HK1116824A - Instrument for heating and cooling - Google Patents

Description

Apparatus for heating and cooling

Technical Field

The subject of the invention is an instrument for heating and cooling an object in a controlled manner, a method for achieving a thermal profile, a method for amplifying nucleic acids, a system for heating a liquid and a system for determining 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.

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.

Methods for heating compositions of matter are also known. For example, in US2002/0061588 a method of heating a nucleic acid by attaching the nucleic acid to a nanoparticle and applying energy to the nanoparticle is described. With this heat, the nucleic acid hybrid on the surface of the modulator is denatured, and one of the strands can be separated into the surrounding liquid. However, this method is inefficient in terms of heating and amplification.

At present, heating of PCR mixtures is mainly performed with Peltier elements with active heating and cooling. These peltier elements require complex electronics compared to systems with active heating and passive cooling.

A method of heating a mixture containing a dye using a pulsed laser is disclosed in US 2004/0129555.

A method for heating microtubes (microtubes) with resistive or inductive heating is disclosed in US 6633785.

A micromachined reaction chamber made of silicon is disclosed in US 6602473. The device has inlet and outlet ports and can be used to perform PCR reactions when inserted into an instrument. The system does not allow for sensitive and rapid temperature control.

In WO98/39487 an appliance for holding a device having a chamber is disclosed, the appliance comprising heating or cooling plates arranged on opposite side walls of a flat device when the device is inserted into the appliance.

The temperature variations provided by the prior art instruments are relatively slow. Thus, there is a need for more rapid amplification of nucleic acids.

Disclosure of Invention

A first subject of the invention is an apparatus for heating and cooling an object in a controlled manner, comprising, in the following order:

a substantially flat temperature sensor element;

a substantially flat rigid thermally conductive substrate;

a substantially flat resistive heater;

a substantially flat barrier layer; and

and cooling the element.

A second subject of the invention is a method for implementing a thermal profile in an apparatus, comprising:

heating and cooling the device in the apparatus of the invention.

A third subject of the invention is a system for determining nucleic acids in a sample, comprising an apparatus according to the invention and a device comprising said sample.

A fourth subject of the invention is a method for amplifying a nucleic acid, comprising:

a) providing a mixture of liquids comprising nucleic acids in a device in an apparatus of the invention; and

b) subjecting the sample in the device to thermal cycling.

A fifth subject of the invention is a system for heating a mixture, comprising:

a device comprising one or more chambers for containing the mixture; and

the instrument of the present invention.

Drawings

Figure 1 schematically shows an example assembly of the instrument of the invention.

FIG. 2 shows the temperature profile set on the instrument to generate the PCR curve.

Figure 3 shows in graphical form the results of two experiments carried out by means of the apparatus of the invention.

Reference numbers:

device for loading a sample 2 in an instrument 1, protective layer 3 for a sensor

Temperature sensor structure/component 4 heat conducting substrate 5 heater 6

Heater blanket 7 thermal isolation layer 8 cooling element 9

Heat transfer surface 10

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. This enables a very sensitive detection of the target nucleic acid.

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.

This process can be carried out using the apparatus of the present invention. For this purpose, the sample containing the nucleic acids is contained as a cooled and heated object in the chamber of a device which has been inserted or is to be inserted into the apparatus of the invention. Hereinafter, the more general term "object" will be replaced by the preferred and exemplary term "device".

The first component of the instrument of the invention is at least one substantially flat temperature sensor element. In this context, substantially flat means that the sensor comprises a surface which does not rise above 1mm, more preferably above 0.1mm, above its average surroundings. This has the advantage that the surface of the device to be heated is in good thermal contact with the sensor element and adjacent layers. It is designed to measure the temperature at the location where it is arranged. Such elements are well known to those skilled in the art and are preferably resistive elements. 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, such as Heraeus Sensor Technology (Kleinos the im, Germany), JUMO GmbH & Co. KG (Fulda, Germany) or Innovative Sensor Technology IST AG (Wattwil, Switzerland). The element has a connector for permanently or reversibly connecting the sensor element to an electrical wire leading to the control unit. The sensor element can be manufactured according to known methods. 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.

Preferably, the temperature sensor element is protected against mechanical and chemical damage by a cover layer. The cover layer is preferably made of glass, preferably with a thickness between 1 μm and 25 μm. It is preferably fabricated by thick film deposition techniques well known in the art. Furthermore, the layer preferably has a low electrical conductivity and a high thermal conductivity.

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 device containing the sample. Preferably, the contact surface of the sensor element comprising the protective cover layer is in close contact with the contact surface of the device. Due to this defined arrangement of the instrument and the device, the temperature in the sample can be deduced with great certainty from the temperature measured in the sensor element.

The results of the temperature measurement are used to control the heating and cooling process in the instrument.

The second essential component of the instrument of the invention is 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. Furthermore, the substrate is flat since 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 third essential component of the apparatus of the invention is a heater. The heater is preferably substantially flat, more preferably a resistance heater. Such heaters are well known in the art. Preferably, the heater is a layer of a material having a high electrical resistance, for example selected from the groupSelecting: ruthenium oxide, silver, gold, platinum, copper, palladium, or other metals. The material is most preferably ruthenium oxide. Preferably, the thickness of this layer is preferably between 10 μm and 30 μm, more preferably between 15 μm and 20 μm. Preferably, the heater has a heating intensity of 15 and 40W/cm²In the meantime.

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.

The fourth basic component is a substantially flat insulating layer which separates the heater from the cooling element. The function of the insulating layer is to provide a suitable heat transfer from the heater to the cooling element and vice versa. On the one hand, the heat transfer must be high enough to ensure adequate cooling of the device, and therefore of the sample, by the layers separating it from the cooling element, but on the other hand, the heat transfer must be low enough not to hinder rapid heating of the sample by the heater during the transient phase (heat ramp) from lower to higher temperatures. The thickness of the isolation layer may be adapted to achieve a higher heating ramp section and a lower cooling ramp section, or vice versa. Therefore, the thermal conductivity should be between 0.5W/(m × K) and 2W/(m × K), more preferably between 0.8W/(m × K) and 1.2W/(m × K). These values obviously depend on the power density of the heater, the thickness of the insulating layer and the cooling capacity of the cooling element. Preferred values for these parameters are heater power densities of 40W/cm²The thickness of the spacer layer was 200 μm.

The material of the barrier layer is preferably selected from the group consisting of: epoxy or glass-ceramic. More preferred are combinations of specific materials, such as alumina particles in epoxy resins. Most preferred are alumina compositions in a glue or epoxy. Preferably, the particulate material is uniformly distributed in the layer.

This layer can in turn be applied in a known manner on an adjacent layer. Preferably, the material is applied as a paste and then sintered between 150 and 180 ℃ for 1.5 to 2 hours to cure. Such epoxy-based layers can withstand temperatures of up to 450 ℃.

In certain aspects, the barrier layer may also be comprised of multiple layers. In a particular embodiment, the isolation layer comprises three layers, wherein the first and third layers are the isolation layers described above and the intermediate layer is an intermediate layer composed of a thermally conductive material such as a metal, ceramic, crystal, or polymer (e.g., aluminum, copper, steel, aluminum oxide, aluminum nitride, silicon carbide, silicon nitride, sapphire, diamond, polyimide). Such an embodiment is advantageous because two such thin isolation layers can be applied technically more easily and shows an increased manufacturing tolerance by this simplified manufacturing method. Furthermore, the intermediate layer is preferably made of a heat conducting material, thereby ensuring a uniform temperature distribution in the layers, also indicating an increased manufacturing tolerance by the simplified manufacturing method.

The fifth essential component of the apparatus of the invention is a cooling element. The purpose of the cooling element is to efficiently conduct heat away from other layers, in particular the insulating layer. The cooling element is therefore preferably formed from a metal, such as aluminium, in the form of a block with a large surface in order to enhance the flow of thermal energy 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 a preferred embodiment, the heater is protected by a cover layer on the side facing the isolation layer. The cover layer may be made of a material selected from the group consisting of: ceramics, glass ceramics and polymers, preferably made of glass ceramics.

The device used with the apparatus and/or method of the invention is a container for holding a mixture containing a sample under the conditions of the method. Thus, the device should be heat resistant to the amount and type of heat provided to the mixture, resistant to the reagents contained in the mixture, and sealed so that the mixture cannot escape from the container.

The basic components of the assembled exemplary instrument 1 are shown in fig. 1, along with some preferred additional components. The device 2 contains a sample. It is arranged in close proximity to the heat transfer surface 10 of the protective cover 3 of the sensor. The sensor element 4 separates the substrate 5 from the device. The adjacent element is a heater 6. The heater cover layer 7 protects the heater and is opposite the thermal isolation layer 8 and the cooling element 9.

The use of the apparatus of the invention preferably involves the efficient control of temperature to ensure the achievement of a temperature profile, preferably the achievement of repeated temperature cycling, which is useful for thermal cycling (e.g. in PCR). The temperature and heat control preferably comprises:

-measuring the temperature of said sample in the device using a sensor element;

-comparing the measured temperature with the temperature that is desired to be reached in the sample;

-applying heat to the sample via the heater element to increase the temperature when the temperature of the sample is below the desired temperature, or to maintain the temperature in the sample when the temperature of the sample is the same as the desired 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. The unit for controlling the heater is called a heat controller.

Due to the flat sensor element, the measurement of the temperature is very fast and does not require a large amount of electronics. The algorithm required for comparing the measured temperature with the desired temperature is also quite simple and a simple PID (proportional-integral-derivative) control algorithm known in the art is sufficient.

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 (for example by pulse width modulation), or 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, this is done by using a control unit contained in the instrument that receives temperature measurements from the sensor and commands the heater to not heat or to heat continuously or intermittently until the desired temperature is reached. More specifically, the temperature in the liquid can be determined using measurements made by a temperature sensor in contact with the device containing the sample and knowledge of the physical state of the interaction. In order to control the desired temperature profile in the liquid over time, the PID control algorithm will set the required heating/cooling power in order to obtain the correct temperature at the desired point in time, taking into account the desired temperature and the temperatures measured at the minimum time intervals. The temperature sensor in contact with the device containing the sample will detect the temperature in a known manner, i.e. for the sensor in contact with the device, the detected temperature is proportional to the designed transverse temperature intensity distribution over the entire contact surface. Mechanical contact between the instrument and the device is considered insufficient when a lower than expected temperature is measured at the sensor in contact with the device. When the measured temperature and the expected temperature are related to each other, the mechanical contact is considered to be in operation. In another embodiment, a second sensor element may be used to determine the temperature in the sample and assess contact between the instrument and the device. In this case, the resolution of the measured temperature is twice that of the case with only one sensor, and the risk of obtaining an inappropriate temperature in the sample is significantly reduced.

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 of the present invention is preferably a resistance 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 device in such a way that the device is inside the coil, depending on the design of the coil.

Another embodiment of the invention is a method for implementing a thermal profile in an apparatus, comprising:

heating and cooling the device in the apparatus of the invention.

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.

Preferably, the method of the invention further comprises cooling the device by means of a cooling element of the type described above. Preferably, the cooling is performed by subjecting the apparatus, more preferably the cooling element, to a flow of a fluid, preferably a gas (e.g. air) for a fin structure or a buried heat pipe.

Another embodiment of the invention is a system for determining nucleic acid in a sample comprising an apparatus of the invention and a device containing or designed to receive said sample. This system can be used in the method of the invention. Thus, a system preferably comprises reagents and consumables for performing the assay, and optionally may be automated by including a robot for handling the device and/or sample. The device can be inserted in the system in order to ensure proper application of current and cooling capacity to the components of the instrument and ultimately heating and cooling the device in its active position.

Another embodiment of the present invention is a method for amplifying a nucleic acid, comprising: providing a mixture of liquids comprising said nucleic acid in a device of the apparatus of the invention; and subjecting the sample in the device to thermal cycling.

Another embodiment of the invention is a system for heating a mixture, the system comprising: a device comprising one or more chambers for containing said mixture; and an apparatus according to the invention.

Example 1

Manufacture of the apparatus of the invention

In a first step, a thin-film temperature sensor element, which is made of platinum and is commercially available from Heraeus, is coated on a ceramic substrate, which is commercially available from CeramTec AG (Plochingen, Germany). Here, the ceramic substrate was made of alumina and had a thickness of 635 μm. The sensor element is protected by a cover layer made of glass-ceramic with a thickness of 20 μm. This step is carried out on a coating machine known in the art. In a second manufacturing step, the thick film heater is made 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 of the substrate. The thick film layer is also protected by a cover layer made of glass-ceramic and having a thickness of 20 μm. Once the substrate is at least coated with the thin-film layer, it can be processed in a further step which determines the thickness of the isolating layer and thus the thermodynamic behaviour. 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. The cooling block is made of copper and comprises a flow channel with an inlet and an outlet for fluid cooling, and is in contact (thermal bonding) with a barrier layer of a multi-compound (with adhesive properties) that still remains viscous. In the thermal bonding step at a temperature of 180 deg.c, the cooling block is bonded to the heater layer side with a spacer layer of a certain thickness.

Example 2

PCR Using the Instrument of the invention

Using the thermocycler described in example 1, multiple PCR runs were performed by the commercially available Light Cycler Parvo B19 kit (Cat No 3246809, 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 a template. The temperature profile shown in FIG. 2 was set to generate a PCR curve. The temperature slope is chosen such that the PCR efficiency is still good, but the thermocycler will handle a much faster slope, e.g. 20 ℃/s.

The results of two experiments are shown in fig. 3 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).

Claims (16)

1. An apparatus for heating and cooling an object in a controlled manner, the apparatus comprising in the following order:

a substantially flat temperature sensor element;

a substantially flat rigid thermally conductive substrate;

a substantially flat resistive heater;

a substantially flat barrier layer; and

and cooling the element.

2. The instrument of claim 1, wherein the sensor element comprises a resistive element and a cover layer, the cover layer protecting the resistive element from direct contact with the environment, and the cover layer having a thickness between 1 μ ι η and 25 μ ι η.

3. The apparatus of claim 2, wherein the cover layer has an object contact surface that reflects a surface shape of the object and faces the sensor contact surface of the object.

4. An instrument according to any preceding claim, wherein the sensor element has a thickness of between 0.01 μm and 10 μm, preferably between 0.8 μm and 1.2 μm.

5. An instrument according to any preceding claim, wherein the thermally conductive substrate is between 0.1mm and 10mm thick.

6. An instrument according to any preceding claim, wherein the thermally conductive substrate is made of an electrically isolating material.

7. An instrument as claimed in any preceding claim, wherein the heater is between 10 μm and 30 μm thick.

8. An apparatus according to any preceding claim, further comprising a thermal controller.

9. A method for implementing thermal profiling in an apparatus, comprising:

heating the device in an apparatus as claimed in any one of claims 1 to 8.

10. The method of claim 9, further comprising cooling the device.

11. The method of claim 10, wherein the cooling is performed by subjecting the instrument to a flow of fluid.

12. The method of claim 11, wherein the fluid is a liquid or a gas.

13. The method of any of claims 9 to 12, wherein the thermal profile comprises repeated thermal cycling.

14. A system for determining nucleic acid in a sample, comprising the instrument of any one of claims 1 to 8 and a device containing the sample.

15. A method for amplifying a nucleic acid, comprising:

a) providing a mixture of liquids comprising nucleic acids in a device in an apparatus according to any one of claims 1 to 8; and

b) subjecting the sample in the device to thermal cycling.

16. A system for heating a mixture, comprising:

a device comprising one or more chambers for containing the mixture; and

an apparatus as claimed in any one of claims 1 to 8.