge are in contact. Also, the surface may be formed by a material forming the sensor element, or by a cover layer covering the sensor element. This embodiment allows measuring most of the thermal image of the cartridge chamber with the sensor element of the device in order to obtain a representative average of the temperature over the whole interface between the cartridge chamber and the chamber contact surface of the device.

In another embodiment the device of the system comprises at least two sensor elements, wherein a first sensor element is located on a surface of the device enabling interaction of the chamber contact surface with the first sensor element and a second substantially flat temperature sensor element is located on a surface of the device enabling simultaneous interaction of said cartridge body contact surface with said second sensor element. An advantage of this embodiment is that the average temperature over the whole interface between the cartridge chamber and the chamber contact surface of the device can be determined more accurately. In a particular embodiment, the two sensors may be used to measure a laterally extending temperature gradient across the contact surface between the cartridge and the device. This embodiment has the advantage that the laterally distributed heat intensity gradient over the contact surface can be monitored and the temperature difference in the liquid in the chamber can be compensated for by taking into account the gradient, so that the temperature within the liquid can be more accurately determined and maintained.

In certain embodiments of the system of the present invention, the cartridge may comprise a plurality of chambers for holding the mixture. In the system, the device further comprises a plurality of sensor elements. Preferably, the sensor elements are arranged such that for each chamber of the device a first sensor element is located on a surface of the device facing the contact surface of the cartridge, thereby enabling the cartridge body contact surface or chamber contact surface of a particular chamber to interact with the first sensor element. Thus, in this embodiment, the device comprises one sensor element for each chamber of the cartridge. Furthermore, in some embodiments, a first sensor element for each chamber is located on a surface of the device that enables the particular chamber contact surface to interact with the first sensor element, and a second substantially flat temperature sensor element is located on a surface of the device that enables the cartridge body contact surface to simultaneously interact with the second sensor element. Here, it is preferred that the first sensor element may be substantially similar to the shape of the specific chamber. The latter device may also be used to detect and heat multiple cartridges with one device, each cartridge comprising one chamber. In all these embodiments, it is important that the determined interaction of the cartridge or cartridges with the device will be very accurate and precise in being in a predetermined position. These embodiments of the system are used to perform multiple reactions in parallel simultaneously within the same system in controlled and monitored ways in different chambers, and thus can be used for high throughput applications. In this way, lateral heat flow can be monitored and compensated for even when the device comprises a plurality of sensors or sensor pairs on the same thermally conductive substrate, and the sensors are in contact with one cartridge having a plurality of chambers, or with a plurality of cartridges each having at least one chamber.

In the system of the invention, the heater layer of the device is preferably made of the same material as the sensor element or of a material that can be processed under similar manufacturing conditions as the sensor element. In particular, materials such as platinum, nickel or mixtures of platinum or nickel with other noble metals may be used. In certain embodiments, the heater layer is less than 30 μm thick. In one particular embodiment, the heater and sensor elements may be positioned in the same layer. An advantage of this embodiment is that the device comprising the heater and the sensor element in the same layer can be manufactured with relatively low complexity and relatively easy. In such an embodiment, the heater and sensor elements, although embedded in the same layer, may be two distinct and separate components mounted on the layer.

The device of the present invention also includes a thermally conductive substrate. The sensor element is located on a surface of the heat conducting substrate facing the contact surface of the cartridge. The heater may be located on the same surface of the heat conducting substrate as the sensor element or, in a preferred embodiment, on the opposite surface of the heat conducting substrate to the sensor element, in both embodiments the sensor element is directed towards the contact surface of the cartridge. Thus, the heater and/or the sensor element may be mounted on the thermally conductive substrate by the above-described method, or may be embedded in the surface of the material forming the thermally conductive substrate.

In a particular embodiment, the sensor element may be used as a heater, thereby enabling the detection of temperature and subsequent application of heat to the mixture as a heater layer by a heat pulse when the temperature is below a specified temperature. Furthermore, the first substantially flat sensor element and the heater layer may be combined to form one combined sensor/heater element. In this embodiment, the heater layer is identical to the first substantially flat temperature sensor element, and thus, the combined heater/sensor element can be used to alternate heating and temperature sensing cycles. This embodiment is particularly advantageous when used in applications where only few heating operations are required (e.g., in isothermal applications where it is particularly desirable to maintain a constant temperature), where the combined heater/sensor element senses the temperature in the chamber of the cartridge and can be used for shorter heating pulses when the temperature in the chamber is below a specified temperature.

In a preferred embodiment, the system of the present invention is used to amplify nucleic acids in a sample.

A cross-sectional view through the basic components of an exemplary system of the present invention is shown in fig. 1. Fig. 1A shows a cross-sectional view of a cartridge 1, the cartridge 1 having a chamber 2 for containing a mixture and a cartridge body 4. The box further comprises: a covering layer 3, wherein the covering layer 3 covers the box body and the chamber and has the functions of protection and heat conduction; and a contact surface 5 for contacting the device. The contact surfaces include a chamber contact surface 6 and a cartridge contact surface 7. Fig. 1B shows a cross-sectional view of a device 11, which device 11 comprises a first sensor element 12 and a second sensor element 13. These sensor elements are mounted to a heat conducting substrate 15 and protected by a cover layer 14 for the sensor elements. Furthermore, the device comprises a heater layer 16, which heater layer 16 is mounted on a surface of the heat conducting substrate 15, which heater layer 16 is located on a surface opposite to the sensor elements 12, 13. Figure 1C shows a cross-sectional view through the system when the cartridge 1 and the device 11 form a defined physical interaction. The contact between the cartridge 1 and the device 11 is made such that the first sensor element 12 interacts with the cartridge body contact surface 7 and the second sensor element 13 is located entirely within the chamber contact surface 6. Fig. 2 shows a cross-sectional view through the basic components of an exemplary system C, wherein the cartridge a contains a plurality of cartridge chambers 2 for containing a mixture, while the device B comprises a first sensor element 12 and a second sensor element 13 for each chamber of the cartridge. Fig. 2A shows a cross-sectional view of a cartridge 1, the cartridge 1 having two chambers 2 for containing a mixture and a cartridge body 4. The box further comprises: a cover layer 3 which covers the box body and the chamber and has protection and heat conduction functions; and a contact surface 5 for contacting the device. The contact surfaces include a chamber contact surface 6 and a cassette contact surface 7 for each particular chamber. Fig. 2B shows a cross-sectional view of the device 11, the device 11 comprising a first sensor element 12 and a second sensor element 13 for each chamber of the cartridge. These sensor elements are mounted on a heat conducting substrate 15 and protected by a cover layer 14 for the sensor elements. Furthermore, the device comprises a heater layer 16, which heater layer 16 is mounted on a surface of the heat conducting substrate 15 and arranged on a surface opposite to the sensor elements 12, 13. Figure 2C shows a cross-sectional view through the system when the cartridge 1 and the device 11 form a defined physical interaction. The contact between the cartridge 1 and the device 11 is made such that each first sensor element 12 interacts with the cartridge body contact surface 7 and each second sensor element 13 is located entirely within the chamber contact surface 6 of each particular chamber.

Another embodiment of the invention is an apparatus for heating a cartridge containing a chamber in a controlled manner, the apparatus comprising: at least one substantially flat temperature sensor element arranged parallel to a cross-section of the cartridge chamber; a heat conductive substrate; and a heater layer. Wherein the sensor element is located on a surface of the heat conducting substrate facing the chamber of the cartridge, thereby enabling the cartridge to interact with the sensor element. The heater layer may be located on the same surface of the heat conducting substrate as the sensor element or, in a preferred embodiment, the heater is located on the opposite surface of the heat conducting substrate to the sensor element, with the sensor element facing the chamber of the cartridge. In a particular embodiment, the sensor element is substantially similar to the cross-sectional shape of the cartridge chamber. This embodiment is advantageous because it enables the thermal image of the cartridge chamber to be measured by the sensor element of the device when the sensor element is in physical contact with the cartridge chamber and enables the temperature of the entire interface between the cartridge chamber and the sensor element of the device to be averaged. Furthermore, this embodiment enables monitoring whether the contact between the cartridge chamber and the sensor element of the device extends over the entire interface or whether a part of the cartridge chamber is not in physical contact with the chamber contact surface of the device. In a particular embodiment of the device, the sensor element extends along a cross-section of the cartridge chamber more than 10%, preferably more than 25%, more preferably more than 40% of the cross-section. Thus, when the sensor element and the cartridge chamber physically interact, the sensor element interacts with at least 10%, preferably at least 25%, more preferably at least 40% of the surface of the cartridge chamber. In yet another embodiment, the sensor element surface in contact with the chamber contact surface may not be entirely filled with the sensor structure, but may also be formed as a ring or other shape suitable for imaging and averaging the temperature within the liquid in terms of a laterally extending thermal intensity distribution, geometrical characteristics and mechanical rigidity or deformability of the chamber seal. Thus, the material forming the sensor element may be in direct contact with the material of the cartridge chamber or may be in indirect contact when the sensor element and/or the cartridge is covered with a cover layer. In a further embodiment the sensor element is of a bifilar construction and has a thickness of between 0.01 μm and 10 μm, preferably between 0.8 μm and 1.2 μm. The heat conducting substrate is preferably between 0.1mm and 5mm thick and may be made of an electrically isolating material.

Fig. 3 shows a cross-section through two different exemplary embodiments of the device according to the invention. Fig. 3A shows a cross-sectional view of a device comprising a first sensor element 12 and a second sensor element 13. The sensor elements are mounted on a heat conducting substrate 15 and protected by a cover layer 14 for the sensor elements. Furthermore, the device comprises a heater layer 16 mounted on the surface of the heat conducting substrate 15 and on the surface opposite to the sensor elements 12, 13. Fig. 3B shows a second embodiment of the device, which also comprises two sensor elements 12, 13. Unlike the first embodiment, the two sensor elements are embedded in the surface of the material forming the heat conductive substrate 15. The two sensor elements 12, 13 are protected by a cover layer 14 for the sensor elements, and a heater layer 16 is mounted on the surface of the heat conducting substrate 15 opposite the sensor elements.

Fig. 4 shows a top view of various exemplary embodiments of the device. In all the embodiments shown, the sensor elements have connectors 17 for permanently or reversibly connecting these elements with electrical wires leading to the control unit. In fig. 4A, the device comprises a first sensor element 12 and a second sensor element 13, which are located and mounted on a thermally conductive substrate, as described above. The first sensor element 12 is arranged to allow direct physical interaction of said first sensor element with the cartridge inside the cartridge body contact surface and outside the chamber contact surface, while the second sensor element 13 is arranged to allow direct physical interaction of said second sensor element within the chamber contact surface of the cartridge. The connectors 17 are shown in three different exemplary locations on the surface of the thermally conductive substrate 15, but may be randomly distributed on the surface to suit the particular requirements of the instrument in which the device is to be incorporated.

Fig. 4B shows a further embodiment of the device comprising only one sensor element. In the first diagram of fig. 4B, the device comprises a first sensor element 12, the first sensor element 12 being positioned such that the sensor element is capable of direct physical interaction with the cartridge body contacting surface of the cartridge. In the second figure, the device comprises a first sensor element 12, the first sensor element 12 being positioned such that it is capable of direct physical interaction in the chamber contact surface of the cartridge. In a particular embodiment, the first sensor element substantially resembles the cross-sectional shape of the cartridge chamber. In the third figure, the cartridge contains two sensor elements 18, the two sensor elements 18 having the same function as one sensor element, but being independently controllable and operable. The two sensor elements 18 are arranged to enable direct physical interaction in the chamber contact surface of the cartridge. An advantage of the latter embodiment is the redundancy of the sensor elements. Thus, when one sensor element is inoperable, the temperature may still be detected by the second sensor element. This embodiment is preferred for applications where accuracy of temperature is highly required. Furthermore, one of the sensor elements may be used as a sensor element, while the second sensor element may be used as a heater.

Fig. 4C shows an embodiment of the device in which the first sensor element is constituted by two or more sensor elements 18 which can be controlled and operated independently, the first sensor element being positioned such that it enables a direct physical interaction of the first sensor element within the chamber contact surface of the cartridge. This embodiment also has the advantage of redundancy of the sensor elements. Thus, when one sensor element is inoperable, the temperature may still be detected by the other sensor elements. Also, one of the sensor elements may be used as a heater, and the other sensor elements may be used as sensor elements.

The first diagram of figure 4D shows an embodiment of the device comprising a first sensor element 12 positioned to enable direct physical interaction of the first sensor element with the cartridge inside the cartridge body contact surface and outside the chamber contact surface, and a combined sensor/heater element 19. This embodiment is advantageously used for applications where multiple cycles with large temperature differences are performed and the heater layer is not sufficient to perform this heating in the right time. Here, the combined sensor/heater element may be used as a backup and backup. In a particular embodiment, the combined sensor/heater element 19 is a heater layer of the device. This embodiment is advantageously used for applications where only a small temperature difference needs to be applied (e.g. isothermal applications). An embodiment of the device is shown in the second diagram of fig. 4D, in which the first sensor element is formed by two or more separate sensor elements.

Another embodiment of the invention is a method for implementing and controlling a thermal profile in a system, comprising:

-heating a cartridge containing a mixture in a chamber by means of the device of the system of the invention; and

-controlling the heating process of the mixture and the temperature of the mixture with the first and/or second sensor element of the apparatus.

The thermal profile is the series of temperatures to be achieved in the sample. Preferably, all temperatures of the profile are above room temperature, more preferably between 37 and 98℃, most preferably between 40 and 96℃. The profile may be an ascending profile, wherein the temperatures increase over time, or a descending profile, wherein the temperatures decrease over time. Most preferred is a profile with maximum and minimum temperatures, i.e. temperature increase and decrease. In the most preferred embodiment of the invention, the thermal profile comprises repeated thermal cycling, which is required for PCR. These thermal cycles will include: a maximum temperature that allows the double-stranded nucleic acid to be denatured into single strands; and a minimum temperature, thereby allowing annealing of single-stranded nucleic acids into double strands. In yet another embodiment, the thermal profile may be a rising profile, wherein the temperatures rise over time and will remain constant for a determined period of time at one or more determined temperature levels. This embodiment can be used, for example, to melt and denature double or multiple strands of DNA by the application of heat and to determine a DNA melting curve. In another embodiment, the thermal profile may be a constant profile, wherein the temperature will remain constant for a determined period of time at one or more determined temperature levels. This embodiment can be used for isothermal applications, such as rolling circle amplification of polymerases (e.g., Phi 29).

The heating process and the temperature of the mixture contained in the chamber of the cartridge are controlled by means of sensor elements to ensure the performance of a temperature profile, preferably the performance of repeated temperature cycles (such as thermal cycles used in e.g. PCR), comprising:

-measuring the temperature of the mixture in the chamber of the cartridge with the first and/or second sensor element of the device;

-comparing the measured temperature with a specified temperature that is desired to be reached in the mixture;

-applying heat to the mixture through the heater layer to increase the temperature when the temperature of the mixture is below a specified temperature, or to maintain the temperature in the mixture when the temperature of the mixture is the same as the specified temperature.

Thus, in a very preferred mode, the invention comprises controlling and adjusting the heating process by means of a computer program in dependence on the temperature of the liquid. A unit for controlling the heater, comparing the measured temperature with a designated temperature, and applying heat to the mixture is referred to as a heat control unit. Here, the thermal control unit includes at least an applicator/active input to the system (i.e., heater/cooler), a sensor (i.e., temperature sensor element), and a closed-loop algorithm (e.g., PID) for adjusting the temperature to a specified level. The algorithm required to compare the measured temperature with the specified temperature is rather simple and straightforward. Thus, a PID (proportional-integral-derivative) control algorithm known in the art, which includes a formula for describing the physical interaction between the device and the liquid in the cartridge chamber, can be used for the closed-loop regulation. This closed-loop PID regulation with z-transform equation is illustrated in fig. 5, where "h" is a time interval, e.g. 5ms, 10ms, 20ms, 50ms, 100ms, "Ti" is an integration time constant for the PID regulator "k (z)", and "Td" is a differentiation time constant of the PID regulator. In the formula of the PID controller k (z), "Kp" is a proportional term, "ui (z) ═ h/Ti)/(z-1)" is an integral term, and "ud (z) ═ Td (z-1)/(h x z)" is a differential term. The Z-transform equation is an equation in the frequency domain that describes a discrete function in the frequency domain. The laplace transform is used to transform the analog equation from the time domain to a corresponding analog function in the frequency domain.

Preferably, the software used in the analytical instrument of the invention reads out the signal (e.g. the temperature sensor signal) at predetermined time intervals, so that only discrete time information can be processed. Therefore, a continuous analog function in the frequency domain must be transformed into a discrete form in the frequency domain. The resulting function (discrete form in the frequency domain) can itself be easily changed back to a discrete recursive function in the time domain.

This enables to check the stability of the regulator k (z) and the physical interaction h (z) of the thermocycler with the liquid in the chamber in the z-transform of the closed loop function cl (z). Also, the function formed in the discrete time domain is a recursive function. This recursive form is very advantageous for the algorithm when combined with a PID regulator (see also "Control Systems Engineering (third edition), Norman s. Therefore, when the temperature sensor is read out at fixed time intervals, the heating/cooling power of the heater layer can be measured.

The temperature of the liquid in the chamber of the cartridge can be determined using the measurement data of the sensor element when the sensor element is in physical contact with the contact surface of the cartridge and taking into account the determined parameter describing the physical interaction of the cartridge with the device. In order to control the time-varying specified temperature profile in the liquid, the PID control algorithm will set the required heating/cooling power of the heater element so as to obtain the default temperature at the required point in time taking into account the specified temperature and the measured temperature of the last measured time interval mentioned above. In an embodiment comprising two sensor elements, the sensor element in contact with the chamber contact surface will detect the temperature in a known manner, i.e. for the sensor element in contact with the cartridge contact surface, its detected temperature is proportional to the designed lateral temperature intensity distribution over the entire contact surface. When a lower temperature than expected is measured at the sensor element of the chamber contact surface, the mechanical contact between the device and the chamber contact surface of the cartridge is considered insufficient. Whereas the mechanical contact between the device and the cartridge is considered to be not suitable when a lower temperature than expected is measured at the sensor element in contact with the cartridge body contact surface. Thus, an analysis instrument comprising the system will output error information for increasing the reliability of the analysis results at the beginning of the measurement. On the other hand, the mechanical contact is considered to be in operation when the temperatures measured by the two sensor elements are both related to each other and are both within the expected range. Thus, in an embodiment comprising two sensor elements, the resolution of the measured temperature is twice as high as when there is only one sensor element, and therefore the risk of undesired aberrations in the temperature in the liquid is significantly reduced. The system is therefore used for internal control of mechanical contacts and results in more reliable results, which is particularly important for in vitro diagnostic applications.

Moreover, when the sensor element is particularly flat, the temperature measurement is very fast and does not require a large number of electronic components.

The heat can be applied in any known manner by means of a heater, for example by continuously applying an electric current to a resistive heater, or by inputting the heat in current pulses, or by using an alternating current. The pulse length or current magnitude required to achieve a particular temperature increase can be determined in simple experiments by determining the temperature in an example sample and varying the current magnitude and/or pulse length for a given cooling capacity.

Preferably, the heating is performed by contact heating. Contact heating is heating in which a heat medium is brought into contact with a material to be heated so that energy can flow from the heat medium to the material through a contact surface therebetween. The heater layer of the present invention is preferably a resistive heater. The resistance heating utilizes the effect that the resistance of the small-diameter wire generates energy loss due to heat when current flows therethrough. One preferred design is a heating coil with a predetermined resistance for resistance heating. The coil may be formed by a wire or it may be designed in other ways, for example as a conductor of any material on a printed circuit board or on a substrate, for example ceramic or polyimide. One option is that the coil is formed on a suitable substrate by thin film or thick film techniques. The coil may be located at the bottom, top or side of the container or even surround the box in such a way that the box is inside the coil, depending on the design of the coil.

Preferably, the method of the present invention further comprises cooling the cartridge. Preferably, said cooling is performed by subjecting the system, more preferably a cooling element comprised in the system, to a flow of a fluid, preferably a gas (e.g. air) for the fin structure or the buried heat pipe. The purpose of the cooling element is to efficiently conduct heat away from the system, and in particular the device. The cooling element is therefore preferably made of a good thermal conductor, for example a ceramic compound or a metal such as aluminium, in the form of a block with a large surface in order to increase the heat energy flow into the surrounding environment. The surface can be increased by providing fins on the metal block (passive cooling), optionally by increasing convection around the cooling element by a fan (active cooling). Instead of fins, liquid (e.g. water) cooling may be used, or a buried (in metal block) heat pipe with fins at the other end may be utilized.

In another embodiment of the present invention, there is provided a method for amplifying nucleic acid using the system of the present invention, which comprises:

-providing a sample comprising nucleic acids in a chamber of the cartridge;

-subjecting the sample in the chamber of the cartridge to thermal cycling.

Another embodiment of the invention is an instrument for performing a biological assay comprising heating a sample in a controlled manner, the instrument comprising at least a system of the invention, wherein said device is positioned in the instrument so as to be capable of a predetermined physical interaction with said cartridge when said cartridge is inserted into the instrument and in contact with said device. The apparatus may further comprise: an excitation unit and a detection unit for analyzing a sample contained in the cartridge and performing a heating operation; as well as reagents and consumables, for performing the assay, and optionally the instrument may also be automated by including a robot for processing the cartridge and/or the sample. In the instrument, the cartridge and the device are brought into physical contact in a defined manner so as to ensure that the cartridge and the device perform a suitable predetermined physical interaction. Thus, the sensor element of the device is positioned in contact with the cartridge in the contact surface (chamber contact surface or cartridge body contact surface).

In yet another embodiment, the system of the present invention is used to implement and monitor thermal gradient profiles on the cartridge.

Example 1

Manufacture of the device of the invention

In a first step, two thin-film temperature sensor elements made of platinum and available from the company Heraeus are coated on a ceramic substrate made of alumina, which is available from the company CeramTec AG (Plochingen, germany). Here, the ceramic substrate was made of alumina, had a thickness of 635 μm, and was protected by a protective layer made of glass ceramic (having a thickness of 20 μm). This step is performed on a coating machine.

In a second fabrication step, the thick film heater is constructed on the other, opposite side of the substrate. To this end, a thin film of ruthenium oxide (thickness 20 μm) was coated on the opposite side. The thick film layer is also protected by a protective layer, which is also made of glass-ceramic (thickness 20 μm). Once the substrate is at least coated with a thin film layer, it can be processed in a further step which determines the thickness of the isolating layer and thus the thermodynamic behaviour of the isolating layer. The barrier layer was deposited in a definite shape on the protective layer of the heater by means of a screen printing method known in the art to form a thickness of 100 μm by means of an Epoxy glue solution available from Epoxy Technology Inc. Also, a cooling block is mounted on the isolation layer. The cooling block is brought into contact with a barrier layer of a multi-compound (with adhesive properties) which still remains viscous. In the final sintering step at a temperature of 180 c, the cooling block is glued to the heater layer side with a determined thickness of the isolating layer.

Example 2

Using the system of the present invention to achieve PCR and monitor the temperature of the liquid in the cartridge chamber

Using the thermocycler described in example 1, various PCR runs were performed by the commercially available Light Cycler Parvo B19 kit (Cat No3246809, Roche diagnostics GmbH, Germany) for real-time PCR detection, following the instructions provided by the manufacturer in the kit, and using the Light Cycler Parvo B19Standard as template. The temperature profile shown in FIG. 6 was set to generate a PCR curve. The temperature slope is chosen such that the PCR efficiency is still good, while the thermocycler will handle a much faster slope, e.g. 20 ℃/s.

The results of two experiments are shown in fig. 7 in the form of curves measured on a test plate with the thermocycler, using the temperature sensor, and using a test plate real-time fluorescence photometer capable of exciting and measuring the fluorescent substances described in the Light Cycler ParvoB19 kit (Roche Diagnostics GmbH, germany).

In fig. 8, an example of all required temperatures for a PID (proportional-integral-derivative) temperature regulation algorithm (as shown in fig. 5) is shown as part of one particular cycle (see the first cycle in fig. 6). Two temperature sensors, set temperature and real-time predicted temperature calculated on the instrument are shown. The temperatures measured by the two sensor elements show similar deviations from the set temperature, indicating that the thermal contact between the chamber contact surface and the sensor contact surface is in acceptable operating conditions. After all temperatures were recorded, the real-time predicted temperature in the liquid-filled chamber was verified by recalculating the predicted temperature. The real-time predicted temperature and the recalculated predicted temperature are well matched, i.e. the adjustment algorithm works correctly.

Claims

1. A system for heating and cooling a mixture in a controlled manner, comprising a cartridge and a device,

the cartridge comprises at least:

a chamber for containing the mixture; and

a contact surface for contacting the device, having a chamber contact surface and a cartridge contact surface;

a first substantially flat temperature sensor element;

a heat conductive substrate; and

a heater layer;

2. The system of claim 1, wherein the first sensor element is located on a surface of the device allowing physical interaction of the first sensor element with the chamber contact surface, and a second substantially flat temperature sensor is located on a surface of the device allowing physical interaction of the second sensor element with the cartridge body contact surface simultaneously.

3. The system of any one of claims 1 and 2, wherein the first sensor element is located on a surface of the device allowing physical interaction of the first sensor element with the chamber contact surface of the cartridge and is substantially similar to the shape of the chamber.

4. A system according to any one of claims 1 to 3, wherein the first sensor element is located on a surface of the device so as to allow physical interaction of the first sensor element with the chamber contact surface of the cartridge, the surface of the first sensor element being in contact with the cartridge throughout the extent of the chamber contact surface and the surface of the first sensor element occupying at least 10% of the chamber contact surface.

5. The system of any one of claims 1 to 4, wherein the first and/or second sensor elements have a bifilar structure.

6. The system of any one of claims 1 to 5, wherein the first and/or second sensor element also acts as a heater element.

7. The system of any one of claims 1 to 6, wherein any one of the sensor elements comprises a resistive element and a cover layer protecting the resistive element from direct contact with the environment and having a thickness of less than 25 μm.

8. The system according to any one of claims 1 to 7, wherein the thickness of the sensor element is between 0.01 μm and 10 μm, preferably between 0.8 μm and 1.2 μm.

9. The system of any one of claims 1 to 8, wherein the thickness of the thermally conductive substrate is between 5mm and 0.1 mm.

10. The system of any one of claims 1 to 9, wherein the thermally conductive substrate is made of an electrically insulating material.

11. A system according to any one of claims 1 to 10, wherein the heater has a thickness of less than 30 μm and is located on a surface of the thermally conductive substrate opposite the sensor element with the sensor element facing the contact surface of the cartridge.

12. The system of any one of claims 1 to 10, wherein the first substantially flat sensor element is combined with the heater layer to form one combined sensor/heater element.

13. A system according to any one of claims 1 to 10, wherein the first sensor element is constituted by two or more sensor elements which can be controlled and operated independently, the first sensor element being arranged to enable direct physical interaction of the first sensor element within the chamber contact surface of the cartridge when the device is brought into physical contact with the cartridge.

14. An apparatus for heating a cartridge comprising a chamber in a controlled manner, said apparatus comprising at least from top to bottom one on top of the other in the following order:

at least one substantially flat temperature sensor element;

a heat conductive substrate; and

a heater layer;

15. The apparatus of claim 14, wherein the sensor element is substantially similar to the cross-sectional shape of the chamber.

16. The apparatus of any one of claims 14 to 15, wherein the sensor element extends along the cross-section over 10% of the cross-section.

17. The device of any one of claims 14 to 16, wherein the sensor element has a bifilar structure.

18. The device according to any of claims 14 to 17, wherein the thickness of the sensor element is between 0.01 μm and 10 μm, preferably between 0.8 μm and 1.2 μm.

19. The apparatus of any one of claims 14 to 18, wherein the substrate has a thickness of between 5mm and 0.1 mm.

20. The system of any one of claims 14 to 19, wherein: the heat conducting substrate is made of an electric insulating material.

21. A method for implementing and controlling thermal profiles in a system, comprising:

heating a cartridge containing a mixture in a chamber by means of a system according to any one of claims 1 to 13; and

the heating process and the temperature of the mixture are controlled by means of the first and/or second sensor elements of the device.

22. The method of claim 21, wherein controlling the heating process and the temperature of the mixture with the sensor element comprises:

measuring the temperature of the mixture in the chamber of the cartridge with the first and/or second sensor element of the device;

comparing the measured temperature to a specified temperature that the mixture is intended to reach; and

heat is applied to the mixture through the heater layer to increase the temperature when the temperature of the mixture is lower than a designated temperature, or to maintain the temperature in the mixture when the temperature of the mixture is the same as the designated temperature.

23. The method of claim 22, wherein the comparing of the measured temperature to the specified temperature and the applying of heat to the mixture are performed by a heat control unit.

24. The method of any of claims 21 to 23, further comprising cooling the cartridge.

25. The method of claim 24, wherein the cooling is performed by subjecting the system to a flow of a fluid, the fluid being a liquid or a gas.

26. The method of any one of claims 21 to 25, wherein the thermal profile comprises repeated thermal cycles.

27. A method of amplifying nucleic acids using the system of any one of claims 1 to 13, comprising:

a) providing a sample comprising nucleic acids in a chamber of the cartridge;

b) subjecting the sample in the chamber of the cartridge to thermal cycling.

28. An instrument for performing a biological assay comprising heating a sample in a controlled manner, the instrument comprising at least a system according to any one of claims 1 to 13, wherein the device is located within the instrument to allow a defined and predetermined physical interaction with the cartridge when the cartridge is inserted into the instrument and brought into contact with the device.

29. Use of the system of any one of claims 1 to 13 for achieving a thermal profile in a device.

30. Use of the system of any one of claims 1 to 13 for amplifying nucleic acids in a sample.